WO2002002778A1

WO2002002778A1 - Nucleotide sequences which code for the mdha gene from corynebacterium glutamicum

Info

Publication number: WO2002002778A1
Application number: PCT/EP2001/006928
Authority: WO
Inventors: Douwe Molenaar; Michel Eduard Van Der Rest; Andre Drysch
Original assignee: Degussa Ag
Priority date: 2000-07-04
Filing date: 2001-06-20
Publication date: 2002-01-10
Also published as: ATE312929T1; DE60115900T2; DE10032350A1; WO2002002778A8; EP1297148A1; US6995002B2; KR100904744B1; US7067288B2; ES2254459T3; KR20030013521A; DE60115900D1; EP1297148B1; US20020037568A1; CN1440458A; AU2001279674A1; US20040142454A1

Abstract

The invention relates to polynucleotides from coryneform bacteria which code for the mdhA gene and comprise polynucleotide sequences, chosen from the group consisting of polynucleotide which is identical to the extent of at least 70 % to a polynucleotide which codes for a polypeptide which comprises the amino acid sequence of SEQ ID No. 3, polynucleotide which codes for a polypeptide which comprises an amino acid sequence which is identical to the extent of at least 70 % to the amino acid sequence of SEQ ID No. 3, polynucleotide which is complementary to the polynucleotides of a), b) or c) [sic], and polynucleotide comprising at least 15 successive nucleotides of the polynucleotide sequences of a), b) or c); and a process for the fermentative preparation of L-amino acids using coryneform bacteria, in which at least the mdhA gene is present in attenuated form, and the use of the polynucleotide sequences as hybridization probes.

Description

: NEW NUCLEOTIDE SEQUENCES WHICH CODE FOR THE MDHA GENE

The invention provides nucleotide sequences from coryneform bacteria which code for the mdhA gene and a process for the fermentative preparation of amino acids, in particular L-lysine, by attenuation of the mdhA gene.

Prior Art

L-Amino acids, in particular lysine, are used in human medicine and in the pharmaceuticals^' industry, in the foodstuffs industry and very particularly in animal nutrition.

It is known that amino acids are prepared by fermentation from strains of coryneform bacteria, in particular • Corynebacterium glutamicum. Because of their great importance, work is constantly being undertaken to improve the preparation processes. Improvements to the process can relate to fermentation measures, such as, for example, stirring and supply of oxygen, or the composition of the nutrient media, such as, for example, the sugar concentration during the fermentation, or the working up to the product form by, for example, ion exchange chromatography, or the intrinsic output properties of the microorganism itself.

Methods of utagenesis, selection and mutant selection are used to improve the output properties of these microorganisms. Strains which are resistant to antimetabolites or are auxotrophic for metabolites of regulatory importance and which produce amino acids are obtained in this manner.

Methods of the recombinant DNA technique have also been employed for some years for improving the strain of Corynebacterium strains which produce L-amino acids. Object of the Invention

The inventors had the object of providing new measures for improved fermentative preparation of amino acids, in particular L-lysine.

Description of the Invention

L-Amino acids, in particular lysine, are used in human medicine and in the pharmaceuticals industry, in the foodstuffs industry and very particularly in animal nutrition. There is therefore a general interest in providing new improved processes for the preparation of amino acids, in particular L-lysine.

The invention provides an isolated polynucleotide from coryneform bacteria, comprising a polynucleotide sequence which codes for the mdhA gene, chosen from the group consisting of

a) polynucleotide which is identical to the extent of at least 70% to a polynucleotide which codes for a polypeptide which comprises the amino acid sequence of SEQ ID No. 3,

b) polynucleotide which codes for a polypeptide which comprises an amino acid sequence which is identical to the extent of at least 70% to the amino acid sequence of SEQ ID No. 3,

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

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

the polypeptide preferably having the activity of malate dehydrogenase. The invention also provides an isolated polypeptide with malate dehydrogenase activity, which comprises the amino acid sequence according to SEQ ID No. 1.

The invention also provides the polynucleotide according to claim 1, this preferably being a DNA which is capable of replication, comprising:

(i) the nucleotide sequence shown in SEQ ID no. 2, or

(ii) at least one sequence which corresponds to sequence (i) within the range 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) sense mutations of neutral function in (i) .

The invention also provides:

a DNA which is capable of replication and comprises the nucleotide sequence as shown in SEQ ID No. 2,

a vector containing the polynucleotide as claimed in claim 1, point d, in particular pEMmdhAint, deposited in E. coli DSM 13494 on 18.05.2000 at the DSMZ [German Collection of Microorganisms and Cell Cultures] , Braunschweig (Germany) ,

and coryneform bacteria which contain a deletion or insertion in the mdhA gene, in particular using the vector pEMmdhAint.

The invention also provides polynucleotides which substantially comprise a polynucleotide sequence, which are obtainable by screening by means of hybridization of a corresponding gene library, which comprises the complete mdhA gene with the polynucleotide sequence corresponding to SEQ ID No. 2, with a probe which comprises the sequence of the polynucleotide mentioned, according to SEQ ID no. 2 or a fragment thereof, and isolation of the DNA sequence mentioned.

Polynucleotides, which contain sequences, according to the invention, are suitable as hybridization probes for RNA, cDNA and DNA, in order to isolate, in the full length, nucleic acids, or polynucleotides or genes which code for malate dehydrogenase or to isolate those nucleic acids or polynucleotides or genes which have a high similarity of sequence with that of the malate dehydrogenase gene.

