WO2004113550A1 - Process for producing l-glutamic acid - Google Patents

Process for producing l-glutamic acid Download PDF

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Publication number
WO2004113550A1
WO2004113550A1 PCT/JP2004/008807 JP2004008807W WO2004113550A1 WO 2004113550 A1 WO2004113550 A1 WO 2004113550A1 JP 2004008807 W JP2004008807 W JP 2004008807W WO 2004113550 A1 WO2004113550 A1 WO 2004113550A1
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Prior art keywords
protein
seq
amino acid
glutamic acid
activity
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PCT/JP2004/008807
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French (fr)
Japanese (ja)
Inventor
Gen Nonaka
Takako Saitou
Yoko Kuwabara
Kazuhiko Matsui
Shinichi Sugimoto
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Ajinomoto Co., Inc.
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Publication of WO2004113550A1 publication Critical patent/WO2004113550A1/en

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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
    • C12P13/14—Glutamic acid; Glutamine

Definitions

  • the present invention relates to the fermentation industry, and more particularly, to a method for producing L-gnoretamic acid and a bacterium used for the method.
  • L-glutamic acid is widely used as a seasoning raw material and the like.
  • L-glutamic acid has been industrially produced by a fermentation method using a coryneform bacterium belonging to the genus Brevibataterum, which has the ability to produce L-gnoretamic acid.
  • a coryneform bacterium belonging to the genus Brevibataterum which has the ability to produce L-gnoretamic acid.
  • a strain isolated from the natural world or an artificial mutant thereof is used in order to improve the productivity.
  • An object of the present invention is to provide a novel technique for improving L-glutamic acid productivity in the production of L-glutamic acid using a coryneform bacterium.
  • the inventors of the present invention conducted a process of conducting research on genes involved in acid resistance of coryneform bacteria. Thus, they found that by increasing the activity of a gene product of unknown function, which was named ORF1554, the ability of coryneform bacteria to produce L-gnoretamic acid could be improved, leading to the completion of the present invention. Was.
  • the present invention is as follows.
  • a method for producing L-gnoretamic acid comprising culturing a coryneform bacterium capable of producing L-gnoretamic acid in a medium, producing and accumulating L-glutamic acid in the culture, and collecting L-glutamic acid from the culture. 3. The method according to claim 1, wherein the bacterium is modified so that the activity of the protein shown in the following (A) or (B) is increased.
  • the bacterium increases the copy number of a gene encoding the protein shown in (A) or (B), or encodes the protein shown in (A) or (B) in the bacterial cell.
  • the method according to (1) or (2), wherein the activity of the protein in the cell is increased by modifying the expression control sequence of the gene so that the expression of the gene is enhanced.
  • a coryneform bacterium which has an ability to produce L-gnoretamic acid and has been modified so that the activity of the protein shown in the following (A) or (B) is increased.
  • A a protein having the amino acid sequence of SEQ ID NO: 2;
  • B the amino acid sequence of SEQ ID NO: 2, comprising an amino acid sequence containing one or several amino acid substitutions, deletions, insertions, or additions, and an activity of improving the ability of a coryneform bacterium to produce L-gnoretamic acid;
  • FIG. 1 is a diagram showing the growth and L-glutamic acid yield of a Brevibataterium 'ratatofamentum sucA-deficient strain in which RF1554 and ORF1554 * were amplified.
  • the coryneform bacterium of the present invention is a coryneform bacterium which has an ability to produce L-glutamic acid and has been modified so that the activity of the protein shown in the following (A) or (B) is increased.
  • the protein of (A) or (B) may be referred to as “ ⁇ RF1554 product”, and the DNA encoding the protein may be referred to as “ ⁇ RF1554”.
  • Karoe the ORF1554 including an expression control sequence up port such as a motor which is adjacent to the ⁇ _RF sometimes for convenience called a "0 RF1554".
  • the "coryneform bacterium” also includes a bacterium which has been conventionally classified into the genus Brevibataterium, and which is now classified into the genus Corynebateterium (Int. J. Syst. Bacteriol., 41, 255 (1981)), and also includes bacteria of the genus Brevibataterirum, which is very closely related to Corynebacterium. Examples of such coryneform bacteria include the following.
  • L_glutamic acid-producing ability refers to the ability to accumulate L-gnoretamic acid in a medium when the coryneform bacterium of the present invention is cultured. This L-glutamic acid-producing ability may be a property of a wild type coryneform bacterium or a property imparted or enhanced by breeding.
  • coryneform bacterium having the ability to produce L-glutamic acid include the following strains.
  • Brevibataterum 'Flavum AJ11573 See Japanese Unexamined Patent Publication No. 56-151495 Brevibataterum' Flavam AJ12210 (FERM P-8123) Japanese Patent Application Laid-Open No.
  • Modified to increase the activity of an ORF1554 product in a cell means that the activity per cell is higher than that of a non-modified strain, for example, a wild-type coryneform bacterium.
  • a non-modified strain for example, a wild-type coryneform bacterium.
  • the wild-type coryneform bacterium to be compared is, for example, Brevibataterium 'ratatophamentum ATCC13869.
