DE10021829A1 - New nucleotide sequences coding for the pgsA2 gene - Google Patents

Abstract

The invention relates to a genetically modified coryneform bacterium whose pgsA2 gene is enhanced, and to an isolated polynucleotide which codes for the CDP-diacylglycerol-glycerol-3-phosphate 3 phosphatidyltransferase from coryneform bacteria, as well as to a process for the fermentative production of L-glycerol. Amino acids enhancing the pgsA2 gene in the bacteria and the use of the polynucleotide as a primer or hybridization probe.

Description

The invention relates to genetically modified coryneform bacteria for which CDP-diacylglycerol-glycerol 3-phosphate 3 phosphatidyltransferase coding Nucleotide sequences and methods for fermentative Production of amino acids, in particular L-lysine, under Use of coryneform bacteria in which the pgsA2- Gene responsible for the CDP-diacylglycerol-glycerol-3-phosphate 3 Phosphatidyltransferase encoded (EC 2.7.8.5), amplified becomes.

Amino acids, especially L-lysine, find in the Human medicine and in the pharmaceutical industry, but especially in animal nutrition, application.

It is known that amino acids by fermentation of Strains coryneformer bacteria, in particular Corynebacterium glutamicum. Because of the Great importance is constantly attached to the improvement of Manufacturing process worked. process improvements can fermentation measures such. B. stirring and supply of oxygen, or the composition of the Nutrient media such. B. the sugar concentration during the Fermentation, or the work-up to product form by z. As ion exchange chromatography or the intrinsic Performance characteristics of the microorganism itself affect.

To improve the performance characteristics of this Microorganisms become methods of mutagenesis, selection and mutant selection applied. In this way you get Strains that are resistant to antimetabolites such. B. the Lysine analog S- (2-aminoethyl) -cysteine or auxotrophic for are regulatory metabolites and L-amino acids such as B. produce L-lysine.  

For some years now, methods of recombinant DNA technology for strain improvement amino acid producing strains of Corynebacterium used by one amplifies individual amino acid biosynthesis genes and investigated the effect on amino acid production. Reviews for this can be found among others 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)).

Object of the present invention was to provide new tools for improved fermentative production of amino acids, in particular L-lysine.

This task is solved by a genetic engineering altered, coryneform bacterium whose gene pgsA2, the for the CDP-diacylglycerol-glycerol-3-phosphate 3 Phosphatidyltransferase encoded, is enhanced.

Amino acids, especially L-lysine, find in the Human medicine, in the pharmaceutical industry and especially in animal nutrition application. It exists therefore a general interest in it, new improved Process for the preparation of amino acids, in particular L- Lysine, provide.

If in the following L-lysine or lysine are mentioned, are so that not only the base, but also the salts such. B. Lysine monohydrochloride or lysine sulfate.

The invention relates to a genetically modified coryneformes bacterium in which its gene pgsA2, which is the CDP-diacylglycerol-glycerol-3-phosphate 3 phosphatidyltrans ferase encoded, is amplified.  

The term "reinforcement" describes in this context the increase in intracellular activity of one or of several enzymes in a microorganism that are caused by the corresponding DNA are encoded.

Reinforcement can be done using various manipulations the bacterial cell can be reached.

To achieve reinforcement, in particular one Overexpression, the copy number of the corresponding genes can be increased, one can use a strong promoter, or it may be the promoter and regulatory region or the Ribosome binding site located upstream of the Structural gene is mutated. In the same way act expression cassettes upstream of the Structural gene to be installed. By inducible Promoters it is additionally possible to express in the Progress of fermentative L-lysine production increase. It can also be used for a gene corresponding enzyme is encoded with a high activity. By measures to extend the lifetime of the m-RNA the expression is also improved. In addition, will by preventing the degradation of the enzyme also the Total enzyme activity increased. If necessary, you can These measures can also be combined as desired.

The microorganisms that are the subject of the present Invention, L-amino acids, in particular L-lysine, from glucose, sucrose, lactose, fructose, maltose, Molasses, starch, cellulose or glycerol and ethanol produce. It may be representative coryneformer Bacteria in particular of the genus Corynebacterium act. In the genus Corynebacterium is in particular the Art Corynebacterium glutamicum to call in the professional world known for their ability to use L-amino acids to produce.  

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

Corynebacterium glutamicum ATCC13032
Corynebacterium acetoglutamicum ATCC15806
Corynebacterium acetoacidophilum ATCC13870
Corynebacterium thermoaminogenes FERM BP-1539
Corynebacterium molassecola ATCC17965
Brevibacterium flavum ATCC14067
Brevibacterium lactofermentum ATCC13869 and
Brevibacterium divaricatum ATCC14020

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

Corynebacterium glutamicum FERM-P 1709
Brevibacterium flavum FERM-P 1708
Brevibacterium lactofermentum FERM-P 1712
Corynebacterium glutamicum FERM-P 6463
Corynebacterium glutamicum FERM-P 6464 and
Corynebacterium glutamicum DSM5715.

