KR20020097244A - New nucleotide sequences which code for the pgsA2 gene - Google Patents

Abstract

The present invention, genetically modified coryneform bacteria, augmented with the pgsA2 gene; Polynucleotides isolated from coryneform bacteria, encoding CDP-diacylglycerol-glycerol-3-phosphate 3-phosphatidyltransferase; By fermenting pgsA2 gene in bacteria to ferment L-amino acids; And the use of such polynucleotides as primers or hybridization probes.

Description

New nucleotide sequences which code for the pgsA2 gene}

The present invention relates to genetically modified coryneform bacteria, nucleotide sequences encoding CDP-diacylglycerol-glycerol-3-phosphate 3-phosphatidyltransferase, and CDP-diacylglycerol-glycerol-3-phosphate 3-phosphatidyltransferase Provided are methods for fermentatively preparing amino acids, particularly L-lysine, using coryneform bacteria enriched with the encoding pgsA2 gene (EC 2.7.8.5).

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

Amino acids are known to be produced by fermentation from Coryneform bacteria, in particular Corynebacterium glutamicum strains. Due to their enormous importance, efforts have been made to improve the manufacturing process. Improvements in this method can be achieved by means of fermentation, eg, agitation and oxygenation, or composition of the nutrient medium, eg, sugar concentration during fermentation, or post-treatment of the product, eg, by ion exchange chromatography, or It may be related to the inherent productivity of the microorganisms themselves.

Mutagenesis, selection and mutant screening are used to improve the productivity of these microorganisms. Produces L-amino acids, such as L-lysine, which is resistant to antimetabolites, such as lysine homologue S- (2-aminoethyl) -cysteine, or is nutritionally important for regulatoryly important metabolites A strain to be obtained is obtained by the method described above.

By amplifying each amino acid biosynthetic gene and studying its effect on amino acid production, recombinant DNA techniques have been used for years to improve the amino acid producing Corynebacterium strains. An overview paper on this is described in particular by Kinoshita, "Glutamic Acid Bacteria", 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).

It is an object of the present invention to provide novel means for improved fermentative preparation of amino acids, in particular L-lysine.

This object is achieved by genetically modified coryneform bacteria which are enriched in the pgsA2 gene encoding CDP-diacylglycerol-glycerol-3-phosphate 3-phosphatidyltransferase.

Amino acids, in particular L-lysine, are used in the human medicine and pharmaceutical industries, especially in animal feed. Therefore, it is of general interest to provide new and improved methods for producing amino acids, especially L-lysine.

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

The present invention provides a genetically modified coryneform bacterium enriched in the pgsA2 gene encoding CDP-diacylglycerol-glycerol-3-phosphate 3-phosphatidyltransferase.

In this regard, the term "enhanced" means increasing the intracellular activity of one or more enzymes in a microorganism encoded by the corresponding DNA.

Enhancement can be achieved through various manipulations of bacterial cells.

To achieve augmentation, particularly overexpression, one may increase the number of copies of the corresponding gene, use a strong promoter, or mutate the ribosomal binding site upstream of the promoter and regulatory region or structural gene. Expression cassettes introduced upstream of the structural gene also work in the same way. Inducible promoters may further increase expression during fermentative L-lysine preparation. In addition, a gene encoding a corresponding enzyme having high activity can be used. Expression can also be improved by means of extending the life of m-RNA. Moreover, enzyme activity is increased overall by preventing degradation of the enzyme. In addition, these means can be arbitrarily combined as needed.

The microorganisms provided by the present invention can produce L-amino acids, especially L-lysine, from glucose, sucrose, lactose, fructose, maltose, molasses, starch or cellulose, or from glycerol and ethanol. Representative examples of these microorganisms are coryneform bacteria, especially those of the genus Corynebacterium. Among the genus Corynebacterium, mention may be made in particular of the Corynebacterium glutamicum species whose L-amino acid production capacity is known to those skilled in the art.

Suitable strains among the genus Corynebacterium, in particular Corynebacterium glutamicum species, include, for example, known wild type strains:

Corynebacterium glutamicum ATCC 13032,

Corynebacterium acetoglutamicum ATCC 15806,

Corynebacterium acetoacidophilum ATCC 13870,

Corynebacterium thermoaminogenes FERM BP-1539,

Corynebacterium melassecola ATCC 17965,

Brevibacterium flavum ATCC 14067,

Brevibacterium lactofermentum ATCC 13869 and

Brevibacterium divaricatum ATCC 14020; And

L-lysine-producing mutants or strains 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 DSM 5715.

