EP1307476A1 - Nukleotidsequenzen die für das mete-gen kodieren - Google Patents

Nukleotidsequenzen die für das mete-gen kodieren

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Publication number
EP1307476A1
EP1307476A1 EP01967191A EP01967191A EP1307476A1 EP 1307476 A1 EP1307476 A1 EP 1307476A1 EP 01967191 A EP01967191 A EP 01967191A EP 01967191 A EP01967191 A EP 01967191A EP 1307476 A1 EP1307476 A1 EP 1307476A1
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
EP01967191A
Other languages
English (en)
French (fr)
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 DE10109689A external-priority patent/DE10109689A1/de
Application filed by Degussa GmbH filed Critical Degussa GmbH
Publication of EP1307476A1 publication Critical patent/EP1307476A1/de
Ceased legal-status Critical Current

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Classifications

    • 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
    • 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
    • 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

Definitions

  • amino acids such as e.g. L- methionine
  • the inventors had the object of providing new measures for improved fermentative preparation of amino acids, in particular L-methionine .
  • the invention provides an isolated polynucleotide from coryneform bacteria, comprising a polynucleotide sequence which codes for the metE 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 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),
  • polypeptide preferably having the activity of homocysteine methyltransferase I.
  • the invention also provides the above-mentioned polynucleotide, this preferably being a DNA which is capable of replication, comprising:
  • the invention also provides
  • 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
  • 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 I or to isolate those nucleic acids or polynucleotides or genes which have a high similarity of sequence with that of the homocysteine methyltransferase I 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 I 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 I, and also those which are at least 70%, preferably at least 80% and in particular which are at least 90% to 95% identical to 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- methionine, using coryneform bacteria which in particular already produce amino acids, and in which the nucleotide sequences which code for the metE gene are enhanced, in particular over-expressed.
  • 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. 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
  • L-amino acid-producing mutants or strains prepared therefrom such as, for example, the L-methionine-producing strain
  • the new metE gene from C. glutamicum which codes for the enzyme homocysteine methyltransferase I (EC 2.1.1.14) has been isolated.
  • E. coli Escherichia coli
  • the setting up of gene libraries is described in generally known textbooks and handbooks. The textbook by innacker: Gene und Klone, Amsterdam Einbowung 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).
  • TJ rt ⁇ 3 O ⁇ - tr rt tr ⁇ ⁇ — D tr TJ ⁇ 3 CO .
  • TJ ⁇ l cx CD 3 ft) O ⁇ li ⁇ cu ⁇ - ⁇ ii — 0) o tc 3 ⁇ li ⁇ 3 ⁇ > ⁇ ⁇ 0 OJ ⁇ CO 3 ⁇ 3 rt rt cx CO o rt rt • •>.
  • amino acids in particular L-methionine
  • amino acids in particular L- methionine
  • amino acids in particular L-methionine
  • metE gene for one or more genes chosen from the group consisting of
  • the term "attenuation" in this connection describes the reduction or elimination of the intracellular activity of one or more enzymes (proteins) in a microorganism which are coded by the corresponding DNA, for example by using a weak promoter or using a gene or allele which codes for a corresponding enzyme with a low activity or inactivates the corresponding gene or enzyme (protein) , and optionally combining these measures.
  • 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.
  • metE gene it may furthermore be advantageous, for the production of amino acids, in particular L-methionine, to eliminate undesirable side reactions, (Nakayama: “Breeding of Amino Acid Producing Micro-organisms", in: Overproduction of Microbial Products, Krumphanzl, Sikyta, Vanek (eds.), Academic Press, London, UK, 1982) .
  • amino acids in particular L-methionine
  • 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 repeated fed batch 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 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.
  • 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, ⁇ le, 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. M ⁇ ller 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 animal feedstuffs additive according to the invention comprises at least the predominant proportion of the further substances, in particular organic substances, which are formed or added and are present in solution in the fermentation broth, where these have not been separated off by suitable processes.
  • the biomass can be separated off to the extent of up to 70%, preferably up to 80%, preferably up to 90%, preferably up to 95%, and particularly preferably up to 100%.
  • up to 20% of the biomass preferably up to 15%, preferably up to 10%, preferably up to 5%, particularly preferably no biomass is separated off.