Another object of the present invention is an isolated polynucleotide from coryneform bacteria containing a polynucleotide sequence selected from the group

• a) polynucleotide which is at least 70% homologous with a polynucleotide encoding a polypeptide, the the amino acid sequence of SEQ ID no. 2 contains
• b) polynucleotide encoding a polypeptide having a Amino acid sequence containing at least 70% homologous is linked to the amino acid sequence of SEQ ID NO. 2,
• c) polynucleotide that is complementary to Polynucleotides of a) or b), and  
• d) polynucleotide containing at least 15 successive nucleotides of the polynucleotide sequence from a), b) or c).

"Homolog" in the context of the present application is a Polynucleotide sequence to the sequence of the invention then, if they are in their base composition and sequence at least 70%, preferably at least 80%, especially preferably at least 90% with the sequence according to the invention matches. Under a "homologous protein" according to the present invention proteins are understood which have an amino acid sequence associated with the Amino Acid Sequence Encoded by the Gene pgsA2 (SEQ ID NO: 1) at least 70%, preferably at least 80%, is encoded most preferably at least 90% match, wherein "to agree" is to be understood as meaning that corresponding amino acids are either identical, or it are mutually homologous amino acids. When "homologous amino acids" refers to those that are in their properties, in particular with regard to charge, Hydrophobicity, steric properties, etc.

The invention furthermore relates to a polynucleotide as described above, which is preferably a replicable DNA comprising:

• a) the nucleotide sequence shown in SEQ ID no. 1, or
• b) at least one sequence corresponding to the sequence (i) in Framework of degeneration of the genetic code corresponds to, or
• c) at least one sequence identical to that for the sequence (i) or (ii) complementary sequence hybridized, and optionally
• d) functionally neutral mutations in (i) that belong to same or a homologous amino acid lead.

Other items are
a preferably recombinant polynucleotide which is replicable in coryneform bacteria and has the nucleotide sequence SEQ ID NO. 1 comprises
a polynucleotide encoding a polypeptide having the amino acid sequence SEQ ID NO. 2 comprises
a vector containing the DNA sequence of C. glutamicum coding for the pgsA2 gene, contained in the vector pJC1pgsA2 deposited in Corynebacterium glutamicum under the number 13251,
and coryneform bacteria serving as the host cell, which contain the vector or in which the pgsA2 gene is amplified.

The invention also polynucleotides are the the complete gene with the polynucleotide sequence according to SEQ ID no. Contain 1 or fragments thereof and by screening by hybridization of a corresponding gene bank with a probe containing the sequence of mentioned polynucleotide according to SEQ ID no. 1 or one Contains fragment thereof, and isolation of said DNA Sequence are available.

Polynucleotide sequences according to the invention are also known as Hybridization probes suitable for RNA, cDNA and DNA to to isolate full-length cDNA useful for the CDP diacylglycerol-glycerol-3-phosphate 3 phosphatidyltrans encode ferase and to isolate such cDNA or genes that a high similarity with the sequence of the CDP diacylglycerol-glycerol-3-phosphate 3 phosphatidyltrans have ferase gene.

Polynucleotide sequences according to the invention are also suitable as a primer for the polymerase chain reaction (PCR), for the production of DNA suitable for CDP-diacylglycerol  Glycerol-3-phosphate 3 phosphatidyltransferase proteins coded.

Such oligonucleotides serving as probes or primers can be more than 30, preferably up to 30, especially preferably up to 20, most preferably at least 15 contain consecutive nucleotides. Are suitable also oligonucleotides with a length of at least 40 or 50 nucleotides.

"Isolated" means from its natural environment separated out.

"Polynucleotide" generally refers to Polyribonucleotides and polydeoxyribonucleotides, wherein it unmodified RNA or DNA or modified RNA or DNA can act.

By "polypeptides" is meant peptides or proteins, the two or more linked via peptide bonds Contain amino acids.

The polypeptides of the invention include a polypeptide according to SEQ ID no. 2, in particular those with the biological activity of CDP-diacylglycerol-glycerol-3 Phosphate 3 phosphatidyltransferase protein and also those that are at least 70% homologous with the Polypeptide according to SEQ ID no. 2, preferably at least 80% and especially those with at least 90% to 95% homology with the polypeptide according to SEQ ID no. 2 and the named Have activity.

The invention also relates to a method for fermentative production of amino acids, in particular L- Lysine, using coryneform bacteria, which in particular already produce an amino acid, and in those coding for the pgsA2 gene nucleotide sequences amplified, in particular overexpressed.  

In the present invention is the first time for the CDP-diacylglycerol-glycerol-3-phosphate 3 phosphatidyltrans ferase coding pgsA2 gene of C. glutamicum.

