EP1297149A2

EP1297149A2 - Gpsa gene from corynebacteria and use thereof in synthesis of l-amino acids

Info

Publication number: EP1297149A2
Application number: EP01962683A
Authority: EP
Inventors: Madhavan Nampoothiri; Brigitte Bathe; Lothar Eggeling; Hermann Sahm
Original assignee: Forschungszentrum Juelich GmbH; Degussa GmbH
Current assignee: Forschungszentrum Juelich GmbH; Evonik Operations GmbH
Priority date: 2000-07-01
Filing date: 2001-05-15
Publication date: 2003-04-02
Also published as: AU2001283819A1; WO2002002777A2; WO2002002777A3

Abstract

This invention relates to a coryneform bacterium, the gpsA gene of which is enhanced, to an isolated polynucleotide which encodes glycerol-3-phosphate dehydrogenase from coryneform bacteria, and also to a process for the fermentative production of L-amino acids with enhancement of the gpsA gene in the bacteria.

Description

New nucleotide sequences which encode the gpsA gene

This invention relates to nucleotide sequences which encode glycerol-3-phosphate dehydrogenase, and to coryneform bacteria in which the gpsA gene which encodes glycerol-3-phosphate dehydrogenase is enhanced, and also relates to a process for the fermentative production of L- a ino acids, particularly L-glutamate, using coryneform bacteria in which the gpsA gene is enhanced.

Amino acids, particularly L-lysine and L-glutamate, are used in human medicine, in the pharmaceutical industry, in the food industry, and are used in particular in animal nutrition.

It is known that amino acids are produced by the fermentation of strains of coryneform bacteria, particularly Corynebacterium glutamicυm . Due to the considerable importance of these amino acids, attempts are continuously being made to improve the production process. Process improvements can involve fermentation technology measures, such as stirring and supplying with oxygen for example, or can relate to the composition of the culture media, such as the sugar concentration during fermentation for example, or to work-up to give the desired form of product by ion exchange chromatography for example, or to the intrinsic production properties of the microorganism itself.

Methods of mutagenesis, selection and mutant selection are employed in order to improve the production properties of these microorganisms. In this manner, strains are obtained which are resistant to antimetabolites, such as the lysine analogon S- (2-aminoethyl) -cysteine for example, or which are auxotrophic for metabolites of regulatory importance, and which produce L-amino acids such as L-lysine or L- gluta ate for example. Moreover, for some years methods of recombinant DNA technology have been used to improve strains of Corynebacterium which produce amino acids. This has been achieved by amplifying individual amino acid biosynthesis genes and investigating the effect on amino acid production. Review articles on this topic, amongst other sources, are those by Kinoshita C"Glutamic Acid Bacteria", in: Biology of Industrial Microorganisms, Demain and Solomon (Eds.), Benjamin Cummings, London, UK, 1985, 115- 142), Hilliger (BioTec 2, 40-44 (1991)), Eggeling (Amino Acids 6:261-272 (1994)), Jetten and Sinskey (Critical Reviews in Biotechnology 15, 73-103 (1995)) and Sahm et al. (Annals of the New York Academy of Science 782, 25-39 (1996) ) .

The object of the present invention was to provide new process adjuvants for improving the fermentative production of amino acids, particularly L-lysine and L- glutamate.

This object is achieved by a coryneform bacterium, the gpsA gene of which, which encodes glycerol-3-phosphate dehydrogenase, is enhanced.

Amino acids, particularly L-lysine and L-glutamate, are used in human medicine, in veterinary medicine, in the pharmaceutical industry and particularly in the food industry. There is therefore a general interest in the provision of new, improved processes for producing amino acids, particularly L-glutamate.

When L-lysine or lysine or L-glutamate or glutamate are mentioned below, this means not only the bases but also the salts thereof.

The present invention relates to a coryneform bacterium, in which the gpsA gene thereof, which encodes glycerol-3- phosphate dehydrogenase, is enhanced. In this connection, the term "enhancement" means increasing the intracellular activity of one or more enzymes in a microorganism which are encoded by the corresponding DNA.

Enhancement can be achieved with the aid of various manipulations of the bacterial cell.

In order to achieve enhancement, particularly over- expression, the number of copies of the corresponding gene can be increased, a strong promoter can be used, or the promoter- and regulation region or the ribosome binding site which is situated upstream of the structural gene can be mutated. Expression cassettes which are incorporated upstream of the structural gene act in the same manner. In addition, it is possible to increase expression in the course of the fermentative production of L-lysine- or L- glutamate by employing inducible promoters . A gene can also be used which encodes a corresponding enzyme with a high activity. Expression can also be improved by measures for extending the life of the m-RNA. Furthermore, enzyme activity as a whole is increased by preventing the degradation of the enzyme. Moreover, these measures can optionally be combined in any desired manner.

The microorganisms to which the present invention relates can produce L-amino acids, particularly L-lysine and L- glutamate, from glucose, saccharose, lactose, fructose, maltose, molasses, starch or cellulose, or from glycerol and ethanol. They can be representatives of coryneform bacteria, particularly of the genus CoryneJacterium. A bacterium of the genus CoryneJbacterium which should be mentioned in particular is the Corynebacterium glutamicum species, which is known to those skilled in the art for its capacity of producing L-amino acids.

Examples of suitable strains of the genus Corynebacterium, particularly of the Corynebacterium glutamicum species, are the known wild-type strains Corynebacterium glutamicum ATCC13032 Corynebacterium acetoglutamicum ATCC15806 Corynebacterium acetoacidophilum ATCC13870 Corynebacterium thermoaminogenes FERM BP-1539 Corynebacterium melassecola ATCC17965

Brevibacterium flavum ATCC14067 Brevibacterium lactofermentum ATCC13869, and Brevibacterium divaricatum ATCC14020,

and L-lysine-producing mutants or strains which are produced therefrom, such as

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 DSM5715.

