KR20020097245A - Nucleotides sequences coding for the cdsA gene - Google Patents

Abstract

The present invention amplifies the genetically modified coryneform bacteria amplified by the cdsA gene, polynucleotides isolated from coryneform bacteria encoding phosphatidate cytydilyl transferase, and the cdsA gene to ferment L-amino acids in the bacteria. And to the use of polynucleotides as primers or hybridization probes.

Description

Nucleotides sequences coding for the cdsA gene

Amino acids, especially L-lysine, are used in the human medicine and pharmaceutical industries, but especially in animal feed.

Amino acids are known to be produced by fermentation of strains of Coryneform bacteria, in particular Corynebacterium glutamicum. Due to their great importance, there have been ongoing efforts to improve the manufacturing process. Improvements in the method yield the product by means associated with fermentation techniques, eg, agitation and oxygen supply, or composition of a nutrient medium such as, for example, sugar concentration during fermentation, or, for example, ion exchange chromatography. After treatment, or inherent performance characteristics of the microorganisms themselves.

Performance characteristics of these microorganisms are improved using mutagenesis, screening and mutation screening. In this way, it is resistant to an antimetabolite such as lysine analog S- (2-aminoethyl) cysteine, or it is nutritionally constitutive to an amino acid that is important for regulation and is L-amino acid such as L-lysine. Obtain a strain to produce.

For many years, recombinant DNA technology methods have also been used to improve the amino acid producing Corynebacterium strains by amplifying each biosynthetic gene and investigating its effect on amino acid production. An overview of this content is described in particular by Kinoshita ("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. (Annuals) of the New York Academy of Science 782, 25-39 (1996)).

The present invention relates to amino acids, in particular L-, using a genetically modified coryneform bacterium, a nucleotide sequence encoding phosphatidate citidilyl transferase, and a coryneform bacterium amplified with the cdsA gene encoding phosphatidate citidilyl transferase. Provided are methods for preparing lysine fermentatively.

Figure 1: Map of plasmid pJC1cdsA

Abbreviations and names are defined as follows:

Orf2, rep: plasmid-encoded replication origin, seed. Glutamicum (from pH1519)

cdsA: Mr. CdsA (phosphatidate citidilyl transferase) gene from glutamicum ATCC13032

Kan: kanamycin resistance gene

NarI: restriction site of the restriction enzyme NarI

SalI: restriction site of the restriction enzyme SalI

SgrAI: restriction site of the restriction enzyme SgrAI

Bst1107: restriction site of the restriction enzyme Bst1107

NheI: restriction site of the restriction enzyme NheI

XhoI: restriction site of the restriction enzyme XhoI.

ClaI: restriction site of the restriction enzyme ClaI

BstEII: restriction site of the restriction enzyme BstEII

EcoRI: restriction site of the restriction enzyme EcoRI

Figure 2:

Seed at 40 ° C. Proliferation of Glutamicum ATCC 13032 and ATCC 13032 / pJCcdsA

OD: optical density

The present invention aims to provide new auxiliaries for improved fermentative preparation of amino acids, in particular L-lysine.

This object is achieved by a genetically modified coryneform bacterium amplified with the cdsA gene encoding phosphatidate cydylyl transferase.

Amino acids, in particular L-lysine, are used in human medicine, in particular in the pharmaceutical industry and especially in animal feed. Thus, there has been general interest in providing novel improved methods for preparing amino acids, especially L-lysine.

When L-lysine or lysine is mentioned below, it means not only a base but also a salt, for example lysine monohydrochloride or lysine sulfate.

The present invention provides a genetically modified coryneform bacterium amplified with the cdsA gene encoding phosphatidate cydylyl transferase.

In this regard, the term “amplification” refers to an increase in the intracellular activity of one or more enzymes in a microorganism encoded by the corresponding DNA.

Amplification can be accomplished by manipulation of various bacterial cells.

Amplification, in particular overexpression, can be accomplished by increasing the number of copies of the corresponding gene, using strong promoters or by mutating a regulatory region or ribosomal-binding site located on top of the structural gene. Expression cassettes incorporated on top of the structural genes work in the same way. Inducible promoters may also increase expression during fermentative L-lysine production. Genes encoding corresponding enzymes with enhanced activity may also be used. In addition, expression is improved by means of extending the life of m-RNA. In addition, enzyme activity is generally increased by preventing degradation of the enzyme. Optionally, such means can be used freely.

The microorganisms provided by the present invention can produce L-amino acids, especially L-lysine, from glucose, sucrose, lactose, fructose, maltose, molasses, starch, cellulose, or glycerol and ethanol. The microorganism may include representative coryneform bacteria of the genus Corynebacterium in particular. Among the genus Corynebacterium, mention may be made specifically of the Corynebacterium glutamicum species known to the person skilled in the art for the ability to produce L-amino acids.

Suitable strains of the genus Corynebacterium, in particular Corynebacterium glutamicum species, are for example the following 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

As mutants or strains producing L-lysine prepared therefrom, for example,

Corynebacterium glutamicum FERM-P 1709,

Brevibacterium plaboom FERM-P 1708,

Brevibacterium lactofermentum FERM-P 1712,

Corynebacterium glutamicum FERM-P 6463,

Corynebacterium glutamicum FERM-P 6464 and

Corynebacterium glutamicum DSM5715.