In addition, the present invention,

a) a polynucleotide homologous in a range of at least 70% to a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2,

b) a polynucleotide encoding a polypeptide comprising an amino acid sequence homologous to at least 70% of 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 comprising a polynucleotide sequence selected from the group consisting of polynucleotides comprising at least 15 contiguous nucleotides in the polynucleotide sequence of a), b) or c).

In the context of the present application, when the polynucleotide sequence coincides with the sequence according to the invention in the range of at least 70%, preferably at least 80%, particularly preferably at least 90% in base composition and sequence, the polynucleotide sequence is Is “homology” with a sequence according to the invention. According to the present invention, “homologous protein” refers to an amino acid sequence that matches at least 70%, preferably at least 80%, particularly preferably at least 90%, of the amino acid sequence encoded by the pgsA2 gene (SEQ ID NO: 1). As understood as having proteins, "matches" are understood to mean that the corresponding amino acids are the same or are amino acids that are homologous to each other. Those amino acids that correspond to the properties of amino acids, in particular charge, hydrophobicity, steric properties, etc., are called "homologous amino acids".

In addition, the present invention,

(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) Provided above is a polynucleotide as described above, preferably replicable DNA, comprising a mutant of neutral function in sequence (i) which induces identical or homologous amino acids.

In addition, the present invention,

A recombinant polynucleotide capable of replicating in coryneform bacteria, comprising the nucleotide sequence of SEQ ID NO: 1, preferably

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

A seed encoding the pgsA2 gene contained in the vector pJC1pgsA2 deposited as Corynebacterium glutamicum under accession no. A vector containing the DNA sequence of glutamicum and

Coryneform bacteria that act as host cells, containing vectors or enhanced pgsA2 genes are provided.

The present invention also provides a polynucleotide comprising a complete gene having a polynucleotide sequence corresponding to SEQ ID NO: 1 or a fragment thereof, which comprises a sequence of the mentioned polynucleotide according to SEQ ID NO: 1 of the corresponding gene library or a fragment thereof Obtainable by hybridization with a probe comprising, and by separation of the mentioned DNA sequences.

In addition, the polynucleotide sequence according to the present invention separates the full-length cDNA encoding CDP-diacylglycerol-glycerol-3-phosphate 3-phosphatidyltransferase, and CDP-diacylglycerol-glycerol-3-phosphate 3-phosphatidyl It is suitable as a hybridization probe for RNA, cDNA and DNA for isolating cDNAs or genes having a high degree of similarity with the sequence of the transferase gene.

The polynucleotide sequences according to the invention are also suitable as primers for polymerase chain reaction (PCR) for the preparation of DNA encoding CDP-diacylglycerol-glycerol-3-phosphate 3-phosphatidyltransferase protein.

Such oligonucleotides which act as probes or primers may comprise at least 30, preferably at most 30, particularly preferably at most 20, very particularly preferably at least 15 consecutive nucleotides. Oligonucleotides of 40 or 50 or more nucleotides in length are also suitable.

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

"Polynucleotide" generally refers to polyribonucleotides and polydeoxyribonucleotides, which may be unmodified RNA or DNA, or modified RNA or DNA.

A "polypeptide" is understood to be a peptide or protein comprising two or more amino acids joined via peptide bonds.

The polypeptide according to the invention is at least 70% with the polypeptide according to SEQ ID NO: 2, in particular the polypeptide having the biological activity of CDP-diacylglycerol-glycerol-3-phosphate 3-phosphatidyltransferase, and the polypeptide according to SEQ ID NO: 2. Homologous to the range, preferably homologous to the range of at least 80%, homologous to the range of at least 90 to 95% and including the above-mentioned activities.

The present invention also provides a method for fermentatively producing amino acids, in particular L-lysine, in particular using coryneform bacteria which have already produced amino acids and which have been enriched in the nucleotide sequence encoding the pgsA2 gene, in particular overexpressed. .

C., encoding CDP-diacylglycerol-glycerol-3-phosphate 3-phosphatidyltransferase. The pgsA2 gene of glutamicum is first described herein.