The present invention further relates to a polynucleotide which is isolated from coryneform bacteria, containing a polynucleotide sequence selected from the group comprising

a) a polynucleotide, at least 70 % of which is homologous with a polynucleotide which encodes a polypeptide which contains the amino acid sequence of SEQ ID No.2,

b) a polynucleotide which encodes a polypeptide which comprises an amino acid sequence, at least

70 % of which is homologous with the amino acid sequence of SEQ ID No. 2,

c) a polynucleotide which is complementary to the polynucleotides of a) or b) , and

d) a polynucleotide containing at least 15 successive nucleotides of the polynucleotide sequence of a) , b) or c) . In the context of the present Application, a polynucleotide sequence is "homologous" with the sequence according to the invention if at least 70%, preferably at least 80%, most preferably at least 90% of its base composition and base sequence corresponds to the sequence according to the invention. According to the invention, a "homologous protein" is to be understood to comprise proteins which contain an amino acid sequence at least 70 % of which, preferably at least 80 % of which, most preferably at least 90 % of which, corresponds to the amino acid sequence which is encoded by the gpsA gene (SEQ ID No.l), wherein "corresponds" is to be understood to mean that the corresponding amino acids are either identical or are mutually homologous amino acids. The expression "homologous amino acids" denotes those which have corresponding properties, particularly with regard to their charge, hydrophobic character, steric properties, etc.

The present invention also relates to a polynucleotide as described above, which is preferably a replicable DNA containing:

(i) the nucleotide sequence shown in SEQ ID No. 1, or

(ii) at least one sequence which corresponds to sequence

(i) in the context of the degeneration of the genetic code, or

(iii) at least one sequence which hybridizes with the sequence complementary to sequence (i) or (ii) , and optionally

(iv) functionally neutral mutations in (i) which result in the same or a homologous amino acid.

The present invention further relates to

a replicable polynucleotide which comprises or consists of the nucleotide sequence of SEQ ID No. 1, a polynucleotide sequence which encodes a polypeptide which comprises or consists of the amino acid sequence of SEQ ID No. 2,

a vector containing the DNA sequence of C. glutamicum which encodes the gpsA gene, contained in the vector (plasmid) pJClgpsA deposited as a Corynebacterium glutamicum with the number DSM 13493,

and coryneform bacteria which serve as a host cell and which contain the vector or in which the gpsA gene is enhanced.

The present invention also relates to polynucleotides which contain the complete gene with the polynucleotide sequence corresponding to SEQ ID No. 1 or fragments thereof, and which can be obtained by screening by means of the hybridization of a corresponding gene bank with a probe which contains the sequence of said polynucleotide corresponding to SEQ ID No. 1 or a fragment thereof, and isolation of said DNA sequence.

Polynucleotide sequences according to the invention are suitable as hybridization probes for RNA, cDNA and DNA, in order to isolate the complete length of cDNA which encodes glycerol-3-phosphate dehydrogenase and in order to isolate those cDNAs or genes which exhibit a high degree of similarity to the sequence of the glycerol-3-phosphate dehydrogenase gene.

Polynucleotide sequences according to the invention are also suitable as primers for polymerase chain reaction (PCR) for the production of DNA which encodes glycerol-3- phosphate dehydrogenase.

Oligonucleotides such as these, which serve as probes or primers, can contain more than 30, preferably up to 30, more preferably up to 20, most preferably at least 15 successive nucleotides. Oligonucleotides with a length of at least 40 or 50 nucleotides are also suitable.

The term "isolated" means separated from its natural environment .

The term "polynucleotide" refers in general to polyribonucleotides and polydeoxyribonucleotides, and can denote an unmodified RNA or DNA or a modified RNA or DNA.

The term "polypeptides" is to be understood to mean peptides or proteins which contain two or more amino acids which are bound via peptide bonds.

The polypeptides according to invention include polypeptides corresponding to SEQ ID No. 2, particularly those with the biological activity of glycerol-3-phosphate dehydrogenase, and also includes those, at least 70 % of which, preferably at least 80% of which, are homologous with the polypeptide corresponding to SEQ ID No. 2, and most preferably those which exhibit a homology of least 90 % to 95 % with the polypeptide corresponding to SEQ ID No. 2 and which have said activity.

The invention also relates to a process for the fermentative production of L-amino acids, particularly L- lysine and L-glutamate, using coryneform bacteria which in particular already produce an amino acid and in which the nucleotide sequences which encode the gpsA gene are enhanced, and in particular are over-expressed.

In the present invention, the gpsA gene of C. glutamicum which encodes glycerol-3-phosphate dehydrogenase (EC 1.1.1.94) is demonstrated for the first time.