To isolate the pgsA2 gene or other genes of C. glutamicum first becomes a gene bank of this Microorganism created in E. coli. The creation of Genebanks is in well-known textbooks and Written down manuals. As an example, be that Textbook by Winnacker: Genes and Clones, An Introduction to genetic engineering (Verlag Chemie, Weinheim, Germany, 1990) or the handbook of Sambrook et al .: Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989). A well-known gene bank is the E. coli K-12 strain W3110, described by Kohara et al. (Cell 50, 495-508 (1987)) in λ vectors. Bathe et al. (Molecular and General Genetics, 252: 255-265, 1996) describe a gene bank of C. glutamicum ATCC13032, the with the aid of the cosmid vector SuperCos I (Wahl et al., 1987, Proceedings of the National Academy of Sciences USA, 84: 2160-2164) in the E. coli K-12 strain NM554 (Raleigh et al., 1988, Nucleic Acids Research 16: 1563-1575). Börmann et al. (Molecular Microbiology 6 (3), 317-326 (1992)) again describe a gene bank of C. glutamicum ATCC13032 using the cosmid pHC79 (scab and Collins, Gene 11, 291-298 (1980)). For the production of a Genbank of C. glutamicum in E. coli can also use plasmids such as pBR322 (Bolivar, Life Sciences, 25, 807-818 (1979)) or pUC9 (Vieira et al., 1982, Gene, 19: 259-268) become. Suitable hosts are in particular such E. coli Strains which are restriction and recombination defective. An example of this is the strain DH5αmcr, by Grant et al. (Proceedings of the National Academy of Sciences USA, 87 (1990) 4645-4649). The help of cosmids cloned long DNA fragments can then again in common, for sequencing subcloned suitable vectors and then sequenced  be, as it is z. In Sanger et al. (Proceedings of the National Academy of Sciences of the United States of America, 74: 5463-5467, 1977).

In this way, the new coding for the gene pgsA2 DNA sequence of C. glutamicum obtained as SEQ ID NO. 1 Part of the present invention is. It was also from the present DNA sequence with those described above Methods the amino acid sequence of the corresponding protein derived. In SEQ ID no. 2 is the resulting Amino acid sequence of the pgsA2 gene product shown.

Coding DNA sequences consisting of SEQ ID no. 1 through the degeneracy of the genetic code are also part of the invention. In the same way DNA sequences identified by SEQ ID NO. 1 or parts of SEQ ID No. 1 hybridize, part of the invention. In the Experts are also conservative amino acid exchanges such as B. Exchange of glycine for alanine or of Aspartic acid against glutamic acid in proteins as "Sense mutations" known to none fundamental change in the activity of the protein lead, d. H. are functionally neutral. It is also known that changes at the N- and / or C-terminus of a protein its function does not significantly affect or this even stabilize. Details can be found on the Expert among others 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 in known textbooks of Genetics and molecular biology. Amino acid sequences that correspondingly from SEQ ID no. 2, are also part of the invention.

Similarly, DNA sequences SEQ ID NO. 1 or parts of SEQ ID no. 1 hybridize, part of the Invention. Finally, DNA sequences are part of  Invention produced by the polymerase chain reaction (PCR) be made using primers that are from SEQ ID no. 1 result. Have such oligonucleotides typically at least 15 nucleotides in length.

Instructions for the identification of DNA sequences by means of Hybridization is found among others in the art Manual "The DIG System Users Guide for Filters Hybridization "the company Boehringer Mannheim GmbH (Mannheim, Germany, 1993) and Liebl et al. (International Journal of Systematic Bacteriology (1991) 41: 255-260). Instructions for the Amplification of DNA Sequences using the polymerase chain reaction (PCR) the expert finds among others in the manual of Gait: Oligonucleotide synthesis: a practical approach (IRL Press, Oxford, UK, 1984) and Newton and Graham: PCR (Spectrum Academic Publishing House, Heidelberg, Germany, 1994).

In working on the present invention could be noted that coryneform bacteria after Enhancement of the pgsA2 gene in an improved way Amino acids, especially L-lysine, produce.

The considered genes or gene constructs can either be in Plasmids with different copy numbers are present or be integrated and amplified in the chromosome. alternative may also overexpress the genes involved by changing the composition of the media and Cultural guidance can be achieved.

Instructions for this, the expert finds among others Martin et al. (Bio / Technology 5, 137-146 (1987)) Guerrero et al. (Gene 138, 35-41 (1994)), Tsuchiya and Morinaga (Bio / Technology 6, 428-430 (1988)), to Eikmanns et al. (Gene 102, 93-98 (1991)), in the European Patent Specification EPS 0 472 869, in US Pat. No. 4,601,893 Schwarzer and Puhler (Bio / Technology 9, 84-87 (1991)) Remscheid et al. (Applied and Environmental Microbiology  60, 126-132 (1994)), LaBarre et al. (Journal of Bacteriology 175, 1001-1007 (1993)), in the patent application WO 96/15246 to Malumbres et al. (Gene 134, 15-24 (1993)), Japanese Patent Laid-Open Publication JP-A-10-229891, Jensen and Hammer (Biotechnology and Bioengineering 58, 191-195 (1998)), to Makrides (Microbiological Reviews 60: 512-538 (1996)) and in well-known textbooks of genetics and molecular biology.

By way of example, the pgsA2 gene according to the invention was obtained with the aid of of plasmids overexpressed.