Seed. To isolate the pgsA2 gene or other genes of glutamicum, the gene library of the microorganism is primarily derived from E. coli. Construct in E. coli. The construction of gene libraries is generally described in known textbooks and handbooks. Textbooks [Winnacker: Gene und Klone, Eine Einfuhrung in die Gentechnologie (Gene and Clones, An Introduction to Genetic Engineering) (Verlag Chemie, Weinheim, Germany, 1990)] or handbook [Sambrook et al .: Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989) may be mentioned by way of example. Well-known gene libraries are described in E. coli constructed in the λ vector according to Kohara et al., Cell 50, 495-508 (1987). Gene library of E. coli K-12 strain W3110. Bathe et al., Molecular and General Genetics, 252: 255-265, 1996, describe the cosmid vector SuperCos I (Wahl et al., 1987, Proceedings of the National Academy of Science USA, 84: 2160). -2164]. Seed constructed in E. coli K-12 strain NM554 (Raleigh et al., 1988, Nucleic Acids Research 16: 1563-1575). A genetic library of glutamicum ATCC 13032 is described. In addition, Bormann et al., Molecular Microbiology 6 (3), 317-326 (1992) are obtained using cosmid pHC79 (Hohn and Collins, Gene 11, 291-298 (1980)). Mr. Han. A genetic library of glutamicum ATCC 13032 is described. this. Mr. Colais. To prepare a gene library of glutamicum, pBR322 (Bolivar, Life Sciences, 25, 807-818 (1979)) or pUC9 (Vieira et al., 1982, Gene, 19: 259-268) and The same plasmid can also be used. Restriction-defective and recombinant-defective E. coli. E. coli strains are particularly suitable as hosts. An example of such a strain is strain DH5αmcr described in Grant et al., Proceedings of the National Academy of Sciences USA, 87 (1990) 4645-4649. Subsequently, long chain DNA fragments cloned using cosmid as described in Sanger et al., Proceedings of the National Academy of Sciences of the United States of America 74: 5463-5467, 1977 are then sequenced. Subcloning into a conventional vector suitable for may then be sequenced.

C, which encodes the pgsA2 gene and which is a component of the present invention as SEQ ID NO: 1. A novel DNA sequence of glutamicum is obtained by the method described above. In addition, the amino acid sequence of the corresponding protein is derived from the present DNA sequence by the method described above. The resulting amino acid sequence of the pgsA2 gene product is shown in SEQ ID NO: 2.

In addition, the coding DNA sequence generated from SEQ ID NO: 1 due to the degeneracy of the genetic code is also a component of the invention. In the same way, DNA sequences that hybridize with SEQ ID NO: 1 or a portion of SEQ ID NO: 1 are also components of the present invention. Conservative amino acid replacement, ie, glycine to alanine or aspartic acid to glutamic acid in proteins is known among those skilled in the art as "sense mutations" that do not fundamentally change the activity of the protein, ie, are neutral in function. It is also known that changes on the N and / or C terminus of a protein can substantially stabilize or rather stabilize its action. Those skilled in the art will find relevant information 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 textbooks of genetics and molecular biology. Amino acid sequences obtained from SEQ ID NO: 2 in a corresponding manner are also components of the invention.

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

One skilled in the art will specifically refer to the guidelines for the identification of DNA sequences by hybridization [see “The DIG System Users Guide for Filter Hybridization” from Boehringer Mannheim GmbH (Mannheim, Germany, 1993); And Liebl et al., International Journal of Systematic Bacteriology (1991) 41: 255-260. Those skilled in the art will specifically refer to the instructions for DNA sequence amplification using polymerase chain reaction (PCR) (Gait, Oligonucleotide synthesis: A Practical Approach (IRL Press, Oxford, UK, 1984)); And Newton & Graham: PCR (Spektrum Akademischer Verlag, Heidelberg, Germany, 1994).

During the study of the present invention, it was found that coryneform bacteria produce amino acids, in particular L-lysine, in an improved manner after enhancement of the pgsA2 gene.

The gene (s) constructs under study can be present in plasmids of varying numbers of copies, or integrated and enhanced within the chromosome. Alternatively, overexpression of the gene of interest can be achieved by changing the composition and culture process of the medium.

A person skilled in the art will, in particular, refer to the guidance in this regard, see Martin et al. Guerrero et al., Gene 138, 35-41 (1994); Tsuchiya and Morinaga, Bio / Technology 6, 428-430 (1988); Eikmanns et al., Gene 102, 93-98 (1991); EP 0 472 869; US Patent 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); 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); And Makrides, Microbiological Reviews 60: 512-538 (1996), and known textbooks of genetics and molecular biology.

By way of example, the pgsA2 gene according to the invention is overexpressed using plasmids.

Suitable plasmids are those cloned 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 the cryptic 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 [US-A 5,158,891] ] Based plasmids can be used in the same manner.

