WO2002018430A2 - Sequences nucleotidiques codant les genes metr et metz - Google Patents

Sequences nucleotidiques codant les genes metr et metz Download PDF

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WO2002018430A2
WO2002018430A2 PCT/EP2001/008221 EP0108221W WO0218430A2 WO 2002018430 A2 WO2002018430 A2 WO 2002018430A2 EP 0108221 W EP0108221 W EP 0108221W WO 0218430 A2 WO0218430 A2 WO 0218430A2
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gene
codes
polynucleotide
methionine
amino acid
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PCT/EP2001/008221
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WO2002018430A8 (fr
WO2002018430A3 (fr
Inventor
Brigitte Bathe
Walter Pfefferle
Klaus Huthmacher
Christian RÜCKERT
Jörn Kalinowski
Alfred Pühler
Michael Binder
Dieter Greissinger
Georg Thierbach
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Degussa Ag
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Priority claimed from DE10109688A external-priority patent/DE10109688A1/de
Application filed by Degussa Ag filed Critical Degussa Ag
Priority to AU8198401A priority Critical patent/AU8198401A/xx
Priority to EP01960503A priority patent/EP1313757A2/fr
Publication of WO2002018430A2 publication Critical patent/WO2002018430A2/fr
Publication of WO2002018430A3 publication Critical patent/WO2002018430A3/fr
Publication of WO2002018430A8 publication Critical patent/WO2002018430A8/fr

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    • 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
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • 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