  • organic substances include organic by-products which are optionally produced, in addition to the L-methionine, and optionally discharged by the microorganisms employed in the fermentation.
  • L-amino acids chosen from the group consisting of L-lysine, L-valine, L-threonine, L- alanine or L-tryptophan.
  • vitamins chosen from the group consisting of vitamin Bl (thiamine) , vitamin B2 (riboflavin) , vitamin B5 (pantothenic acid), vitamin B6 (pyridoxine) , vitamin B12 (cyanocobalamin) , nicotinic acid/nicotinamide and vitamin E (tocopherol) .
  • organic substances including L-methionine and/or D-methionine and/or the racemic mixture D,L-methionine, can also be added, depending on requirements, as a concentrate or pure substance in solid or liquid form during a suitable process step.
  • organic substances mentioned can be added individually or as mixtures to the resulting or concentrated fermentation broth, or also during the drying or granulation process. It is likewise possible to add an organic substance or a mixture of several organic substances to the fermentation broth and a further organic substance or a further mixture of several organic substances during a later process step, for example granulation.
  • 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.
  • 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
  • auxiliary substances chosen from the group consisting of silicas, silicates, stearates, grits and bran to the substances obtained according to a) to d) for stabilization and to increase the storability; or
  • 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
  • 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) .
  • chromosomal DNA was isolated by the method of Eikmanns et al. (Microbiology 140: 1817 -1828 (1994) ) .
  • metA gene library Accession Number AF052652
  • metE SEQ ID No. 1
  • PCR polymerase chain reaction
  • metA-EVP5 metA-EVP5
  • metA-EVP3 metA-EVP3
  • metE-EVP5 metE-EVP5 :
  • the primers shown were synthesized by MWG-Biotech AG (Ebersberg, 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 Pwo-Polymerase from Roche Diagnostics GmbH (Mannheim, Germany) . With the aid of the polymerase chain reaction, the primers allow amplification of a DNA fragment 1161 bp in size, which carries the metA gene, and a DNA fragment 2286 bp in size, which carries the metE gene.
  • the primer metA-EVP5 contains the sequence for the cleavage site of the restriction endonuclease EcoRI, the primer metA-EVP3 the cleavage site of the restriction endonuclease BamHI, the primer metE-EVP5 the cleavage site of the restriction endonuclease Bglll and the primer metE- EVP3 the cleavage site of the restriction endonuclease Sail, which are marked by underlining in the nucleotide sequence shown above.
  • the metA fragment 1161 bp in size was cleaved with the restriction endonucleases EcoRI and BamHI, and the metE fragment 2286 bp in size was cleaved with the restriction . endonucleases Bglll and Sail.
  • the two batches were separated by gel electrophoresis and the fragments metA (approx. 1150 bp) and metE (approx. 2270 bp) were then isolated from the agarose gel with the QiaExII Gel Extraction Kit (Product No. 20021, Qiagen, Hilden, Germany) .
  • 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 EcoRI and BamHI and then dephosphorylated with shrimp alkaline phosphatase (Roche Diagnostics GmbH, Mannheim, Germany, Product Description SAP, Product No. 1758250) .
  • the metA fragment approx. 1150 bp in size isolated from the agarose gel in example 3.1 and cleaved with the restriction endonucleases BamHI and EcoRI was mixed with the vector pZ8-l prepared in this way 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 ⁇ mcr (Hanahan, In: DNA cloning. A Practical Approach. Vol. I. IRL-Press, Oxford, Washington DC, USA). Selection li ⁇ - tc ! ⁇ ⁇ xi OJ ⁇ > rt o ⁇ D H IT" •XJ ⁇ -3 X ⁇ ⁇ 1-3 o . , rt ⁇ 3 CO ii ⁇ - tc ! ⁇ ) ) 0 ) ⁇ > rt o
  • the vectors pCREmetA and pCREmetAE obtained in example 3.2 and 3.3 were 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 been supplemented with 25 mg/1 kana ycin. Incubation was carried out for 2 days at 33°C.
  • Plasmid DNA was isolated from in each case one transformant by conventional methods (Peters-Wendisch et al., 1998, Microbiology 144, 915-927) and checked by restriction cleavage. The resulting strains were called ATCC13032/pCREmetA and ATCC13032/ ⁇ CREmetAE.
  • the C. glutamicum strains ATCCl3032/pCREmetA and ATCC13032/pCREmetAE obtained in example 3 were cultured in a nutrient medium suitable for the production of methionine and the methionine content in the culture supernatant was determined.
  • the strains were first incubated on an agar plate with the corresponding antibiotic (brain-heart agar with kanamycin (50 mg/1) ) for ' 24 hours at 33°C. Starting from this agar plate culture, in each case a preculture was seeded (10 ml medium in a 100 ml conical flask) . The complete medium Cglll was used as the medium for the precultures.
  • 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 these precultures such that the initial OD (660 nm) of the main cultures was 0.1.
  • Medium MM was used for the main cultures.