The invention also

(a) a polynucleotide that is at least 70% identical to the polynucleotide encoding a polypeptide containing the amino acid sequence of SEQ ID NO: 2,

(b) a polynucleotide encoding a polypeptide containing an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO: 2,

(c) a polynucleotide complementary to the polynucleotide of (a) or (b), and

(d) a polynucleotide isolated from a coryneform bacterium containing a polynucleotide sequence selected from the group consisting of polynucleotides containing at least 15 consecutive nucleotides in the polynucleotide sequence of (a), (b) or (c) above To provide.

For the purposes of this application, a polynucleotide sequence is selected from the sequence according to the invention if the base composition and sequence thereof match at least 70%, preferably at least 80%, particularly preferably at least 90% of the sequences according to the invention. Homology ". According to the invention, a "homologous protein" is a protein having an amino acid sequence which matches at least 70%, preferably at least 80%, particularly preferably at least 90%, with an amino acid sequence encoded by the cdsA gene (SEQ ID NO: 1). It should be understood that "match" includes those amino acids whose corresponding amino acids are identical or homologous to each other. "Homologous amino acids" are, in particular, amino acids having corresponding properties on charge, hydrophobicity or conformational properties.

In addition, the present invention preferably

(i) the nucleotide sequence set forth in SEQ ID NO: 1

(ii) one or more sequences corresponding to sequence (i) within the degeneracy range of the genetic code, or

(iii) one or more sequences that hybridize with the sequence complementary to sequence (i) or (ii) and optionally

(iv) a polynucleotide sequence as described above comprising a replicable DNA containing a functionally neutral mutation in (i) resulting in identical or homologous amino acids.

The invention also

Preferably a recombinant polynucleotide capable of replicating in coryneform bacteria, comprising the nucleotide sequence set forth in SEQ ID NO: 1,

A polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2,

A seed encoding the cdsA gene contained in the vector pJC1cdsA deposited as Corynebacterium glutamicum under No. 13252. A vector containing the DNA sequence of C. glutamicum and

Coryneform bacteria containing the vector or acting as host cells with amplified cdsA gene are provided.

The present invention comprises a complete gene having a polynucleotide sequence according to SEQ ID NO: 1 or a fragment thereof, hybridization of a probe containing a suitable gene library with a sequence of polynucleotides according to SEQ ID NO: 1, or a fragment thereof and a DNA sequence referred to Polynucleotides obtainable by screening by separation of are provided.

The polynucleotide sequence according to the present invention is an RNA, cDNA for isolating a full-length cDNA encoding phosphatidate citidilyl transferase and for separating such a cDNA or gene exhibiting a high level of similarity with the sequence of phosphatidate citidil transferase. And also as hybridization probes for DNA.

The polynucleotide sequences according to the invention are further suitable as primers for polymerase chain reaction (PCR) for preparing DNA encoding phosphatidate cytidilyl transferase.

Such oligonucleotides which act as probes or primers may contain at least 30, preferably at most 30, particularly preferably at most 20, very particularly preferably at least 15 consecutive nucleotides. Also suitable are oligonucleotides having a length of 40-50 or more nucleotides.

"Isolated" means to be separated from its natural environment.

In general, "polynucleotide" refers to polyribonucleotides and polydeoxyribonucleotides into which RNA or DNA may be unmodified or modified.

"Polypeptide" means a peptide or protein containing two or more amino acids joined via peptide bonds.

The polypeptide according to the invention is at least 70% homologous, preferably at least 80% homologous to the polypeptide according to SEQ ID NO: 2, in particular to the polypeptide having the biological activity of the phosphatidate cydylyl transferase, and also to the polypeptide according to SEQ ID NO: 2 Adult polypeptides, and polypeptides according to SEQ ID NO: 2, in particular having a 90-95% homology and exhibiting the abovementioned activities.

The present invention relates, in particular, to a fermentative process for the preparation of amino acids, in particular L-lysine, using coryneform bacteria in which the amino acid has been produced beforehand and the nucleotide sequence encoding the cdsA gene has been amplified, in particular overexpressed.

The present invention firstly encodes phosphatidate cydylyl transferase. The cdsA gene of glutamicum is described.

Seed. The cdsA gene or other genes from glutamicum may first be constructed from a library of these microorganisms. Isolation by constructing in E. coli. The construction of gene libraries is generally described in known textbooks and manuals. Examples include textbooks [Winnacker: Gene und Klone, Eine Einfuhrung in die Gentechnologie (Verlag Chemie, Weinheim, Germany, 1990) or handbook [Sambrook et al .: Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratory) Press, 1989) One very well-known gene library is the one constructed in the λ-vector by Kohara et al. (Cell 50, 495-508 (1987)). A gene library of E. coli K-12 strain W3110. Bathe et al., Molecular and General Genetics, 252: 255-265, 1996, described in the cosmid vector SuperCos I (Wahl et al., 1987, Proceedings of the National Academy of Sciences USA 84: 2160-2164, using E. coli K-12 strain NM554 (Raleigh et al., 1988, Nucleic Acids Research 16: 1563-1575). A genetic library of constructed C. glutamicum ATCC13032 is described. Joe: Bormann et al. (Molecular Microbiology 6 (3), 317-326 (1992)) describe C. glutamim using cosmid pHC79 (Hohn and Collins, Gene 11, 291-298 (1980)). The genetic library of Qum ATCC13032 is described: The library of C. glutamicum in E. coli is either pBR322 [Bolivar, Life Sciences, 25, 807-818 (1979)] or pUC9 [Vieira et al. al., 1982, Gene, 19: 259-268. Suitable hosts are particularly E. coli with restriction deletions and recombinant deletions. E. coli strains. One example of such a strain is the DH5αmcr strain described in Grant et al., Proceedings of the National Academy of Sciences USA, 87 (1990) 4645-4649. The long DNA fragment cloned with cosmid as an aid can then be cloned into a conventional vector suitable for sequencing and then described, for example, in Sanger et al., Proceedings of the National Academy of Sciences of the United States of America, 74: 5463-5467, 1977).

