EP1320616A2 - Gensequenzen für sige von corynebacterium glutamicum - Google Patents

Gensequenzen für sige von corynebacterium glutamicum

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Publication number
EP1320616A2
EP1320616A2 EP01965132A EP01965132A EP1320616A2 EP 1320616 A2 EP1320616 A2 EP 1320616A2 EP 01965132 A EP01965132 A EP 01965132A EP 01965132 A EP01965132 A EP 01965132A EP 1320616 A2 EP1320616 A2 EP 1320616A2
Authority
EP
European Patent Office
Prior art keywords
gene
codes
polynucleotide
sequence
sige
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Withdrawn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
EP01965132A
Other languages
English (en)
French (fr)
Inventor
Bettina Möckel
Thomas Hermann
Mike Farwick
Michael Binder
Walter Pfefferle
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.)
Evonik Operations GmbH
Original Assignee
Degussa GmbH
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Filing date
Publication date
Priority claimed from DE10126422A external-priority patent/DE10126422A1/de
Application filed by Degussa GmbH filed Critical Degussa GmbH
Publication of EP1320616A2 publication Critical patent/EP1320616A2/de
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/34—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Corynebacterium (G)
    • 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
    • C12N1/205—Bacterial isolates
    • 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
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/15—Corynebacterium

Definitions

  • the invention provides nucleotide sequences from coryneform bacteria which code for the sigE gene and a process for the fermentative preparation of amino acids using bacteria in which the sigE gene is enhanced.
  • L-Amino acids are used in human medicine and in the pharmaceuticals industry, in the foodstuffs industry and especially 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 process can relate to fermentation measures, such as, for example, stirring and supply of oxygen, or the composition of the nutrient media, such as, for example, the sugar concentration during the fermentation, or the working up to the product form by, for example, ion exchange chromatography, or the intrinsic output properties of the microorganism itself.
  • fermentation measures such as, for example, stirring and supply of oxygen, or the composition of the nutrient media, such as, for example, the sugar concentration during the fermentation, or the working up to the product form by, for example, ion exchange chromatography, or the intrinsic output properties of the microorganism itself.
  • Methods of mutagenesis, selection and mutant selection are used to improve the output properties of these microorganisms. Strains which are resistant to antimetabolites or are auxotrophic for metabolites of regulatory importance and produce amino acids are obtained in this manner.
  • the inventors had the object of providing new measures for improved fermentative preparation of amino acids.
  • L-amino acids or amino acids are mentioned in the following, this means one or more amino acids, including their salts, chosen from the group consisting of L- asparagine, L-threonine, L-serine, L-glutamate, L-glycine, L-alanine, L-cysteine, L-valine, L-methionine, L- isoleucine, L-leucine, L-tyrosine, L-phenylalanine, L- histidine, L-lysine, L-tryptophan and L-arginine. Lysine is particularly preferred.
  • the invention provides an isolated polynucleotide from coryneform bacteria, comprising a polynucleotide sequence which codes for the sigE gene, chosen from the group consisting of
  • polynucleotide which is identical to the extent of at least 70% to a polynucleotide which codes for a polypeptide which comprises the amino acid sequence of SEQ ID No. 2,
  • polynucleotide which codes for a polypeptide which comprises an amino acid sequence which is identical to the extent of at least 70% to the amino acid sequence of SEQ ID No. 2,
  • polynucleotide comprising at least 15 successive nucleotides of the polynucleotide sequence of a) , b) or c), the polypeptide preferably having the activity of sigma factor E.
  • the invention also provides the above-mentioned polynucleotide, this preferably being a DNA which is capable of replication, comprising:
  • the invention also provides
  • a polynucleotide in particular DNA, which is capable of replication and comprises the nucleotide sequence as shown in SEQ ID No. 1;
  • polynucleotide which codes for a polypeptide which comprises the amino acid sequence as shown in SEQ ID No. 2;
  • a vector containing the polynucleotide according to the invention in particular a shuttle vector or plasmid vector, and
  • coryneform bacteria which contain the vector or in which the sigE gene is enhanced.
  • the invention also provides polynucleotides which substantially comprise a polynucleotide sequence, which are obtainable by screening by means of hybridization of a corresponding gene library of a coryneform bacterium, which comprises the complete gene or parts thereof, with a probe which comprises the sequence of the polynucleotide according to the invention according 'to SEQ ID No.l or a fragment thereof, and isolation of the polynucleotide sequence mentioned.