  • the “activity for improving the ability of coryneform bacterium to produce L-gnoretamic acid” refers to the activity of such an ORF1554 product. Specifically, when a strain of a coryneform bacterium that has been modified to overexpress the ORF1554 product in excess of the wild-type or non-modified strain is cultured in a culture medium, L-gnoretamine is higher than the wild-type or non-modified strain. If the amount of acid accumulated in the medium is high or the production rate of L-glutamic acid is high, it can be said that the modified strain has improved L-glutamic acid-producing ability.
  • Enhancement of the activity of the ORF1554 product in coryneform bacterium cells is achieved by enhancing the expression of ORF1554. Enhancement of the expression level of the gene can be achieved by increasing the copy number of ORF1554.
  • the ORF1554 fragment is ligated to a vector that functions in the bacterium, preferably a multicopy vector, to produce a recombinant DNA, which is then introduced into a host capable of producing L-glutamic acid to transform the DNA. do it.
  • a transformant may be obtained by introducing the recombinant DNA into a wild-type coryneform bacterium, and then imparting L-gnoretamic acid-producing ability to the transformant.
  • any of genes derived from coryneform bacteria and genes derived from other organisms such as bacteria belonging to the genus Escherichia can be used.
  • ORF1554 of Brevibataterum 'ratatophamentum' has been clarified according to the present invention (SEQ ID NO: 1), and a gene putatively homologous to ORF1554 of Corynebacterium glutamicum also includes Since the sequence has already been determined (DDBJ / EMBL / GenBank accession # AP005277-302), a coryneform bacterium was prepared using primers prepared based on those nucleotide sequences, for example, the primers shown in SEQ ID NOS: 9 and 10. P method using chromosomal DNA of type III (PCR: polymerase chain reaction; et al, Trends Genet. 5, 185 (1989)) to obtain ORF1554 and its adjacent regions. Homologs of ORF1554 of other microorganisms can be obtained in a similar manner.
  • Chromosomal DNA can be obtained from a DNA donor bacterium by, for example, the method of Saito and Miura (H.
  • the ORF1554 amplified by the PCR method is connected to a vector DNA capable of autonomous replication in cells of Escherichia coli and / or coryneform bacteria to prepare a recombinant DNA, which is introduced into Escherichia coli. If you keep it, later operations will be slow.
  • Vectors capable of autonomous replication in Escherichia coli cells include pUC19, pUC18, pHSG299,
  • a vector that functions in a coryneform bacterium is, for example, a plasmid that can autonomously replicate in a coryneform bacterium. Specific examples include the following.
  • a DNA fragment having the ability to autonomously replicate a plasmid in a coryneform bacterium is taken out, and inserted into the above-mentioned vector for Escherichia coli.
  • shuttle vectors examples include the following.
  • the microorganisms holding each solid and the accession number of the international depositary organization are shown in parentheses.
  • PAJ655 Escherichia Cori AJ11882 (FERM BP-136) Coryneha, Cterium 'Dartamicum SR8201 (ATCC39135)
  • These vectors are obtained from the deposited microorganism as follows. The cells collected during the logarithmic growth phase were lysed using lysozyme and SDS, centrifuged at 30,000 X g, and the lysate was obtained. The supernatant obtained was added with polyethylene glycol, and the cesium chloride-etidium bromide equilibrium was added. Separate and purify by density gradient centrifugation.
  • the recombinant DNA prepared as described above may be introduced into coryneform bacteria according to a transformation method that has been reported so far. For example, as described in Escherichia coli K-12, a method of increasing the permeability of DNA by treating recipient cells with calcium chloride (Mandel, M. and Higa'AJ Mol. Biol., 53, 159 (1970)), and a method for preparing DNA and introducing competent cells from cells in the growth stage (Duncan, CH, Wilson, GA and Young, FE, Gene, 1, 153 (1977)).
  • the recombinant DNA can be transformed into protoplasts or sperm plasts that readily incorporate the recombinant DNA, as is known for Bacillus subtilis, actinomycetes and yeast. (Uhang, S. and uhoen, SN, Molec. Uen. Enet., 168, 1 ⁇ ⁇
  • coryneform bacteria can be transformed by the electric pulse method (Japanese Patent Application Laid-Open No. 2-207791).
  • ORF1554 can also be achieved by causing ORF1554 to exist in multiple copies on the chromosomal DNA of a coryneform bacterium. On the chromosomal DNA of coryneform bacteria
  • RF1554 in multiple copies, homologous recombination is performed using a sequence present in multiple copies on chromosomal DNA.
  • a sequence present in multiple copies on chromosomal DNA repetitive DNA and inverted 'repeat existing at the end of a transposable element can be used.
  • ORF1554 can be mounted on a transposon, transferred, and introduced into multiple copies on chromosomal DNA.
  • enhancement of the ORF1554 product activity can also be achieved by replacing an expression regulatory sequence such as the promoter of ORF1554 on chromosomal DNA or a plasmid with a strong one.
  • an expression regulatory sequence such as the promoter of ORF1554 on chromosomal DNA or a plasmid
  • the lac promoter, the t ⁇ promoter, the trc promoter and the like are known as strong promoters.