Definitions

  • the invention provides nucleotide sequences from coryneform bacteria which code for the metR and metZ genes and a process for the fermentative preparation of amino acids, in particular L-methionine, by attenuation of the metR and/or metZ gene.
  • L-Amino acids in particular ethionine, 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 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
  • the composition of the nutrient media such as, for example, the sugar concentration during the fermentation
  • 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 which produce amino acids, such as e.g. L-methionine, are obtained in this manner.
  • Methods of the recombinant DNA technique have also been employed for some years for improving the strain of Corynebacterium strains which produce L-amino acids, 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.
  • 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.
  • the invention provides isolated polynucleotides from coryneform bacteria, which comprise the polynucleotide sequences which code for the metR and/or metZ genes, 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. 3,
  • polynucleotide which is complementary to the polynucleotides of a) , b) c) or d) , and
  • polynucleotide comprising at least 15 successive nucleotides of the polynucleotide sequence of a) , b) , c) , d) or e) ,
  • polypeptides according to a) or c) preferably having the activity of the transcription activator MetR and the polypeptides according to b) or d) preferably having the activity of O-succinylhomoserine sulfhydrylase (MetZ) .
  • the invention also provides the above-mentioned polynucleotides, these preferably being DNAs which are capable of replication, comprising:
  • the invention also provides: a 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 or SEQ ID No. 3,
  • coryneform bacteria in which the metR gene and/or the metZ gene is or are attenuated, in particular by deletion, insertion or base exchange.
  • 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. 1 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 the transcription activator MetR and/or 0- succinylhomoserine sulfhydrylase or to isolate those nucleic acids or polynucleotides or genes which have a high similarity with the sequence of the transcription activator MetR gene and/or that of the O-succinylhomoserine sulfhydrylase 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 the transcription activator MetR and/or O-succinylhomoserine sulfhydrylase 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.
  • 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 according to the invention include the polypeptides according to SEQ ID No. 2 and SEQ ID No. 3, in particular those with the biological activity of the transcription activator MetR and of O-succinylhomoserine sulfhydrylase, 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 polypeptides according to SEQ ID No. 2 and SEQ ID No. 3 and have the activities mentioned.
  • Polypeptides are understood as meaning peptides or proteins which comprise two or more amino acids bonded via peptide bonds.
  • the invention moreover provides a process for the fermentative preparation of amino acids, in particular methionine, using coryneform bacteria which in particular already produce the amino acids, and in which the nucleotide sequences which code for the metR gene and/or for the metZ gene are attenuated, in particular eliminated or expressed at a low level.
  • 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.
  • the microorganisms which the present invention provides can prepare L-amino acids, in particular 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 Corynebacteriu . 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 Corynebacterium glutamicum ATCC13032 Corynebacterium acetoglutamicu ATCC15806 Corynebacterium acetoacidophilum ATCC13870 Corynebacterium melassecola ATCC17965 Corynebacterium thermoaminogenes FERM BP-1539 Brevibacterium flavum ATCC14067 Brevibacterium lactofermentum ATCC13869 and Brevibacterium divaricatum ATCC14020
  • L-amino acid-producing mutants or strains prepared therefrom such as, for example, the L-methionine-producing strain
  • 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.
  • 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)).
  • Coding DNA sequences which result from SEQ ID No. 1 by the degeneracy of the genetic code are also 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. Information in this context can be found by the expert, inter alia, in Ben-Bassat et al.
  • 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.
  • the hybridization takes place under stringent conditions, i.e. only hybrids in which the probe and target sequence, i.e. the polynucleotides treated with the probe, are at least 70% identical are formed. It is known that the stringency of the hybridization, including the washing steps, is influenced or determined by varying the buffer composition, the temperature and the salt concentration. The hybridization reaction is preferably carried out under a relatively low stringency compared with the washing steps (Hybaid Hybridisation Guide, Hybaid Limited, Teddington, UK, 1996) .
  • a 5x SSC buffer at a temperature of approx. 50 - 68°C, for example, can be employed for the hybridization reaction.
  • Probes can also hybridize here with polynucleotides which are less than 70% identical to the sequence of the probe. Such hybrids are less stable and are removed by washing under stringent conditions. This can be achieved, for example, by lowering the salt concentration to 2x SSC and optionally subsequently 0.5x SSC (The DIG System User's Guide for Filter Hybridisation, Boehringer Mannheim, Mannheim, Germany, 1995) a temperature of approx. 50 - 68°C being established. It is optionally possible to lower the salt concentration to 0. Ix SSC.