  • 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 100 ml conical flasks 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.
  • chromosomal DNA was isolated by the method of Eikmanns et al. (Microbiology 140: 1817 -1828 (1994)). Starting from the nucleotide sequence of the methionine biosynthesis gene metY (DE: 10043334.0) of C. glutamicum ATCC13032, the following oligonucleotides were chosen for the polymerase chain reaction (PCR) (see SEQ ID No. 7 and SEQ ID No. 8) :
  • metY-EVP5 metY-EVP5 :
  • metY-EVP3 metY-EVP3 :
  • the primers shown were synthesized by MWG-Biotech AG (Ebersberg, 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 Pwo-Polymerase from Roche Diagnostics GmbH (Mannheim, Germany) . With the aid of the polymerase chain reaction, the primers allow amplification of a DNA fragment 1341 bp in size, which carries the metY gene.
  • the primer metY-EVP5 contains the sequence for the cleavage site of the restriction endonuclease Sail and the primer metY-EVP3 the cleavage site of the restriction endonuclease Nsil, which are marked by underlining in the nucleotide sequence shown above.
  • the metY fragment 1341 bp in size was cleaved with the restriction endonucleases Sail and Nsil.
  • the batch was separated by gel electrophoresis and the fragment metY (approx. 1330 bp) was then isolated from the agarose gel with the QiaExII Gel Extraction Kit (Product No. 20021, Qiagen, Hilden, Germany) .
  • the plasmid pCREmetA described in example 3.2 was cleaved completely with the restriction enzymes Sail and Pstl and then dephosphorylated with shrimp alkaline phosphatase (Roche Diagnostics GmbH, Mannheim, Germany, Product Description SAP, Product No. 1758250) .
  • the metY fragment approx. 1330 bp in size isolated from the agarose gel in example 5.1 and cleaved with the restriction endonucleases Sail and Nsil was mixed with the vector pCREmetA prepared in this way 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 ⁇ 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, cx Hi li ⁇ - tc •x.
  • DSMZ German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany
  • the vector pCREmetAY obtained in example 5.3 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 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 been 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 (Peters-Wendisch et al., 1998, Microbiology 144, 915-927) and checked by restriction cleavage. The resulting strain was called ATCC13032pCREmetAEY.
  • strain C. glutamicum ATCC13032/pCREmetAEY constructed by the process described in example 4 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-1 was used as the medium for the preculture.
  • 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
  • the medium MM-2 which has the following composition, was used for the main culture:
  • Vitamin B12 (sterile-filtered) 0.02 mg/1
  • OD optical density
  • the biomass was first separated off from a fermentation broth comprising L-methionine prepared by the process of example 6 and comprising about 39 mg/1 L-methionine.
  • a fermentation broth comprising L-methionine prepared by the process of example 6 and comprising about 39 mg/1 L-methionine.
  • 0.5 1 of the above-mentioned fermentation broth was centrifuged with a laboratory centrifuge of the Biofuge- Stratos type from Heraeus (Dusseldorf, Germany) for 20 minutes at 4,000 rpm and the supernatant from the centrifugation was then purified further by cross-flow ultrafiltration with an MRC polymer membrane of 30kD in an ultrafiltrations unit from ICT GmbH (Bad Homburg, Germany) .
  • the biomass was first separated off from a fermentation broth comprising L-methionine prepared by the process as described under example 6 and comprising about 39.0 mg/1 L- methionine.
  • the fermenter contents of the above- mentioned fermentation broth were centrifuged and subjected to ultrafiltration as described in example 7.
  • a portion of the suspension improved in this way was then lyophilized in a freeze-dryer of the type LYOVAC GT 2 from Leybold (Cologne, Germany) .
  • the product comprising L- methionine prepared in this manner had a content of 70 wt.% L-methionine and was free-flowing.
  • the remaining portion of the suspension improved in this way was treated by means of spray drying in a laboratory spray dryer of the B ⁇ chi-190 type from Buchi-Labortechnik GmbH (Constance, Germany) at an intake temperature of 170°C, a starting temperature of 105°C, a pressure difference of -40 mbar and an air flow rate of 600 NL/h.
  • the product comprising L-methionine prepared in this manner had a content of 70 wt.% L-methionine and was free-flowing.
  • the remaining portion of the biomass-containing broth was treated by means of spray drying in a laboratory spray dryer of the B ⁇ chi-190 type from Biichi-Labortechnik GmbH (Constance, Germany) at an intake temperature of 170°C, a starting temperature of 105°C, a pressure difference of - 40 mbar and an air flow rate of 600 NL/h.
  • the product comprising L-methionine prepared in this way had a content of 65 wt.% L-methionine and was free-flowing.
  • Km Resistance gene for kanamycin
  • metE metE gene of C. glutamicum
  • metY metY gene of C. glutamicum
  • metA metA gene of C. glutamicum
  • rrnB-TlT2 Terminator T1T2 of the rrnB gene of E. coli