  • Polynucleotides which comprise the sequences according to the invention are suitable as hybridization probes for RNA, cDNA and DNA, in order to isolate, in the full length, nucleic acids or polynucleotides or genes which code for sigma factor E or to isolate those nucleic acids or polynucleotides or genes which have a high similarity of sequence with that of the sigE gene.
  • Polynucleotides which comprise the sequences according to the invention are furthermore suitable as primers with the aid of which DNA of genes which code for sigma factor E can be prepared by the polymerase chain reaction (PCR) .
  • PCR polymerase chain reaction
  • oligonucleotides which serve as probes or primers comprise at least 30, preferably at least 20, very particularly preferably at least 15 successive nucleotides. Oligonucleotides which have a length of at least 40 or 50 nucleotides are also suitable.
  • 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.
  • the polynucleotides according to the invention include a polynucleotide according to SEQ ID No. 1 or a fragment prepared therefrom and also those which are at least 70%, preferably at least 80% and in particular at least 90% to 95% identical to the polynucleotide according to SEQ ID No. 1 or a fragment prepared therefrom.
  • Polypeptides are 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 sigma factor E, and also those which are at least 70%, preferably at least 80% and in particular at least 90% to 95% identical to the polypeptide according to SEQ ID No. 2 and have the activity mentioned.
  • the invention furthermore relates to a process for the fermentative preparation of amino acids chosen from the group consisting of L-asparagine, L-threonine, L-serine, L- glutamate, L-glycine, L-alanine, L-cysteine, L-valine, L- methionine, L-isoleucine, L-leucine, L-tyrosine, L- phenylalanine, L-histidine, L-lysine, L-tryptophan and L- arginine using coryneform bacteria which in particular already produce amino acids and in which the nucleotide sequences which code for the sigE gene are enhanced, in particular over-expressed.
  • amino acids chosen from the group consisting of L-asparagine, L-threonine, L-serine, L- glutamate, L-glycine, L-alanine, L-cysteine, L-valine, L- methionine, L-isoleu
  • enhancement in this connection describes the increase in the intracellular activity of one or more enzymes in a microorganism which are .coded by the corresponding DNA, for example by increasing the number of copies of the gene or allele or of the genes or alleles, using a potent promoter or using a gene or allele which codes for a corresponding enzyme having a high activity, .and optionally combining these measures.
  • the microorganisms which the present invention provides can produce L-amino acids 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 (C. glutamicum) , are in particular the known wild-type strains
  • E. coli Escherichia coli
  • the setting up of gene libraries is described in generally known textbooks and handbooks. The textbook by Winnacker: Gene und Klone, Amsterdam Einf ⁇ hrung in die Gentechnologie (Verlag Chemie, Weinheim, Germany, 1990) , or the handbook by Sambrook et al.: Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989) may be mentioned as an example.
  • a well-known gene library is that of the E. coli K-12 strain W3110 set up in ⁇ vectors by Kohara et al. (Cell 50, 495 -508 (1987)).
  • plasmids such as pBR322 (Bolivar, Life Sciences, 25, 807-818 (1979)) or p ⁇ C9 (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 in turn be subcloned in the usual vectors suitable for sequencing and then sequenced, 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 resulting DNA sequences can then be investigated with known algorithms or sequence analysis programs, such as e.g. that of Staden (Nucleic Acids Research 14, 217- 232(1986)), that of Marck (Nucleic Acids Research 16, 1829- 1836 (1988)) or the GCG program of Butler (Methods of Biochemical Analysis 39, 74-97 (1998)).
  • known algorithms or sequence analysis programs such as e.g. that of Staden (Nucleic Acids Research 14, 217- 232(1986)), that of Marck (Nucleic Acids Research 16, 1829- 1836 (1988)) or the GCG program of Butler (Methods of Biochemical Analysis 39, 74-97 (1998)).
  • the new DNA sequence of C. glutamicum which codes for the sigE gene and which, as SEQ ID No. 1, is a constituent of the present invention has been found.
  • the amino acid sequence of the corresponding protein has furthermore been derived from the present DNA sequence by the methods described above.
  • the resulting amino acid sequence of the sigE 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 furthermore 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 furthermore known that changes on the N and/or C terminus of a protein cannot substantially impair or can even stabilize the function thereof.
  • 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.
  • Suitable plasmids are those which are replicated 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 .
