EP1278865A1 - Für das pgsa2-gen kodierende nukleotidsequenzen - Google Patents

Für das pgsa2-gen kodierende nukleotidsequenzen

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Publication number
EP1278865A1
EP1278865A1 EP01940367A EP01940367A EP1278865A1 EP 1278865 A1 EP1278865 A1 EP 1278865A1 EP 01940367 A EP01940367 A EP 01940367A EP 01940367 A EP01940367 A EP 01940367A EP 1278865 A1 EP1278865 A1 EP 1278865A1
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EP
European Patent Office
Prior art keywords
gene
codes
polynucleotide
sequence
pgsa2
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP01940367A
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English (en)
French (fr)
Inventor
Madhavan Nampoothiri
Bettina Möckel
Walter Pfefferle
Lothar Eggeling
Hermann Sahm
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Forschungszentrum Juelich GmbH
Evonik Operations GmbH
Original Assignee
Forschungszentrum Juelich GmbH
Degussa GmbH
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Application filed by Forschungszentrum Juelich GmbH, Degussa GmbH filed Critical Forschungszentrum Juelich GmbH
Publication of EP1278865A1 publication Critical patent/EP1278865A1/de
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1288—Transferases for other substituted phosphate groups (2.7.8)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/08—Transferases for other substituted phosphate groups (2.7.8)
    • C12Y207/08005—CDP-diacylglycerol-glycerol-3-phosphate 3-phosphatidyltransferase (2.7.8.5)

Definitions

  • the invention provides -genetically modified coryneform bacteria, nucleotide sequences which code for CDP- diacylglycerol-glycerol-3-phosphate 3- phosphatidyltransferase and processes for the fermentative preparation of amino acids, in particular L-lysine, using coryneform bacteria in which the pgsA2 gene, which codes for CDP-diacylglycerol-glycerol-3-phosphate 3- phosphatidyltransferase (EC 2.7.8.5), is enhanced.
  • Amino acids in particular L-lysine, are used in human medicine and in the pharmaceuticals industry, but in particular in animal nutrition.
  • amino acids are prepared by fermentation from strains of coryneform bacteria, in particular Corynebacterium glutamicum . Because of their great importance, work is constantly being undertaken to improve the preparation processes. Improvements to the processes can relate to fermentation measures, such as e. g. stirring and supply of oxygen, or the composition of the nutrient media, such as e. g. the sugar concentration during the fermentation, or the working up to the product form by e. g. ion exchange chromatography, or the intrinsic output properties of the microorganism itself.
  • fermentation measures such as e. g. stirring and supply of oxygen, or the composition of the nutrient media, such as e. g. the sugar concentration during the fermentation, or the working up to the product form by e. g. ion exchange chromatography, or the intrinsic output properties of the microorganism itself.
  • Methods of mutagenesis, selection and mutant selection are used to improve the output properties of these microorganisms.
  • Strains which are resistant to antimetabolites such as e. g. the lysine analogue S-(2- aminoethyl) -cysteine, or are auxotrophic for metabolites of regulatory importance and produce L-amino acids, such as e. g. L-lysine, are obtained in this manner.
  • the object of the present invention was to provide new aids for improved fermentative preparation of amino acids, in particular L-lysine.
  • This object is achieved by a genetically modified coryneform bacterium, the pgsA2 gene of which, which codes for CDP-diacylglycerol-glycerol-3-phosphate 3- phosphatidyltransferase, is enhanced.
  • Amino acids in particular L-lysine
  • L-lysine are used in human medicine, in the pharmaceuticals industry and in particular in animal nutrition. There is therefore a general interest in providing new improved processes for the preparation of amino acids, in particular L-lysine.
  • the invention provides a genetically modified coryneform bacterium, in which its pgsA2 gene, which codes for CDP- diacylglycerol-glycerol-3-phosphate 3- phosphatidyltransferase, is enhanced.
  • Enhancement can be achieved with the aid of various manipulations of the bacterial cell.