Suitable plasmids are those which are in coryneform Bacteria are replicated and expressed. numerous known plasmid vectors such. 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)) are based on the cryptic plasmids pHM1519, pBL1 or pGA1. Other Plasmid vectors such. For example, those based on pCG4 (US-A 4,489,160), or pNG2 (Serwold-Davis et al., FEMS Microbiology Letters 66, 119-124 (1990)), or pAG1 (US Pat. No. 5,158,891) can be used in the same way become.

An example of a plasmid by means of which the pgsA2 gene can be overexpressed is pJC1pgsA2 ( FIG. 1), which is located on the E. coli C. glutamicum shuttle vector pJC1 (Cremer et al., 1990, p. Molecular and General Genetics 220: 478-480) and contains the coding for the pgsA2 gene DNA sequence of C. glutamicum. It is contained in strain DSM5715 / pJC1pgsA2.

In addition, such plasmid vectors are suitable with the aid of one of the procedure of gene amplification by Integration into the chromosome can apply, just like it for example, Remscheid et al. (Applied and Environmental Microbiology 60, 126-132 (1994))  Duplication or amplification of the hom-thrB operon has been described. This method becomes the complete one Gene is cloned into a plasmid vector which is in a host (typically E. coli), but not in C. glutamicum can replicate. Suitable vectors are, for example, pSUP301 (Simon et al., Bio / Technology 1, 784-791 (1983)), pK18mob or pK19mob (Schäfer et al., Gene 145, 69-73 (1994)), pGEM-T (Promega corporation, Madison, WI, USA), pCR2.1-TOPO (Shuman (1994) Journal of Biological Chemistry 269: 32678-84; US Pat. No. 5,487,993), pCR® Blunt (Invitrogen, Groningen, the Netherlands; Bernard et al., Journal of Molecular Biology, 234: 534-541 (1993)) or pEM1 (shrink et al. 1991, Journal of Bacteriology 173: 4510-4516) Question. The plasmid vector containing the gene to be amplified is subsequently by conjugation or Transformation into the desired strain of C. glutamicum transferred. The method of conjugation is for example in Schäfer et al. (Applied and Environmental Microbiology 60, 756-759 (1994)). Methods for Transformation are, for example, in Thierbach et al. (Applied Microbiology and Biotechnology 29, 356-362 (1988)), Dunican and Shivnan (Bio / Technology 7, 1067-1070 (1989)) and Tauch et al. (FEMS Microbiological Letters 123, 343-347 (1994)). After homologous recombination by means of a "cross over" event contains the resulting strain at least two copies of the relevant Gene.

In addition, it can be used for the production of amino acids, in particular L-lysine be advantageous, in addition to the pgsA2 gene one or more enzymes of the respective biosynthetic pathway, glycolysis, anaplerotic, citric acid cycle or amino acid export overexpress.  

Thus, for example, for the production of L-lysine one or more of the genes selected from the group can be used simultaneously

• The dapA- coding for the dihydrodipicolinate synthase Gen (EP-B 0 197 335), or
• - that for the succinyldiaminopimelate desuccinylase coding dapE gene, or
• - that for a feed back resistant aspartate kinase coding lysC gene (Kalinowski et al., (1990), Molecular and General Genetics 224, 317-324), or
• - that for the glyceraldehyde-3-phosphate dehydrogenase coding gap gene (Eikmanns (1992), Journal of Bacteriology 174: 6076-6086), or
• The tpi gene coding for the triose phosphate isomerase (Eikmanns (1992), Journal of Bacteriology 174: 6076-6086), or
• The pgk gene coding for the 3-phosphoglycerate kinase (Eikmanns (1992), Journal of Bacteriology 174: 6076-6086), or
• The pyc gene coding for the pyruvate carboxylase (DE-A-198 31 609), or
• - the mqo- coding for malate quinone oxidoreductase Gen (Molenaar et al., European Journal of Biochemistry 254, 395-403 (1998)), or
• - The lysE gene encoding lysine export (DE-A-195 48 222)

amplified, in particular overexpressed or amplified become.  

In addition, it may be advantageous for the production of amino acids, in particular L-lysine, in addition to the amplification of the pgsA2 gene simultaneously

• - The coding for the phosphoenolpyruvate carboxykinase pck gene (DE 199 50 409.1, DSM13047) and / or
• The pgj coding for the glucose-6-phosphate isomerase Gene (US 09 / 395,478, DSM12969) and / or
• - The poxB gene coding for pyruvate oxidase (DE 199 51 975.7)

mitigate.

It can also be used for the production of amino acids, especially L-lysine, be beneficial, in addition to the Overexpression of the pgsA2 gene undesired side reactions (Nakayama: "Breeding of Amino Acid Producing Microorganisms ", in: Overproduction of Microbial Products, Krumphanzl, Sikyta, Vanek (eds.), Academic Press, London, UK, 1982).

The microorganisms produced according to the invention can continuous or discontinuous in the batch process (Batch cultivation) or in the fed batch (feed method) or repeated fed batch process (repetitive feed process) for the purpose of producing amino acids, in particular L-lysine to be cultured. A summary about well-known cultivation methods are in the textbook of Chmiel (Bioprocess Engineering 1. Introduction to Bioprocess Engineering (Gustav Fischer-Verlag, Stuttgart, 1991)) or in the textbook Storhas (bioreactors and peripheral facilities (Vieweg-Verlag, Braunschweig / Wiesbaden, 1994)).