A seed encoding the cdsA gene, provided by the present invention as SEQ ID NO: 1. New DNA sequences from glutamicum are obtained in this manner. In addition, the amino acid sequence of the corresponding protein is derived from the DNA sequence using the method described above. SEQ ID NO: 2 shows the resulting amino acid sequence of the product of the cdsA gene.

Also provided by the present invention is a coding DNA sequence resulting from SEQ ID NO: 1 due to the degeneracy of the genetic code. DNA sequences that hybridize with SEQ ID NO: 1 or a portion of SEQ ID NO: 1 are likewise provided by the present invention. Conservative substitutions of amino acids in proteins, for example, substitution of glycine with alanine or substitution of aspartic acid with glutamic acid, do not cause a fundamental change in the activity of the protein, ie, as a functionally neutral "sense mutation". Known to them. It is also known that changes in the N and / or C terminus of a protein can substantially or even stabilize its function. Those skilled in the art will find information related to this, in particular, in Ben-Bassat et al. (Journal of Bacteriology 169: 751-757 (1987)), O'Regan et al. (Gene 77: 237-251 (1989). )), Sahin-Toth et al. (Protein Sciences 3: 240-247 (1994)), Hochuli et al. (Bio / Technology 6: 1321-1325 (1988)) and known genetics and molecular biology textbooks. Can be. Amino acid sequences resulting from SEQ ID NO: 2 in a corresponding manner are also provided by the present invention.

DNA sequences that hybridize with SEQ ID NO: 1 or a portion of SEQ ID NO: 1 are likewise provided by the present invention. Finally, DNA sequences prepared by polymerase chain reaction (PCR) using oligonucleotide primers obtained from SEQ ID NO: 1 are also provided by the present invention. Such oligonucleotides typically have a length of at least 15 nucleotides.

Those skilled in the art specifically refer to the guidelines on the identification of DNA sequences by hybridization ["The DIG System Users Guide for Filter Hybridization, Firma Boehringer Mannheim GmbH, Mannheim, Germany, 1993] and literature [Ref. Liebl et al., International Journal of Systematic Bacteriology (1991) 41: 255-260. Those skilled in the art will specifically refer to instructions for amplifying DNA sequences using polymerase chain reaction (PCR). Gait, Oligonucleotide synthesis: a practical approach, IRL Press, Oxford, UK, 1984 and handbook [Gait: Oligonucleotide synthesis: a practical approach (IRL Press, Oxford, UK, 1984) and Newton & Graham, PCR (Spektrum Akademischer Verlag, Heidelberg, Germany, 1994).

During the study, it was demonstrated that coryneform bacteria produce amino acids, especially L-lysine, in an improved manner when the cdsA gene is amplified.

The gene or gene construct contemplated can be present in the plasmid in various copies or inserted into the chromosome and amplified. Alternatively, overexpression of related genes may be achieved by modifying the composition and culture conditions of the medium.

One skilled in the art will find relevant guidance in particular in Martin et al. (Bio / Technology 5, 137-146 (1987), Tsuchiya and Morinaga, Bio / Technology 6, 428-430 (1988), Eikmanns et al., Gene 102, 93-98 (1991); European Patent EPS 0 472 869 , US Pat. No. 4,601,893, Schwarzer and Puhler (Bio / Technology 9, 84-87 (1991)), Reinscheid et al. (Applied and Environmental Microbiology 60, 126-132 (1994)), LaBarre et al. (Journal of Bacteriology 175, 1001-1007 (1993)), patent application WO 96/15246, Malumbres et al. (Gene 134, 15-24 (1993)), JP-A-10-229891, Jensen and Hammer (Biotechnology and Bioengineering 58, 191-195 (1998)), Makrides (Microbiological Reviews 60: 512-538 (1996)) and well-known genetics and molecular biology textbooks.

For example, the cdsA gene according to the present invention is overexpressed with the aid of a plasmid.

Suitable plasmids are plasmids that are replicated and expressed in coryneform bacteria. Many known plasmid vectors such as pZ1 (Menkel et al., Applied and Environmental Microbiology (1989) 64: 549-554), pEKEx1 (Eikmanns et al., Gene 102: 93-) 98 (1991) or pHS2-1 (Sonnen et al., Gene 107: 69-74 (1991)) is based on latent plasmid pHM1519, pBL1 or pGA1. Other plasmid vectors such as pCG4 [US-A-4,489,160], pNG2 [Serwold-Davis et al., FEMS Microbiology Letters 66, 119-124 (1990)] or pAG1 [Ref. Plasmids based on US-A-5,158,891 can be used in the same manner.