In order to isolate the gpsA gene or other genes of C. glutamicum, a gene bank of this microorganism is first of all constructed in E. coli . The construction of gene banks is described in generally known textbooks and handbooks. Examples thereof include the textbook by Winnacker: Gene und Klone, Eine Einfϋhrung in die Gentechnologie (Verlag Chemie, Weinheim, Germany, 1990) or the Handbook by Sambrook et al. : Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989) . One very well known gene bank is that of the E. coli K-12 strain W3110, which was constructed by Kohara et al. (Cell 50, 495-508 (1987)) in λ- ectors . Bathe et al . (Molecular and General Genetics, 252:255-265, 1996) described a gene bank of C. glutamicum ATCC13032, which with the aid of the cosmid vector SuperCos I (Wahl et al., 1987, Proceedings of the National Academy of Sciences USA, 84:2160-2164) was constructed in the E. coli K-12 strain NM554 (Raleigh et al., 1988, Nucleic Acids Research 16:1563-1575. Bormann et al. (Molecular Microbiology 6(3), 317-326 (1992)) in turn describe a gene bank of C. glutamicum ATCC13032 using the cosmid pHC79 (Hohn and Collins, Gene 11, 291-298 (1980)). Plasmids such as pBR322 (Bolivar, Life Sciences, 25, 807- ,818 (1979)) or pUC9 (Vieira et al., 1982, Gene, 19:259- 268) can also be used for the production of a gene bank of C. glutamicum in E. coli . Those E . coli strains which are restriction- and recombination-deficient are particularly suitable as hosts. One example thereof is the strain DH5αmcr, which was described by Grant et al. (Proceedings of the National Academy of Sciences USA, 87 (1990) 4645- 4649) . Long DNA fragments which are cloned with the aid of cosmids can subsequently again be subcloned in common vectors which are suitable for sequencing and can then be sequenced, as described by Sanger et al. (Proceedings of the National Academy of Sciences of the United States of America, 74:5463-5467, 1977).

In this manner, a new DNA sequence of C. glutamicum has been obtained which encodes the gpsA gene and which as SEQ ID No. 1 forms part of the present invention. Moreover, the amino acid sequence of the corresponding protein has been derived from the present DNA sequence using the methods described above. The resulting amino acid sequence of the gpsA gene product is illustrated in SEQ ID No. 2. The invention also relates to coding DNA sequences which result from SEQ ID No. 1 by degeneration of the genetic code. In the same manner, the invention further relates to DNA sequences which hybridize with SEQ ID No. 1 or with parts of SEQ ID No. 1. Moreover, one skilled in the art is also aware of conservative amino acid replacements such as the replacement of glycine by alanine or of aspartic acid by glutamic acid in proteins as "sense mutations" which do not result in any fundamental change in the activity of the protein, i.e. which are functionally neutral. It is also known that changes at the N- and/or C-terminus of a protein do not substantially impair the function thereof, and may even stabilise said function. Amongst other sources, one skilled in the art will find information on this topic in the articles by Ben-Bassat et al. (Journal of Bacteriology 169:751-757 (1987)), by O'Regan et al . (Gene 77:237-251 (1989)), by Sahin-Toth et al. (Protein Sciences 3:240-247 (1994)), by Hochuli et al . (Bio/Technology 6:1321-1325 (1988)) and in known textbooks on genetics and molecular biology. The present invention also relates to amino acid sequences which result in a corresponding manner from SEQ ID No. 2.

In the same manner, the present invention also relates to DNA sequences which hybridize with SEQ ID No. 1 or with parts of SEQ ID No. 1. Finally, the present invention relates to DNA sequences which are produced by polymerase chain reaction (PCR) using oligonucleotide primers which result from SEQ ID No. 1. Oligonucleotides of this type typically have a length of at least 15 nucleotides.

Amongst other sources, one skilled in the art will find instructions for the identification of DNA sequences by means of hybridization in the Handbook "The DIG System User' s Guide for Filter Hybridization" published by Boehringer Mannheim GmbH (Mannheim, Germany, 1993) and in the article by Liebl et al . (International Journal of

Systematic Bacteriology (1991) 41: 255-260). Amongst other sources, one skilled in the art will find instructions for the amplification of DNA sequences with the aid of polymerase chain reaction (PCR) in the Handbooks by Gait: Oligonucleotides synthesis: a practical approach (IRL Press, Oxford, UK, 1984) and by Newton and Graham: PCR

(Spektrum Akademischer Verlag, Heidelberg, Germany, 1994) .

The work which has been carried out on the present invention has enabled it to be ascertained that, after enhancement of their gpsA gene has been effected, coryneform bacteria produce amino acids, particularly L- lysine and L-glutamate, in an improved manner.

The genes or gene constructs concerned can either be present with different numbers of copies in plasmids, or can be integrated and amplified in the chromosome. Alternatively, over-expression of the gene concerned can be effected by changing the composition of the medium and by changing the way in which cultivation is effected.

Amongst other sources, one skilled in the art will find instructions on this topic in the articles by Martin et al. (Bio/Technology 5, 137-146 (1987)), by Guerrero et al. (Gene 138, 35-41 (1994)), by Tsuchiya and Morinaga (Bio/Technology 6, 428-430 (1988)), by Eikmanns et al. (Gene 102, 93-98 (1991)), in European Patent Specification EPS 0 472 869, in US Patent 4,601,893, in the articles by Schwarzer and Puhler (Bio/Technology 9, 84-87 (1991), by Reinscheid et al. (Applied and Environmental Microbiology 60, 126-132 (1994)), by LaBarre et al. (Journal of Bacteriology 175, 1001-1007 (1993)), in Patent Application WO 96/15246, in the article by Malumbres et al . (Gene 134, 15 - 24 (1993)), in Japanese laid-open Patent

Specification JP-A-10-229891, in the articles by Jensen and Hammer (Biotechnology and Bioengineering 58, 191-195 (1998)), by Makrides (Microbiological Reviews 60:512-538 (1996) ) and in known textbooks on genetics and molecular biology. For example, the gpsA gene according to the invention has been over-expressed with the aid of plasmids .

Suitable plasmids are those which are replicated and expressed in coryneform bacteria. Numerous known plasmid vectors such as pZl (Menkel et al., Applied and

Environmental Microbiology (1989) 64: 549-554), pEKExl (Eik anns et al., Gene 102:93-98 (1991)) or pHS2-l (Sonnen et al., Gene 107:69-74 (1991)) are based on the cryptic plasmids pHM1519, pBLl or pGAl . Other plasmid vectors, such as those which are based on pCG4 (US-A 4,489,160), or pNG2 (Serwold-Davis et al . , FEMS Microbiology Letters 66, 119-124 (1990)), or pAGl (US-A 5,158,891), can be used in the same manner.