The culture medium to be used must suitably Claims of the respective strains suffice. descriptions of culture media of various microorganisms are in Manual of Methods for General Bacteriology  American Society for Bacteriology (Washington D.C., USA, 1981). As a carbon source can sugar and Carbohydrates such. Glucose, sucrose, lactose, Fructose, maltose, molasses, starch and cellulose, oils and Fats such as Soybean oil, sunflower oil, peanut oil and Coconut oil, fatty acids such. For example, palmitic acid, stearic acid and linoleic acid, alcohols such as. As glycerol and ethanol and organic acids such. As acetic acid can be used. These substances can be used singly or as a mixture become. As a nitrogen source, organic nitrogen containing compounds such as peptones, yeast extract, Meat extract, malt extract, corn steep liquor, Soybean meal and urea or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, Ammonium carbonate and ammonium nitrate can be used. The Nitrogen sources may be singly or as a mixture be used. As phosphorus source can phosphoric acid, Potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts are used. The culture medium must also contain salts of metals such as As magnesium sulfate or iron sulfate, for the Growth are necessary. Finally, essential Growth substances such as amino acids and vitamins in addition to the above mentioned substances. The culture medium In addition, suitable precursors may be added. The mentioned feedstocks can be used to culture in the form of a added one-off approach or appropriately be fed during cultivation.

For pH control of the culture basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or Ammonia water or acidic compounds such as phosphoric acid or sulfuric acid used in a suitable manner. to Foam processing control can use anti-foaming agents z. B. fatty acid polyglycol esters are used. to Maintaining the stability of plasmids can the Medium suitable selective substances such. B.  Antibiotics are added. To aerobic conditions maintain oxygen or oxygen containing gas mixtures such. B. air into the culture entered. The temperature of the culture is usually at 20 ° C to 45 ° C, and preferably at 25 ° C to 40 ° C. The Culture is continued until a maximum is reached Lysine has formed. This goal is usually within 10 hours to 160 hours.

The analysis of L-lysine can be done by anion exchange chromatography followed by ninhydrin Derivatization done, as in Spackman et al. (Analytical Chemistry, 30, (1958), 1190).

The following microorganism has been deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany) under the Budapest Treaty:

• - Corynebacterium glutamicum strain DSM5715 / pJC1pgsA2 as DSM13251.

The inventive method is used for fermentative Production of amino acids, in particular L-lysine.

Examples

The present invention will be described below with reference to Embodiments explained in more detail.

example 1 Preparation of a Genomic Cosmid Gene Bank Corynebacterium glutamicum ATCC 13032

Chromosomal DNA from Corynebacterium glutamicum ATCC 13032 was isolated as described by Tauch et al. (1995, Plasmid 33: 168-179) isolated and partially digested with the restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg, Germany, product description Sau3AI, Code no. 27-0913-02). The DNA fragments were dephosphorylated with shrimp alkaline phosphatase (Roche Molecular Biochemicals, Mannheim, Germany, product description SAP, code no. 1758250). The DNA of the cosmid vector SuperCos1 (Wahl et al. (1987) Proceedings of the National Academy of Sciences USA 84: 2160-2164), obtained from Stratagene (La Jolla, USA, Product Description SuperCos1 Cosmid Vector Kit, Code no. 251301) was cleaved with the restriction enzyme XbaI (Amersham Pharmacia, Freiburg, Germany, product description XbaI, Code no. 27-0948-02) and also dephosphorylated with shrimp alkaline phosphatase. Subsequently, the cosmid DNA was cleaved with the restriction enzyme BamHI (Amersham Pharmacia, Freiburg, Germany, product description BamHI, Code no. 27-0868-04). The cosmid DNA treated in this way was mixed with the treated ATCC 13032 DNA and the mixture was mixed with T4 DNA ligase (Amersham Pharmacia, Freiburg, Germany, product description T4 DNA ligase, code no. 27-0870-04). treated. The ligation mixture was then packaged in phage using Gigapack II XL Packing Extract (Stratagene, La Jolla, USA, Product Description Gigapack II XL Packing Extract, Code no. 200217). To infect E. coli strain NM554 (Raleigh et al 1988, Nucleic Acid Research 16: 1563-1575), the cells were taken up in 10 mM MgSO ₄ and mixed with an aliquot of the phage suspension. Infection and titering of the cosmidbank were performed as described by Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor), where the cells were plated on LB agar (Lennox, 1955, Virology, 1: 190) with 100 mg / L ampicillin. After overnight incubation at 37 ° C, recombinant single clones were selected.

