EP1307475A1 - Sequences nucleotidiques codant pour le gene meth - Google Patents

Sequences nucleotidiques codant pour le gene meth

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Publication number
EP1307475A1
EP1307475A1 EP01965135A EP01965135A EP1307475A1 EP 1307475 A1 EP1307475 A1 EP 1307475A1 EP 01965135 A EP01965135 A EP 01965135A EP 01965135 A EP01965135 A EP 01965135A EP 1307475 A1 EP1307475 A1 EP 1307475A1
Authority
EP
European Patent Office
Prior art keywords
gene
codes
polynucleotide
methionine
sequence
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.)
Ceased
Application number
EP01965135A
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German (de)
English (en)
Inventor
Brigitte Bathe
Bettina Möckel
Walter Pfefferle
Klaus Huthmacher
Christian RÜCKERT
Jörn Kalinowski
Alfred Pühler
Michael Binder
Dieter Greissinger
Georg Thierbach
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 DE10109687A external-priority patent/DE10109687A1/de
Application filed by Degussa GmbH filed Critical Degussa GmbH
Publication of EP1307475A1 publication Critical patent/EP1307475A1/fr
Ceased 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1003—Transferases (2.) transferring one-carbon groups (2.1)
    • C12N9/1007—Methyltransferases (general) (2.1.1.)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/142—Amino acids; Derivatives thereof
    • 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
    • C12P13/12—Methionine; Cysteine; Cystine
    • 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
    • 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
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