Polynucleotides, which contain sequences according to the invention, are furthermore suitable as primers with the aid of which DNA of genes which code for malate dehydrogenase can be prepared with the polymerase chain reaction (PCR) .

Such oligonucleotides which serve as probes or primers comprise at least 30, preferably at least 20, very particularly preferably at least 15 successive nucleotides. Oligonucleotides which have a length of at least 40 or 50 nucleotides are also suitable.

"Isolated" means separated out of its natural environment.

"Polynucleotide" in general relates to polyribonucleotides and polydeoxyribonucleotides, it being possible for these to be non-modified RNA or DNA or modified RNA or DNA.

"Polypeptides" are understood as meaning peptides or proteins which comprise two or more amino acids bonded via peptide bonds.

The polypeptides according to the invention include the polypeptides according to SEQ ID No. 3, in particular those with the biological activity of malate dehydrogenase, and also those which are at Least 70%, preferably at least 80% and in particular at least 90% to 95% identical to the polypeptide according to SEQ ID No. 3 and have the activity mentioned.

The invention moreover provides a process for the fermentative preparation of amino acids, in particular lysine, using coryneform bacteria which in particular • already produce the amino acids, and in hich the nucleotide sequences which code for the mdhA gene are attenuated, in particular eliminated or expressed at a low level.

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

The microorganisms which the present invention provides can prepare amino acids, in particular lysine, from glucose, sucrose, lactose, fructose, maltose, molasses, starch, cellulose or from glycerol and ethanol. They can be representatives of coryneform bacteria, in particular of the genus Corynebacterium. Of the genus Corynebacterium, there may be mentioned in particular the species Corynebacterium glutamicum, which is known among experts for its ability to produce L-amino acids.

Suitable strains of the genus Corynebacterium, in particular of the species Corynebacterium glutamicum, are in particular the known wild-type strains

Corynebacterium glutamicum ATCC13032

Corynebacterium acetoglutamicum ATCC15806 Corynebacterium acetoacidophilum ATCC13870 Corynebacterium elassecola ATCC17965 Corynebacterium thermoa inogenes FERM BP-1539 Brevibacterium flavum ATCC14067 Brevibacterium lactofermentum ATCC13869 and Brevibacterium divaricatum ATCC14020

and L-amino acid-producing mutants, or strains, prepared therefrom, 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 ^•

Corynebacterium glutamicum DM58-1 j •

Corynebacterium glutamicum DG52-5 Corynebacterium glutamicu 'DSM5714 and

Corynebacterium glutamicum DSM1Z866.

The new mdhA gene of C. glutamicum which codes for the enzyme malate dehydrogenase (EC 1.1.1.37) has been found.

For this, the malate dehydrogenase enzyme protein is first purified to homogeneity by chromatographic methods. Methods and instructions for protein purification and preparation are described e.g. in the textbook by Schleifer and ensink: Practical Methods in Molecular Biology (Springer Verlag, Berlin, Germany, 1981) ; in the handbook by Harris and Angal: Protein Purification Methods: A Practical

Approach (IRL Press, Oxford, UK, 1989) ; in the textbook by Scopes: Protein Purification: Principles and Practice, 3^rd ed. (Springer Verlag, New York, USA, 1993), and in generally known textbooks and handbooks. The pure enzyme protein can then be broken down into peptides by treatment with suitable enzymes, such as, for example, trypsin or chymotrypsin. The amino acid sequence of these peptides can be determined by the method of N-terminal sequencing described by Edman (Archives of Biochemistry 22, 475 (1949) ) . It is likewise possible to determine the N- terminal amino acids of the purified enzyme protein directly. Methods and instructions for protein sequencing are described, for example, in Smith: Protein Sequencing Protocolls [sic]: Methods in Molecular Biology, Vol. 64 and Vol. 112 (Humana Press, Totowa, NJ, USA, 1996) and in Kamp et al.: Protein Structure Analysis: Preparation, Characterization, and Microsequencing (Springer Verlag. New York, NY, USA, 1997) . In this manner the amino acid sequence of the malate dehydrogenase enzyme protein can be determined in part or in full, depending on the outlay. The 20 N-terminal amino acids of the malate dehydrogenase enzyme protein isolated are shown in SEQ ID No. 1.

Utilizing the known use of codons for coryneform bacteria (Malumbres et al. (Gene 134, 15 - 24 (1993)), synthetic oligonucleotides can be synthesized and can be employed as primers for amplification of the corresponding chromosomal DNA segments by means of the polymerase chain . reaction (PCR) . Instructions for this are to found by the expert, inter alia, for example in the handbook by Gait:

Oligonukleotide [sic] synthesis: a practical approach (IRL Press, Oxford, UK, 1984) and in Newton and Graham: PCR (Spektrum Akademischer Verlag, Heidelberg Germany, 1994) . The DNA fragment of the sod gene obtained in this manner is then cloned by known methods, as described e. g. in

Sambrook et al.: Molecular Cloning: A Laboratory Manual 2^nd ed. (Cold Spring Harbor Laboratory Press, USA, 1989) and can be employed .as. a probe for the search for the complete gene, including its 5' and 3' flanks,- in gene libraries.