One example of a plasmid that can be used to overexpress the cdsA gene is E. coli-C. Seed encoding the cdsA gene based on the glutamicum shuttle vector pJC1 (Cremer et al., 1990, Molecular and General Genetics 220: 478-480). There is pJC1cdsA (FIG. 1) containing the DNA sequence of glutamicum. It is contained in strain DSM5715 / PJC1cdsA.

Further suitable plasmid vectors are described, for example, for the replication and amplification of the hom-thrB operon, as described by Reinscheid et al., Applied and Environmental Microbiology 60, 126-132 (1994). Plasmid vector used to perform gene amplification by insertion into chromosomes. In this method, the complete gene can be replicated in the host (typically E. coli), It can be cloned into plasmid vectors that cannot be replicated in glutamicum. Vectors that can be considered include, for example, pSUP301 (Simon et al., Bio / Technology 1, 784-791 (1983)), pK18mob or pK19mob [Schafer et al., Gene 145, 69-73 (1994)], pGEM-T from Promega corporation, Madison, Wis., USA, pCR2.1-TOPO [Shuman (1994). Journal of Biological Chemistry 269: 32678-84; US-A 5,487,993], PCR ^R Blunt [manufactured by Invitrogen, Groningen, Netherlands; Bernard et al., Journal of Molecular Biology, 234: 534-541 (1993) or pEM1 (Schrumpf et al., 1991, Journal of Bacteriology 173: 4510-4516). The desired strain C. was then conjugated or transformed with the plasmid vector containing the gene to be amplified. Transform into glutamicum. Conjugation methods are described, for example, in Schafer et al., Applied and Environmental Microbiology 60, 756-759 (1994). Transformation methods are described, for example, in Thierbach et al., Applied Microbiology and Biotechnology 29, 356-362 (1988), Dunican and Shivnan (Bio / technology 7, 1067-1070 (1989)) and Tauch et al. (FEMS Microbiological Letters 123, 343-347 (1994)). After homologous recombination using "crossing over", the resulting strain contains two or more copies of that gene.

To produce amino acids, especially L-lysine, it may also be advantageous to amplify or overexpress not only the cdsA gene, but also one or more enzymes of a particular biosynthetic pathway, glycolysis, complement metabolism, citric acid cycle or amino acid excretion.

Thus, to produce L-lysine, it may be advantageous to simultaneously amplify, in particular overexpress or amplify one or more genes selected from the group consisting of, for example:

DapA gene encoding dihydropicolinate synthase (EP-B 0 197 335),

DapE gene encoding succinyldiaminopimelate disuccinase,

LysC gene encoding feed back resistant aspartate kinase [Kalinowski et al. (1990), Molecular and General Genetics 224, 317-324;

Gap gene encoding glyceraldehyde-3-phosphate dehydrogenase (Eikmanns (1992), Journal of Bacteriology 174: 6076-6086),

Tpi gene encoding triocell phosphate isomerase (Eikmanns (1992), Journal of Bacteriology 174: 6076-6086),

Pgk gene encoding 3-phosphoglycerate kinase (Eikmanns (1992), Journal of Bacteriology 174: 6076-6086),

Pyc gene encoding pyruvate carboxylase [DE-A-19831609],

Malate: mqo gene encoding quinone oxidoreductase (Molenarr et al., European Journal of Biochemistry 254, 395-403 (1998)) or

LysE gene encoding lysine excretion (DE-A-195 48 222).

In addition, to produce amino acids, especially L-lysine, it may be advantageous to attenuate the following genes simultaneously, in addition to amplification of the cdsA gene:

Pck gene encoding phosphoenol pyruvate carboxykinase (see DE 199 59 409.1, DSM 13047) and / or

Pgi gene encoding glucose 6-phosphate isomerase (see US 09 / 396,478, DSM 12969) and / or

PoxB gene encoding pyruvate oxidase (DE 1995 1 975.7).

In order to produce L-amino acids, especially L-lysine, it may also be advantageous to inhibit unwanted side reactions, in addition to overexpression of the cdsA gene. See Nakayama: "Breeding of Amino Acid Producing Micro-organisms"; Overproduction of Microbial Products, Krumphanzl, Sikyta, Vanek (eds.), Academic Press, London, UK, 1982.

For the production of amino acids, in particular L-lysine, microorganisms prepared according to the invention can be cultured continuously or discontinuously using a batch process or a fed batch process or a repeated fed batch process. A summary of known culture methods can be found in textbooks by Chmiel (Bioprozesstechnik 1. Einfuhrung in die Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991)) or textbooks by Storas [Bioreaktoren und periphere Einrichtungen (Vieweg Verlag, Braunschweig / Wiesbaden, 1994).