Definitions

  • the invention provides nucleotide sequences from coryneform bacteria which code for the metH gene and a process for the fermentative preparation of amino acids, in particular L- methionine, using bacteria in which the metH gene is enhanced.
  • L-Amino acids in particular L-methionine, are used in human medicine and in the pharmaceuticals industry, in the foodstuffs industry and very particularly in animal nutrition.
  • amino acids are prepared by fermentation from strains of coryneform bacteria, in particular Corynebacteriu gluta icum. 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 chro atography, 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 chro atography, or the intrinsic output properties of the microorganism itself.
  • Strains which are resistant to antimetabolites such as e.g. the methionine analogue ⁇ - methyl-methionine, ethionine, norleucine, N- acetylnorleucine, S-trifluoromethylhomocysteine, 2-amino-5- heprenoitic acid, seleno-methionine, methionine- sulfoxi ine, methoxine, 1-aminocyclopentane-carboxylic acid, or are auxotrophic for metabolites of regulatory importance and produce amino acids, such as e.g. L- methionine, are obtained in this manner.
  • antimetabolites such as e.g. the methionine analogue ⁇ - methyl-methionine, ethionine, norleucine, N- acetylnorleucine, S-trifluoromethylhomocysteine, 2-amino-5- heprenoitic acid, sel
  • Methods of the recombinant DNA technique have also been employed for some years for improving the strain of Corynebacterium strains which produce L-amino acid, by amplifying individual amino acid biosynthesis genes and investigating the effect on the amino acid production.
  • the inventors had the object of providing new measures for improved fermentative preparation of amino acids, in particular L-methionine.
  • the salts such as e.g. methionine hydrochloride or methionine sulfate are also meant, by this.
  • the invention provides an isolated polynucleotide from coryneform bacteria, comprising a polynucleotide sequence which codes for the metH 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,
  • polypeptide which is complementary to the polynucleotides of a) or b) , and d) polynucleotide comprising at least 15 successive nucleotides of the polynucleotide sequence of a) , b) or c), the polypeptide preferably having the activity of ho ocysteine methyltransferase II.
  • the invention also provides the above-mentioned polynucleotide, this preferably being a DNA which is capable of replication, comprising:
  • the invention also provides
  • a vector containing the polynucleotide according to the invention in particular a shuttle vector or plasmid vector, and and coryneform bacteria serving as the host cell, which contain the vector or in which the metH 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, which comprises the complete gene with the polynucleotide sequence corresponding to SEQ ID No. 1, 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.
  • 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 homocysteine methyltransferase II or to isolate those nucleic acids or polynucleotides or genes which have a high similarity of sequence with that of the homocysteine methyltransferase II 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 homocysteine methyltransferase II can be prepared by the polymerase chain reaction (PCR) .
  • PCR polymerase chain reaction
  • Such 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. Oligonucleotides with a length of at least 100, 150, 200, 250 or 300 nucleotides are optionally 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 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 homocysteine methyltransferase II, and also those which are at least
  • the invention moreover provides a process for the fermentative preparation of amino acids, in particular L- methionine, using coryneform bacteria which in particular already produce amino acids, and in which the nucleotide sequences which code for the metH gene are enhanced, in particular over-expressed.
  • the term "enhancement” in this connection describes the increase in the intracellular activity of one or more enzymes (proteins) in a microorganism which are coded by the corresponding DNA, for example by increasing the number of copies of the gene or genes, using a potent promoter or using a gene or allele which codes for a corresponding enzyme (protein) having a high activity, and optionally combining these measures.
  • the activity or concentration of the corresponding protein is in general increased by at least 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400% or 500%, up to a maximum of 1000% or 2000%, based on the starting microorganism.
  • the microorganisms which the present invention provides can prepare L-amino acids, in particular L-methionine, 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.
  • Suitable strains of the genus Corynebacterium in particular of the species Corynebacterium glutamicum (C. glutamicum) , are in particular the known wild-type strains
  • L-amino acid-producing mutants or strains prepared therefrom such as, for example, the L-methionine-producing strain
  • the new metH gene from C. glutamicum which codes for the enzyme homocysteine methyltransferase II (EC 2.1.1.13) has been isolated.
  • 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)).
  • 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.
  • 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.
  • PCR polymerase chain reaction
  • coryneform bacteria produce amino acids, in particular L-methionine, in an improved manner after over-expression of the metH gene.
  • the number of copies of the corresponding genes can be increased, 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-methionine production.