It is likewise possible to isolate the complete gene, including its 5' and 3' flanks, by the method of "plasmid rescue". In this method, a fragment of the gene of interest, which has been obtained., for example, in the manner described above, is cloned in a plasmid vector which is not replicative for coryneform bacteria. The plasmid vector containing the gene fragment is incorporated or integrated into the target gene of the host by transformation and subsequent homologous recombination. The target gene is thereby marked. The chromosomal DNA of the strain marked in this way is then isolated and digested with a suitable restriction enzyme. Suitable restriction enzymes are, in particular, those which do not cleave within the vector DNA employed. The resulting DNA fragments are circularized by treatment with ligase and a host suitable for replication of the plasmid vector, typically Escherichia coli, is transformed with the ligation mixture. Plasmid DNA is isolated from the transformants and the cloned DNA sections are sequenced. The method of "plasmid rescue" is described, for example, by Niaudet et al. (Gene 19, 277-284 (1982)). Methods of 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) or in the protocol of Zimmerman et al. (BioTechniques 17:302 (1994)).

The resulting DNA sequences can then be investigated with known algorithms or sequence analysis programs, such as e.g. that of Staden (Nucleic Acids Research 14, 217- 232(1986)), that of Marck (Nucleic Acids Research 16, 1829- 1836 (1988)) or the GCG program of Butler (Methods of Biochemical Analysis 39, 74-97 (1998)).

The new DNA sequence of C. glutamicum which codes for the mdhA gene and which, as SEQ ID No. 2, is a constituent of the present invention has been found in this manner. The amino acid sequence of the corresponding translation product or gene product has moreover been derived from the present DNA sequence by the methods described above. The resulting amino acid sequence of the mdhA gene product is shown in SEQ ID No. 3. It is known (0" Regan et al., Gene 77, 237-251 (1989)), that the amino acid methionine or formyl-methionine coded by the start codon ATG is removed from various proteins by enzymes of the host.

Coding DNA sequences which result from SEQ ID No. 2 by the degeneracy of the genetic code are also a constituent of the invention. In the same way, DNA sequences which hybridize with SEQ ID No. 2 or parts of SEQ ID No. 2 are a constituent of the invention. Finally, DNA sequences which are prepared by the polymerase chain reaction (PCR) using primers which result from SEQ ID No. 2 are a constituent of the invention.

Instructions for identifying DNA sequences by means of hybridization can be found by the expert, inter alia, in the handbook "The DIG System Users Guide for Filter Hybridization" from Boehringer Mannheim GmbH (Mannheim, Germany, 1993) and in Liebl et al. (International Journal of Systematic Bacteriology (1991) 41: 255-260) . Instructions for amplification of DNA sequences with the aid of the polymerase chain reaction (PCR) can be found by the expert, inter alia, in the handbook by Gait: Oligonukleotide [sic] synthesis: a pract «ical approach (IRL

Press, Oxford, UK, 1984) and in Newton and Graham: PCR (Spektru Akademischer Verlag, Heidelberg, Germany, 1994) .

It has been found that coryneform bacteria produce amino acids, in particular L-lysine, in an improved manner after attenuation of the mdhA gene.

To achieve an attenuation, either the expression of the mdhA gene or the catalytic properties of the enzyme proteins can be reduced or eliminated. The two measures can optionally be combined.

The reduction in gene expression can take place by suitable culturing or by genetic modification (mutation) of the signal structures of gene expression. Signal structures of gene expression are, for example, repr-essor genes, activator genes, operators, promoters, attenuators, ribosome binding sites, the start codon and terminators. The expert can find information on this e. g. in the 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)), Vasicova et al. (Journal of Bacteriology 181: 6188 (1999)) and in known textbooks of genetics and molecular biology, such as e. g. the textbook by Knippers ("Molekulare Genetik [Molecular Genetics]", 6th edition, Georg Thieme Verlag, Stuttgart, Germany, 1995) or that by Winnacker ("Gene und Klone [Genes and Clones]", VCH Verlagsgesellschaft, Weinheim, Germany, 1990) .

Mutations which lead to a change or reduction in the catalytic properties of enzyme proteins are known from the prior art; examples which may be mentioned are the works by Qiu and Goodman (Journal of Biological Chemistry 272: 8611- 8617 (1997)), Sugimoto et al. (Bioscience Biotechnology and Biochemistry 61: 1760-1762 (1997)) and Mδckel ("Die Threonindehydratase aus Corynebacterium glutamicum: Aufhebung der ^"allosterischen Regulation und Struktur des Enzyms [Threonine dehydratase from Corynebacterium glutamicum: Cancelling the allosteric regulation and structure of the enzyme]", Reports from the Julich Research Centre, Jϋl-2906, ISSN09442952, Julich, Germany, 1994). Comprehensive descriptions can be found in known textbooks of genetics and molecular biology, such as e. g. that by Hagemann ("Allgemeine Genetik [General Genetics]", Gustav Fischer Verlag, Stuttgart, 1986) .

Possible mutations are transitions, transversions, insertions and deletions. Depending on the effect of the amino acid exchange on the enzyme activity, missense mutations or nonsense mutations are referred to. Insertions or deletions of at least one base pair in a gene lead to frame shift mutations, as a consequence of which incorrect amino acids are incorporated or translation is interrupted prematurely. Deletions of several codons typically lead to a complete loss of the enzyme activity. Instructions on generation of • such mutations are prior art and can be found in known textbooks of genetics and molecular biology, such as e. g. the textbook by Knippers ("Molekulare Genetik [Molecular Genetics]", 6th. edition, Georg Thieme Verlag, Stuttgart, Germany, 1995), that by Winnacker ("Gene und Klone [Genes and Clones]", VCH Verlagsgesellschaft, Weinhei , Germany, 1990) or that by Hagemann ("Allgemeine Genetik [General Genetics]", Gustav Fischer Verlag, Stuttgart, 1986) .