An example of a plasmid that can be used to overexpress the pgsA2 gene is pJC1pgsA2 (FIG. 1), Coli-C. Seed encoding the pgsA2 gene, based on the glutamicum shuttle vector pJC1 (Cremer et al., 1990, Molecular and General Genetics 220: 478-480). It contains the DNA sequence of glutamicum. It is contained in strain DSM5715 / pJC1pgsA2.

To replicate or enhance the hom-thrB operon, genes are enhanced by incorporation into the chromosome, as described, for example, in Reinscheid et al., Applied and Environmental Microbiology 60, 126-132 (1994). Plasmids that can help to help are also suitable. In this method, the complete gene is cloned into a plasmid vector that can be replicated in the host (typically E. coli) but not in Corynebacterium glutamicum. Possible vectors 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 [Source: 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 (Invitrogen, Groningen, Holland; Bernard et al., Journal of Molecular Biology, 234: 534-541 (1993)) or pEM1 (Schrumpf et al., 1991, Journal of Bacteriology) 173: 4510-4516. Then, the plasmid vector containing the gene to be enhanced is transferred into the desired Corynebacterium glutamicum strain by conjugation or transformation, as described in Schafer et al. al., Applied and Environmental Microbiology 60, 756-759 (1994) .Transformation methods are described 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) .. Strains generated after homologous recombination by “crossover” events Contains two or more copies of the gene.

In addition, in the preparation of amino acids, especially L-lysine, it may be advantageous to amplify or overexpress one or more enzymes involved in specific biosynthetic pathways, glycolysis, complement metabolism, citric acid cycles or amino acid translocation in addition to the pgsA2 gene.

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

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

DapE gene encoding succinyl diaminopimelate desuccinase,

LysC gene encoding feed-bag 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 triose 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 (DE-A-19831609) encoding pyruvate carboxylase,

Mqo gene encoding malate-quinone oxidoreductase (Molenaar et al., European Journal of Biochemistry 254, 395-403 (1998)) or

LysE gene, which encodes lysine transmigration (DE-A-195 48 222).

In addition to enhancing the pgsA2 gene, simultaneous attenuation of the following genes may be advantageous in the preparation of amino acids, especially L-lysine:

Pck gene encoding phosphoenol pyruvate carboxykinase [DE 199 50 409.1; DSM 13047], and / or

Pgi gene encoding glucose 6-phosphate isomerase [US 09 / 396,478; DSM 12969], and / or

PoxB gene encoding pyruvate oxidase [DE: 1995 1975.7].

In addition to overexpressing the pgsA2 gene, eliminating undesirable side reactions may be advantageous in the production of amino acids, particularly L-lysine. Nakayama: "Breeding of Amino Acid Producing Micro-organisms", Overproduction of Microbial Products, Krumphanzl, Sikyta, Vanek (eds.), Academic Press, London, UK, 1982.

To prepare amino acids, in particular L-lysine, the microorganisms prepared according to the invention are cultured continuously or discontinuously in a batch process (batch cultivation), or in a batch feed (batch process) or in a repeated feed batch process (repeated fed batch process). can do. An overview of known culture methods can be found in the textbook [Chmiel, Bioprozesstechnik 1. Einfuhrung in die Bioverfahrenstechnik {Bioprocess Technology 1. Instruction to Bioprocess Technology (Gustav Fischer Verlag, Stuttgart, 1991)}; Storhas, Bioreaktoren und periphere Einrichtungen {Bioreactors and Peripheral Equipment (Vieweg Verlag, Braunschweig / Wiesbaden, 1994)}.

The culture medium used should meet the needs of the particular strain in a suitable manner. Information on culture media for various microorganisms is described in the manual ("Manual of Methods for General Bacteriology", The American Society for Bacteriology (Washington D.C., USA, 1981)).

Sugars and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose, fats and oils, for example soybean oil, sunflower oil, peanut oil and coconut oil, fatty acids, for example , Palmitic acid, stearic acid and linoleic acid, alcohols such as glycerol and ethanol, and organic acids such as acetic acid can be used as the carbon source. These materials can be used individually or as a mixture.

Organic nitrogen-containing compounds such as peptone, yeast extract, meat extract, malt extract, corn steep liquor, soy flour and urea, or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and Ammonium nitrate can be used as the nitrogen source. Nitrogen sources can be used individually or as a mixture.

Phosphoric acid, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or corresponding sodium-containing salts may be used as the phosphorus source. In addition, the culture medium should contain metal salts essential for growth, such as magnesium sulfate or iron sulfate. Finally, essential growth substances such as amino acids and vitamins can be used in addition to the substances mentioned above. In addition, suitable precursors may be added to the culture medium. The starting material can be added to the medium in a single batch or fed in a suitable manner during the cultivation.