  • the number of copies of the corresponding genes can be increased, a potent promoter can be used, or the promoter and regulation region or the ribosome binding site upstream of the structural gene can be mutated.
  • Expression cassettes which are incorporated upstream of the structural gene act in the same way.
  • inducible promoters it is additionally possible to increase the expression in the course of fermentative L-lysine production.
  • a gene which codes for a corresponding enzyme with a high activity The expression is likewise improved by measures to prolong the life of the m-RNA.
  • the enzyme activity is also increased overall by preventing the degradation of the enzyme. These measures can optionally also be combined as desired.
  • the microorganisms which the present invention provides can prepare L-amino acids, in particular L-lysine, from glucose, sucrose, lactose, fructose, maltose, molasses, starch, cellulose or from glycerol and ethanol. They can be representatives of coryneform bacteria, in particular of the genus Corynebacterium. Of the genus Corynebacterium, there may be mentioned in particular the species Corynebacterium glutamicum, which is known among experts for its ability to produce L-amino acids.
  • Suitable strains of the genus Corynebacterium, in particular of the species Corynebacterium glutamicum, are, for example, the known wild-type strains
  • the present invention also provides an isolated polynucleotide from coryneform bacteria, comprising a polynucleotide sequence chosen from the group consisting of
  • polynucleotide which is homologous to the extent of at least 70 % with a polynucleotide which codes for a polypeptide which comprises the amino acid sequence of SEQ ID No. 2,
  • polynucleotide which codes for a polypeptide which comprises an amino acid sequence which is homologous to the extent of at least 70% with the amino acid sequence of SEQ ID No. 2,
  • polynucleotide comprising at least 15 successive nucleotides of the polynucleotide sequence of a) , b) or c) .
  • a polynucleotide sequence is "homologous" to the sequence according to the invention if it coincides in its base composition and sequence with the sequence according to the invention to the extent of at least 70%, preferably at least 80%, particularly preferably at least 90%.
  • a "homologous protein” is to be understood as proteins which have an amino acid sequence which coincide with the amino acid sequence coded by the pgsA2 gene (SEQ ID No.l) to the extent of at least 70 %, preferably at least 80 %, particularly preferably at least 90 %, "coincide” being understood as meaning that either the corresponding amino acids are identical or they are amino acids which are homologous to one another. Those amino acids which correspond in their properties, in particular in respect of charge, hydrophobicity, steric properties etc., are called “homologous amino acids”.
  • the invention also provides a polynucleotide as described above , this preferably being a DNA which is capable of replication, comprising:
  • the invention also provides a preferably recombinant polynucleotide which is capable of replication in coryneform bacteria and comprises the nucleotide sequence SEQ ID No. 1,
  • coryneform bacteria serving as the host cell, which contain the vector or in which the pgsA2 gene is enhanced.
  • the invention also provides polynucleotides which comprise the complete gene with the polynucleotide sequence corresponding to SEQ ID No. 1 or fragments thereof, and which are obtainable by screening by means of hybridization of a corresponding gene library with a probe which comprises the sequence of the polynucleotide mentioned, according to SEQ ID No. 1, or a fragment thereof, and isolation of the DNA sequence mentioned.
  • Polynucleotide sequences according to the invention are also suitable as hybridization probes for RNA, cDNA and DNA, in order to isolate, in the full length, cDNA which code for CDP-diacylglycerol-glycerol-3-phosphate 3- phosphatidyltransferase and to isolate those cDNA or genes which have a high similarity with the sequence of the CDP- diacylglycerol-glycerol-3-phosphate 3- phosphatidyltransferase gene.
  • Polynucleotide sequences according to the invention are furthermore suitable as primers for the polymerase chain reaction (PCR) , for the preparation of DNA which codes for CDP-diacylglycerol-glycerol-3-phosphate 3- phosphatidyltransferase proteins.