  • These alterations in the expression control sequence may be combined with increasing the copy number of ORF1554.
  • the replacement of the expression control sequence can be performed, for example, in the same manner as the gene replacement using a temperature-sensitive plasmid.
  • temperature-sensitive plasmids of coryneform acid bacteria include p48K and pSFKT2 (see JP-A-2000-262288), pHSC4 (see French Patent Publication No. 196676767, and JP-A-5-7491). Can be
  • the ORF1554 used in the present invention has one or several amino acid substitutions, deletions, insertions, or additions at one or more positions, as long as the ORF1554 product activity of the encoded protein is not impaired. Including those encoding the ORF1554 product.
  • the term “several” differs depending on the position and type of the amino acid residue in the three-dimensional structure of the protein, but specifically 2 to 30, preferably 2 to 20, and more preferably 2 to 10 Individual.
  • the mutation of the ORF1554 product described above is a conservative mutation that maintains the activity of the ORF1554 product. Substitutions are changes in which at least one residue in the amino acid sequence has been removed and another residue inserted therein.
  • Amino acids that replace the original amino acids of the ORF1554 product and are considered conservative substitutions include Ala to ser or thr substitutions, arg to gln, his or to: asn force, et al. Glu, gln, lys , His or asp, asp power, asn, glu or gin, cys power, ser or fala, gin power, asn, glu, lys, his, asp or larg Replacement of glu force with gly, asn, gln, lys or iasp, replacement of giy with pro, his force, replacement of asn, lys, gln, arg or tyr, replacement of ile force with leu, met, val or substitution with phe, substitution with leu force, ile, met, val or phe, substitution of lys force with asn, glu, gln, his or arg, substitution of met force with ile, leu,
  • substitution of t ⁇ , tyr, met, ile or leu substitution of ser for thr or ala, substitution of thr for ser or ala, substitution of t ⁇ for phe or tyr, tyr force, etc. his, phe or trp And val to met, ile or leu.
  • ORF1554 product having the amino acid substitution as described above include, for example, an ORF1554 product having an amino acid sequence in which the gnoletamic acid residue at position 81 of SEQ ID NO: 2 is substituted with a glycine residue. Is mentioned.
  • the DNA encoding the protein substantially the same as the ORF1554 product as described above may be obtained by substituting, deleting, inserting, attaching, or removing amino acid residues at a specific site by, for example, site-directed mutagenesis. It can be obtained by modifying the nucleotide sequence of ORF1554 so as to include an inversion.
  • the modified DNA as described above can also be obtained by a conventionally known mutation treatment. Examples of the mutation treatment include a method in which DNA before the mutation treatment is treated in vitro with hydroxylamine or the like; — A method of treating with a mutagen commonly used in mutagenesis treatment such as nitrosoguanidine (NTG) or nitrous acid.
  • DNA having the above mutation is expressed in a suitable cell, and the activity of the expression product is examined, whereby a DNA encoding a protein substantially identical to the ORF1554 product can be obtained.
  • DNA encoding the ORF1554 product having a mutation or a cell carrying the same can be obtained, for example, from the nucleotide sequence of nucleotides 749 to 1414 in the nucleotide sequence of SEQ ID NO: 1 in the sequence listing.
  • a DNA encoding a protein having an ORF1554 product activity that hybridizes with a probe having the base sequence or a part thereof under stringent conditions is obtained.
  • stringent conditions refers to conditions under which a so-called specific hybrid is formed and a non-specific hybrid is not formed. Although it is difficult to quantify these conditions clearly, as an example, DNAs with high homology, for example, 50% or more, preferably 70% or more, more preferably 80% or more, and particularly preferably 90% or more . / o or more, and most preferably 95% or more DNAs having homology are hybridized with each other, and DNA having lower homology is not hybridized with DNA, or under conditions of washing of ordinary Southern hybridizations. There are 60. Conditions for hybridization at a salt concentration corresponding to C, 1 ⁇ SSC, 0.1% SDS, preferably 0.1 ⁇ SSC, 0.1% SDS.
  • a partial sequence of the nucleotide sequence of SEQ ID NO: 1 can also be used as a probe.
  • a probe can be prepared by PCR using an oligonucleotide prepared based on the nucleotide sequence of SEQ ID NO: 1 as a primer and a DNA fragment containing the nucleotide sequence of SEQ ID NO: 1 as a type II.
  • the conditions for washing the hybridization include 50 ° C., 2 ⁇ SSC, and 0.1% SDS.
  • the DNA encoding the protein substantially identical to the ORF1554 product may be, for example, an amino acid sequence represented by SEQ ID NO: 2, preferably 50% or more, more preferably 70% or more, and even more preferably 80% or more. %, Particularly preferably 90% or more, most preferably 95% or more, and DNA encoding a protein having ORF1554 product activity.
  • coryneform bacterium of the present invention has been modified so that the activity of the ORF1554 product is increased, and further, the activity of an enzyme that catalyzes L-glutamic acid biosynthesis is enhanced. ,.