To adjust the pH of the medium, basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water, or acid compounds such as phosphoric acid or sulfuric acid can be used in a suitable manner. Defoamers such as fatty acid polyglycol esters can be used to control foam generation. In order to maintain the stability of the plasmid, a suitable substance which optionally acts, for example antibiotics, can be added to the medium. To maintain an aerobic state, oxygen or an oxygen-containing gas mixture, such as air, may be added to the medium. The temperature of the medium is usually 20 ° C to 45 ° C, preferably 25 ° C to 40 ° C. The culture is continued until the maximum amount of lysine is produced. This goal is typically achieved within 10 to 160 hours.

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

The following microorganisms have been deposited with the Deutsche Sammlung fur Mikroorganismen und Zellkulturen (DSMZ) = German Collection of Microorganisms and Cell Cultures in Braunschweig, Germany:

Corynebacterium glutamicum strain DSM5715 / pJC1pgsA2 (Accession No .: DSM 13251)

The process according to the invention is used to fermentally prepare amino acids, in particular L-lysine.

The present invention will be described in more detail below through specific embodiments.

Example 1

Preparation of Genomic Cosmid Gene Library from Corynebacterium glutamicum ATCC 13032

Chromosomal DNA from Corynebacterium glutamicum ATCC 13032 was isolated as described in Tauch et al., 1995, Plasmid 33: 168-179, and the restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg, Germany, Product Description Sau3AI, Code no. 27-0913-02). DNA fragments are dephosphorylated with shrimp alkaline phosphatase (Roche Molecular Biochemicals, Mannheim, Germany, Product Description SAP, Code no. 1758250). Cosmid vector SuperCos1 (available from Stratagene, La Jolla, USA, Product Description SuperCos1 Cosmid Vector Kit, Code no. 251301), Wahl et al. (1987) Proceedings of the National Academy of Sciences USA 84, 2160-2164] were digested with the restriction enzyme XbaI (Amersham Pharmacia, Freiburg, Germany, Product Description XbaI, Code no. 27-0948-02), and similarly with shrimp alkali. Dephosphorylation with phosphatase. Cosmid DNA is then cleaved with the restriction enzyme BamHI (Amersham Pharmacia, Freiburg, Germany, Product Description BamHI, Code no. 27-0868-04). Cosmid DNA treated in this manner is mixed with treated ATCC 13032 DNA and the batch is T4-DNA-ligase (Amersham Pharmacia, Freiburg, Germany, Product Description T4 DNA ligase, Code no. 27-0870-04) To be processed. The ligation mixture is then packed into phage using Gigapack II XL Packing Extract (Stratagene, La Jolla, USA, Product Description Gigapack II XL Packing Extract, Code no. 200217). this. To infect E. coli strain NM554 (Raleigh et al., 1988, Nucleic Acid Research 16: 1563-1575), cells are immersed in 10 mM MgSO ₄ and mixed with aliquots of phage suspension. Cells were plated on LB agar containing 100 μg / L Ampicillin (Lennox, 1955, Virology, 1: 190) to determine infection and titer of cosmid libraries. See Sambrook et al. , 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor. After incubation at 37 ° C. overnight, recombinant individual clones are selected.