  • the expression is likewise improved by measures to prolong the life of the m- RNA.
  • the enzyme activity is also increased by preventing the degradation of the enzyme protein.
  • the genes or gene constructs can either be present in plasmids with a varying number of copies, or can be integrated and amplified in the chromosome. Alternatively, an over- expression of the genes in question can furthermore be achieved by changing the composition of the media -and the culture procedure.
  • 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- 73 (1994)), pGEM-T (Promega corporation, Madison, WI, USA), pCR2.1-TOPO (Shuman (1994) .
  • amino acids in particular L-methionine
  • amino acids in particular L- methionine
  • amino acids in particular L-methionine
  • metH gene for one or more genes chosen from the group consisting of
  • the activity or concentration of the corresponding protein is in general reduced to 0 to 50%, 0 to 25%, 0 to 10% or 0 to 5% of the activity or concentration of the wild-type protein.
  • the 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-methionine.
  • batch culture batch culture
  • feed process fed batch
  • repetitive feed process repetition feed process
  • a summary of known culture methods is described in the textbook by Chmiel (Bioreatechnik 1. Einbowung in die Biovonstechnik (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas (Bioreaktoren und periphere bamboo (Vieweg Verlag, Braunschweig/Wiesbaden, 1994) ) .
  • 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 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.
  • Organic and inorganic sulfur-containing compounds such as, for example, sulfides, sulfites, sulfates and thiosulfates, can be used as a source of sulfur, in particular for the preparation of methionine.
  • 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 medium.
  • 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 fermentation broths obtained in this way in particular containing L-methionine, usually have a dry weight of 7.5 to 25 wt.% and contain L-methionine. It is furthermore also advantageous if the fermentation is conducted in a sugar- limited procedure at least at the end, but in particular over at least 30% of the duration of the fermentation. That is to say, the concentration of utilizable sugar in the fermentation medium is reduced to > 0 to 3 g/1 during this period.
  • the fermentation broth prepared in this manner is then further processed.
  • all or some of the biomass can be removed from the fermentation broth by separation methods, such as e.g. centrifugation, filtration, decanting or a combination thereof, or it can be left completely in this.
  • This broth is then thickened or concentrated by known methods, such as e.g. with the aid of a rotary evaporator, thin film evaporator, falling film evaporator, by reverse osmosis, or by nanofiltration.
  • This concentrated - fermentation broth can then be worked up by ". ' methods' of freeze drying, spray drying, spray granulation or by other processes to give a preferably free-flowing, finely divided powder.
  • This free-flowing, finely divided powder can then in turn by converted by suitable compacting or granulating processes into a coarse-grained, readily free-flowing, storable and largely dust-free product.
  • suitable compacting or granulating processes into a coarse-grained, readily free-flowing, storable and largely dust-free product.
  • organic or inorganic auxiliary substances or carriers such as starch, gelatin, cellulose derivatives or similar substances, such as are conventionally used as binders, gelling agents or thickeners in foodstuffs or feedstuffs processing, or further substances, such as, for example, silicas, silicates or stearates.
  • Free-flowing is understood as meaning powders which flow unimpeded out of the vessel with the opening of 5 mm (millimeters) of a series of glass outflow vessels with outflow openings of various sizes (Klein, Seifen, Ole, Fette, Wachse 94, 12 (1968)).
  • finely divided means a powder with a predominant content (> 50 %) with a particle size of 20 to 200 ⁇ m diameter.
  • coarse-grained means products with a predominant content (> 50 %) with a particle size of 200 to 2000 ⁇ m diameter.
  • dust-free means that the product contains only small contents ( ⁇ 5 %) with particle sizes of less than 20 ⁇ m diameter.
  • the particle size determination can be carried out with methods of laser diffraction spectrometry. The corresponding methods are described in the textbook on “Teilchengr ⁇ entown in der Laborpraxis” by R. H. Muller and R. Schuhmann, Academicliche Verlagsgesellschaft Stuttgart (1996) or in the textbook “Introduction to Particle Technology” by M. Rhodes, Verlag Wiley & Sons (1998).
  • “Storable” in the context of this invention means a product which can be stored for up to 120 days, preferably up to 52 weeks, particularly preferably 60 months, without a ' substantial loss ( ⁇ 5%) of methionine occurring.
  • the product can be absorbed on to an organic or inorganic carrier substance which is known and conventional in feedstuffs processing, such as, for example, silicas, silicates, grits, brans, meals, starches, sugars or others, and/or mixed and stabilized with conventional thickeners or binders.
  • feedstuffs processing such as, for example, silicas, silicates, grits, brans, meals, starches, sugars or others, and/or mixed and stabilized with conventional thickeners or binders.
  • the product can be brought into a state in which it is stable to digestion by animal stomachs, in particular the stomach of ruminants, by coating processes ("coating") using film-forming agents, such as, for example, metal carbonates, silicas, silicates, alginates, stearates, starches, gums and cellulose ethers, as described in DE-C-4100920.