  • PCR polymerase chain reaction
  • Such oligonucleotides which serve as probes or primers can comprise more than 30, preferably up to 30, particularly preferably up to 20, very particularly preferably at least 15 successive nucleotides. Oligonucleotides which have a length of at least 40 or 50 nucleotides are also suitable.
  • Polynucleotide in general relates to polyribonucleotides and polydeoxyribonucleotides, it being possible for these to be non-modified RNA or DNA or modified RNA or DNA.
  • Polypeptides is understood as meaning peptides or proteins which comprise two or more amino acids bonded via peptide bonds .
  • polypeptides according to the invention include a polypeptide according to SEQ ID No. 2, in particular those with the biological activity of the CDP-diacylglycerol- glycerol-3-phosphate 3-phosphatidyltransferase protein, and also those which are homologous to the extent of at least 70 % with the polypeptide according to SEQ ID No. 2, and preferably are homologous to the extent of at least 80% and in particular to the extent of at least 90 % to 95 % with the polypeptide according to SEQ ID no. 2, and have the activity mentioned.
  • the invention moreover provides a process for the fermentative preparation of amino acids, in particular L- lysine, using coryneform bacteria which in particular already produce an amino acid, and in which the nucleotide sequences which code for the pgsA2 gene are enhanced, in particular over-expressed.
  • the pgsA2 gene of C. glutamicum which codes for CDP- diacylglycerol-glycerol-3-phosphate 3- phosphatidyltransferase is described for the first time in the present invention.
  • a gene library of this microorganism is first set up in E. coli . The setting up of gene libraries is described in generally known textbooks and handbooks.
  • glutamicum ATCC13032 which was set up 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). Bormann et al. (Molecular Microbiology 6(3), 317-326)) (1992)) in turn describe a gene library of C. glutamicum ATCC13032 using the cosmid pHC79 (Hohn and Collins, Gene 11, 291-298 (1980)). To prepare a gene library of C. glutamicum in E.
  • coli it is also possible to use plasmids such as pBR322 (Bolivar, Life Sciences, 25, 807-818 (1979)) or pUC9 (Vieira et al., 1982, Gene, 19:259-268).
  • Suitable hosts are, in particular, those E. coli strains which are restriction- and recombination- defective.
  • An example of these is the strain DH5 ⁇ mcr, which has been described by Grant et al. (Proceedings of the National Academy of Sciences USA, 87 (1990) 4645-4649) .
  • the long DNA fragments cloned with the aid of cosmids can then in turn be subcloned and subsequently sequenced in the usual vectors which are suitable for sequencing, such as is described e. g. by Sanger et al. (Proceedings of the National Academy of Sciences of the United States of America, 74:5463-5467, 1977).
  • the new DNA sequence of C. glutamicum ' which codes for the pgsA2 gene and which, as SEQ ID No. 1, is a constituent of the present invention, was obtained in this manner.
  • the amino acid sequence of the corresponding protein has moreover been derived from the present DNA sequence by the methods described above.
  • the resulting amino acid sequence of the pgsA2 gene product is shown in SEQ ID No. 2.
  • Coding DNA sequences which result from SEQ ID No. 1 by the degeneracy of the genetic code are also a constituent of the invention.
  • DNA sequences which hybridize with SEQ ID No. 1 or parts of SEQ ID No. 1 are a constituent of the invention.
  • Conservative amino acid exchanges such as e. g. exchange of glycine for alanine or of aspartic acid for glutamic acid in proteins, are moreover known among experts as "sense mutations" which do not lead to a fundamental change in the activity of the protein, i.e. are of neutral function. It is moreover known that changes on the N and/or C terminus of a protein cannot substantially impair the function thereof or can even stabilize this.
  • DNA sequences which hybridize with SEQ ID No. 1 or parts of SEQ ID No. 1 are a constituent of the invention.
  • DNA sequences which are prepared by the polymerase chain reaction (PCR) using primers which result from SEQ ID NO. 1 are a constituent of the invention.
  • PCR polymerase chain reaction
  • Such oligonucleotides typically have a length of at least 15 nucleotides.