A common 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 of interest is cloned in a plasmid vector which can replicate in a host (typically E. coli) , but not in C. glutamicum. Possible vectors are, for example, _,pSUP301 (Simon et al., Bio/Technology 1, 784-791 (1983)), pKlδmob or pKl9mob (Schafer et al., Gene 145, 69- 73 (1994)), pK18mobsacB or pK19mobsacB (Jager et al . , Journal of Bacteriology 174: 5462-65 (1992)), pGEM-T (Promega corporation, Madison, WI, USA), pCR2.1-TOPO (Shuman (1994). Journal of Biological Chemistry 269:32678- 84; US Patent 5,487,993), pCR®Blunt (Invitrog n, 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 central part of the coding region of the gene is then transferred into the desired strain of C. glutamicum by conjugation or transformation. The method of conjugation is. described, for example, by Schafer et al. (Applied and Environmental Microbiology 60, 756-759 (1994)). Methods for transformation are described, for example, by Thierbach et al. (Applied Microbiology and Biotechnology 29, 356-362 (1988)), Dunican and Shivnan (Bio/Technology 7, 1067-1070 (1989)) and Tauch et al. (FEMS Microbiological Letters 123, 343-347 (1994)). After homologous recombination by means of a "cross-over" event, the coding region of the gene in question is interrupted by the vector sequence and two incomplete alleles are obtained, one lacking the 3' end and one lacking the 5' end. This method has been used, for example, by Fitzpatrick et al. (Applied Microbiology and Biotechnology 42, 575-580 (1994)) to eliminate the recA gene of C. glutamicum.

Figure 1 shows by way of example the plasmid vector . pEMmdhAint, with the aid of which the mdhA gene can be disrupted or eliminated.

In the method of gene replacement, a mutation, such as e.g. a deletion, insertion or base exchange, is established in vitro in the gene of interest. The allele prepared is in turn cloned in a vector which is not replicative for C. glutamicum and this is then transferred into the desired host of C. glutamicum by transformation or conjugation. After homologous recombination by means of a first "crossover" event which effects integration and a suitable second "cross-over" event which effects excision in the target gene or in the target sequence, the incorporation of the mutation or of the allele is achieved. This method was used, for example, by Peters-Wendisch (Microbiology 144, 915 - 927 (1998)) to eliminate the pyc gene of C. glutamicum by a deletion.

A deletion, insertion or a base exchange can be incorporated into the mdhA gene in this manner.

In addition, it may be advantageous for the production of L-amino acids, in particular L-lysine, to enhance, in particular to over-express, one or more enzymes of the particular biosynthesis pathway, of glycolysis, of anaplerosis, of the pentose phosphate cycle or of amino acid export, in addition to attenuation of the mdhA gene.

Thus, for example, for the preparation of L-lysine, at the same time one or more of the genes chosen from the group consisting of

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

• the eno gene which codes for enolase (DE: 19947791.4),

• the zwf gene which codes for the zwf gene product (JP-A- 09224661),

• the pyc gene which codes for pyruvate carboxylase (Eikmanns (1992), Journal of Bacteriology 174:6076-6086), • the lysE gene which codes for lysine export (DE-A-195 48 222)

can be enhanced, in particular, over-expressed.

It may furthermore be advantageous for the production of amino acids, in particular L-lysine, in addition to the attenuation of the mdhA gene, at the same time for one or more of the genes chosen from the group consisting of

• the pck gene which codes for phosphoenol pyruvate carboxykinase (DE 199 50 409.1, DSM 13047),

• the pgi gene which codes for glucose 6-phosphate isomerase (US 09/396,478, DSM 12969),

• the poxB gene which codes for pyruvate oxidase

(DE:1995 1975.7, DSM 13114)

to be attenuated. In addition to attenuation of the mdhA gene it may furthermore be advantageous, for the production of amino ^• acids, in particular L-lysine, to eliminate undesirable side reactions, (Nakayama: "Breeding of Amino Acid Producing Micro-organisms", in: Overproduction of Microbial Products, Krumphanzl, Sikyta, Vanek (eds.), Academic Press, London, UK, 1982) .

The invention also provides the microorganisms prepared according to the invention, and these can be cultured continuously or discontinuously in the batch process (batch culture) or in the fed batch (feed process) or repeated fed batch process (repetitive feed process) for the purpose of production of L-amino acids, in particular L-lysine. A summary of known culture methods are [sic] described in the textbook by Chmiel (Bioprozesstechnik 1. Einfϋhrung in die Bioverfahrenstechnik [Bioprocess Technology 1. Introduction to Bioprocess Technology (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 meet the requirements of the particular strains in a suitable manner. Descriptions of culture media for various microorganisms are contained in the handbook "Manual of Methods for General Bacteriology" of the American Society for Bacteriology (Washington D.C., USA, 1981). Sugars and carbohydrates, such as e.g. glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose, oils and fats, such as, for example, soya oil, sunflower . oil, groundnut oil and coconut fat, 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, can be used as the source of carbon. These substance can be used individually or as a mixture. 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, ammoni m phosphate, ammonium carbonate and ammonium nitrate, can be used as the source of nitrogen. The sources of nitrogen can be used individually or as a mixture. Phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium- containing salts can be used as the source of phosphorus. The culture medium must furthermore comprise salts of metals, such as, for example, magnesium sulfate or iron sulfate, which are necessary for growth. Finally, essential growth substances, such as amino acids and vitamins, can be employed in addition to the abovementioned substances. Suitable precursors can moreover be added to the culture medium. The starting substances mentioned can be added to the culture in the form of a single batch, or can be fed in during the culture in a suitable manner.