One example of a plasmid with the aid of which the gpsA gene can be over-expressed is pJClgpsA (Figure 1) , which is based on the E. coli - C. glutamicum shuttle vector pJCl (Cremer et al., 1990, Molecular and General Genetics 220: 478 - 480) and which contains the DNA sequence of C. glutamicum which encodes the gpsA gene. This is contained in the strain DSM5715/pJClgpsA.

Also suitable are those plasmid vectors by means of which the process of gene amplification by integration in the chromosome can be employed, such as that described, for example, by Reinscheid et al. (Applied and Environmental Microbiology 60, 126-132 (1994)) for the duplication or amplification of the hom-thrb operon. In this method, the complete gene is cloned in a plasmid vector which can replicate in a host (typically E. coli ) , but which cannot replicate in C. glutamicum . Examples of suitable vectors include pSUP301 (Simon et al., Bio/Technology 1, 784-791

(1983)), pKlδmob or pK19mob (Schafer et al . , Gene 145, 69- 73 (1994)), pGEM-T (Promega Corporation, Madison, WI, USA), pCR2.1-TOPO (Shuman (1994). Journal of Biological Chemistry 269:32678-84; US-A 5,487,993), pCR^®Blunt (Invitrogen, Groningen, Holland; Bernard et al., Journal of Molecular Biology, 234: 534-541 (1993)) or pEMl (Schrumpf et al, 1991, Journal of Bacteriology 173:4510- 4516) . The plasmid vector which contains the gene to be amplified is subsequently converted by conjugation or transformation into the desired strain of C. glutamicum . The conjugation method is described, for example, by Schafer et al. (Applied and Environmental Microbiology 60, 756-759 (1994)). Transformation methods are described, for example, by Thierbach et al . (Applied Microbiology and Biotechnology 29, 356-362 (1988)), by Dunican and Shivnan (Bio/Technology 7, 1067-1070 (1989)) and by Tauch et al. (FEMS Microbiological Letters 123, 343-347 (1994)). After homologous recombination by means of a "cross over" occurrence, the resulting strain contains at least two copies of the gene concerned.

Moreover, apart from the gpsA gene, it may be advantageous for the production of amino acids, particularly L- glutamate, to intensify or over-express one or more genes which encode enzymes of the biosynthesis route employed, of glycolysis, of anaplerosis, of the citric acid cycle or of amino acid export.

Thus, for the production of L-lysine, for example, one or more genes selected from the following group can be simultaneously enhanced, and in particular can be over- expressed or amplified:

• the dapA gene which encodes dihydrodipicolinate synthase (EP-B 0 197 335), or

• the dapE gene which encodes succinyl diaminopimelate desuccinylase, or

• the lysC gene which encodes feed-back resistant aspartate kinase (Kalinowski et al. (1990), Molecular and General Genetics 224, 317-324), or • the gap gene which encodes glyceraldehyde-3-phosphate dehydrogenase (Eikmanns (1992), Journal of Bacteriology 174:6076-6086), or

• the tpi gene which encodes triose phosphate iso erase (Eikmanns (1992), Journal of Bacteriology 174:6076- 6086), or

• the pgk gene which encodes 3-phosphoglycerate kinase

(Eikmanns (1992), Journal of Bacteriology 174:6076- 6086) , or

• the pyc gene which encodes pyruvate carboxylase (DE-A- 19831609), or

• simultaneously, the mqo gene which encodes malate- quinone oxidoreductase (Molenaar et al., European Journal of Biochemistry 254, 395-403 (1998)), or

• the lysE gene which encodes lysine export (DE-A-195 48 222) .

Furthermore, for the production of L-glutamate, for example, one or more genes selected from the following group can be simultaneously enhanced, and in particular can be over-expressed or amplified:

• the gdh gene which encodes glutamate-dehydrogenase (DE: 19907347.3), and/or

• the pyc gene which encodes pyruvate carboxylase

(Peters-Wendisch et al.(1998), Microbiology 144: 915- 927) .

Moreover, for the production of L-lysine it may be advantageous if, in addition to the enhancement of the gpsA gene:

• the pck gene which encodes phosphoenol pyruvate carboxykinase (DE 199 50 409.1, DSM 13047) and/or • the pgi gene which encodes glucose-6-phosphate isomerase (US 09/396,478, DSM 12969)

is attenuated.

Furthermore, for the production of L-glutamate it may be advantageous if, in addition to the enhancement of the gpsA gene :

• the odhA gene which encodes α-ketoglutarate dehydrogenase (WO 9534672 Al 951221*), or

• the dtsRl gene which encodes DtsRl protein (WO 952324 Al 950831*) , or

• the dtsR2 gene which encodes DtsR2 protein (WO 9902692A Al 990121*) ,

is simultaneously attenuated.

Moreover, for the production of amino acids, particularly L-lysine and L-glutamate, it may be advantageous if, in addition to the over-expression of the gpsA gene, unwanted secondary reactions are suppressed (Nakayama: "Breeding of Amino Acid Producing Micro-organisms", in: Overproduction of Microbial Products, Krumphanzl, Sikyta, Vanek (Eds.), Academic Press, London, UK, 1982) .