  • PCR polymerase chain reaction
  • coryneform bacteria produce amino acids, in particular L-lysine, in an improved manner after enhancement of the pgsA2 gene.
  • genes or gene constructs under consideration can either be present in plasmids with a varying number of copies, or can be integrated and enhanced in the chromosome. Alternatively, an over-expression of the genes in question can moreover be achieved by changing the composition of the media and the culture procedure.
  • the pgsA2 gene according to the invention was over-expressed with the aid of plasmids.
  • Suitable plasmids are those which are replicated and expressed in coryneform bacteria.
  • Numerous known plasmid vectors such as e. g. pZl (Menkel et al., Applied and Environmental Microbiology (1989) 64: 549-554), pEKExl (Eikmanns et al., Gene 102:93-98 (1991)) or pHS2-l (Sonnen et al., Gene 107:69-74 (1991)) are based on the cryptic plasmids pHM1519, pBLl or pGAl.
  • Other plasmid vectors such as e. g.
  • pJClpgsA2 (figure 1) , which is based on the E. coli - C. glutamicum shuttle vector pJCl (Cremer et al., 1990, Molecular and General Genetics 220: 478 - 480) and contains the DNA sequence of C. glutamicum which codes for the pgsA2 gene. It is contained in the strain DSM5715/pJClpgsA2.
  • Plasmid vectors which are moreover suitable are those with the aid of which the process of gene enhancement by integration into the chromosome can be used, as has been described, for example, by Reinscheid et al. (Applied and Environmental Microbiology 60, 126-132 (1994)) for duplication or enhancement of the hom-thrB operon.
  • the complete gene is cloned in a plasmid vector which can replicate in a host (typically E. coli) , but not in C. glutamicum.
  • Possible vectors are, for example, pSUP301 (Simon et al., Bio/Technology 1, 784-791 (1983)), ⁇ K18mob or pKl9mob (Schafer et al., Gene 145, 69-73 (1994)), pGEM-T (Promega corporation, Madison, WI, USA), pCR2.1-TOPO (Shuman (1994).
  • the plasmid vector which contains the gene to be enhanced is then transferred into the desired strain of C. glutamicum by conjugation or transformation.
  • the method of conjugation is described, for example, by Schafer et al. (Applied and Environmental Microbiology 60, 756-759 (1994)). Methods for transformation are described, for example, by Thierbach et al.
  • amino acids in particular L-lysine
  • microorganisms prepared according to the invention can be cultured continuously or discontinuously in the batch process (batch culture) or in the fed batch (feed process) or repeated fed batch process (repetitive feed process) for the purpose of production of amino acids, in particular L- lysine.
  • batch culture batch culture
  • feed process fed batch
  • repetitive feed process repetition feed process
  • the culture medium to be used must meet the requirements of the particular strains in a suitable manner. Descriptions of culture media for various microorganisms are contained in the handbook “Manual of Methods for General Bacteriology” of the American Society for Bacteriology (Washington D.C., USA, 1981).
  • Sugars and carbohydrates such as e. g. glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose, oils and fats, such as e. g. soya oil, sunflower oil, groundnut oil and coconut fat, fatty acids, such as e. g. palmitic acid, stearic acid and linoleic acid, alcohols, such as e. g. glycerol and ethanol, and organic acids, such as e. g. acetic acid, can be used as the source of carbon. These substance can be used individually or as a mixture.
  • oils and fats such as e. g. soya oil, sunflower oil, groundnut oil and coconut fat
  • fatty acids such as e. g. palmitic acid, stearic acid and linoleic acid
  • alcohols such as e. g. glycerol and ethanol
  • organic acids such as e. g. acetic
  • DSMZ German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany
  • the process according to the invention is used for the fermentative preparation of amino acids, in particular L- lysine.
  • the ligation mixture was then packed in phages with the aid of Gigapack II XL Packing Extracts (Stratagene, La Jolla, USA, Product Description Gigapack II XL Packing Extract, Code no. 200217) .