Example 2 Isolation and sequencing of the pgsA2 gene

The cosmid DNA of a single colony was treated with the Qiaprep Spin Miniprep Kit (Product No. 27106, Qiagen, Hilden, Germany) isolated according to the manufacturer and with the Restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg, Germany, product description Sau3AI, product no. 27-0913-02) partially split. The DNA fragments were with Shrimp alkaline phosphatase (Roche Molecular Biochemicals, Mannheim, Germany, Product Description SAP, Product No. 1758250) dephosphorylated. To Gelelektrophoretischer separation was carried out the isolation the cosmid fragments in the size range of 1500 to 2000 bp with the QiaExII Gel Extraction Kit (Product No. 20021, Qiagen, Hilden, Germany). The DNA of the sequencing vector pZero-1 purchased from Invitrogen (Groningen, Netherlands, Product Description Zero Background Cloning Kit, Product No. K2500-01) was mixed with the restriction enzyme BamHI (Amersham Pharmacia, Freiburg, Germany, Product Description BamHI, Product No. 27-0868-04) split. The Ligation of Cosmidfragmente in the Sequencing vector pZero-1 was prepared as described by Sambrook et al. (1989, Molecular Cloning: A laboratory Manual, Cold Spring  Harbor) described, wherein the DNA mixture with T4 ligase (Pharmacia Biotech, Freiburg, Germany) Was incubated overnight. This ligation mixture was into the E. coli strain DH5αMCR (Grant, 1990, Proceedings of the National Academy of Sciences USA, 87: 4645-4649) by electroporation (Tauch et al. 1994, FEMS Microbiol Letters, 123: 343-7) and on LB- Agar (Lennox, 1955, Virology, 1: 190) with 50 mg / L zeocin plated. The plasmid preparation of the recombinant Clones were carried out with the Biorobot 9600 (Product No. 900200, Qiagen, Hilden, Germany). The sequencing was done according to the dideoxy chain termination method of Sanger et al. (1977, Proceedings of the National Academy of Sciences USA, 74: 5463-5467) with modifications according to Zimmermann et al. (1990, Nucleic Acids Research, 18: 1067). It became the "RR dRhodamine Terminator Cycle Sequencing Kit" by PE Applied Biosystems (Product No. 403044, Weiterstadt, Germany) used. The gel electrophoretic Separation and analysis of the sequencing reaction took place in a "Rotophore NF Acrylamide / Bisacrylamide" Gel (29: 1) (Product No. A124.1, Roth, Karlsruhe, Germany) with the "ABI Prism 377 "sequencer from PE Applied Biosystems (Weiterstadt, Germany).

The obtained crude sequence data were then under Application of the Staden program package (1986, Nucleic Acids Research, 14: 217-231), version 97-0. The Single sequences of pZero1 derivatives became one coherent contig assembled. The computer-aided Coding area analysis was carried out with the program XNIP (Staden, 1986, Nucleic Acids Research, 14: 217-231). Further analyzes were carried out with the "BLAST search programs" (Altschul et al., 1997, Nucleic Acids Research, 25: 3389- 3402), against the non-redundant database of the "National Center for Biotechnology Information "(NCBI, Bethesda, MD, USA).  

The nucleotide sequence obtained is shown in SEQ ID no. 1 shown. Analysis of the nucleotide sequence revealed open reading frame of 291 base pairs, which as pgsA2- Gene was designated. The pgsA2 gene encodes a protein of 97 amino acids.

Example 3 Cloning of the pgsA2 gene in vector pJC1

Chromosomal DNA from Corynebacterium glutamicum ATCC 13032 was isolated as described by Tauch et al. (1995, Plasmid 33: 168-179). With the aid of the polymerase chain reaction, a DNA fragment carrying the pgsA2 gene was amplified. For this purpose, the following primers were used:

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 for the Cleavage site of the restriction enzyme XbaI (underlined Nucleotides). The primers shown were from the company MWG Biotech (Ebersberg, Germany) and synthesized the standard PCR method of Innis et al., (PCR protocol, A guide to methods and applications, 1990, Academic Press) the PCR reaction was performed. The primers enable the Amplification of an approximately 749 bp DNA fragment, which carries the pgsA2 gene from Corynebacterium glutamicum.

After gel electrophoresis separation took place Isolation of the PCR fragment from the agarose gel with the QiaExII Gel Extraction Kit (Product No. 20021, Qiagen, Hilden, Germany).

The PCR fragment obtained in this way was labeled with the Restriction enzyme XbaI completely cleaved. The approx. 749 bp large pgsA2 fragment was removed from the agarose gel with the  QiaExII Gel Extraction Kit (Product No. 20021, Qiagen, Hilden, Germany).

As a vector was the E. coli - C. glutamicum shuttle vector pJC1 (Cremer et al., 1990, Molecular and General Genetics 220: 478-480). This plasmid was also completely cleaved with the restriction enzyme XbaI and then with shrimp alkaline phosphatase (Roche Molecular Biochemicals, Mannheim, Germany, Product Description SAP, Product No. 1758250) dephosphorylated.