The microorganisms which are produced according to the invention can be cultivated batch-wise or continuously, e.g. by a batch process (batch cultivation) or by a fed batch process (feed process) or by a repeated fed batch process (repetitive feed process) , for the purpose of producing amino acids, particularly L-glutamate. A review of known methods of cultivation is given in the textbook by Chmiel (Bioprozesstechnik 1. Einfiihrung in die Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991)) and in the textbook by Storhas (Bioreaktoren und periphere Einrichtungen (Vieweg Verlag, Brunswick/Wiesbaden, 1994) ) . The culture medium which is used must fulfil the requirements of the strains concerned in a suitable manner. Descriptions of culture media for various microorganisms are given in the Handbook "Manual of Methods for General Bacteriology" published by the

American Society for Bacteriology (Washington D.C., USA, 1981) . Suitable sources of carbon include sugar and carbohydrates such as glucose, saccharose, lactose, fructose, maltose, molasses, starch and cellulose, oils and fats such as soya oil, sunflower oil, peanut oil and cocoa fat, fatty acids such as palmitic acid, stearic acid and linoleic acid, alcohols such as glycerol and ethanol, and organic acids such as acetic acid. These substances can be used individually or in admixture. Suitable sources of nitrogen include compounds which contain organic nitrogen, such as peptone, yeast extract, meat extract, malt extract, corn steep liquor, soya bean flour and urea, and inorganic compounds such as ammonium sulphate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. These sources of nitrogen can be used individually or in admixture. Phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate, or the corresponding sodium-containing salts, can be used as sources of phosphorus. In addition, the culture medium must contain salts of metals such as magnesium sulphate or iron sulphate which are necessary for growth. Finally, essential growth promoting substances such as amino acids and vitamins can be used in addition to the aforementioned substances. Moreover, suitable precursors can be added to the culture medium. The aforementioned substances which are used can be added to the culture in the form of a single batch or can be supplied in a suitable manner during cultivation.

Basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or aqueous ammonia, or acidic compounds such as phosphoric acid or sulphuric acid are used in a suitable manner in order to control the pH of the culture. Anti-foa ing agents such polyglycol esters of fatty acids can be used to control the generation of foam. In order to maintain the stability of plasmids, suitable substances with a selective action, such as antibiotics, can be added to the medium. In order to maintain aerobic conditions, ^■ oxygen or oxygen-containing gas mixtures such as air are passed into the culture. The temperature of the culture normally ranges from^'20°C to 45°C and is preferably 25°C to 40°C. Cultivation is continued until a maximum of glutamate has been formed. This target is normally reached within 10 hours to 160 hours.

The following microorganism has been deposited in the German Collection of Microorganisms and Cell Cultures (DSMZ, Brunswick, Germany) in accordance with the Budapest Convention:

• Corynebacterium glutamicum strain DSM5715/pJClgpsA as DSM 13493.

The process according to the invention can be employed for the fermentative production of amino acids, particularly L-lysine and L-glutamate.

Legends to the Figure:

Figure 1: Map of the plasmid pJClgpsA

The numbers of base pairs are given as approximate values which can be obtained within the limits of reproducibility. The abbreviations and descriptions used have the following meanings:

Orf2,rep plasmid-coded replication origin C. glutamicum (of pHM1519) gpsA: gpsA (glycerol-3-phosphate dehydrogenase) gene from C. glutamicum ATCC13032

Kan: kanamycin-resistant gene Xbal: cleavage site of the restriction enzyme Xba_l

Pstl: cleavage site of the restriction enzyme Pst_l

Xhol: cleavage site of the restriction enzyme Xhol

S al: cleavage site of the restriction enzyme Smal

Bglll: cleavage site of the restriction enzyme Bglll

EcoRI: cleavage site of the restriction enzyme EcoRI

Examples

The present invention is described in more detail below with reference to examples of embodiments.

Example 1

Production of a genomic cosmid gene bank 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 was 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 SuperCosl (Wahl et al . (1987) Proceedings of the National Academy of Sciences USA 84:2160-2164), purchased from Stratagene (La Jolla, USA, product description SuperCosl cosmid vector Kit, Code no. 251301) was cleaved with the restriction enzyme Xbal (Amersham Pharmacia, Freiburg, Germany, product description Xbal, Code no. 27-0948-02) and was likewise dephosphorylated with shrimp alkaline phosphatase. The cosmid DNA was subsequently cleaved with the restriction enzyme BamHI (Amersham Pharmacia, Freiburg, Germany, product description BamHI, Code no. 27-0868-04) . The cosmid DNA which was treated in this manner was mixed with the treated ATCC 13032-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 mix was subsequently packed in phages with the aid of Gigapack II XL Packing Extract (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 Research 16:1563-1575), the cells were taken up in 10 mM MgS0₄ and were mixed with an aliquot of the phage suspension. Infection and titration of the cosmid bank were effected as described by Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor) , with the cells being plated out on to LB agar (Lennox, 1955, Virology, 1:190) with 100 mg/1 ampicillin. After incubation overnight at 37 °C, recombinant individual clones were selected.

Example 2

Isolation and sequencing of the gpsA gene

The cosmid DNA of a single .colony was isolated using a Qiaprep Spin Miniprep Kit (Product No. 27106, Qiagen, Hilden, Germany) in accordance with the manufacturer's instructions and was 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 separation by gel electrophoresis, cosmid fragments of the order of 1500 to 2000 bp were isolated using a QiaExII Gel Extraction Kit (Product No. 20021, Qiagen, Hilden, Germany) . The DNA of the sequencing vector, pZero-1, purchased from Invitrogen (Groningen, Holland, 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) . Ligation of the cosmid fragments in the sequencing vector 5 pZero-1 was effected as described by Sambrook et al.