  • Polynucleotide fragments which are, for example, at least 70% or at least 80% or at least 90% to 95% identical to the sequence of the probe employed can be isolated by increasing the hybridization temperature stepwise from 50 to 68°C in steps of approx. 1 - 2°C. Further instructions on hybridization are obtainable on the market in the form of so-called kits (e.g. DIG Easy Hyb from Roche Diagnostics GmbH, Mannheim, Germany, Catalogue No. 1603558) .
  • kits e.g. DIG Easy Hyb from Roche Diagnostics GmbH, Mannheim, Germany, Catalogue No. 1603558
  • PCR polymerase chain reaction
  • coryneform bacteria produce amino acids, in particular L-methionine, in an improved manner after attenuation of the metR and/or metZ gene.
  • either the expression of the metR and/or of the metZ gene or the catalytic properties of the enzyme proteins can be reduced or eliminated.
  • the two measures can optionally be combined.
  • the reduction in gene expression can take place by suitable culturing or by genetic modification (mutation) of the signal structures of gene expression.
  • Signal structures of gene expression are, for example, repressor genes, activator genes, operators, promoters, attenuators, ribosome binding sites, the start codon and terminators.
  • the expert can find information on this e.g. in the patent application WO 96/15246, in Boyd and Murphy (Journal of Bacteriology 170: 5949 (1988)), in Voskuil and Chambliss (Nucleic Acids Research 26: 3548 (1998), in Jensen and Hammer (Biotechnology and Bioengineering 58: 191 (1998)), in Patek et al.
  • a central part of the coding region of the gene of interest 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)), pKl ⁇ mobsacB or pK19mobsacB (Jager et al., Journal of Bacteriology 174: 5462-65 (1992)), pGEM-T (Promega corporation, Madison, WI, USA), pCR2.1-TOPO (Shuman (1994).
  • the plasmid vector which contains the central part of the coding region of the gene is then transferred into the desired strain of C. glutamicum by conjugation or transformation.
  • the method of conjugation is described, for example, by Schafer et al. (Applied and Environmental Microbiology 60, 756-759 (1994)). Methods for transformation are described, for example, by Thierbach et al.
  • a mutation such as e.g. a deletion, insertion or a base exchange
  • the allele prepared is in turn cloned in a vector which is not replicative for C. glutamicum and this is then transferred into the desired host of C. glutamicum by transformation or conjugation.
  • a first "cross over” event which effects integration
  • a suitable second "cross-over” event which effects excision in the target gene or in the target sequence
  • the incorporation of the mutation or of the allele is achieved.
  • This method was used, for example, by Peters-Wendisch et al. (Microbiology 144, 915 - 927 (1998)) to eliminate the pyc gene of C. glutamicum by a deletion.
  • a deletion, insertion or a base exchange can be incorporated into the metR gene or the metZ gene in this manner.
  • 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.
  • L- methionine in addition to the attenuation of the metR and/or metZ gene, for one or more genes chosen from the group consisting of
  • 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 L-amino acids, in particular L-methionine.
  • batch culture batch culture
  • feed process fed batch
  • repetitive feed process repetition feed process
  • the culture medium to be used must meet the requirements of the particular strains in a suitable manner. Descriptions of culture media for various microorganisms are contained in the handbook “Manual of Methods for General Bacteriology” of the American Society for Bacteriology (Washington D.C., USA, 1981).
  • Sugars and carbohydrates such as e.g. glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose, co ⁇ " O CO CO 3 £U 3 ⁇ CO 3 rt ⁇ ⁇ v TJ ⁇ Hi O ⁇ ⁇ TJ CO ⁇ O H- ⁇ o O Hi o o ⁇ ⁇ 0 ) ⁇ H- ⁇ ⁇ P- O ⁇ ⁇ O l- 1 - " 1-5 0> o H 01 to ⁇ ⁇ H- ⁇ H ⁇ O i-s H- 1 O ⁇ - ⁇ -
  • 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
  • the process according to the invention is used for the fermentative preparation of amino acids, in particular L- methionine.
  • 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 Ba HI (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 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) . This ligation mixture was then electroporated (Tauch et al.
  • the plasmid preparation of the recombinant clones was carried out with 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 Zimmermann et al. (1990, Nucleic Acids Research, 18:1067).
  • the "RR dRhodamin Terminator Cycle Sequencing Kit” from PE Applied Biosystems Product No. 403044, Rothstadt, Germany) was used.
  • the raw sequence data obtained were then processed using the Staden program package (1986, Nucleic Acids Research, 14:217-231) version 97-0.
  • the individual sequences of the pZerol derivatives were assembled to a continuous contig.
  • the computer-assisted coding region analysis was prepared with the XNIP program (Staden, 1986, Nucleic Acids Research, 14:217-231).
  • the resulting nucleotide sequence is shown in SEQ ID No. 1. Analysis of the nucleotide sequence showed two open reading frames of 567 base pairs and 1146 base pairs, which were called the metR gene and metZ gene. The metR gene codes for a protein of 189 amino acids, the metZ gene codes for a protein of 382 amino acids.
  • chromosomal DNA was isolated from the strain ATCC13032 by the method of Tauch et al. (Plasmid 33:168-179 (1995) ) .
  • the oligonucleotides described below were chosen for generation of the metR-metZ deletion allele by means of the polymerase chain reaction (PCR) by the gene Soeing method (Horton, Molecular Biotechnology 3: 93-98 (1995)).