Example 2

Isolation and Sequencing of the pgsA2 Gene

Cosmid DNA of each colony was isolated using a Qiaprep Spin Miniprep Kit (Product No. 27106, Qiagen, Hilden, Germany) according to the manufacturer's instructions, and the restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg, Germany, Product Description Sau3AI, Partly with Product No. 27-0913-02). DNA fragments are dephosphorylated with shrimp alkaline phosphatase (Roche Molecular Biochemicals, Mannheim, Germany, Product Description SAP, Product No. 1758250). After separation by gel electrophoresis, cosmid fragments ranging in size from 1500 to 2000 bp are separated with a QiaExII gel extraction kit (Product No. 20021, Qiagen, Hilden, Germany). DNA of the sequencing vector pZero-1 (Invitrogen, Groningen, Holland, Product Description Zero Background Cloning Kit, Product No. K2500-01) was restricted to the restriction enzyme BamHI (Amersham Pharmacia, Freiburg, Germany, Product Description BamHI, Product). No. 27-0868-04). Linkage of cosmid fragments in the sequencing vector pZero-1 is performed as described in Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, in which case a DNA mixture. Is incubated overnight with T4 ligase (Pharmacia Biotech, Freiburg, Germany). The linking mixture was then electroporated by Tauch et al. 1994, by FEMS Microbiol Letters 123: 343-7. LB agar containing 50 mg / l zeocine, transformed by introduction into E. coli strain DH5αMCR (Grant, 1990, Proceedings of the National Academy of Sciences USA, 87: 4645-4649), Lennox, 1955, Virology , 1: 190). Plasmid preparation of recombinant clones is performed using Biorobot 9600 (Product No. 900200, Qiagen, Hilden, Germany). Sequencing was described by dideoxy chain-termination by Sanger et al., 1977, Proceedings of the National Academies of Sciences USA, 74: 5463-5467. Zimmermann et al., 1990, Nucleic Acids Research 18: 1067]. "RR dRhodamin Terminator Cycle Sequencing Kit" (purchased from PE Applied Biosystems; Product No. 403044, Weiterstadt, Germany). Analysis of the separation and sequencing reactions by gel electrophoresis was carried out using a "Rotiphoresis NF acrylamide / bisacrylamide" gel (29: 1) using an "ABI Prism 377" sequencer (PE Applied Biosystems, Weiterstadt, Germany). (Product No. A124.1, Roth, Karlsruhe, Germany).

The raw sequence data obtained is then processed using the Staden program package (1986, Nucleic Acids Research, 14: 217-231) version 97-0. Each sequence of the pZero1 derivative is assembled into consecutive contigs. Computer-assisted encryption domain analysis can be found in the XNIP program [Staden, 1986, Nucleic Acids Research. 14: 217-231. For further analysis, see the "BLAST Search Program" for non-redundant databanks of the "National Biotechnology Information Center" (NCBI, Bethesda, MD, USA) [Altschul et al., 1997, Nucleic Acids Research. 25: 3389-3402.

The obtained nucleotide sequence is shown in SEQ ID NO: 1. Analysis of this nucleotide sequence revealed an open reading frame of 291 base pairs called the pgsA2 gene. The pgsA2 gene encodes a 97 amino acid protein.

Example 3

Cloning of the pgsA2 Gene in the Vector pJC1

Chromosome DNA is isolated from Corynebacterium glutamicum 13032 as described in Tauch et al., 1995, Plasmid 33: 168-179. DNA fragments carrying the pgsA2 gene are amplified using polymerase chain reaction. To do this, use the following primers:

5'-TGC TCT AGA CGT CCG TCG AGA GGT TTT TAG G-3 '

5'-TGC TCT AGA CCC CGC CAG ATT CTC CGA CAT-3 '

Both oligonucleotides carry the sequence (underlined nucleotide) for the cleavage site of the restriction enzyme XbaI. The primers presented are synthesized by MWG Biotech (Ebersberg, Germany) and PCR reactions are described in Innis et al., PCR protocol. A guide to methods and applications, 1990, Academic Press]. The primers can be used to amplify DNA fragments of size 749 bp that carry the pgsA2 gene from Corynebacterium glutamicum.

After separation by gel electrophoresis, PCR fragments are separated from agarose gels using a QiaExII gel extraction kit (Product No. 20021, Qiagen, Hilden, Germany).

PCR fragments obtained in this manner are completely cleaved using the restriction enzyme XbaI. A pgsA2 fragment of about 749 pb in size is separated from the agarose gel using a QiaExII gel extraction kit (Product No. 20021, Qiagen, Hilden, Germany).

this. Coli-C. The glutamicum shuttle vector pJC1 (Cremer et al., 1990, Molecular and General Genetics 220: 478-480) is used as the vector. The plasmid is also digested completely with restriction enzyme XbaI and then dephosphorylated with shrimp alkaline phosphatase (Roche Molecular Biochemicals, Mannheim, Germany, Product Description SAP, Product No. 1758250).

The pgsA2 fragment obtained in this way is mixed with the prepared vector pJC1 and the batch is treated with T4 ligase (Amersham Pharmacia, Freiburg, Germany, Product Description T4-DNA-Ligase, Code no. 27-0870-04). . The linkage batch is E. coli strain DH5α [Hanahan, DNA cloning. A practical approach. Vol. I. Transform in IRL-Press, Oxford, Washington DC, USA. Plasmid-bearing cells are selected by plating transgenic batches on LB agar (Lennox, 1995, Virology, 1: 190) containing 50 mg / L kanamycin. After overnight incubation at 37 ° C., recombinant individual clones are selected. Using a Qiaprep Spin Miniprep Kit (Product No. 27106, Quiagen, Hilden, Germany) according to the manufacturer's instructions, plasmid DNA was isolated from the transformants, digested with restriction enzyme EcoRI and subjected to subsequent agarose gel electrophoresis. To confirm the plasmid. The resulting plasmid is named pJC1pgsA2.