  • Examples of enzymes that catalyze L-glutamic acid biosynthesis include gnoretamic acid dehydrogenase, gnoretamine synthetase, glutamate synthase, isoquenate dehydrogenase, aconitate hydratase, citrate synthase, phosphoenolpyruvate carboxylase, phosphoenolpyruvate synthase, enolase, and the like.
  • Phosphoglyceromutase Phosphoglyceromutase, phosphoglycerate kinase, glyceraldehyde_3_phosphate dehydrogenase, triosephosphate isomerase, fructosebisphosphate aldolase, phosphofructokinase, glucose Phosphate isomerase and the like.
  • an enzyme that catalyzes a reaction that diverges from the L-glutamic acid biosynthetic pathway to produce a compound other than L-glutamic acid is reduced or lost.
  • Enzymes that catalyze the reaction that diverges from the biosynthetic pathway of L-daltamate to produce compounds other than L-glutamic acid include polyketoglutarate dehydrogenase (KKGDH), isocitrate lyase, and acetyl phosphate transferase.
  • Acetic acid kinase acetohydroxyacid synthase, acetolactate synthase, acetyl formate transferase, lactate dehydrogenase, glutamate decarboxylase, 1-pyrroline dehydrogenase, and the like.
  • KGDH is preferred.
  • the KGDH is encoded by the sucA gene.
  • Coryneform bacteria in which the sucA gene has been disrupted are described in detail in W095 / 34672 International Publication Pamphlet and JP-A-7-834672.
  • the sucA gene-disrupted strain may be worse than that of the parent strain, it is preferable to select a strain that has good growth using an appropriate medium.
  • the medium include a CM2B plate (10 g / L polypeptone, 10 g / L yeast extratato, 5 g / L NaCl, 10 / ig / L biotin, 20 g / L agar, pH 7.0).
  • the coryneform bacterium obtained as described above is cultured in a medium, L-glutamic acid is produced and accumulated in the medium, and L-gnoretamic acid is collected from the medium to efficiently produce L-gnoretamic acid. be able to.
  • a conventional medium containing a carbon source, a nitrogen source, inorganic salts, and, if necessary, organic trace nutrients such as amino acids and vitamins is used.
  • Either a synthetic medium or a natural medium can be used.
  • the carbon source and nitrogen source used in the medium any type may be used as long as the strain to be cultured can be used.
  • the carbon source sugars such as glucose, glycerol, fructose, sucrose, maltose, mannose, galactose, starch hydrolyzate and molasses are used.
  • organic acids such as acetic acid and citric acid, ethanol and the like are used. Alcohols may be used alone or in combination with other carbon sources. Used for
  • ammonia ammonium salts such as ammonium sulfate, ammonium carbonate, ammonium chloride, ammonium phosphate, and ammonium acetate, or nitrates are used.
  • Examples of the organic trace nutrients include amino acids, vitamins, fatty acids, nucleic acids, and peptones, casamino acids, yeast extracts, and soybean protein decomposed products containing these, and nutrients that require amino acids for growth. When an auxotrophic mutant is used, it is preferable to supplement the required nutrients.
  • inorganic salts phosphates, magnesium salts, calcium salts, iron salts, manganese salts and the like are used.
  • aeration culture is performed while controlling the fermentation temperature to 20 45 ° C and the pH to 59. If the pH drops during cultivation, neutralize with an alkali such as ammonia gas for adding calcium carbonate. By vigorously culturing for 10 hours to 120 hours, a significant amount of L-glutamic acid is accumulated in the culture solution.
  • an alkali such as ammonia gas for adding calcium carbonate.
  • the method of collecting L-glutamic acid from the culture solution after completion of the culture may be performed according to a known recovery method. For example, it is collected by removing the cells from the culture solution and then concentrating and crystallization.
  • the wild strain ATCC13869 strain of Brevibata terium 'ratatophamentum' was mutated with the following mutations using the mutation agents N-methyl_N and nitro-N-nitrosoguanidine (NTG).
  • N-methyl_N and nitro-N-nitrosoguanidine NTG.
  • the cells were shake-cultured until (OD) was about 0.7. After culturing the cells, use 50 mM phosphate buffer
  • the screening method is as follows.
  • the continuously cultured cells were cultured in MM / MES medium (pH: 5.7 (adjusted with KOH)) (20 g / L glucose, 10 g / L (NH) SO, lg / L KHPO, 0.4 g / L MgSO-7H0,
  • Chromosomal DNA was prepared from each of the strains of Brevibataterium 'ratatophamentum 16-1, 16-20, and 15-11 obtained as described above, and partially degraded with the restriction enzyme Sau3AI. A 6 kbp DNA fragment was purified. These DNA fragments were introduced into the BamHI site of a plasmid vector (pSFK6) capable of autonomous replication in both Escherichia coli and Corynebacterium bacteria. Genomic libraries with about 14,000 clones for the 16-1 strain, about 7,000 clones for the 16-20 strain, and about 14,000 clones for the 15-11 strain were obtained.