(1989, Molecular Cloning: A laboratory Manual, Cold Spring Harbor) , the DNA mixture being incubated overnight with T4 ligase (Pharmacia Biotech, Freiburg, Germany) . This ligation mix was subsequently transferred into E. coli 0 strain DH5αMCR (Grant, 1990, Proceedings of the National Academy of Sciences U.S.A., 87:4645-4649) by means of electroporation (Tauch et al. 1994, FEMS Microbiol Letters, 123:343-7) and the batch was plated out on LB agar (Lennox, 1955, Virology, 1:190) with 50 mg/1 zeocin.

15 The plasmid was prepared from the recombinant clone using a Biorobot 9600 (Product No. 900200, Qiagen, Hilden, Germany) . Sequencing was effected by the dideoxy chain termination method of Sanger et al. (1977, Proceedings of the National Academy of Sciences U.S.A., 74:5463-5467)

2.0 with modifications according to Zimmermann et al . (1990, Nucleic Acids Research, 18:1067). The "RR d-Rhodamine Terminator Cycle Sequencing Kit" of PE Applied Biosystems (Product No. 403044, Weiterstadt, Germany) was used for this purpose. Separation by gel electrophoresis and

25 analysis of the sequencing reaction were effected in a "rotiphoresis NF acrylamide/bisacrylamide" gel (29:1) (Product No. A124.1, Roth, Karlsruhe, Germany), using the "ABI Prism 377" sequencing device of PE Applied Biosystems (Weiterstadt, Germany) .

30 The raw sequence data which were obtained were subsequently processed using the Staden software package (1986, Nucleic Acids Research, 14:217-231) Version 97-0. The individual sequences of the pZerol derivatives were assembled to form a coherent contig. Computer-aided

35 analysis was performed using the XNIP program (Staden, 1986, Nucleic Acids Research, 14:217-231). Further analyses were performed using "BLAST search programs" (Altschul et al., 1997, Nucleic Acids Research, 25:3389- 3402), compared with the non-redundant databank of the National Center for Biotechnology Information (NCBI, Bethesda, MD, USA) .

The nucleotide sequence obtained is illustrated in SEQ ID No. 1. Analysis of the nucleotide sequence showed the presence of an open reading frame comprising 996 base pairs, which was designated as the gpsA gene. The gpsA gene encodes a protein comprising 332 amino acids (SEQ ID No.2).

Example 3

Cloning the gpsA gene in the vector pJCl

Chromosomal DNA from Corynebacterium glutamicum ATCC 13032 was isolated as described by Tauch et al. (1995, Plasmid 33:168-179) . A DNA fragment bearing the gpsA gene was amplified by polymerase chain reaction. The following primers were used for this purpose:

5^{^}-TGC TCT AGA TGC GGG TGG CTT GGG ACA T-3

5^N-TGC TCT AGA ACG ACT GCG ACG CGG ACT TTT C-3

The primers illustrated were synthesised by MWG Biotech

(Ebersberg, Germany) and the PCR reaction was carried out by the standard PCR method of Innis et al. (PCR protocol. A guide to methods and applications, 1990, Academic Press) . The primers enabled amplification to be effected of a DNA fragment with a size of about 1193 bp and bearing the gpsA gene of Corynebacterium glutamicum.

After separation by gel electrophoresis, the PCR fragment was isolated from the agarose gel using a QiaExII Gel Extraction Kit (Product No. 20021, Qiagen, Hilden, Germany) . The E. coli - C. glutamicum shuttle vector pJCl (Cremer et al., 1990, Molecular and General Genetics 220: 478 - 480) was used as a vector. This plasmid was completely cleaved with the restriction enzyme BamHI, was treated with Klenow polymerase (Roche Diagnostics GmbH, Mannheim, Germany) and was subsequently dephosphorylated with shrimp alkaline phosphatase (Roche Diagnostics GmbH, Mannheim, Germany, product description SAP, Product No. 1758250) .

The gpsA fragment obtained in this manner was mixed with the prepared vector pJCl and was ligated with the aid of a SureClone Ligation Kit (Amersham Pharmacia Biotech, Uppsala, Sweden) according to the manufacturer's instructions. The ligation batch was transformed in the E. coli strain DH5α (Hanahan, in: DNA cloning. A practical approach. Vol. I. IRL Press, Oxford, Washington DC, USA). Plasmid-bearing cells were selected by plating out the transformation batch on LB agar (Lennox, 1955, Virology, 1:190) with 50 mg/1 kanamycin. After incubation overnight at 37 °C, recombinant individual clones were selected. Plasmid DNA was isolated from a transformant using a Qiaprep Spin Miniprep Kit (Product No. 27106, Qiagen, Hilden, Germany) according to the manufacturer's instructions and was cleaved with the restriction enzyme Xbal in order to investigate the plasmid by subsequent agarose gel electrophoresis. The plasmid obtained was designated as pJClgpsA.

Example 4

Transformation of the strains ATCC13032 and DSM5715 with the plasmid pJClgpsA

The C. glutamicum strains ATCC13032 and DSM5715 were transformed with the plasmid pJClgpsA using the electroporation method described by Liebl et al. (FEMS Microbiology Letters, 53:299-303 (1989)). The transfor ants were selected on LBHIS agar consisting of 18.5 g/1 brain-heart infusion bouillon, 0.5 M sorbitol, 5 g/1 bacteriological trypton, 2.5 g/1 bacteriological yeast extract, 5 g/1 NaCl and 18 g/1 bacteriological agar which was supplemented with 25 mg/1 kanamycin. Incubation was effected for 2 days at 33°C.

Plasmid DNA was isolated from each transformant by the usual methods (Peters-Wendisch et al . , 1998, Microbiology, 144, 915 - 927), was cut with the restriction endonuclease Xbal and the plasmid was investigated by subsequent agarose gel electrophoresis. The strains obtained were designated as ATCC13032/ρJClgpsA and DSM5715/pJClgpsA.