The culture medium to be used must suitably meet the requirements of the particular strain. Various culture media for microorganisms are described in "Manual of Methods for General Bacteriology" from the American Society for Bacteriology (Washington D.C., USA, 1981). Carbon sources that can be used include sugars and carbohydrates (e.g. glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose), oils and fats (e.g. soybean oil, sunflower oil, peanut oil and coconut oil), fatty acids (Eg palmitic acid, stearic acid and linoleic acid), alcohols such as glycerol and ethanol and organic acids such as acetic acid. These materials can be used individually or as a mixture. Nitrogen sources that can be used are nitrogen containing organic compounds (e.g. peptone, yeast extract, meat extract, malt extract, corn steep liquor, soy flour and urea) or inorganic compounds (e.g. ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and Ammonium nitrate). The nitrogen source can be used alone or as a mixture. Phosphorus sources that can be used are phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium containing salts. The culture medium should also contain metal salts necessary for propagation (eg magnesium sulfate or iron sulfate). Finally, in addition to the substances mentioned above, essential growth-promoting substances such as amino acids and vitamins can be used. In addition, suitable precursors may be added to the culture medium. The nutritional substances mentioned can be added to the culture as a single batch or can be fed as appropriate during the culture.

Basic pH (eg sodium hydroxide, potassium hydroxide, ammonia or aqueous ammonia) or acidic compound (eg phosphoric acid or sulfuric acid) is appropriately used to adjust the pH of the culture. Antifoams, for example fatty acid polyglycol esters, can be used to control foam. Plasmid stability can be maintained by the addition of a suitable selective agent, for example an antibiotic, to the medium. To maintain aerobic conditions, an oxygen or oxygen containing gas mixture, such as air, is introduced into the culture. The temperature of the culture is usually 20 ° C to 45 ° C and preferably 25 ° C to 40 ° C. The culture is continued until the maximum amount of lysine is formed. This object is typically achieved within 10 to 16 hours.

Analysis of L-lysine can be performed using anion exchange chromatography followed by ninhydrin derivatization as described in Spackman et al., Analytical Chemistry, 30, (1958), 1190. .

The following microorganisms were deposited in Deutsche Sammlung fur Mikrorganismen und Zellkulturen (DSMZ) in Braunschweig, Germany:

Corynebacterium glutamicum strain DSM5715 / pJC1cdsA as DSM 13252.

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

The invention is illustrated in more detail by the following examples.

Example 1

Preparation of Genomic Cosmid Gene Library from Corynebacterium glutamicum ATCC 13032

Chromosome DNA was isolated from Corynebacterium glutamicum ATCC 13032 and described by restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg) as described in Tauch et al., 1995, Plasmid 33: 168-179. , Germnay, product name Sau3AI, code number 27-0931-02). DNA fragments are dephosphorylated with shrimp alkaline phosphatase (Source: Roche Molecular Biochemicals, Mannheim, Germany, trade name SAP, code no. 1758250). Cosmid vector SuperCos1 (Wahl et al. (1987) Proceedings of the National Academy of Sciences USA 84: 2160-2164; From Stratagene, La Jolla, USA, product name SuperCos1 Cosmid Vector Kit, code number 251301], was digested with restriction enzyme XbaI (manufacturer: Amersham Pharmacia, Freiburg, Germany, product name XbaI, code number 27-0948-02) It is also dephosphorylated with shrimp alkaline phosphatase. Cosmid DNA is then digested with the restriction enzyme BamHI (manufactured by Amersham Pharmacia, Freiburg, Germany, product name BamHI, code no. 27-0868-04). Cosmid DNA treated in this manner is mixed with treated ATCC 13032 DNA and the batch is treated with T4 DNA ligase (manufactured by Amersham Pharmacia, Freiburg, Germany, trade name: T4 DNA Ligase, code no. 27-0870-04). do. The ligation mixture is then packed in phage using Gigapack II XL Packing Extract (Stratagene, La Jolla, USA, product name: Gigapack II XL Packing Extract, code no. 200217). this. E. coli strain NM554 [Raleigh et al. 1988, Nucleic Acid Research 16: 1563-1575] suspend cells in 10 mM MgSO ₄ and mix them with aliquots of phage suspension to infect. Cosmid libraries are infected and titered as described in Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, where cells contain LB containing 100 mg / L ampicillin. Agar (Lennox, 1955, Virology, 1: 190). After incubation at 37 ° C. overnight, each recombinant clone is selected.