  • Gigapack II XL Packing Extracts Stratagene, La Jolla, USA, Product Description Gigapack II XL Packing Extract, Code no. 200217.
  • the cells were taken up in 10 mM MgS0 and mixed with an aliquot of the phage suspension.
  • the infection and titering of the cosmid library were carried out as described by Sambrook et al.
  • the cosmid DNA of an individual colony was isolated with the Qiaprep Spin Miniprep Kit (Product No. 27106, Qiagen, Hilden, Germany) in accordance with the manufacturer's instructions and partly cleaved with the restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg, Germany, Product Description Sau3AI, Product No. 27-0913-02) .
  • the DNA fragments were dephosphorylated with shrimp alkaline phosphatase (Roche Molecular Biochemicals, Mannheim,
  • the raw sequence data obtained were then processed using the Staden program package (1986, Nucleic Acids Research, 14:217-231) version 97-0.
  • the individual sequences of the pZerol derivatives were assembled to a continuous contig.
  • the computer-assisted coding region analysis was prepared with the XNIP program (Staden, 1986, Nucleic Acids Research, 14:217-231). Further analyses were carried out with the "BLAST search program" (Altschul et al., 1997, Nucleic Acids Research,- 25:3389-3402), against the non- redundant databank of the "National Center for
  • the resulting nucleotide sequence is shown in SEQ ID No. 1. Analysis of the nucleotide sequence showed an open reading frame of 291 base pairs, which was called the pgsA2 gene. The pgsA2 gene codes for a protein of 97 amino acids.
  • Chromosomal DNA from Corynebacterium glutamicum ATCC 13032 was isolated as described by Tauch et al. (1995, Plasmid 33:168-179). A DNA fragment which carries the pgsA2 gene was amplified with the aid of the polymerase chain reaction. The following primers were used for this:
  • Both oligonucleotides carry the sequence for the cleavage site of the restriction enzyme Xbal (nucleotides underlined) .
  • the primers shown were synthesized by MWG Biotech (Ebersberg, Germany) synthesized and the PCR reaction was carried out by the standard PCR method of Innis et al., (PCR protocol. A guide to methods and applications, 1990, Academic Press) .
  • the primers allow amplification of a DNA fragment of approx 749 bp in size, which carries the pgsA2 gene from Corynebacterium glutamicum.
  • the PCR fragment was isolated from the agarose gel with the QiaExII Gel Extraction Kit (Product No. 20021, Qiagen, Hilden, Germany) .
  • the PCR fragment obtained in this manner was cleaved completely with the restriction enzyme Xbal.
  • the pgsA2 fragment approx 749 bp in size was isolated from the agarose gel with the QiaExII Gel Extraction Kit (Product No. 20021, Qiagen, Hilden, Germany) .
  • the E. coli - C. glutamicum shuttle vector pJCl (Cremer et al., 1990, Molecular and General Genetics 220: 478 -480) was used as the vector. This plasmid was also cleaved completely with the restriction enzyme Xbal and then dephosphorylated with shrimp alkaline phosphatase (Roche Molecular Biochemicals, Mannheim, Germany, Product Description SAP, Product No. 1758250) .
  • the pgsA2 fragment obtained in this manner was mixed with the prepared vector pJCl and the batch was treated with T4 DNA ligase (Amersham Pharmacia, Freiburg, Germany, Product Description T4-DNA-Ligase, Code no.27-0870-04) .
  • the ligation batch was transformed in the E. coli strain DH5 ⁇ (Hanahan, In: DNA cloning. A practical approach. Vol. I. IRL-Press, Oxford, Washington DC, USA) . Selection of plasmid-carrying cells was made by plating out the transformation batch on LB agar (Lennox, 1955, Virology, 1:190) with 50 mg/1 kanamycin.