Basic compounds, such as sodium hydroxide, potassium hydroxide, ammonia or aqueous ammonia, or acid compounds, such as phosphoric acid or sulfuric acid, can be employed in a suitable manner to control the pH. Antifoams, such as, for example, fatty acid polyglycol esters, can be employed to control the development of foam. Suitable substances having a selective action, such as, for example, antibiotics, can be added to the medium to maintain the stability of plasmids. To maintain aerobic conditions, oxygen or oxygen-containing gas mixtures, such as, for example, air, are introduced into the culture. The temperature of the culture is usually 20°C to 45°C, and preferably 25°C to 40°C. Culturing is continued until a maximum of the desired product has formed. This target is usually reached within 10 hours to 160 hours.

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

The following microorganism was deposited at the Deutsche Sammlung fur Mikroorganismen und Zellkulturen (DSMZ = German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) in accordance with the Budapest Treaty on 18.05.2000:

• Escherichia coli DH5αmcr/pEMmdhAint as DSM13494

The process according to the invention is used for the fermentative preparation of amino acids, in particular L-lysine.

The present invention is explained in more detail in the following with the aid of embodiment examples.

The isolation of plasmid DNA from Escherichia coli and all techniques of restriction, Klenow and alkaline phosphatase treatment were carried out by the method of Sambrook et al. (Molecular cloning. A Laboratory Manual (1989) Cold Spring Harbour Laboratory Press, Cold Spring Harbor, NY, USA) . Methods for transformation of Escherichia coli are also described in this handbook.

The composition of the usual nutrient media, such as LB or TY medium, can also be found in the handbook by Sambrook et al.

The malate dehydrogenase activity was determined as described by Sanwal (Journal of Biological Chemistry 244 (7) .1831-1837 (1969)) and Smith (Methods of Enzymatical

Analysis, 1985, ed. 3, volume 3 (Bergmeyer et al. (eds.)), VCH Verlagsgesellschaft, Weinhei , Germany) . Protein concentrations were measured by the method of Smith et al. (Analytical Biochemistry 150:76-85 (1985)).

Example 1

Isolation and purification of malate dehydrogenase from Corynebacterium glutamicum ATCC 13032

Four times, 100 ml 2 x TY were inoculated with in each case a colony of the strain Corynebacterium glutamicum ATCC 13032 and in each case cultured in 250 ml conical flasks for 16 hours at 30°C and 200 revolutions per minute. The cells were washed twice in buffer A (50 mM K phosphate, pH 7.5, 1 mM dithiothreitol, 2 mM ethylenediaminetetraacetic acid) at 4°C, and resuspended in 20 ml of the same buffer. The cells were broken down three times in a precooled French Pressure Cell from Spectronic Unica (Rochester, NY, USA) under 165 MPa. The cell debris was then sedimented in a centrifuge at 4°C for 10 minutes at 75600 x g. The malate dehydrogenase protein was purified in three steps.

Step 1

Streptomycin sulfate precipitation: ι The supernatant was removed and further treated with a streptomycin sulfate precipitation. For this, a 10 per cent (weight/volume) streptomycin sulfate solution was slowly added, while stirring at 0°C, until a final concentration of 0.75% was reached. The batch was incubated for 15 minutes on ice and then centrifuged for 10 minutes at 4°C and 15000 x-g. The supernatant was then introduced into a dialysis hose pretreated in accordance with the manufacturers instructions (Medicell, London, UK) and dialysed twice in each case in 1 1 buffer B (10 mM K phosphate, pH 7.5, 1 mM dithiothreitol, 2 mM ethylenediaminetetraacetic acid) for 1.5 hours at 4°C. The dialysate was then centrifuged for 15 minutes at 4°C and 75600 x g. Step 2

Anion exchanger chromatography:

The supernatant from step 1 was removed and introduced on to an anion exchanger column of the type "Resource Q" (1 ml column volume, Amersham Pharmacia, Freiburg, Germany) equilibrated with 4 column volumes of buffer B. The column was flushed with 4 column volumes of buffer B and then eluted with a linear gradient from buffer B to buffer C (10 mM K phosphate, pH 7.5, 1 mM dithiothreitol, 2 mM ethylenediaminetetraacetic acid, 1 M NaCl) in 15 column volumes. The anion exchanger chromatography was carried out at 20°C and the fractions were collected at 4°C. The malate dehydrogenase was eluted at approx. 0.5 M NaCl. The fractions which contained malate dehydrogenase activity were combined and then desalinated by means of gel filtration over PD-10 columns (Amersham Pharmacia, Freiburg, Germany) with ice-cold buffer D (10 mM K phosphate, pH 7.5) in accordance with the manufacturer's instructions .

Step 3

Dyestuff affinity chromatography:

Less than 10 g total protein from step 2 were introduced on to a column filled with 2.5 ml of the column material "Reactive Brown 10" (Sigma, Deisenhofen, Germany) and equilibrated with 12.5 ml buffer D. The column was flushed with 15 ml ice-cold buffer D and the malate dehydrogenase was eluted with 2.5 ml buffer E (10 mM K phosphate, pH 7.5. 1 mM NADH) . The eluate was desalinated by means of gel filtration over PD-10 columns as described above.