Example 2

Isolation and Sequencing of the cdsA Gene

Cosmid DNA was isolated from each colony using the Qiaprep Spin Miniprep Kit (product no .: 27160, manufactured by Qiagen, Hilden, Germany) according to the manufacturer's instructions, and the restriction enzyme Sau3AI (manufacturer) : Amersham Pharmacia, Freiburg, Germnay, product name: Sau3AI, code number: 27-0931-02). DNA fragments are dephosphorylated with shrimp alkaline phosphatase (Source: Roche Molecular Biochemicals, Mannheim, Germany, trade name SAP, code no. 1758250). After separation by gel electrophoresis, cosmid fragments of size 1500 to 2000 bp are separated using a QiaExII gel extraction kit (product number: 20021, manufactured by Qiagen, Hilden, Germany). DNA of the sequencing vector pZero-1 (purchased from Invitrogen, Groningen, Netherlands, product name: Zero Background Cloning Kit, product number: K2500-01) was restricted to BamHI (manufacturer: Amersham Pharmacia, Freiburg, Germany, product name BamHI, code number) 27-0868-04). Cosmid fragments are described in literature; Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, were linked into the sequencing vector pZero-1 and the DNA mixture was T4 ligase from Pharmacia Biotech, Freiburg, Germany. Incubate overnight with. The linking mixture is then e. Electroporation into E. coli strain DH5αMCR [Grant, 1990, Proceedings of the National Academy of Sciences USA, 87: 4645-4649] and Tauch et al., 1994, FEMS Microbiol Letters, 123: 343-7 ], LB agar containing 50 mg / ml zeocin (Lennox, 1955, Virology, 1: 190). Plasmids of recombinant clones are prepared using Biorobot 9600 (product number: 90020, manufactured by Qiagen, Hilden, Germany). Sequencing is described by Zimmermann et al. (1990, Nucleic Acids Research, 18: 1067), Sanger et al., 1977, Proceedings of the National Academy of Sciences. U.S.A., 74: 5463-5467]. RR dRhodamin Terminator Cycle Sequencing Kit (manufactured by PE Applied Biosystems, Weiterstadt, Germany, product no .: 403044). Analysis of isolation and sequencing reactions by gel electrophoresis was carried out using an "ABI Prism 377" sequencer (PE Applied Biosystems, Weiterstadt, Germany) using a "Rotiforesis NF acrylamide / bisacrylamide" gel (29: 1) (product no. A124.1, Roth, Karlsruhe, Germany).

The raw sequence data obtained is then processed using the Staden software package (1986, Acids Research, 14: 217-231) version 97-0. Integrate each sequence of pZero1 derivatives into a contiguous arrangement. Computer assisted encryption range analysis is performed using the XNIP program (Staden, cf. 1986, Nucleic acids Rearch, 14: 217-231). For the affluent database of the National Center for Biotechnology Information (NCBI, Bethesda, MD, USA), the "BLAST search program" [Altschul et al., 1997, Nucleic Acids Research, 25: 3389-3402] for further analysis.

The obtained nucleotide sequence is described in SEQ ID NO: 1. Analysis of the nucleotide sequence shows an open reading frame of 891 base pairs designated as cdsA genes. The cdsA gene encodes a protein of 297 amino acids.

Example 3

Cloning of the cdsA Gene into Vector pJC1

Chromosome DNA is isolated from Corynebacterium glutamicum ATCC 13032 as described in Tauch et al., (1995, Plasmid 33: 168-179). DNA fragments carrying the cdsA gene are amplified using polymerase chain reaction. The following primers are used for this purpose:

5'-CGC GGA TCC GTG GCC CAA GCT TTA CGA CGG ATA C-3 '

5'-CGC GGA TCC GGC TCG CAA GGA AAA GGA ACT GAT-3 '

Both oligonucleotides carry the sequence (underlined nucleotide) for the cleavage site of the restriction enzyme BamHI. The primers described were synthesized by MWG Biotech Co., Ltd. (Eversweg, Germany), so PCR reactions were described in Innis et al., PCR protocol. A guide to methods and applications, 1990, Academic Press]. This primer causes the 1095 bp DNA fragment with the cdsA gene from Corynebacterium glutamicum to be amplified.

After separation by gel electrophoresis, PCR fragments are separated from agar gels using a QiaExII gel extraction kit (product number: 20021, manufactured by Qiagen, Hilden, Germany).

PCR fragments obtained in this manner are digested thoroughly with restriction enzyme BamHI. 1087 bp cdsA fragments are isolated from agar gels using a QiaExII gel extraction kit (product number: 1087, manufactured by Qiagen, Hilden, Germany).

The vector used is this. Coli-C. Glutamicum shuttle vector pJC1 (Cremer et al, 1990, Molecular and General Genetics 220: 478-480). The plasmid is also digested completely with restriction enzyme BamHI and then dephosphorylated with shrimp alkaline phosphatase (source: Roche Molecular Biochemicals, Mannheim, Germany, trade name SAP, code no. 1758250).

The cdsA fragment obtained in this manner is mixed with the prepared pJC1 vector and the batch is treated with T4 DNA ligase (manufactured by Amersham Pharmacia, Freiburg, Germany, trade name: T4 DNA Ligase, code no. 27-0870-04). Followed by a connection batch. E. coli strain DH5α [Hanahan: DNA cloning. A practical approach. Vol. I. Transform in IRL-Press, Oxford, Washington DC, USA. Plasmid-containing cells are selected by plating transfection batches in LB agar containing 50 mg / l kanamycin. After incubation at 37 ° C. overnight, each recombinant clone is selected. Plasmid DNA was isolated from the transformants using the Quiaprep Spin Miniprep Kit (Product No .: 27160, manufactured by Qiagen, Hilden, Germany) according to the manufacturer's instructions, and the plasmid was subjected to subsequent agarose gel electrophoresis. Cut with restriction enzyme to confirm. The resulting plasmid is named pJC1cdsA.

Example 4

Transformation of strain DSM5715 with plasmid pJC1cdsA

Strain DSM5715 is then transformed with plasmid pJC1cdsA using the electroporation method described in Liebl et al., FEMS Microbiology Letters, 53: 299-303 (1989). Transformants were screened with 18.5 g / l of brain-heart fusion culture, 5 g / l of Bakto tryptone, 2.5 g / l of Bakto yeast extract, 5 g / l of NaCl and 18 g / l of Agar agar supplemented with 25 mg / l of kanamycin. Performed in LBHIS agar. Incubate at 33 ° C. for 2 days.