The pgsA2 fragment thus obtained was ligated with the prepared vector pJC1 mixed and the batch with T4 DNA ligase (Amersham Pharmacia, Freiburg, Germany, Product Description T4 DNA Ligase, Code no. 27-0870-04) treated. The ligation mixture was added to the E. coli strain DH5α (Hanahan, In: DNA cloning. A practical approach. Vol. IRL-Press, Oxford, Washington DC, USA). The selection of plasmid-carrying cells was carried out by Plating the transformation mixture on LB agar (Lennox, 1955, Virology, 1: 190) with 50 mg / l kanamycin. To Incubation overnight at 37 ° C were recombinant Single clones selected. Plasmid DNA was from a Transformant with the Qiaprep Spin Miniprep Kit (Product No. 27106, Qiagen, Hilden, Germany) according to manufacturer's instructions isolated and cleaved with the restriction enzyme XbaI to the plasmid by subsequent agarose gel electrophoresis to check. The resulting plasmid became pJC1pgsA2 called.  

Example 4 Transformation of strain DSM5715 with the plasmid pJC1pgsA2

The strain DSM5715 was subjected to the plasmid pJC1pgsA2 Application of the von Liebl et al., (FEMS Microbiology Letters, 53: 299-303 (1989)) transformed. The selection of the transformants took place on LBHIS agar consisting of 18.5 g / l Brain-Heart infusion Boullion, 0.5M sorbitol, 5g / l Bacto tryptone, 2.5g / l Bacto-Yeast extract, 5 g / l NaCl and 18 g / l Bacto agar, the supplemented with 25 μg / ml kanamycin. The Incubation was for 2 days at 33 ° C.

Plasmid DNA was made from a transformant according to the usual Isolated methods (Peters-Wendisch et al., 1998, Microbiology, 144, 915-927), with the Restriction endonuclease EcoRI cut; around the plasmid by subsequent agarose gel electrophoresis check. The obtained strain became DSM5715 / pJC1pgsA2 called.

The strain DSM5715 / pJC1pgsA2 was used by Deutsche Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany) according to the Budapest Treaty deposited as DSM13251.

Example 5 Production of lysine

The C. glutamicum strain obtained in Example 5 DSM5715 / pJC1pgsA2 was in a production for lysine cultivated suitable nutrient medium and the lysine content in the Culture supernatant determined.  

For this purpose, the strain was first incubated on agar plate with the appropriate antibiotic (brain heart agar with kanamycin (50 ug / ml)) for 24 hours at 33 ° C. Starting from this agar plate culture, a preculture was inoculated (10 ml of medium in the 100 ml Erlenmeyer flask). The medium used for the preculture was the complete medium CgIII.

Medium CgIII NaCl 2.5 g / l Bacto-peptone 10 g / l Bacto-yeast extract 10 g / l Glucose (separately autoclaved) 2% (w / v)

The pH was adjusted to pH 7.4 set.

To this was added kanamycin (25 mg / L). The preculture was incubated for 16 hours at 33 ° C at 240 rpm on a shaker. From this preculture, a major culture was inoculated so that the initial OD (660 nm) of the main culture was 0.1. The medium MM was used for the main culture.

Medium MM CSL (Corn Steep Liquor) 5 g / l MOPS (morpholinopropanesulfonic acid) 20 g / l Glucose (separately autoclaved) 50 g / l (NH ₄ ) ₂ SO ₄ 25 g / l KH ₂ PO ₄ 0.1 g / l MgSO ₄ .7H ₂ O 1.0 g / l CaCl ₂ .2H ₂ O 10 mg / 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 L-leucine 0.1 g / l CaCO ₃ 25 g / l

CSL, MOPS and saline were adjusted to pH 7 with ammonia water and autoclaved. Subsequently, the sterile substrate and vitamin solutions were added and the dry autoclaved CaCO ₃ .

The cultivation is carried out in 10 ml volume in a 100 ml Erlenmeyer flask with harassment. It was kanamycin (25 μg / ml) added. The cultivation was carried out at 33 ° C and 80% humidity.

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

Table 1 shows the result of the experiment.  

Table 1

Legend to the picture

Fig. 1: Map of the plasmid pJC1pgsA2

The abbreviations and designations used have the following meaning.

oriCg: plasmid-encoded origin of replication C. glutamicum (from pHM1519)
pgsA2: pgsA2 (CDP-diacylglycerol-glycerol-3-phosphate-phosphatidyltransferase) gene from C. glutamicum ATCC13032
Kan: Kanamycin resistance gene
BamHI: Interface of the restriction enzyme BamHI
EcoRI: restriction enzyme EcoRI site
HindIII: restriction enzyme HindIII site
SalI: Site of the restriction enzyme SalI
SmaI: Interface of the restriction enzyme SmaI
XbaI: Restriction enzyme XbaI site

SEQUENCE LISTING

Claims (19)