  • Plasmid DNA was isolated from a transformant with the Qiaprep Spin Miniprep Kit (Product No. 27106, Qiagen, Hilden, Germany) in accordance with the manufacturer's instructions and cleaved with the restriction enzyme Xbal to check the plasmid by subsequent agarose gel electrophoresis. The resulting plasmid was called pJClpgsA2.
  • the strain DSM5715 was transformed with the plasmid pJClpgsA2 using the electroporation method described by Liebl et al., (FEMS Microbiology Letters, 53:299-303 (1989) ) . Selection of the transformants took place on LBHIS agar comprising 18.5 g/1 brain-heart infusion broth, 0.5M sorbitol, 5 g/1 Bacto-tryptone, 2.5 g/1 Bacto-yeast extract, 5 g/1 NaCl, 18 g/1 Bacto-agar, which had been supplemented with 25 ⁇ g/ml kanamycin. Incubation was carried out for 2 days at 33°C.
  • Plasmid DNA was isolated from a transformant by conventional methods (Peters-Wendisch et al., 1998, Microbiology, 144, 915 -927) and cut with the restriction endonuclease EcoRI; in order to check the plasmid by subsequent agarose gel electrophoresis. The resulting strain was called DSM5715/pJClpgsA2.
  • DSMZ German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany
  • the C. glutamicum strain DSM5715/pJClpgsA2 obtained in example 5 was cultured in a nutrient medium suitable for the production of lysine and the lysine content in the culture supernatant was determined.
  • the strain was first incubated on an agar plate with the corresponding antibiotic (brain-heart agar with kanamycin (50 ⁇ g/ml) for 24 hours at 33°C.
  • a preculture was seeded (10 ml medium in a 100 ml conical flask) .
  • the complete medium Cglll was used as the medium for the preculture.
  • Kanamycin 25 mg/1 was added to this.
  • the preculture was incubated for 16 hours at 33°C at 240 rpm on a shaking machine.
  • a main culture was seeded from this preculture such that the initial OD (660nm) of the main culture was 0.1.
  • Medium MM was used for the main culture.
  • MOPS morpholinopropanesulfonic 20 g/1 acid
  • the CSL, MOPS and the salt solution were brought to pH 7 with aqueous ammonia and autoclaved.
  • the sterile substrate and vitamin solutions were then added, as well as the CaC0 3 autoclaved in the dry state.
  • Culturing is carried out in a 10 ml volume in a 100 ml conical flask with baffles. Kanamycin (25 ⁇ g/ml) was added. Culturing was carried out at 33°C and 80% atmospheric humidity. After 24 hours, the OD was determined at a measurement wavelength of 660 nm with a Biomek 1000 (Beckmann Instruments GmbH, Kunststoff) . The amount of lysine formed was determined with an amino acid analyzer from Eppendorf- BioTronik (Hamburg, Germany) by ion exchange chromatography and post-column derivatization with ninhydrin detection.
  • FIG. 1 Map of the plasmid pJClpgsA2
  • oriCg Plasmid-coded replication origin C. glutamicum (of pHMl519 )
  • pgsA2 pgsA2 CDP-diacylglycerol-glycerol-3-phosphate phosphatidyltransferase gene from C. glutamicum ATCC13032
  • Kan Kanamycin resistance gene
  • Hindlll Cleavage site of the restriction enzyme Hindlll

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EP01940367A 2000-05-04 2001-04-26 Für das pgsa2-gen kodierende nukleotidsequenzen Withdrawn EP1278865A1 (de)

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DE10021829A DE10021829A1 (de) 2000-05-04 2000-05-04 Neue für das pgsA2-Gen kodierende Nukleotidsequenzen
DE10021829 2000-05-04
PCT/EP2001/004704 WO2001083766A1 (en) 2000-05-04 2001-04-26 New nucleotide sequences which code for the pgsa2 gene

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US6448037B1 (en) * 1998-02-20 2002-09-10 Smithkline Beecham Corporation PgsA
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KR20020097244A (ko) 2002-12-31
DE10021829A1 (de) 2001-11-08

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