Step 4

Dyestuff affinity chromatography:

Step 3 was repeated. The protein purified in this way was stored at -20°C. For stabilization of the activity, the sample was supplemented with 1 mg/ml bovine serum albumin. The supplementation with bovine serum albumin was omitted in the case of samples employed for determination of the N- terminal sequence of the malate dehydrogenase protein. A Lambda-10 spectrophotometer from Perkin Elmer (Foster City, CA, USA) was used for measuring the activity of the purified enzyme. The purified malate dehydrogenase had a maximum specific oxalacetate-reducing activity of 1200 to 1300 μmol/min and mg protein at a concentration of 0.1 mM oxalacetate and 0.3 mM NADH.

The purity of the malate dehydrogenase isolated was determined by means of SDS-polyacrylamide gel electrophoresis. The analysis showed that the purified malate dehydrogenase was present as a homogeneous protein with -an apparent molecular weight of 33kDa.

Example 2

Determination of the N-position amino acid sequence

The N-position amino acid sequence of the purified malate dehydrogenase protein was determined by Edman degradation (Edman, Molecular Biology Biochemistry Biophysics 8:211- 55(1970)) by means of the automatic sequencer model 476A from PE Biosystems (Foster City, CA, USA) . The resulting amino acid sequence (see also SEQ ID No. 1) was:

Asn-Ser-Pro-Gln-Asn-Val-Ser-Thr-Lys-Lys-Val-Thr-Val-Thr- Gly-Ala-Ala-Gly-Gln-Ile.

Example 3

Preparation of a vector with a copy of an internal fragment of the mdhA gene

A segment of the mdhA gene was amplified by means of the polymerase chain reaction (PCR) starting from chromsomal [sic] DNA of the strain ATCC13032 and then cloned. With. the aid of the N-position amino acid sequence determined in example 2, the degenerated primer Pi was discarded. This primer had the sequence:

5 ' -AARGTYACYGTYACYGGY-3 ' . ,

^" The primers [sic] P2, which had the following sequence, 35 employed as the second degenerated primer:

5 ' -CGRTTRTGRTCVARRCG-3 ' .

The abbreviation R stands for the nucleobases A or G, the abbreviation Y stands for C or T and the abbreviation V stands for A, G or C.

The primers shown were synthesized by MWG Biotech (Ebersberg, Germany) . The PCR reaction was carried out by the standard PCR method of Innis et al., (PCR protocols. A guide to methods and applications, 1990, Academic Press, New York, USA) . Genomic DNA of Corynebacterium glutamicum ATCC13032, which ^'was^' isolated by the method described by Pospiech and Neumann (Trends in Genetics 11:217-218 (1995)), was used as the template.

In the polymerase chain reaction, a cycle comprising denaturing (30 seconds, 94°C), annealing (60 seconds, 63°C) and synthesis (90 seconds, 72°C) was passed through 30 times. A last synthesis of 10 minutes at 72°C was then carried out. The Taq polymerase from Promega (Madison, WI, USA) and a Thermocycler from Techne (Cambridge, UK) were used.

The DNA fragment of the mdhA gene approx. 470 bp long prepared in this manner was purified with the aid of the QIAQuick PCR Purification Kit from Qiagen (Hilden, Germany) and cloned into the EcoRV cleavage site of the vector pBlueskript II SK(+) (Stratagene, La Jolla, CA, USA). The fragment was then isolated with the aid of the restriction enzymes BamHI and Hindlll and cloned into the vector pEMl treated with the restriction enzymes BamHI and Hindlll (Schrumpf et al. Journal of Bacteriology 173, 4510-4516 (1991) ) in E. coli DHα, selection taking place on LB medium supplemented with 50 μg/ml kanamycin. The plasmid formed in this way was called pEMmdhAint.

Example 4 _ - —

Determination of the sequence of the mdhA gene

In the strain Corynebacterium glutamicum ATCC 13032, the mdhA gene was inactivated with the aid of the plasmid pEMmdhAint.

The strain Corynebacterium glutamicum ATCC 13032 was transformed with the plasmid pEMmdhAint as described by Van der Rest et al (Applied Microbiology and Biotechnology 52,541-545 (1999)). Selection of the transformants was carried ut on LBHIS agar, which had been supplemented with 25 mg/1 kanamycin. LBHIS agar comprises LB medium, which has been supplemented with 18.5 g/1 brain-heart broth (Becton Dickinson, Sparks MD, USA) , 91 g/1 s'orbitol and 15 g/1 agar-agar. The strain ATCCl3032mdhA: :pEMmdhAint, in which the plasmid pEMmdhAint is integrated in the mdhA gene, was formed in. this manner.

Chromosomal DNA was isolated from the strain ATCC13032mdhA: :pEMmdhAint, as described by Pospiech and Neumann (Trends in Genetics 11:217-218 (1995)), and digested completely in two different batches, once with the restriction enzyme Stul and once with the restriction enzyme Xbal . The chromosomal restriction fragments were ligated as described by Niaudet et al. (Gene 19, 277-284 (1982)) and Escherichia coli DH5α-MCR (Grant et al Proceedings of the National Academy of Sciences U.S.A. 87,4645-4649 (1990)) was transformed with the ligation batch. Transformants were selected on LB agar with 50 mg/1 kanamycin. Plasmid DNA was isolated from the transformants and subjected to a restriction analysis. The plasmids pEM dhAint-StuI and pEMmdhAint-Xbal, ^'which, in addition to the internal fragment of the mdhA gene 470 bp long already cloned in the plasmid pEMmdhAint, carried regions of the 5' and of the 3' end of the gene, were identified in this manner. The plasmid pEMmdhAint-StuI, obtained from the Stul digestion, additionally had 0.56 kb on the 5' end and 0.40 kb on the 3' end of the 470 bp region. The plasmid pEMmdhAint-XbaI, obtained from the Xbal digestion, additionally had 1.2 kb on the 3' end of the 470 bp region.