  • pSFK6 plasmid vector
  • the pSFK6 was obtained from a vector for Escherichia coli pHSG399 (see S. Takeshita et al: Gene 61, 63-74 (1987), which can be purchased from Takara Shuzo Co., Ltd.) and the force of Streptococcus fucaris namycin resistance gene.
  • This is a plasmid constructed from the prepared plasmid pKl and the plasmid pAM330 (see US Pat. No. 4,427,773, and Japanese Patent Publication No. 58-67699) extracted from Brevibatadium 'ratatofamentum ATCC13869 (Japanese Patent Laid-Open No. 2000-2000). -262288, U.S. Patent No. 6,303,383).
  • the genomic libraries of each of the 16-1, 16-20, and 15-11 strains were introduced into Brevibataterium ratatofamentum ATCC13869.
  • the plasmid was introduced by the electric pulse method (Japanese Patent Laid-Open No. 2-207791).
  • the transformant was plated on an MM / MES plate at pH 5.7, and after culturing at 31.5 ° C. for 7 to 9 days, a clone having formed a colony was obtained.
  • Hundreds of thousands of clones were searched for each mutant genomic library.
  • Brevibataterium lactofermentum ATCC13869 is not capable of forming colonies on MM / MES plates with ⁇ 5.7 or less. it is conceivable that.
  • Four such clone strengths (clone # D5, clone # F1, clone # F2, clone # H87) were obtained.
  • a part of the base sequence of the genomic DNA fragment on the plasmid of each of the clones # D5, # F1, # F2, and # H87 is shown in SEQ ID NOS: 1, 3, 5, and 7, respectively.
  • Each of these sequences contains an open reading frame (ORF) (ORF1554: nucleotides 749 to 1414 of SEQ ID NO: 1, ORF1249: nucleotides 250 to 2748 of SEQ ID NO: 3, ORF39: nucleotides 55 to 1212 of SEQ ID NO: 5).
  • ORF1059 base number 595 1644 of sequence 7).
  • Each ORF is The amino acid sequences to be assigned are shown in SEQ ID NOs: 2, 4, 6, and 8.
  • nucleotide numbers 1449 to 1455 of SEQ ID NO: 1, nucleotide numbers 3317 to 3326 of SEQ ID NO: 3, and nucleotide numbers 17 to 17 of SEQ ID NO: 5 are sequences derived from PHSG399.
  • a DNA fragment having the nucleotide sequence shown in SEQ ID NO: 1 was transformed into a plasmid vector pSAC4 capable of autonomous replication in both Escherichia coli and Corynebacterium bacteria.
  • the plasmid pD5_2A was prepared.
  • pSAC4 is obtained by digesting plasmid pHM1519 (Miwa, k. Et al., Agric. Biol. Chem., 48 (1984) 2901-2903) that can autonomously replicate in coryneform bacteria with restriction enzymes BamHI and Kpnl.
  • the replication origin Japanese Patent Application Laid-Open No. 5-7491
  • a DNA fragment having the nucleotide sequence shown in SEQ ID NO: 2 (having BglII at the 3 'end and a recognition sequence for Kpnl at the 5' end) was ligated to the BamHI and Kpnl sites of pSAC4, and the plasmid pFl_lB was prepared.
  • a DNA fragment having the nucleotide sequence shown in SEQ ID NO: 3 was inserted into the Xbal site of pSAC4 to prepare a plasmid pF2_2A.
  • a DNA fragment having the nucleotide sequence shown in SEQ ID NO: 4 was inserted into the Smal site of pSAC4 to prepare a plasmid pH87_4A.
  • the above four types of plasmids were each introduced into Brevibataterium 'ratatophamentum ATCC13869. Each transformant was cultured at 31.5 ° C for 5 days on MM / MES plates adjusted to acidic pH, and colony formation was examined. Table 1 shows the results.
  • the primers shown in SEQ ID NOs: 9 and 10 were designed, and a region containing about 750 bp upstream and about 30 bp downstream of the wild-type ORF1554 from Brevibatatellium 'ratatophamentum ATCC13869 was subjected to PCR.
  • pyrobest DNA polymerase (Takara Shuzo Co., Ltd.) was used. After 94 ° C for 5 minutes, 30 cycles of 98 ° C for 5 seconds, 65 ° C for 10 seconds, and 72 ° C for 60 seconds were performed.
  • the obtained 1.5 kb PCR product is cut at the Xbal site designed on both primers, inserted into the Xbal site of the plasmid vector pSAC4, and pD5WT-1 is Produced.
  • L30-2 / pD5_2A strain was produced.
  • the plasmid was introduced by the electric pulse method (Japanese Patent Application Laid-Open No. 2-207791).
  • the L30-2 strain was obtained from a CM2B plate (10 g / L polypeptone, 10 g / L yeast extra) from the sucA gene-deficient strain ⁇ S (B095 / 34672 international publication pamphlet) of Brevibataterium 'ratatophamentum ATCC13869. Tato, 5 g / L NaCl, 10 ⁇ g / L biotin, 20 g / L agar, pH 7.0).