To separate plasmid DNA from the transformants using conventional methods (Peters-Wendisch et al., 1998, Microbiology, 144, 915-927) and to confirm the plasmid by subsequent gel electrophoresis, Cut with restriction endonuclease BamHI. The resulting strain was named DSM5717 / pJC1cdsA and deposited in Deutsche Sammlung fur Mikrorganismen und Zellkulturen (DSMZ, Braunschweig, Germany) as DSM 13252 according to the Budapest Treaty.

Example 5

Preparation of Lysine

Seed obtained in Example 5. Glutamicum strain DSM5715 / pJC1cdsA is incubated in a nutrient medium suitable for the production of L-lysine and the L-lysine content in the culture supernatant is measured.

To this end, the strains are first incubated at 33 ° C. for 24 hours in agar plates containing appropriate antibiotics (brain / heart agar containing kanamycin (50 mg / L)). Starting from the agar plate culture, precultures are inoculated (100 mL of medium in a 100 mL Erlenmeyer flask). Complete medium CgIII is used as the medium for the preculture.

Medium CgIII

NaCl 2.5g / ℓ

Bakto peptone 10g / ℓ

Bacterial Yeast Extract 10g / ℓ

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

The pH value is adjusted to pH 7.4.

Kanamycin (25 mg / L) is added to the medium. The precultures are incubated for 16 hours at 33 ° C. at 240 rpm on a shaker. The main culture is inoculated from the preculture so that the initial OD (660 nm) of the main culture is 0.1. Medium MM is used for the main culture.

Badge MM

CSL (corn immersion liquid) 5 g / ℓ

MOPS (morpholinopropanesulfonic acid) 20 g / ℓ

50 g / l glucose (individually autoclaved)

(NH ₄ ) ₂ SO ₄ 25g / ℓ

KH ₂ PO ₄ 0.1 g / ℓ

MgSO ₄ * 7H ₂ O 1.0 g / ℓ

CaCl ₂ * 2H ₂ O 10mg / ℓ

FeSO ₄ * 7H ₂ O 10 mg / ℓ

MnSO ₄ * H ₂ O 5.0 mg / ℓ

Biotin (sterile-filtered) 0.3 mg / l

Thiamine * HCl (sterile-filtered) 0.2 mg / l

0.1 g / l L-Leucine (sterilized-filtered)

CaCO ₃ 25g / ℓ

CSL, MOPS and salt solution are adjusted to pH 7 with ammonia water and autoclaved. Anhydrous autoclaved CaCO _{3 is} then added with sterile substrate and vitamin solution.

Cultivation is carried out in a 10 ml volume in a 100 ml Erlenmeyer flask equipped with a flow spoiler. Add kanamycin (25 mg / L). Incubate at 33 ° C. and 80% atmospheric humidity.

After 48 hours, the OD is measured using a Biomek 1000 (Beckmann Instruments GmbH, Munich) at a measurement wavelength of 660 nm. The amount of lysine formed is determined by post-column derivatives using ion exchange chromatography and ninhydrin detection using an amino acid analyzer (Eppendorf-BioTronik, Hamburg, Germany).

Table 1 shows the test results.

Strain OD (660) Lysine HClg / ℓ DSM5715 / pJC1cdsA 12.3 14.4 DSM5715 11.9 14.0

Example 6:

Improvement of proliferative properties

Seed using the plasmid pJCcdsA obtained in Example 3. Glutamicum strain ATCC 13032 is transformed. The strain is transformed as described in Example 4 and confirmed by restriction digestion and agarose gel electrophoresis as in Example 4. The strain ATCC 13032 / pJCcdsA obtained is incubated in a nutrient medium suitable for measuring proliferation and the proliferation is measured at various temperatures.

To this end, the strains are incubated for 24 hours at 30 ° C. on an agar plate containing an appropriate antibiotic (brain / heart agar containing kanamycin (5 mg / L)) as described in Example 5. Starting from the agar plate culture, the preculture is inoculated (10 ml of medium in a 100 ml Erlenmeyer flask). Complete medium CgIII described in Example 5 is used as the medium for the preculture. Kanamycin (25 mg / L) is added to the medium. The precultures are incubated for 16 hours at 30 ° C. at 240 rpm on a shaker. The main culture is inoculated from the preculture so that the initial OD (660 nm) of the main culture is 0.7. Medium MM is used for the main culture.

Badge MM

MOPS (morpholinopropanesulfonic acid) 42 g / ℓ

Glucose (individually autoclaved) 40 g / l

(NH ₄ ) ₂ SO ₄ 20g / ℓ

KH ₂ PO ₄ 1.0g / ℓ

K ₂ HPO ₄ 1.0 g / ℓ

MgSO ₄ * 7H ₂ O 0.25 g / ℓ

CaCl ₂ * 2H ₂ O 10mg / ℓ

FeSO ₄ * 7H ₂ O 10 mg / ℓ

MnSO ₄ * H ₂ O 10 mg / ℓ

ZnSO ₄ * H ₂ O 1mg / ℓ

CuSO ₄ 0.2mg / ℓ

NiCl ₂ * 6H ₂ O 0.02 mg / l

Biotin (sterile-filtered) 0.2 mg / l

Protocatecuic Acid (Sterile-Filtered) 30 mg / ℓ

MOPS and salt solution are adjusted to pH 7 with ammonia water and autoclaved. Sterile substrate and vitamin solution are then added.