  • plasmid vectors such as e.g. those based on pCG4 (US-A 4,489,160), or pNG2 (Serwold-Davis et al., FEMS Microbiology Letters 66, 119- 124 (1990)), or pAGl (US-A 5,158,891), can be used in the same manner.
  • Plasmid vectors which are furthermore suitable are also those with the aid of which the process of gene amplification 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 amplification 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)), pKl ⁇ mob or pK19mob (Schafer et al., Gene 145, 69-
  • L-amino acids to enhance, in particular over-express one or more enzymes of the particular biosynthesis pathway, of glycolysis, of anaplerosis, of the citric acid cycle, of the pentose phosphate cycle, of amino acid export and optionally regulatory proteins, in addition to the sigE gene.
  • the invention also provides the microorganisms prepared according to the invention, and these can be cultured continuously or discontinuously in the batch process (batch culture) or in the fed batch (feed process) or repeated fed batch process (repetitive feed process) for the purpose of production of amino acids.
  • batch culture batch culture
  • feed process feed process
  • repetitive feed process repetition feed process
  • 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 substances 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 acid
  • Organic nitrogen-containing compounds such as peptones, yeast extract, meat extract, malt extract, corn steep liquor, soya bean flour and urea
  • inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate, can be used as the source of nitrogen.
  • the sources of nitrogen can be used individually or as a mixture.
  • Phosphoric acid potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium- containing salts can be used as the source of phosphorus.
  • the culture medium must furthermore comprise salts of metals, such as e. g. magnesium sulfate or iron sulfate, which are necessary for growth.
  • essential growth substances such as amino acids and vitamins, can be employed in addition to the above-mentioned substances. Suitable precursors can moreover be added to the culture
  • the starting substances mentioned can be added to the culture in the form of a single batch, or can be fed in during the culture in a suitable manner.
  • Basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or aqueous ammonia, or acid compounds, such as phosphoric acid or sulfuric acid, can be employed in a suitable manner to control the pH of the culture.
  • Antifoams such as e.g. fatty acid polyglycol esters, can be employed to control the development of foam.
  • Suitable substances having a selective action such as e.g. antibiotics, can be added to the medium to maintain the stability of plasmids.
  • oxygen or oxygen-containing gas mixtures such as e.g. air, are introduced into the culture.
  • the temperature of the culture is usually 20°C to 45°C, and .preferably 25°C to 40°C. Culturing is continued until a maximum of the desired product has formed. This target is usually reached within 10 hours to 160 hours.
  • the process according to the invention is used for fermentative preparation of amino acids.
  • DSMZ German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany
  • composition of the usual nutrient media such as LB or TY medium, can also be found in the handbook by Sambrook et al.
  • Chromosomal DNA from Corynebacterium glutamicum ATCC 13032 was isolated as described by Tauch et al. (1995, Plasmid 33:168-179) and partly cleaved 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 Diagnostics GmbH, Mannheim, Germany, Product Description SAP, Code no. 1758250) .
  • the DNA of the cosmid vector SuperCosl (Wahl et al.
  • the cosmid DNA was then cleaved with -the restriction enzyme BamHI (Amersham Pharmacia, Freiburg, Germany, Product Description BamHI, Code no. 27-0868-04) .
  • the cosmid DNA treated in this manner was mixed with the treated ATCC13032 DNA 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 mixture was then packed in phages with the aid of Gigapack II XL Packing Extract (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. (1989, Molecular Cloning: A laboratory Manual, Cold Spring Harbor) , the cells being plated out on LB agar (Lennox, 1955, Virology, 1:190) with 100 mg/1 aitipicillin. After incubation overnight at 37°C, recombinant individual clones were selected.
  • 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 Diagnostics GmbH, Mannheim, Germany, Product Description SAP, Product No. 1758250) .
  • the cosmid fragments in the size range of 1500 to 2000 bp were isolated with the QiaExII Gel Extraction Kit (Product No.
  • This ligation mixture was then electroporated (Tauch et al. 1994, FEMS Microbiol Letters, 123:343-7) into the E. coli strain DH5 ⁇ MCR (Grant, 1990, Proceedings of the National Academy of Sciences U.S.A., 87:4645-4649) and plated out on LB agar (Lennox, 1955, Virology, 1:190) with 50 mg/1 zeocin.
  • the plasmid preparation of the recombinant clones was carried out with the Biorobot 9600 (Product No. 900200, Qiagen, Hilden, Germany) .