Example 4

Transformation of strain DSM5715 with plasmid pJC1pgsA2

Strain DSM5715 is transformed with plasmid pJC1pgsA2 using the electroporation method described in Liebl et al., FEMS Microbiology Letters, 53: 299-303 (1989). The transformants were 18.5 g / l brain-heart leaching culture supplemented with kanamycin 25 µg / l, 0.5 M sorbitol, bacto-tryptone 5 g / l, bacto-yeast extract 2.5 g / l, NaCl 5 g / l and Bac-agar is selected on LBHIS agar comprising 18 g / l. Incubation is carried out at 33 ° C. for 2 days.

Plasmid DNA was isolated from the transformants by conventional methods (Peters-Windisch et al., 1998, Microbiology 144, 915-927), digested with restriction endonuclease EcoRI, and the plasmid was subsequently agar. Check by rose gel electrophoresis. The resulting strain is named DSM5715 / pJC1pgsA2.

Strain DSM5715 / pJC1pgsA2 was deposited in accordance with the Budapest Treaty as DSM 13251 at the international depository institution Deutsche Sammlung fur Mikroorganismen und Zellkulturen (DSMZ) = German Collection of Microorganisms and Cell Cultures, in Braunschweig, Germany.

Example 5

Preparation of Lysine

Seed obtained in Example 4. Glutamicum strain DSM5715 / pJC1pgsA2 is incubated in a nutrient medium suitable for lysine production and then the lysine content in the culture supernatant is measured.

To this end, strains are incubated for 24 hours at 33 ° C. on agar plates containing primarily antibiotics (brain-heart agar containing kanamycin (50 μg / L)). Starting from this agar plate culture, the preculture is seeded (10 ml medium per 100 ml conical flask). Complete medium CgIII is used as the medium for preculture.

Cg III medium

NaCl 2.5g / ℓ

Bakto-Peptone 10g / ℓ

Bacterium-Yeast Extract 10g / ℓ

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

pH is adjusted to 7.4.

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

MM badge

CSL (corn immersion liquid) 5 g / ℓ

MOPS (morpholinopropanesulfonic acid) 20 g / ℓ

Glucose (autoclaved separately) 50 g / l

(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 filtration) 0.3 mg / l

Thiamine * HCl (sterile filtration) 0.2mg / ℓ

0.1 g / l L-leucine (sterile filtration)

CaCO ₃ 25g / ℓ

The pH of the CSL, MOPS and salt solution is adjusted to 7 with aqueous ammonia and then autoclaved. Then, sterile substrate and vitamin solution, and CaCO ₃ autoclaved in anhydrous state are added.

Cultivation is carried out at a 10 ml dose per 100 ml conical flask equipped with baffles. Kanamycin (25 μg / L) is added. Incubation is performed at 33 ° C. and 80% atmospheric humidity.

After 24 hours, the OD is measured at a measurement wavelength of 660 nm using a Biomek 1000 (Beckmann Instruments GmbH, Munich). The amount of lysine formed is measured by post-column derivatization using ion exchange chromatography and ninhydrin detection using an amino acid analyzer (Eppendorf-BioTronik, Hamburg, Germany).

The experimental results are shown in Table 1.

Strain OD (660 nm) Lysine-HCl (g / l) DSM5715 / pJC1pgsA2 12.1 8.60 DSM5715 11.9 7.80

1: Map of plasmid pJC1pgsA2

Abbreviations and names used have the following meanings.

oriCg: Mr. Plasmid-Encoding Replication Origin of Glutamicum (pHM1519)

pgsA2: Mr. PgsA2 (CDP-Diacylglycerol-glycerol-3-phosphate 3-phosphatidyltransferase) Gene from Glutamicum ATCC 13032

Kan: Kanamycin Resistance Gene

BamHI: cleavage site of the restriction enzyme BamHI

EcoRI: cleavage site of restriction enzyme EcoRI

HindIII: cleavage site of the restriction enzyme HindIII

SalI: cleavage site of restriction enzyme SalI

SmaI: cleavage site of the restriction enzyme SmaI

XbaI: cleavage site of restriction enzyme XbaI

Claims (19)