Example 5

Production of L-glutamate using the strain ATCC13032/ pJClgpsA

The C. glutamicum strain ATCC13032/pJClgpsA which was obtained in Example 4 was cultivated in a nutrient medium suitable for the production of glutamate, and the glutamate content in the culture supernatant was determined.

For this purpose, the strain was first incubated on an agar plate with the corresponding antibiotic (brain-heart agar with kanamycin (50 mg/1)) for 24 hours at 33°C. A preliminary culture was inoculated with this agar plate culture (10 ml medium in an 100 ml Erlenmeyer flask) . The complete medium Cglll (2.5 g/1 NaCl, 10 g/1 bacteriological peptone, 10 g/1 bacteriological yeast extract, pH 7.4, 20 g/1 glucose (autoclaved separately)) was used as the medium for the preliminary culture. Kanamycin (25 mg/1) was added to the latter. The preliminary culture was incubated for 16 hours at 33 °C, at 240 rpm on a shaker. A main culture was inoculated with this preliminary culture so that the initial OD (660nm) of the main culture was 0.1. The medium CgXII was used for the main culture. After preliminary cultivation in Cglll medium (Keilhauer et al. 1993, Journal of Bacteriology 175:5595-5603), the strain ATCC13032/pJClgpsA was cultivated in CgXII production medium (Keilhauer et al. 1993, Journal of Bacteriology 175:5595-5603). 4% glucose and 50 mg/1 kanamycin sulphate were added.

To induce glutamate formation, 20 g Tween 60 (P-1629, Sigma-Aldrich, Deisenhofen, Germany) plus 80 ml water were mixed and autoclaved. About 4 hours after inoculation, 75 μl of this Tween solution was added to the culture and cultivation was continued.

Cultivation was effected in a volume of 10 ml in a 100 ml Erlenmeyer flask fitted with baffles. Kanamycin (25 mg/1) was added. Cultivation was conducted at 33°C and 80% atmospheric humidity.

After 48 hours, the OD was determined at a measuring wavelength of 660 n using a Bio ek 1000 (Beckmann Instruments GmbH, Munich) . The quantity of glutamate formed was determined using an amino acid analyser supplied by Eppendorf-BioTronik (Hamburg, Germany) , by ion exchange chromatography and subsequent derivative formation using ninhydrin as a detector.

The results of the experiment are given in Table 1.

Example 6

Production of L-lysine

The C. glutamicum strain DSM5715/pJClgpsA obtained in Example 4 was cultivated in a nutrient medium suitable for the production of lysine, and the lysine content in the culture supernatant was determined.

For this purpose, the strain was first incubated on an agar plate with the corresponding antibiotic (brain-heart agar with kanamycin (50 mg/1)) for 24 hours at 33°C. A preliminary culture was inoculated with this agar plate culture (10 ml medium in an 100 ml Erlenmeyer flask) . The complete medium Cglll (2.5 g/1 NaCl, 10 g/1 bacteriological peptone, 10 g/1 bacteriological yeast extract, pH 7.4, 20 g/1 glucose (autoclaved separately)) was used as the medium for the preliminary culture.

Kanamycin (25 mg/1) was added thereto. The preliminary culture was incubated for 16 hours at 33 °C, at 240 rpm on a shaker. A main culture was inoculated with this preliminary culture so that the initial OD (660nm) of the main culture was 0.1. The medium MM was used for the main culture.

Medium MM

CSL (corn steep liquor) 5 g/1

MOPS (morpholinopropanesulphonic 20 g/1 acid)

glucose (autoclaved separately) 50g/l

(NH₄)₂S0₄ 25 g/1

KH₂P0₄ 0.1 g/1

MgS0₄ * 7 H₂0 1.0 g/1

CaCl₂ * 2 H₂0 10 mg/1

FeS0₄ * 7 H₂0 10 mg/1

MnS0₄ * H₂0 5.0mg/l

Biotin (filtered under sterile 0.3 mg/1

conditions)

thiamine * HC1 (filtered under 0.2 mg/1

sterile conditions)

L-leucine 0.1 g/1

CaC0₃ 25 g/1

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

After 72 hours, the OD was determined at a measuring wavelength of 660 nm using a Biomek 1000 (Beckmann Instruments GmbH, Munich) . The quantity of lysine formed was determined using an amino acid analyser supplied by Eppendorf-BioTronik (Hamburg, Germany) , by ion exchange chromatography and subsequent derivative formation using ninhydrin as a detector.

The results of the experiment are given in Table 2

Table 2

I. IDENTIFICATION OF THE MICROORGANISM

Identification reference given by the DEPOSITOR: Accession number given by the INTERNATIONAL DEPOSITARY AUTHORITY DSM 5715/pJClgpsA

DSM 13493

II. 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).

III. RECEIPT AND ACCEPTANCE

This International Depositary Authority accepts the microorganism identified under I. above, which was received by it on 2000 - 05 - 18 (Date of the original deposit)'.