1. Genetically modified coryneform bacterium, characterized in that its gene pgsA2, which codes for the CDP-diacylglycerol-glycerol-3-phosphate 3 phosphatidyltransferase, is enhanced. 2. Genetically modified, coryneform bacterium after Claim 1, characterized in that the starting point Bacterium (wild type) is selected from the group Corynebacterium glutamicum (ATCC13032), Corynebacterium acetoglutamicum (ATCC15806), Corynebacterium acetoacidophilum (ATCC13870), Corynebacterium thermoaminogenes (FERM BP-1539), Corynebacterium molassecola (ATCC17965), Brevibacterium flavum (ATCC14067), Brevibacterium lactofermentum (ATCC13869) and Brevibacterium divaricatum (ATCC14020), or is selected from the Group Corynebacterium glutamicum FERM-P 1709, Brevibacterium flavum FERM-P 1708, Brevibacterium lactofermentum FERM-P 1712, Corynebacterium glutamicum FERM-P 6463, Corynebacterium glutamicum FERM-P 6464 and Corynebacterium glutamicum DSM5715. 3. Genetically modified, coryneform bacterium after Claim 1, characterized in that the Enhancement of the pgsA2 gene by overexpression of the Gene, in particular by increasing the copy number of the Gene, by selecting a strong promoter or a Regulation region above the reading frame, through Mutation of the promoter, the regulatory region or the Ribosome binding site, by incorporation of a suitable Expression cassette above the structural gene or by incorporation of inducible promoters through which Prolonging the lifetime of the corresponding mRNA, by a reduced degradation of the expressed Proteins or by combining several of these Possibilities.   4. Genetically modified coryneform bacteria after one of claims 1 to 3, characterized in that that the strain is transformed with a plasmid vector and the plasmid vector for the pgsA2 gene carries coding nucleotide sequence. 5. Genetically modified coryneform bacteria after one of claims 1 to 4, characterized that it genotypically the strain Corynebacterium glutamicum DSM13251. 6. An isolated polynucleotide from coryneform bacteria containing a polynucleotide sequence selected from the group

• a) polynucleotide which is at least 70% homologous to a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO. 2 comprises, or consists of,
• b) polynucleotide encoding a polypeptide comprising an amino acid sequence which is at least 70% homologous to the amino acid sequence of SEQ ID NO. 2,
• c) polynucleotide which is complementary to the polynucleotides of a) or b), and
• d) polynucleotide containing at least 15 consecutive nucleotides of the polynucleotide sequence of a), b) or c).

7. polynucleotide according to claim 6, the polynucleotide being a coryneform bacterium is replicable, preferably recombinant DNA. 8. polynucleotide according to claim 6, wherein the polynucleotide is an RNA.   9. A replicable DNA according to claim 7, comprising

• a) the nucleotide sequence shown in SEQ ID no. 1, or
• b) at least one sequence corresponding to the sequence (i) in the context of the degeneracy of the genetic code, or
• c) at least one sequence that hybridizes with the sequence complementary to sequence (i) or (ii), and optionally
• d) functionally neutral mutations in (i) leading to homologous amino acids.

10. polynucleotide sequence according to claim 7, 8 or 9, the encodes a polypeptide having the amino acid sequence SEQ ID no. 2 has included. 11. A process for the fermentative production of L-amino acids, characterized in that one carries out the following steps:

• a) fermentation of L-amino acid-producing coryneform bacteria in which at least the pgsA2 gene or nucleotide sequences coding therefor are amplified, in particular overexpressed,
• b) accumulation of the L-amino acid in the medium or in the cells of the bacteria and
• c) isolate from the L-amino acid.

12. The method according to claim 11, characterized, that is a strain according to any one of claims 1 to 5 starts.   13. The method according to claim 11 or 12, characterized, that additional genes that are a protein of the Biosyntheseweges the desired L-amino acid encode in which bacteria are amplified. 14. The method according to any one of claims 11 to 13, characterized, that metabolic pathways that the formation of the desired Reduce amino acid, in the bacteria at least partially off. 15. The method according to any one of claims 12 to 15, characterized, that the amino acid produced is L- Lysine acts. 16. The method according to any one of claims 11 to 15, characterized in that one fermented for the production of lysine bacteria, in which simultaneously one or more of the genes selected from the group

• a) the dapA gene coding for the dihydrodipicolinate synthase,
• b) the gene for the succinyldiaminopimelate-desuccinylase-encoding dapE gene,
• c) the lysC gene coding for a feed-back resistant aspartate kinase,
• d) the tpi gene coding for the triosephosphate isomerase,
• e) the gap gene coding for the glyceraldehyde-3-phosphate dehydrogenase,
• f) the pgk gene coding for the 3-phosphoglycerate kinase,
• g) the pyc gene coding for pyruvate carboxylase,
• h) the mqo gene coding for the malate: quinone oxidoreductase,
• i) the lysE gene coding for the lysine export is simultaneously amplified, in particular overexpressed or amplified.

17. The method according to any one of claims 11 to 16, characterized in that for the production of L-lysine bacteria are fermented, in which simultaneously one or more of the genes selected from the group

• a) the peck gene coding for the phosphoenolpyruvate carboxykinase,
• b) the pgi gene coding for the glucose-6-phosphate isomerase,
• c) the poxB gene coding for pyruvate oxidase

is weakened. 18. Use of polynucleotide sequences or parts of which according to claim 6 as a primer for the preparation of DNA of genes encoding CDP-diacylglycerol-glycerol-3 Phosphate 3 phosphatidyltransferase encode by the Polymerase chain reaction. 19. Use of polynucleotide sequences according to claim 6 as hybridization probes for the isolation of cDNA or Genes that have high homology with the sequence of pgsA2 gene.