  • orfR 20 see also SEQ ID No. 4 :
  • orfRmetZ del (see also SEQ ID No. 5) :
  • the primers shown were synthesized by MWG Biotech (Ebersberg, Germany) and the PCR reaction was carried out using Pfu polymerase (Stratagene, Product. No. 600135, La Jolla, USA) and a PTC 100 Thermocycler (MJ Research Inc., Waltham, USA) .
  • the primers orfR 20 and metZ 21 contain in each case an inserted cleavage site for the restriction enzyme EcoRI, which are marked by underlining in the nucleotide sequence shown above.
  • the primer orfRmetZ del is composed of two regions of the nucleotide sequence, one' of which bonds in the "upstream” region of metR and includes the first two nucleotides of the start codon ATG, and the other bonds after the stop codon of metZ in the "downstream” region thereof.
  • amplification product 402 bp in size contains the "upstream" region of the met R gene including the first two nucleotides of the start codon ATG, and additionally a 20 bp extension, appended with the oligonucleotide orfRmetZ del, which corresponds to a part of the nucleotide sequence of the "downstream" region of metZ.
  • the amplification product was isolated from the agarose gel with the QiaExII Gel Extraction Kit (Product No. 20021, Qiagen, Hilden, Germany) . It was called metRmetZ del fragment 1 and is shown in SEQ ID No. 7.
  • metRmetZ deletion derivative 982 bp in size was produced with the primer metZ 21 and the purified amplification product metRmetZ del fragment 1, which can bond by means of the 20 bp extension from the oligonucleotide orfRmetZ del in the "downstream" region of metZ to the DNA and function there as a primer. It comprises, between the two EcoRI cleavage sites appended to the primers, 368 bp of the "upstream region" of the metR gene, the first two nucleotides of the start codon ATG and 588 bp of the "downstream" region of the metZ gene, starting with the fourth nucleotide after the stop codon of the metZ gene.
  • metRmetZ del fragment 2 It was called metRmetZ del fragment 2 and is shown in SEQ ID No. 8.
  • the 982 bp metRmetZ deletion derivative obtained in example 3 was incorporated by means of deletion mutagenesis with the aid of the sacB system described by Schafer et al., Gene, 14, 69-73 (1994) into the chromosome of C. glutamicum. This system enables the expert to identify or select allele exchanges which take place by homologous recombination. ... . . .
  • the metRmetZ deletion derivative 982 bp in size obtained in example 3 was cleaved with the restriction endonuclease EcoRI and then isolated from the agarose gel with the QiaExII Gel Extraction Kit (Product No. 20021, Qiagen, Hilden, Germany) and used for ligation with the mobilizable cloning vector pKl ⁇ mobsacB described by Schafer et al., Gene, 14, 69-73 (1994) . This was cleaved beforehand with the restriction enzyme EcoRI, subsequently dephosphorylated with shrimp, alkaline phosphatase (Roche Diagnostics GmbH, Mannheim, Germany, Product Description SAP, Product No.
  • plasmid-carrying cells were made by plating out the transformation batch on LB agar (Sambrock et al., Molecular Cloning: A Laboratory Manual, 2 nd Ed., Cold Spring Harbor, New York, 1989), which had been supplemented with 25 mg/1 kanamycin.
  • Plasmid DNA was isolated from a transformant with the aid of the QIAprep Spin Miniprep Kit from Qiagen and the cloned lrp deletion allele was verified by means of sequencing by MWG Biotech (Ebersberg, Germany) .
  • the plasmid was called pK18mobsacBdeltametRmetZ and is shown in figure 1.
  • the vector pKl ⁇ mobsacBdeltametRmetZ mentioned in example 4 was electroporated by the electroporation method of Tauch et al.,(1989 FEMS Microbiology Letters 123: 343-347) in the strain C. glutamicum ATCC13032.
  • the vector cannot replicate independently in ATCC13032 and is retained in the cell only if it has integrated into the chromosome.
  • the plasmid pKl ⁇ mobsacB contains a copy of the sacB gene, which converts sucrose into levan sucrase, which is toxic to C. glutamicum. Only those clones in which the pKl ⁇ mobsacBdeltametRmetZ integrated has been excised again therefore grow on LB agar with sucrose. In the excision, together with the plasmid either the complete chromosomal copy of the metR and metZ genes can be excised, or the metRmetZ deletion derivative.
  • the plasmid pKl ⁇ mobsacBdeltametRmetZ was marked by the method of "The DIG System Users Guide for Filter Hybridization" of Boehringer Mannheim GmbH (Mannheim, Germany, 1993) using the DIG hybridization kit from Boehringer. Chromosomal DNA of a potential deletion mutant was isolated by the method of Eikmanns et al. (Microbiology 140: 1817-1628 (1994)) and in each case cleaved with the restriction enzymes Hindlll and EcoRI in separate batches-.
  • the fragments formed were separated by agarose gel electrophoresis and hybridized at 68°C with the Dig hybridization kit from Boehringer. With the aid of the fragments formed, it could be shown that the strain ATCC13032 has lost its copies of the metR and metZ genes, and instead the region from the 3rd nucleotide of the metR gene up to and including the 3rd nucleotide after the metZ gene is deleted.
  • the strain was called C. glutamicum ATCC13032deltametRmetZ.
  • the C. glutamicum strain ATCCl3032deltametRmetZ obtained 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 a brain-heart agar plate for 24 hours at 33°C. Starting from this agar plate culture, a preculture was seeded (10 ml medium in a 100 ml conical flask) . The medium MM 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.
  • 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. 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.
  • Figure 1 Map of the plasmid pKl ⁇ mobsacBdeltametRmetZ.