  • film-forming agents such as, for example, metal carbonates, silicas, silicates, alginates, stearates, starches, gums and cellulose ethers, as described in DE-C-4100920.
  • the product described above is suitable as a feedstuffs additive, i.e. feed additive, for animal nutrition.
  • the L-methionine content of the . animal feedstuffs additive ' is conventionally 1 wt.% * to 80 wt.%, preferably 2 wt.% to 80 wt.%, particularly preferably ,4 wt.% to 80 wt.%, and very particularly preferably 8 wt.% to 80 wt.%, based on the dry weight of the animal feedstuffs additive. Contents of 1 wt.% to 60 wt.%, 2 wt.% to 60 wt.%, 4 wt.% to 60 wt.%, 6 wt.% to 60 wt.%, .1 wt.%.
  • the water content of the feedstuffs additive is conventionally up to 5 wt.%, . preferably up to 4 wt.%, and particularly preferably less than 2 wt.%.
  • the invention accordingly also provides a process for the preparation of an L-methionine-containing animal feedstuffs additive from fermentation broths, which comprises the steps
  • L-methionine can be carried out by ion exchange chromatography with subsequent ninhydrin derivation, as described by Spackman et al. (Analytical Chemistry, 30, (1958), 1190).
  • the process according to the invention is used for the fermentative preparation of amino acids, in particular L- methionine.
  • DSMZ German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany
  • DSM 14354 Escherichia coli DH5 ⁇ mcr/pCREmetH as DSM 14354.
  • 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 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 4 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 ampicillin. 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. 20021, Qiagen, Hilden, Germany) .
  • the DNA of the sequencing vector pZero-1 obtained from Invitrogen (Groningen, The Netherlands, Product Description Zero Background Cloning Kit, Product No. K2500-01) was cleaved with the restriction enzyme BamHI (Amersham Pharmacia, Freiburg, Germany, Product Description BamHI, Product No. 27-0868-04) .
  • the ligation of the cosmid fragments in the sequencing vector pZero-1 was carried out as described by Sambrook et al. (1989, Molecular Cloning: A laboratory Manual, Cold Spring Harbor), the DNA mixture being incubated overnight with T4 ligase (Pharmacia Biotech, Freiburg, Germany) .
  • 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 Pwo-Polymerase from Roche Diagnostics GmbH (Mannheim, Germany) .
  • the primers allow amplification of a DNA fragment 3718 bp in size, which carries the metH gene.
  • the primer metH-EVP5 contains the sequence for the cleavage site of the restriction endonuclease Bglll and the primer metH-EVP3 the cleavage site of the restriction endonuclease Sail, which are marked by underlining in the nucleotide sequence shown above.
  • the metH fragment 3718 bp in size was cleaved with the restriction endonucleases Bglll and Sail. The batch was separated by gel electrophoresis and the metH fragment
  • the E. coli - C. glutamicum shuttle expression vector pZ8-l (EP 0 375 889) was used as the base vector for the expression.
  • DNA of the plasmid pZ8-l 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) .
  • metH fragment approx. 3700 bp in size isolated from the agarose gel in example 3.1 and cleaved with the restriction endonucleases Bglll and Sail was mixed with the vector pZ8- 1 prepared in this way and the batch was treated with T4
  • 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). 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. After incubation overnight at 37°C, recombinant individual clones were selected. 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 checked by restriction cleavage.
  • Qiaprep Spin Miniprep Kit Product No. 27106, Qiagen, Hilden, Germany
  • the resulting plasmid was called pCREmetH.
  • DSMZ German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany
  • the vector pCREmetH obtained in example 3.2 was electroporated in the strain C. glutamicum ATCC13032 using the electroporation method described by Liebl et al. (FEMS Microbiology Letters, 53:299-303 (1989)). Selection of the plasmid-carrying cells 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 extract, 5 g/1 NaCl and 18 g/1 Bacto-agar, which had bee.n supplemented with 25 mg/1 kanamycin. Incubation was carried out for 2 days at 33°C.
  • Plasmid DNA was isolated from a transformant by conventional methods (Pete-rs-Wendisch et al., 1998,
  • the C. glutamicum strain ATCC13032/pCREmetH obtained in example 3 was cultured in a nutrient medium suitable for the production of methionine and the methionine 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 (25 mg/1) ) for 24 hours at 33°C.
  • a preculture was seeded (10 ml medium in a 100 ml conical flask) .
  • the medium MM was used as the medium for the preculture .
  • Medium MM
  • MOPS morpholinopropanesulfonic acid
  • Vitamin B12 (sterile-filtered) 0.02 mg/1
  • 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.
  • 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 (660 nm) of the main culture was 0.1.
  • Medium MM was also used for the main culture.
  • Culturing is carried out in a 10 ml volume in a 100 ml conical flask with baffles. Kanamycin (25 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 methionine formed was determined with an amino acid analyzer from Eppendorf- BioTronik (Hamburg, Germany) by ion exchange chromatography and post-column derivation with ninhydrin detection.
  • Km Resistance gene for kanamycin metH: metH gene of C. glutamicum
  • Ptac tac promoter
  • Tl T2: Terminator T1T2 of the rrnB gene of E. coli rep: Plas id-coded replication origin for C. glutamicum (of pHM1519)