  • FIG. 1 shows the yield of L-glutamic acid and the final OD (620 nm, measured by diluting the culture solution 51-fold) with respect to the consumed glucose. It was found that amplification of ORF1554 and ORF1554 * both increased the glutamic acid yield of Brevibataterium 'ratatofamentum by about 4%.
  • the L-glutamic acid-producing ability of Brevibataterium 'ratatofamentum can be improved.

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Abstract

A process for producing L-glutamic acid, comprising culturing coryneform bacteria capable of L-glutamic acid production in a culture medium so as to attain formation and accumulation of L-glutamic acid in a culturing product and collecting L-glutamic acid from the culturing product, wherein use is made of coryneform bacteria modified so as to increase the activity of the following protein: (A) protein having an amino acid sequence of SEQ ID No. 2, or (B) protein composed of an amino acid sequence of SEQ ID No. 2 having undergone substitution, deletion, insertion or addition of one or some amino acids, which protein when introduced in coryneform bacteria, exhibits the activity of enhancing the potency for L-glutamic acid production.

Description

明 細 書  Specification
L一グルタミン酸の製造法  Method for producing L-glutamic acid
技術分野  Technical field
[0001] 本発明は、発酵工業に関し、詳しくは、 Lーグノレタミン酸の製造法及びそれに用いる 細菌に関する。 L一グルタミン酸は調味料原料等として広く用いられている。  The present invention relates to the fermentation industry, and more particularly, to a method for producing L-gnoretamic acid and a bacterium used for the method. L-glutamic acid is widely used as a seasoning raw material and the like.
背景技術  Background art
[0002] 従来、 L一グルタミン酸は、 Lーグノレタミン酸生産能を有するブレビバタテリゥム属ゃコ リネバタテリゥム属に属するコリネ型細菌を用いて発酵法により工業生産されている。 これらのコリネ型細菌は、生産性を向上させるために、 自然界から分離した菌株また は該菌株の人工変異株が用いられている。  [0002] Heretofore, L-glutamic acid has been industrially produced by a fermentation method using a coryneform bacterium belonging to the genus Brevibataterum, which has the ability to produce L-gnoretamic acid. For these coryneform bacteria, a strain isolated from the natural world or an artificial mutant thereof is used in order to improve the productivity.
[0003] また、組換え DNA技術により Lーグノレタミン酸の生合成酵素を増強することによって 、 L一グルタミン酸の生産能を増加させる種々の技術が開示されている。  [0003] Also, various techniques for increasing L-glutamic acid-producing ability by enhancing L-gnoretamic acid biosynthetic enzyme by recombinant DNA technology have been disclosed.
さらに、酸性条件下で L一グルタミン酸生産能を有する微生物を培養液中に蓄積す る L一グルタミン酸を析出させながら発酵を行う方法が開発されている。従来、通常の L-グノレタミン酸生産菌は酸性条件下では生育できなレ、ため、 L-グルタミン酸発酵は 中性で行われてレ、たが、酸性条件下で L一グルタミン酸生産能を有する微生物として 見い出されたェンテロパクター 'アグロメランスを、 pHが L一グルタミン酸が析出する条 件に調整された液体培地で培養することによって、培地中に L一グルタミン酸を析出さ せながら生成蓄積させることができる（欧州特許出願公開第 1078989号)。  Furthermore, a method has been developed in which fermentation is performed while precipitating L-glutamic acid that accumulates in a culture solution a microorganism capable of producing L-glutamic acid under acidic conditions. Conventionally, normal L-gunoletamic acid-producing bacteria cannot grow under acidic conditions, so L-glutamic acid fermentation is carried out under neutral conditions.However, microorganisms capable of producing L-glutamic acid under acidic conditions Culturing in a liquid medium whose pH has been adjusted to the condition that L-glutamic acid precipitates can be produced and accumulated while precipitating L-glutamic acid in the medium (Europe). Patent Application Publication No. 1078989).
[0004] ところで、コリネバタテリゥム.ダルタミカム ATCC13032の全ゲノム配列が決定され、 公開されている（DDBJ/EMBL/GenBank accession # BA000036)。 ORF1554と相同 性の高い推定上の ORFが知られているが（DDBJ/EMBL/GenBank accession # AP005277-302)、その機能は知られてレ、なレ、。  [0004] By the way, the entire genome sequence of Corynebacterium dartamicum ATCC13032 has been determined and published (DDBJ / EMBL / GenBank accession # BA000036). A putative ORF with high homology to ORF1554 is known (DDBJ / EMBL / GenBank accession # AP005277-302), but its function is known.
発明の開示  Disclosure of the invention
[0005] 本発明は、コリネ型細菌を用いた L一グルタミン酸の製造において、 L一グルタミン酸 生産性を向上させる新規な技術を提供することを課題とする。  An object of the present invention is to provide a novel technique for improving L-glutamic acid productivity in the production of L-glutamic acid using a coryneform bacterium.