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Abstract

L'invention concerne des polynucléotides issus de bactéries corynéformes codant les gènes mdhR et mdhZ et renfermant des séquences polynucléotidiques choisis dans le groupe comprenant: a) un polynucléotide identique au moins à 70 % à un polynucléotide codant un polypeptide contenant la séquence d'acides aminés SEQ ID No. 2; b) un polynucléotide identique au moins à 70 % à un polynucléotide codant un polypeptide contenant la séquence d'acides aminés SEQ ID No. 3; c) un polynucléotide codant un polypeptide contenant une séquence d'acides aminés identique à au moins 70 % à la séquence d'acides aminés SEQ ID No 2; d) un polynucléotide codant un polypeptide contenant une séquence d'acides aminés identique au moins à 70 % à la séquence d'acides aminés SEQ ID No 3; e) un polynucléotide complémentaire des polynucléotides susmentionnés aux points a), b), c) ou d); et un polynucléotide comprenant au moins 15 nucléotides successifs des séquences polynucléotidiques décrites aux points a), b), c), d) ou e). L'invention concerne également un procédé de préparation par fermentation d'acides aminés L à l'aide de bactéries corynéformes dans lesquelles au moins les gènes metR et/ou metZ sont présents sous forme atténuée; ainsi que l'utilisation des polynucléotides comprenant les séquences selon l'invention comme sondes d'hybridation.
PCT/EP2001/008221 2000-09-02 2001-07-17 Sequences nucleotidiques codant les genes metr et metz WO2002018430A2 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001000842A2 (fr) * 1999-06-25 2001-01-04 Basf Aktiengesellschaft Genes de corynebacterium glutamicum codant des proteines impliquees dans l'homeostase et adaptation
WO2001000843A2 (fr) * 1999-06-25 2001-01-04 Basf Aktiengesellschaft Genes de corynebacterum glutamicum codant pour des proteines de voie metabolique
EP1108790A2 (fr) * 1999-12-16 2001-06-20 Kyowa Hakko Kogyo Co., Ltd. Nouveaux polynuclétides

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001000842A2 (fr) * 1999-06-25 2001-01-04 Basf Aktiengesellschaft Genes de corynebacterium glutamicum codant des proteines impliquees dans l'homeostase et adaptation
WO2001000843A2 (fr) * 1999-06-25 2001-01-04 Basf Aktiengesellschaft Genes de corynebacterum glutamicum codant pour des proteines de voie metabolique
EP1108790A2 (fr) * 1999-12-16 2001-06-20 Kyowa Hakko Kogyo Co., Ltd. Nouveaux polynuclétides

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Title
DATABASE EBI [Online] EMBL; 24 January 2001 (2001-01-24) POMPEJUS M. ET AL.: "Corynebacterium glutamicum genes encoding proteins involved in homeostasis and adaption" Database accession no. Ac: AX065835 XP002185890 *
DATABASE EMBL [Online] EBI; 24 January 2001 (2001-01-24) POMPEJUS M. ET AL.: "Corynebacterium glutamicum genes encoding proteins involved in homeostasis and adaption" Database accession no. Ac:AX063913 XP002203339 *
FOGLINO M ET AL: "A DIRECT SULFHYDRYLATION PATHWAY IS USED FOR METHIONINE BIOSYNTHESIS IN PSEUDOMONAS AERUGINOSA" MICROBIOLOGY, SOCIETY FOR GENERAL MICROBIOLOGY, READING, GB, vol. 141, no. 2, 1 February 1995 (1995-02-01), pages 431-439, XP002054742 ISSN: 1350-0872 *
FRITSCH PAULA S ET AL: "Role of the RNA polymerase alpha subunits in MetR-dependent activation of metE and metH: Important residues in the C-terminal domain and orientation requirements within RNA polymerase." JOURNAL OF BACTERIOLOGY, vol. 182, no. 19, October 2000 (2000-10), pages 5539-5550, XP002185889 ISSN: 0021-9193 *
HWANG B-J ET AL.: "ANALYSIS OF CORYNEBACTERIUM GLUTAMICUM METHIONINE BIOSYNTHETIC PATHWAY: ISOLATION AND ANALSYSIS OF metB ENCODING CYSTATHIONINE gamma-SYNTHASE" MOL. CELLS, vol. 9, no. 3, 3 February 1999 (1999-02-03), pages 300-308, XP001037229 *
LORENZ EVA ET AL: "MetR-mediated repression of the glyA gene in Escherichia coli." FEMS MICROBIOLOGY LETTERS, vol. 144, no. 2-3, 1996, pages 229-233, XP001038523 ISSN: 0378-1097 *
PARK S-D ET AL: "ISOLATION AND ANALYSIS OF META, A METHIONINE BIOSYNTHETIC GENE ENCODING HOMOSERINE ACETYLTRANSFERASE IN CORYNEBACTERIUM GLUTAMICUM" MOLECULAR AND CELLS, KOREAN SOCIETY FOR MOLECULAR SOCIETY, KR, vol. 8, no. 3, 30 June 1998 (1998-06-30), pages 286-294, XP001002218 *
TATE R ET AL: "THE RHIZOBIUM ETLI METZ GENE IS ESSENTIAL FOR METHIONINE BIOSYNTHESIS AND NODULATION OF PHASEOLUS VULGARIS" MOLECULAR PLANT-MICROBE INTERACTIONS, APS PRESS, ST. PAUL, MN, US, vol. 12, no. 1, January 1999 (1999-01), pages 24-34, XP001038478 ISSN: 0894-0282 *

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