The plasmids obtained were sequenced by Seqlab (Gδttingen, Germany) using the Cycle-Sequencing protocol of Zimmerman et al. (BioTechniques 17:302 (1994)).- The sequences were evaluated with PC/Gene Version 6.60 from Intelligenetics Inc. (Geneva, Switzerland) . The sequence of the mdhA gene cluster is shown in SEQ ID No. 2.

Example 5

Preparation of L-lysine producers with an inactivated mdhA gene

Experiments were carried out with various known lysine producers of C. glutamicum in order to investigate the effect of elimination of the mdhA gene on the production of lysine.

5.1. Preparation of the strains _

The strain MH20-22B is described in EP-B-0435132 and is deposited as DSM5715 in accordance with the Budapest Treaty. The strain DM58-1 is described in EP-B-0358940. The transformant DM58-l/pDM6 described there is deposited as DSM4697 in accordance with the Budapest Treaty. The strain DM58-1 can be prepared from DSM4697 by curing methods, such as, for example, by the method of "plasmid curing" described by Schafer et al. (Journal of Bacteriology, 176, 7309-7319 (1994)). The strain DG 52-5 is described in DE-C- 3823451. The transformant DG 52-5/pZl-asd described there is deposited as DSM4421 in accordance with the Budapest Treaty. The strain DG52-5 can also be prepared from DSM4421 by the method of "plasmid curing" .

The strain Corynebacterium glutamicum MH20-22B was transformed with the plasmid pEMmdhAint as described by Van der Rest et al. (Applied Microbiology and Biotechnology 52,541-545 (1999)). Selection of the transformants was carried out on LBHIS agar, which had been supplemented with 25 mg/1 kanamycin. The strain MH20-22BmdhA: :pEMmdhAint was formed in this manner. Starting from the strains DG52-5 and DM58-1, in each case the strains DG52-5mdhA: :pEMmdhAint and DM58-lmdhA: :pEMmdhAint were formed in the same manner.

5.2. Determination of the malate dehydrogenase activity

To confirm the success of the insertion mutagenesis, the malate dehydrogenase activity was measured in the strains MH20-22B, DM58-1 and DG52-5 and in the insertion mutants MH20-22BmdhA::pEMmdhAint, DG52-5mdhA: :pEMmdhAint and DM58- lmdhA: : pEMmdhAint . For this, in each case a colony of the strains was inoculated on to 50 ml 2 x TY medium and incubated for 16 hours in a 250 ml conical flask at 30°C and 200 revolutions per, minute.' The media for the strains MH20-22BmdhA: :pEMmdhAint, DG52-5mdhA::pEMmdhAint and DM58- lmdhA: : pEMmdhAint additionally received 25 μg/ml kanamycin here. The cultures were centrifuged off for 10 minutes at 10000 x g, washed twice with 50 mM potassium phosphate pH 7.5 and then suspended in 5 ml of the same buffer. The cell suspensions were broken down with two passes through an ice-cold French Press Cell under 165 MPa. The samples were then centrifuged for 10 minutes at 75000 x g and 4°C. The clear supernatant was used as the crude extract for the malate dehydrogenase activity measurements. The malate dehydrogenase was determined, using as the buffer 100 mM 3- amino-1-propanol (pH 9.2) with additionally 4.5 mM MgCl2 [sic] and 2.9 mM NAD⁺. The test batch contained 1 ml of this buffer and 50 μl of the crude ^' extract . The batch was first incubated for 30 minutes at 30°C. Thereafter, the malate dehydrogenase reaction was started by addition of 25 mM neutralized L-malate. The absorption was measured at a wavelength of 340 nm in a Lambda-10 spectrophotometer from PE Biosystems (Foster City, CA, USA) . Measurement with the^' complete reaction batch without L-malate served as a control of the specific NAD⁺ reduction rate.

The starting strains MH20-22B, DM58-1 and. DG52-5 had a specific malate dehydrogenase activity of -301, 380 and 354 nmol/min x mg protein respectively, while the mdhA insertion mutants showed no detectable malate dehydrogenase activity. The lower detection limit here for the activity was 2 nmol/min x mg protein.

Example 6

Preparation of L-lysine ■ ,

The strains described in example 5 were incubated in 2 x TY medium, which had been supplemented with kanamycin (25 μg/ml) in the case of the insertion mutants, for 24 hours at 30°C. For culturing in a liquid medium, the medium Cglll (Keilhauer et al. 1993, Journal of Bacteriology 175: 5595-5603), which had_% additionally been supplemented with kanamycin (25 mg/1) for the insertion mutants, was used. For this, 10 ml medium contained in a 100 ml conical flask were inoculated with a colony of the strain and the culture was then used further as a pre-culture.

CGXII-glucose minimal medium (Keilhauer et al . Journal of Bacteriology 175, 5595-5603 (1993)) was used as the production and test medium. The medium contained no kanamycin. Culturing was carried out in 500 ml conical flasks equipped with a metal spiral with a diameter of 2 cm lying along the edge on the base. These flasks were filled with 60 ml CGXII-glucose minimal medium. The cultures were inoculated with the pre-culture such that the optical density at the start had a value of between 0.5 and 0.6. Culturing was carried out for 72 hours at 30°C. The flasks were shaken with a frequency of 140 revolutions per minute.