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EP01967191A 2000-08-02 2001-07-17 Nukleotidsequenzen die für das mete-gen kodieren Ceased EP1307476A1 (de)

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DE10038023 2000-08-02
DE10038023 2000-08-02
DE10109689A DE10109689A1 (de) 2000-08-02 2001-02-28 Neue für das metE-Gen kodierende Nukleotidsequenzen
DE10109689 2001-02-28
PCT/EP2001/008219 WO2002010208A1 (en) 2000-08-02 2001-07-17 Nucleotide sequences which code for the mete gene

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DE10109685A1 (de) * 2000-09-09 2002-04-11 Degussa Neue für das sahH-Gen kodierende Nukleotidsequenzen
KR100651220B1 (ko) 2004-06-29 2006-11-29 씨제이 주식회사 Ｌ-메씨오닌 생산 균주 및 상기 균주를 이용한ｌ-메씨오닌의 생산방법
FR2879075B1 (fr) 2004-12-15 2007-01-19 Adisseo France Sas Soc Par Act Procede de preparation de granules de principe actif hydrophile par extrusion
FR2879074B1 (fr) * 2004-12-15 2007-08-03 Adisseo France Sas Soc Par Act Granules de principe actif hydrophile
EP3856918A1 (de) * 2018-09-28 2021-08-04 Ajinomoto Co., Inc. Verfahren zur herstellung von l-methionin unter verwendung eines bakteriums

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DE3908201A1 (de) * 1989-03-14 1990-09-27 Degussa Verfahren zur fermentativen herstellung von l-lysin
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WO1993017112A1 (en) * 1992-02-20 1993-09-02 Genencor International, Inc. Biosynthesis of methionine using a reduced source of sulfur
DE19644567A1 (de) * 1996-10-26 1998-04-30 Forschungszentrum Juelich Gmbh Mikrobielle Herstellung von Substanzen aus dem aromatischen Stoffwechsel / II
KR20070087035A (ko) * 1999-06-25 2007-08-27 바스프 악티엔게젤샤프트 대사 경로 단백질을 코딩하는 코리네박테리움 글루타미쿰유전자
JP4623825B2 (ja) * 1999-12-16 2011-02-02 協和発酵バイオ株式会社 新規ポリヌクレオチド

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