  • the sequencing was carried out by the dideoxy chain termination method of Sanger et al. (1977, Proceedings of the National Academy of Sciences U.S.A., 74:5463-5467) with modifications according to Zimmer ann et al. (1990, Nucleic Acids Research, 18:1067).
  • the "RR dRhodamin Terminator Cycle Sequencing Kit” from PE Applied Biosystems (Product No. 403044, Rothstadt,
  • 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 a'nalysis was prepared with the XNIP program (Staden, 1986, Nucleic Acids Research, 14:217-231).
  • the resulting nucleotide sequence is shown in SEQ ID No. 1. Analysis of the nucleotide sequence showed an open reading frame of 651 base pairs, which was called the sigE gene.
  • the sigE gene codes for a protein of 216 amino acids (SEQ ID NO. 2) .
  • SEQ ID NO. 3 The DNA sections lying upstream and downstream of SEQ ID NO. 1, which are shown in SEQ ID NO. 3 and SEQ ID NO. 4, were identified in the same manner.
  • the sigE gene region extended by SEQ ID NO. 3 and SEQ ID NO. 4 is shown in SEQ ID NO. 5.
  • chromosomal DNA was isolated by the method of Eikmanns et al. (Microbiology 140: 1817 -1828 (1994)).
  • the following oligonucleotides were chosen for the polymerase chain reaction (see SEQ ID No. 7 and SEQ ID No. 8) .
  • sigEl 5 "TAG TCA CCA CGG TTA AGC CT 3 N sigE2: 5 GCC TTG GTT CTT ACG AAC TG 3 s
  • the primers shown were synthesized by ARK Scientific GmbH Biosystems (Darmstadt, Germany) and the PCR reaction was carried out by the standard PCR method of Innis et al. (PCR protocols. A guide to methods and applications, 1990, Academic Press) with Taq-Polymerase from Qiagen (Hilden, Germany) . With the aid of the polymerase chain reaction, the primers allow amplification of a DNA fragment approx. 2.03 kb in size, which carries the sigE gene.
  • the amplified DNA fragment of approx. 2.03 kb in size which carries the sigE gene was ligated with the TOPO TA Cloning® Kit from Invitrogen Corporation (Carlsbad, CA, USA) in the vector pCR®2.1TOPO (Bernard et al., Journal of Molecular Biology, 234: 534-541 (1993)).
  • the E. coli strain ToplO (Grant et al., Proceedings of the National Academy of Sciences USA, 87 (1990) 4645-4649) was then transformed with the ligation batch in accordance with the instructions of the manufacturer of the kit (Invitrogen Corporation, Carlsbad, CA, USA) .
  • Plasmid DNA was isolated from a transformant with the aid of the QIAprep Spin Miniprep Kit from Qiagen (Hilden, Germany) and checked by treatment with the restriction enzyme Sphl and EcoRI with subsequent agarose gel electrophoresis (0.8 %) . The DNA sequence of the amplified DNA fragment was checked by sequencing.
  • the plasmid was called pCR2. IsigEexp.
  • the strain was called E. coli ToplO / pCR2. IsigEexp.
  • the E. coli - C. glutamicum shuttle vector was constructed according to the prior art.
  • the vector contains the
  • replication region reg of the plasmid pGAl including the replication effector per (US-A- 5,175,108; Nesvera et al., Journal of Bacteriology 179, 1525-1532 (1997)), the tetracycline resistance-imparting tetA(Z) gene of the plasmid pAGl (US-A- 5,158,891; gene library entry at the National Center for Biotechnology Information (NCBI, Bethesda, MD, USA) with the accession number AF121000), the replication region oriV of the plasmid pMBl (Sutcliffe, Cold Spring Harbor Symposium on Quantitative Biology 43,
  • the lacZ ⁇ gene fragment including the lac promoter and a multiple cloning site (mcs) (Norrander, J.M. et al. Gene 26, 101-106 (1983)) and the mob region of the plasmid RP4 (Simon et al.,(1983) Bio/Technology 1:784-791).
  • the vector constructed was transformed in the E. coli strain DH5 ⁇ (Hanahan, In: DNA cloning. A Practical Approach. Vol. I. IRL-Press, Oxford, Washington DC, USA).