[0006] 本発明者らは、コリネ型細菌の耐酸性に関与する遺伝子に関する研究を行う過程 で、それらのうち ORF1554と名付けられた機能未知の遺伝子産物の活性を上昇させ ることにより、コリネ型細菌の Lーグノレタミン酸生産能を向上させることができることを見 出し、本発明を完成するに至った。 [0006] The inventors of the present invention conducted a process of conducting research on genes involved in acid resistance of coryneform bacteria. Thus, they found that by increasing the activity of a gene product of unknown function, which was named ORF1554, the ability of coryneform bacteria to produce L-gnoretamic acid could be improved, leading to the completion of the present invention. Was.
すなわち本発明は、以下のとおりである。  That is, the present invention is as follows.
(1) L-グノレタミン酸生産能を有するコリネ型細菌を培地に培養し、 L-グルタミン酸を 培養物中に生成蓄積させ、該培養物より L-グルタミン酸を採取する、 L-グノレタミン 酸の製造法において、前記細菌は、下記 (A)又は（B)に示すタンパク質の活性が上 昇するように改変されてレヽることを特徴とする方法。  (1) A method for producing L-gnoretamic acid, comprising culturing a coryneform bacterium capable of producing L-gnoretamic acid in a medium, producing and accumulating L-glutamic acid in the culture, and collecting L-glutamic acid from the culture. 3. The method according to claim 1, wherein the bacterium is modified so that the activity of the protein shown in the following (A) or (B) is increased.
(A)配列番号 2に記載のアミノ酸配列を有するタンパク質。  (A) a protein having the amino acid sequence of SEQ ID NO: 2;
(B)配列番号 2に記載のアミノ酸配列において、 1若しくは数個のアミノ酸の置換、 欠失、揷入又は付加を含むアミノ酸配列からなり、かつ、コリネ型細菌の Lーグノレタミン 酸生産能を向上させる活性を有するタンパク質。  (B) the amino acid sequence of SEQ ID NO: 2, comprising an amino acid sequence containing one or several amino acid substitutions, deletions, insertions or additions, and improves the ability of coryneform bacteria to produce L-gnoretamic acid; A protein having activity.
(2)前記タンパク質力 下記（a)又は (b)に示す DNAによりコードされる (1)の方法。  (2) The method according to (1), which is encoded by the DNA shown in (a) or (b) below.
(a)配列番号 1の塩基番号 749— 1414からなる塩基配列を有する DNA。  (a) DNA having a base sequence consisting of base numbers 749-1414 of SEQ ID NO: 1.
(b)配列番号 1の塩基番号 749— 1414からなる塩基配列とストリンジェントな条件 下でハイブリダィズし、かつ、コリネ型細菌の Lーグノレタミン酸生産能を向上させる活 性を有するタンパク質をコードする DNA。  (b) DNA encoding a protein that hybridizes with a base sequence consisting of base numbers 749 to 1414 of SEQ ID NO: 1 under stringent conditions and has an activity to improve the ability of coryneform bacteria to produce L-gnoretamic acid.
(3)前記細菌は、前記 (A)又は（B)に示すタンパク質をコードする遺伝子のコピー数 を高めること、又は前記細菌細胞内の前記 (A)又は（B)に示すタンパク質をコードす る遺伝子の発現が増強するように同遺伝子の発現調節配列を改変することにより、細 胞内の前記タンパク質の活性が上昇したことを特徴とする (1)又は (2)に記載の方法。 (3) The bacterium increases the copy number of a gene encoding the protein shown in (A) or (B), or encodes the protein shown in (A) or (B) in the bacterial cell. The method according to (1) or (2), wherein the activity of the protein in the cell is increased by modifying the expression control sequence of the gene so that the expression of the gene is enhanced.
(4)前記タンパク質は、配列番号 2の 81位のグノレタミン酸残基がグリシン残基に置換 されたアミノ酸配列を有する (1)一 (3)のいずれかに記載の方法。 (4) The method according to any one of (1) to (3), wherein the protein has an amino acid sequence in which a gnoretamic acid residue at position 81 of SEQ ID NO: 2 is substituted with a glycine residue.
(5)前記細菌は、 ひーケトグルタル酸デヒドロゲナーゼを欠損してレ、ることを特徴とす る (1)一 (4)のレ、ずれかに記載の方法。  (5) The method according to any one of (1) to (4), wherein the bacterium is deficient in sheak toglutarate dehydrogenase.
(6) L-グノレタミン酸生産能を有し、かつ、下記 (A)又は（B)に示すタンパク質の活性 が上昇するように改変されたコリネ型細菌。  (6) A coryneform bacterium which has an ability to produce L-gnoretamic acid and has been modified so that the activity of the protein shown in the following (A) or (B) is increased.
(A)配列番号 2に記載のアミノ酸配列を有するタンパク質。 (B)配列番号 2に記載のアミノ酸配列において、 1若しくは数個のアミノ酸の置換、 欠失、挿入又は付加を含むアミノ酸配列からなり、かつ、コリネ型細菌の Lーグノレタミン 酸生産能を向上させる活性を有するタンパク質。 (A) a protein having the amino acid sequence of SEQ ID NO: 2; (B) the amino acid sequence of SEQ ID NO: 2, comprising an amino acid sequence containing one or several amino acid substitutions, deletions, insertions, or additions, and an activity of improving the ability of a coryneform bacterium to produce L-gnoretamic acid; A protein having
図面の簡単な説明  BRIEF DESCRIPTION OF THE FIGURES
[0008] [図 1]〇RF1554及び ORF1554*を増幅したブレビバタテリゥム 'ラタトフアーメンタム sucA欠損株の生育と L一グルタミン酸収率を示す図。  FIG. 1 is a diagram showing the growth and L-glutamic acid yield of a Brevibataterium 'ratatofamentum sucA-deficient strain in which RF1554 and ORF1554 * were amplified.