After incubation for 72 hours, the optical density of the culture and the lysine concentration in the production medium were determined. i

The optical density (OD) was determined with a Lambda B spectrophotometer from Perkin-Elmer at a wavelength of- 600 nm.

L-Lysine was determined by means of "High Performance Liquid Chromatography", a "Hypersil ODS 5μ" column (dimensions: 125 x 4 mm) from Chromatographie-Service, Langerwehe, Germany, being used. The separation was carried out by means of a linear gradient of mobile phases A:

0.1 mM sodium acetate, pH 7.5 and B: methanol (ratio A:B of 85:15 to 0:100). 5 minutes before application the amino acids were derivatized with ortho-phthalodialdehyde (OPA reagent, Pierce, Rockford IL, USA) . Detection was carried out by measuring the fluorescence with an excitation wavelength of 243 nm and an emission wavelength of 436 nm. The result of the experiment is shown in table 1.

TABLE 1

Brief Description of the Figure:

Figure 1: Map of the plasmid pEMmdhAint.

The abbreviations and designations used have the following meaning. The base pair numbers stated are approximate values obtained in the^' context of reproducibility of measurements .

mdhAint Internal fragment of the mdhA gene, bases 566 to 1035 of Seq. ID. O. 1

Km gene Km resistance gene (Tn5)

oriV E. coli replication origin

oriT Origin for transfer (mobilization) from plasmid RP4

BamHI: Cleavage site of the restriction enzyme BamHI

Hindlll: cleavage site of the restriction enzyme Hindlll

Claims

What is claimed is :

1. An isolated polynucleotide from coryneform bacteria, comprising a polynucleotide sequence which codes for the mdhA gene, chosen from the group consisting of

a) polynucleotide which is identical to the extent of at least 70% to a polynucleotide codes [sic] the amino acid sequence of SEQ ID No. 3,

b) polynucleotide which codes for a polypeptide which comprises an amino acid sequence which is identical to the extent of at least to [sic] 70% to the amino acid sequence of SEQ ID No. 3,

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

the polypeptide preferably having the activity of malate dehydrogenase.

2. A polynucleotide as claimed in claim 1, wherein the polynucleotide is a preferably recombinant DNA which is capable of replication in coryneform bacteria.

3. A polynucleotide as claimed in claim 1, wherein the polynucleotide is an RNA.

4. An isolated polypeptide with malate dehydrogenase activity, which comprises the N-terminal amino acid sequence

Asn Ser Pro Gin Asn Val Ser Thr Lys Lys Val Thr Val Thr Gly Ala 1 5 10 15

Ala Gly Gin lie 20 shown in SEQ ID No. 1.

. A DNA as claimed in claim 2 which is capable of replication, comprising

(i) the nucleotide sequence shown in SEQ ID no. 2, or

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

(iv) sense mutations of neutral function in (i) .

6. A coryneform bacterium in which the mdhA gene is attenuated, preferably eliminated.

7. A process for the preparation of L-amino acids, in particular L-lysine, which comprises carrying out the following steps a) fermentation of the bacteria which produce the desired L-amino acid and in which at least the mdhA gene is attenuated, b) concentration of the desired product in the medium or in the cells of the bacteria and c) isolation of the L-amino acid.

8. A process as claimed in claim 7, wherein bacteria in which further genes of the biosynthesis pathway of the desired L-amino acid are additionally amplified, are employed.

9. A process as claimed in claim 7, wherein bacteria in which the metabolic pathways which reduce the formation of the desired L-amino acid are at least partly eliminated are employed.

10. A process as claimed in claim 7, wherein expression of the polynucleotide (s) which code(s) for the mdhA gene is reduced, in particular eliminated.

11. A process^' as claim^'ed in claim 7, wherein the catalytic properties of the polypeptide (enzyme protein) for which the polynucleotide mdhA codes are decreased.

12. A process as claimed in claim 7, wherein for the preparation of L-amino acids, in particular L-lysine, bacteria in which at the same time one or more of the genes chosen from the group consisting of

12.1 the dapA gene which codes for dihydrodipicolinate synthase,

12.2 the pyc gene which codes for pyruvate carboxylase,

12.3 the eno gene which codes for enolase,

12.4 the zwf gene which codes for the zwf gene product,

12.5 the lysE gene which codes for lysine export,

is or are enhanced, preferably over-expressed, are fermented.

13. A process as claimed in claim 7, wherein at the same time one or more of the genes chosen from the group consisting of:

13.1 the pck gene which codes for phosphoenol pyruvate carboxykinase,

13.2 the pgi gene which codes for glucose 6-phosphate isomerase,

13.3 the poxB gene which codes for pyruvate oxidase is or are attenuated.

14. A process as claimed in one or more of the preceding claims, wherein microorganisms of the genus Corynebacterium glutamicum are employed.

15. A process for discovering RNA, cDNA and DNA in order to isolate nucleic acids, or polynucleotides or genes which code for- malate dehydrogenase or have a high similarity to the sequence of the malate dehydrogenase gene, which comprises employing the polynucleotide sequences as claimed in claims 1 to 4 as hybridization probes.

16. A process as claimed in claim 15, wherein arrays, micro arrays or DNA chips are employed.