A genetically modified coryneform bacterium enriched in a pgsA2 gene encoding CDP-diacylglycerol-glycerol-3-phosphate 3-phosphatidyltransferase. The method of claim 1, wherein the starting bacteria (wild type) are Corynebacterium glutamicum (ATCC 13032), Corynebacterium acetoglutamicum (ATCC 15806), Corynebacterium acetoacedo Corynebacterium acetoacidophilum (ATCC 13870), Corynebacterium thermoaminogenes (FERM BP-1539), Corynebacterium melassecola (ATCC 17965), Brevibacterium platerium (Brevibacterium flavum: ATCC 14067), Brevibacterium lactofermentum (ATCC 13869) and Brevibacterium divaricatum (ATCC 14020), or Corynebacterium glutamicum FERM -P 1709, Brevibacterium plaboom FERM-P 1708, Brevibacterium lactofermentum FERM-P 1712, Corynebacterium glutamicum FERM-P 6463, Corynebacterium Genetically modified coryneform bacteria selected from the group consisting of Umm glutamicum FERM-P 6464 and Corynebacterium glutamicum DSM 5715. 2. The promoter, regulatory region or ribosomal binding site of claim 1, wherein the enhancement of the pgsA2 gene is achieved by overexpression of the gene, in particular by increasing the number of copies of the gene, by selection of a strong promoter or regulatory region on the reading frame. By mutation, by introducing an appropriate expression cassette onto the structural gene, by introducing an inducible promoter, by prolonging the corresponding mRNA lifetime, by reducing expressed proteolysis or by a combination of several of these possibilities Genetically modified coryneform bacteria. The genetically modified coryneform bacterium according to any one of claims 1 to 3, wherein the strain is transformed with a plasmid vector carrying a nucleotide sequence encoding the pgsA2 gene. 5. The genetically modified coryneform bacterium according to claim 1, wherein the genotype corresponds to strain Corynebacterium glutamicum DSM 13251. 6. a) a polynucleotide comprising at least 70% homology with a polynucleotide comprising the amino acid sequence of SEQ ID NO: 2 or encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2, b) a polynucleotide encoding a polypeptide comprising an amino acid sequence homologous to at least 70% of 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 comprising a polynucleotide sequence selected from the group consisting of polynucleotides comprising at least 15 contiguous nucleotides of the polynucleotide sequences of a), b) or c). 7. The polynucleotide of claim 6, which is replicable in coryneform bacteria, preferably recombinant DNA. 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 comprising a mutant of neutral function in sequence (i), leading to homologous amino acids. The polynucleotide sequence of claim 7, which encodes a polypeptide having the amino acid sequence of SEQ ID NO: 2. a) fermenting a coryneform bacterium that produces L-amino acids, at least in which the pgsA2 gene or nucleotide sequence encoding it is enhanced, in particular overexpressed, b) concentrating the L-amino acid in the medium or bacterial cell, and c) fermentatively preparing the L-amino acid, comprising performing the step of separating the L-amino acid. The method of claim 11, wherein the strain as claimed in claim 1 is used. 13. The method of claim 11 or 12, wherein the gene further encodes in the bacterium another gene encoding a protein of the biosynthetic pathway of the desired L-amino acid. 14. The method of any one of claims 11 to 13, wherein at least partially eliminating metabolic pathways in bacteria that reduce the formation of the desired amino acid. The method of claim 11, wherein the amino acid produced is L-lysine. The method according to any one of claims 11 to 15, a) dapA gene encoding a dihydrodipicolinate synthase, b) the dapE gene encoding succinyl diaminopimelate desuccinase, c) the lysC gene, which encodes a feed-bag resistant aspartate kinase, d) a tpi gene encoding triose phosphate 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 for producing lysine by fermenting a bacterium that is simultaneously enriched, in particular overexpressed or amplified, with one or more genes selected from the group consisting of lysE genes encoding lysine outflow. The method according to any one of claims 11 to 16, a) a pck gene encoding phosphoenol pyruvate carboxykinase, b) the pgi gene encoding glucose 6-phosphate isomerase and c) A method for producing L-lysine by fermenting a bacterium simultaneously attenuated by one or more genes selected from the group consisting of poxB genes encoding pyruvate oxidase. A primer for preparing DNA of a gene encoding CDP-diacylglycerol-glycerol-3-phosphate 3-phosphatidyltransferase by polymerase chain reaction, comprising a polynucleotide sequence as claimed in claim 6 or a portion thereof. Usage. Use of a polynucleotide sequence as claimed in claim 6 as a hybridization probe for isolating a cDNA or gene having high homology with the sequence of the pgsA2 gene.