  • Plasmid DNA was isolated from a transformant with the aid of the QIAprep Spin Miniprep Kit from Qiagen and checked by restriction with the restriction enzymes EcoRI and Hindlll and subsequent agarose gel electrophoresis (0.8 %) .
  • the plasmid was called pEC-T18mob2 and is shown in figure 1.
  • the E. coli - C. glutamicum shuttle vector pEC-T18mob2 described in example 3.2 was used as the vector.
  • DNA of this plasmid was cleaved completely with the restriction enzymes BamHI and Sail and then dephosphorylated with shrimp alkaline phosphatase (Roche Diagnostics GmbH, Mannheim, Germany, Product Description SAP, Product No. 1758250) .
  • the sigE gene was isolated from the plasmid pCR2. IsigEexp described in example 3.1. by complete cleavage with the enzymes BamHI and Sail. The sigE fragment 1930 bp in size was isolated from the agarose gel with the QiaExII Gel Extraction Kit (Product No. 20021, Qiagen, Hilden, Germany) .
  • the sigE fragment obtained in this manner was mixed with the prepared vector pEC-T18mob2 and the batch was treated with T4 DNA ligase (Amersham Pharmacia, Freiburg, Germany, Product Description T4-DNA-Ligase, Code no. 27-0870-04) .
  • 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 ⁇ MCR (Hanahan, In: DNA cloning. A Practical Approach. Vol. I. IRL-Press, Oxford, Washington DC, USA).
  • 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 enzymes BamHI and Sail to check the plasmid by subsequent agarose gel electrophoresis.
  • the plasmid obtained was called pEC- T18mob2sigEexp. It is shown in figure 2.
  • the strain DSM5715 was transformed with the plasmid pEC- T18mob2sigEexp 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, 0.5 M sorbitol, 5 g/1 Bacto-tryptone, 2.5 g/1 Bacto-yeast
  • Plasmid DNA was isolated from a transformant by conventional methods (Peters-Wendisch et al . , 1998, Microbiology, 144, 915 -927), cleaved with the restriction endonucleases BamHI and Sail, and the plasmid was checked by subsequent agarose gel electrophoresis. The strain obtained was called DSM5715/pEC-Tl8mob2sigEexp.
  • the C. glutamicum strain DSM5715/pEC-T18mob2sigEexp obtained in example 4 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 tetracycline (5 mg/1)) for 24 hours at 33°C.
  • a pre-culture was seeded (10 ml medium in a 100 ml conical flask) .
  • the complete medium Cglll was used as the medium for the pre-culture.
  • Tetracycline (5 mg/1) was added to this.
  • the pre-culture was incubated for 16 hours at 33°C at 240 rpm on a shaking machine.
  • a main culture was seeded from this pre-culture such that the initial OD (660 nm) of the main culture was 0.05.
  • Medium MM was used for the main culture.
  • MOPS morpholinopropanesulfonic 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. Tetracycline (5 mg/1) was added. Culturing was carried out at 33°C and 80 % atmospheric humidity.
  • 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 derivation with ninhydrin detection.
  • FIG. 1 Map of the plasmid pEC-T18mob2
  • oriV ColEl-similar origin from pMBl
  • RP4mob RP4 mobilization site lacZ-alpha: lacZ gene fragment from E. coli
  • Tet Resistance gene for tetracycline sigE: sigE gene of C. glutamicum
  • BamHI Cleavage site of the restriction enzyme
  • BamHI Sail Cleavage site of the restriction enzyme Sail
  • sigE sigE gene of C. glutamicum

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EP01965132A 2000-09-02 2001-07-14 Gensequenzen für sige von corynebacterium glutamicum Withdrawn EP1320616A2 (de)

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DE10043336 2000-09-02
DE10043336 2000-09-02
DE10126422A DE10126422A1 (de) 2000-09-02 2001-05-31 Für das sigE-Gen kodierende Nukleotidsequenzen
DE10126422 2001-05-31
PCT/EP2001/008146 WO2002018428A2 (en) 2000-09-02 2001-07-14 Sequences which code for the sige gene of corynebacterium glutamicum

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US6156532A (en) * 1995-02-20 2000-12-05 Ajinomoto Co., Inc. Stress-resistant microorganism and method of producing fermentative product
DE19548222A1 (de) * 1995-12-22 1997-06-26 Forschungszentrum Juelich Gmbh Verfahren zur mikrobiellen Herstellung von Aminosäuren durch gesteigerte Aktivität von Exportcarriern
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