IV. 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

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

Address: Mascheroder Weg lb D-38124 Braunschweig

Date: 2000- 05-23

¹ Where Rule 6.4 (d) applies, such date is the date on which the status of international depositary authority was acquired. Form DSMZ-BP/4 (sole page) 0196

I. DEPOSITOR II. IDENTIFICATION OF THE MICROORGANISM

Name: DβguSSa-HϋlS AG Accession number given by the

FA-FE-BT INTERNATIONAL DEPOSITARY AUTHORITY: Address: Kantstr . 2 DSM 13493

33790 Halle/Kύnsebeck Date of the deposit or the transfer': 2000 - 05 - 18

III. VIABILITY STATEMENT

The viability of the microorganism identified under II above was tested on 2000 - 05 - 18 ² . On that date, the said microorganism was

(X)³ viable

( )' no longer viable

IV. CONDITIONS UNDER WHICH THE VIABILITY TEST HAS BEEN PERFORMED⁴

V. INTERNATIONAL DEPOSITARY AUTHORITY

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

Address: Mascheroder Weg lb D-38124 Braunschweig

Date: 2000 - 05 -23

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 10.2(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) 0196

Claims

1. A coryneform bacterium, wherein the gpsA gene thereof, which encodes glycerol-3-phosphate dehydrogenase, is enhanced.

2. A coryneform bacterium according to claim 1, wherein the (wild- type) starting bacterium is selected from the group comprising Corynebacterium glutamicum (ATCC13032) , Corynebacterium acetoglutamicum (ATCC15806) , Corynebacterium acetoacidophilum (ATCC13870) , Corynebacterium thermoaminogenes (FERM BP-1539) , Corynebacterium melassecola (ATCC17965) , Brevibacterium flavum (ATCC14067), Brevibacterium lactofermentum (ATCC13869) and Brevibacterium divaricatum (ATCC14020) , or is selected from the group comprising 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 DSM5715.

3. A coryneform bacterium according to claim 1, wherein the enhancement of the gpsA gene is effected by over- expression of the gene, particularly by increasing the number of copies of the gene, by the selection of a strong promoter or a regulation region above the reading frame, by mutation of the promoter, of the regulation region or of the ribosome binding site, by the incorporation of a suitable expression cassette above the structural gene or by the incorporation of inducible promoters, by extending the life of the corresponding RNA, by reduced degradation of the expressed proteins, or by a combination of a plurality of these options.

4. A coryneform bacterium according to any one of claims 1 to 3, wherein the strain is transformed by a plasmid vector and the plasmid vector bears the nucleotide sequence which encodes the gpsA gene.

5. A coryneform bacterium according to any one of claims 1 to 4, wherein it corresponds genotypically to the strain Corynejacterium glutamicum DSM 13493.

6. A polynucleotide which is isolated from coryneform bacteria, containing a polynucleotide sequence selected from the group comprising

a) a polynucleotide, at least 70 % of which is homologous with a polynucleotide which encodes a polypeptide which comprises the amino acid sequence of SEQ ID No. 2 or which consists thereof,

70 % of which is homologous with the amino acid sequence of SEQ ID No. 2,

d) a polynucleotide containing at least 15 successive nucleotides of the polynucleotide sequence of a) , b) or c) .

7. A polynucleotide according to claim 6, wherein the polynucleotide is a DNA which is replicable in coryneform bacteria, preferably a recombinant DNA.

8. A polynucleotide according to claim 6, wherein the polynucleotide is an RNA.

9. A replicable DNA according to claim 7, containing

(i) the nucleotide sequence shown in SEQ ID No. 1 , or (ii) at least one sequence which corresponds to sequence (i) in the context of the degeneration of the genetic code, or

(iv) functionally neutral mutations in (i) which result in homologous amino acids.

10. A polynucleotide sequence according to claims 7, 8 or 9, which encodes a polypeptide which has the amino acid sequence of SEQ ID No. 2.

11. A process for the fermentative production of L-amino acids, wherein the following steps are carried out:

a) fermentation of coryneform bacteria which produce L-amino acid and in which at least the gpsA gene or nucleotide sequences which encode for the latter are enhanced, and in particular is over- expressed,

b) enrichment of the L-amino acid in the medium or in the cells of the bacteria, and

c) isolation of the L-amino acid.

12. A process according to claim 11, wherein a strain according to any one of claims 1 to 5 is used.

13. A process according to claims 11 or 12, wherein further genes, which encode a protein of the biosynthesis route of the desired L-amino acid, are enhanced in addition in the bacteria.

14. A process according to any one of claims 11 to 13, wherein metabolic pathways which reduce the formation of the desired amino acid are at least partially eliminated in the bacteria.

15. A process according to any one of claims 11 to 14, wherein the amino acid which is produced is L- glutamate or L-lysine.

16. A process according to any one of claims 11 to 15, wherein, for the production of glutamate, bacteria are fermented in which one or more of the genes selected from the group comprising

a) the dapA gene which encodes dihydrodipicolinatesynthase,

b) the dapE gene which encodes succinyldiaminopimelate desuccinylase,

c) the lysC gene which encodes a feedback-resistant aspartate kinase,

d) the tpi gene which encodes triose phosphate isomerase,

e) the gap gene which encodes glyceraldehyde-3- phosphate dehydrogenase,

f) the pgk gene which encodes 3-phosphoglycerate kinase,

g) the pyc gene which encodes pyruvate carboxylase,

h) the mqo gene which encodes malate: quinone oxidoreductase,

i) the lysE gene which encodes lysine export,

is simultaneously enhanced, and in particular is over- expressed or amplified.

7. A process according to any one of claims 11 to 16, wherein, for the production of L-lysine, bacteria are fermented in which one or more genes selected from the group comprising

a) the pck gene which encodes phosphoenol pyruvate carboxykinase,

b) the pgi gene which encodes for glucose-6- phosphate isomerase,

is simultaneously attenuated.

18. Use of polynucleotide sequences or parts thereof according to claim 6 as primers for the polymerase chain reaction for producing the DNA of genes which encode glycerol-3-phosphate dehydrogenase.

19. Use of polynucleotide sequences according to claim 6 as hybridization probes for the isolation of cDNA or of genes which exhibit a high degree of homology with the sequence of the gpsA gene.