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Abstract

L'invention concerne un polynucléotide isolé comprenant une séquence polynucléotidique choisie dans le groupe comprenant: a) un polynucléotide identique à 70 % au moins à un polynucléotide codant pour un polypeptide contenant la séquence d'acides aminés décrite dans SEQ ID No. 2; b) un polynucléotide codant pour un polypeptide contenant une séquence d'acides aminés identique à 70 % au moins à la séquence d'acides aminés décrite dans SEQ ID No. 2; c) un polynucléotide complémentaire des polynucléotides susmentionnés aux points a) ou b); et d) un polynucléotide comprenant au moins 15 nucléotides successifs de la séquence polynucléotidique décrite aux points a), b) ou c). L'invention concerne également des procédés de préparation par fermentation d'acides aminés L à l'aide de bactéries corynéformes dans lesquelles le gène metH au moins est présent sous sa forme renforcée; ainsi que l'utilisation des séquences polynucléotidiques comme sondes d'hybridation.
EP01965135A 2000-08-02 2001-07-17 Sequences nucleotidiques codant pour le gene meth Ceased EP1307475A1 (fr)

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DE10038050 2000-08-02
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DE10109687A DE10109687A1 (de) 2000-08-02 2001-02-28 Neue für das metH-Gen kodierende Nukleotidsequenzen
DE10109687 2001-02-28
PCT/EP2001/008220 WO2002010209A1 (fr) 2000-08-02 2001-07-17 Sequences nucleotidiques codant pour le gene meth

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DE10217058A1 (de) * 2002-04-17 2003-11-27 Basf Ag Verfahren zur Herstellung von schwefelhaltigen Feinchemikalien
DE10222858A1 (de) * 2002-05-23 2003-12-04 Basf Ag Verfahren zur fermentativen Herstellung schwefelhaltiger Feinchemikalien
DE10239082A1 (de) * 2002-08-26 2004-03-04 Basf Ag Verfahren zur fermentativen Herstellung schwefelhaltiger Feinchemikalien
DE10359668A1 (de) 2003-12-18 2005-07-14 Basf Ag Verfahren zur Herstellung von Methionin
DE102004035052A1 (de) 2004-07-20 2006-02-16 Basf Ag Mikroorganismen zur Herstellung von schwefelhaltigen Verbindungen
US20090325244A1 (en) * 2006-10-24 2009-12-31 Basf Se Method of increasing gene expression using modified codon usage
US20100041107A1 (en) 2006-10-24 2010-02-18 Basf Se Method of reducing gene expression using modified codon usage
US8252555B2 (en) 2006-12-29 2012-08-28 Evonik Degussa Ag Nucleic acid encoding a cobalamin-dependent methionine synthase polypeptide
WO2008101857A2 (fr) 2007-02-19 2008-08-28 Evonik Degussa Gmbh Bactérie de type corynéen avec activité formiate-thf-synthétase et/ou clivage de glycocolle
AR086790A1 (es) 2011-06-29 2014-01-22 Metabolic Explorer Sa Un microorganismo para la produccion de metionina con importacion de glucosa mejorada
EP2861726B1 (fr) 2012-06-18 2017-05-31 Evonik Degussa GmbH Microorganisme recombiné destiné à la production fermentative de méthionine
PL3039153T3 (pl) 2013-08-30 2019-02-28 Evonik Degussa Gmbh Mikroorganizm do wytwarzania metioniny o ulepszonej aktywności syntazy metioniny i wypływie metioniny
JP2017525379A (ja) 2014-09-01 2017-09-07 メタボリック エクスプローラー メチオニン排出を改善した発酵によるメチオニン産生の方法及び微生物
PL3331998T3 (pl) 2015-08-07 2020-12-28 Evonik Operations Gmbh Wytwarzanie przez fermentację l-metioniny zależne od tiokarboksylanu białka
MX2018006434A (es) 2015-11-27 2018-09-28 Evonik Degussa Gmbh Metodo para producir l-metionina.
EP3296404A1 (fr) 2016-09-15 2018-03-21 Evonik Degussa GmbH Micro-organisme modifie pour production de methionine
BR112020021859A2 (pt) 2018-05-04 2021-03-09 Ajinomoto Co., Inc. Método para produzir l-metionina ou um sal da mesma
BR112021005543A2 (pt) 2018-09-28 2021-06-29 Ajinomoto Co., Inc. método para produzir l-metionina

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