Incubation is carried out in a 60 ml volume in a 500 ml Erlenmeyer flask equipped with a flow spoiler. Add kanamycin (25 mg / L). Incubate at 40 ° C. OD is measured using Ultrospec 3000 (Pharmacia Biotech, Uppsala, Sweden) at a measurement wavelength of 600 nm. Table 2 shows the test results.

Claims (19)

A genetically modified coryneform bacterium amplified with the cdsA gene encoding phosphatidate cydylyl transferase. The method of claim 1, wherein the starting bacteria (wild type) 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 is selected from the group consisting of ATCC14020, Corynebacterium glutamicum FERM-P 1709, Brevibacterium plaboom FERM-P 1708, Brevibacterium lactofermentum FERM-P 1712, Corynebacterium glutamicum FERM-P 6463, Corynebacterium glutamicum FERM-P 6464 and Corynebacterium glutamicum DSM5715 A genetically modified coryneform bacterium selected from the group consisting of. The method of claim 1, wherein the cdsA gene overexpresses the gene, in particular increases the number of copies of the gene, selects a regulatory region located on top of a strong promoter or reading frame, or promotes a promoter, regulatory region or ribosomal-binding Mutating a site, incorporating a suitable expression cassette on top of a structural gene, incorporating an inducible promoter, prolonging the lifespan of a corresponding mRNA, reducing degradation of expressed protein, or among these possibilities Genetically modified coryneform bacteria that are amplified by combining two or more. The genetically modified coryneform bacterium according to any one of claims 1 to 3, wherein the strain is transformed with a plasmid vector and the plasmid vector has a nucleotide sequence encoding the cdsA gene. The genetically modified coryneform bacterium according to any one of claims 1 to 4, wherein the genotype corresponds to strain Corynebacterium glutamicum DSM 13252. (a) a polynucleotide at least 70% identical to a polynucleotide consisting of or encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, (b) a polynucleotide encoding a polypeptide containing an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO: 2, (c) a polynucleotide complementary to the polynucleotide of (a) or (b), and (d) a polynucleotide isolated from a coryneform bacterium containing a polynucleotide sequence selected from the group consisting of polynucleotides containing at least 15 consecutive nucleotides in the polynucleotide sequence of (a), (b) or (c) above Nucleotides. The polynucleotide of claim 6, which is preferably recombinant DNA capable of replicating in coryneform bacteria. The polynucleotide of claim 6 which is RNA. The method of claim 7, wherein (i) the nucleotide sequence set forth in SEQ ID NO: 1 (ii) one or more sequences corresponding to sequence (i) within the degeneracy range of the genetic code, or (iii) one or more sequences that hybridize with the sequence complementary to sequence (i) or (ii) and optionally (iv) A replicable DNA containing a functionally neutral mutation in (i) resulting in homologous amino acids. A polynucleotide sequence according to any one of claims 7 to 9, encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 2. (a) fermenting an L-amino acid producing coryneform bacterium, wherein at least the cdsA gene or nucleotide sequence encoding it is amplified, in particular overexpressed, (b) accumulating L-amino acids in the medium or cells of the bacterium; and (c) a method of fermentative preparation of L-amino acids, comprising the step of separating the L-amino acids. The method according to claim 11, wherein the strain according to claim 1 is used. The method of claim 11 or 12, wherein additional genes encoding proteins in the biosynthetic pathway of the desired L-amino acid are further amplified in bacteria. The method of claim 11, wherein the metabolic pathway that reduces the formation of the desired amino acid is at least partially inhibited in the bacterium. The method according to any one of claims 11 to 14, wherein the amino acid produced is L-lysine. The method according to any one of claims 11 to 15, for the preparation of lysine, (a) a dapA gene encoding a dihydropicolinate synthase, (b) a dapE gene encoding succinyldiaminopimelate disuccinase, (c) a lysC gene encoding a feed back resistant aspartate kinase, (d) a tpi gene that encodes triophosphate isomerase, (e) a gap gene encoding glyceraldehyde 3-phosphate dehydrogenase, (f) a pgk gene encoding 3-phosphoglycerate kinase, (g) a pyc gene encoding pyruvate carboxylase, (h) the mqo gene encoding malate: quinone oxidoreductase and (i) A method in which at least one of the genes selected from the group consisting of lysE genes encoding lysine excretion is simultaneously amplified, in particular overexpressed or amplified bacteria. The method according to any one of claims 12 to 17, for the production of L-lysine, (a) a pck gene encoding phosphoenolpyruvate carboxykinase, (b) a pgi gene encoding glucose 6-phosphate isomerase or (c) A method in which at least one gene selected from the group consisting of poxB genes encoding pyruvate oxidase is fermented simultaneously with attenuated bacteria. Use of a polynucleotide sequence according to claim 6 or part thereof as a primer for preparing DNA of a gene encoding a phosphatidate cytidilyl transferase by a polymerase chain reaction. Use of a polynucleotide sequence according to claim 6 as a hybridization probe for the isolation of cDNAs or genes showing enhanced homology with sequences of the cdsA gene.