発明を実施するための最良の形態  BEST MODE FOR CARRYING OUT THE INVENTION
[0009] 以下、本発明を詳細に説明する。 Hereinafter, the present invention will be described in detail.
く 1〉本発明のコリネ型細菌  <1> Coryneform bacterium of the present invention
本発明のコリネ型細菌は、 L一グルタミン酸生産能を有し、かつ、下記 (A)又は（B) に示すタンパク質の活性が上昇するように改変されたコリネ型細菌である。  The coryneform bacterium of the present invention is a coryneform bacterium which has an ability to produce L-glutamic acid and has been modified so that the activity of the protein shown in the following (A) or (B) is increased.
(A)配列番号 2に記載のアミノ酸配列を有するタンパク質。  (A) a protein having the amino acid sequence of SEQ ID NO: 2;
(B)配列番号 2に記載のアミノ酸配列において、 1若しくは数個のアミノ酸の置換、 欠失、揷入又は付加を含むアミノ酸配列からなり、かつ、コリネ型細菌の Lーグノレタミン 酸生産能を向上させる活性を有するタンパク質。  (B) the amino acid sequence of SEQ ID NO: 2, comprising an amino acid sequence containing one or several amino acid substitutions, deletions, insertions or additions, and improves the ability of coryneform bacteria to produce L-gnoretamic acid; A protein having activity.
[0010] 以下、前記（A)又は（B)のタンパク質を「〇RF1554産物」、同タンパク質をコードす る DNAを「〇RF1554」とレ、うことがある。また、 ORF1554にカロえ、同〇RFに隣接するプ 口モーター等の発現調節配列を含めて、便宜上「0RF1554」と呼ぶことがある。 Hereinafter, the protein of (A) or (B) may be referred to as “ΔRF1554 product”, and the DNA encoding the protein may be referred to as “ΔRF1554”. Further, Karoe the ORF1554, including an expression control sequence up port such as a motor which is adjacent to the 〇_RF sometimes for convenience called a "0 RF1554".
[0011] 本発明において、「コリネ型細菌」とは、従来ブレビバタテリゥム属に分類されていた 、現在コリネバタテリゥム属に分類された細菌も含み（Int. J. Syst. Bacteriol., 41, 255(1981))、またコリネバタテリゥム属と非常に近縁なブレビバタテリゥム属細菌を含 む。このようなコリネ型細菌の例として以下のものが挙げられる。  [0011] In the present invention, the "coryneform bacterium" also includes a bacterium which has been conventionally classified into the genus Brevibataterium, and which is now classified into the genus Corynebateterium (Int. J. Syst. Bacteriol., 41, 255 (1981)), and also includes bacteria of the genus Brevibataterirum, which is very closely related to Corynebacterium. Examples of such coryneform bacteria include the following.
[0012] コリネバタテリゥム.ァセトァシドフィラム  [0012] Corynebacterium.acetoacidophilum
コリネバタテリゥム.ァセトグルタミカム  Corynebacterium.acetoglutamicum
コリネバタテリゥム 'アル力ノリティカム  Corynebacterium 'Al Force Noricam
コリネバタテリゥム.カルナェ  Corynebate territory Karnaye
コリネバタテリゥム ·ダルタミカム  Corynebate tertiary cam
コリネバタテリゥム 'リリウム コリネバタテリゥム'メラセコーラ Corynebacterium 'Lilium Corynebacterium 'Merase Cola'
コリネバタテリゥム.サーモアミノゲネス Corynebacterium thermoaminogenes
コリネバタテリゥム.ノヽーキユリス Corynebacterium nouriculus
ブレビバタテリゥム.ディバリカタム Brevi Batterium
ブレビバタテリゥム.フラバム Brevi Batteries, Flavum
ブレビバタテリゥム.インマリオフィラム Brevi Batterium Inmario Filum
ブレビバタテリゥム 'ラタトフアーメンタム Brevi Batteries' Ratatov Armamentum
ブレビバタテリゥム.ロゼゥム Brevi Batterium Rosem
ブレビバタテリゥム.サッカロリティカム Brevi Batteries
ブレビバタテリゥム.チォゲ二タリス Brevi Batteries.
コリネバタテリゥム 'アンモニアゲネス Corynebacterium 'Ammonia Genes
ブレビバタテリゥム .アルバム Brevi Batteries Album
ブレビバタテリゥム.セリヌム Brevibataterum.Selinum
ミクロバタテリゥム.アンモニアフィラム Micro Batteries, Ammonia Filam
具体的には、下記のような菌株を例示することができる。 コリネバタテリゥム 'ァセトグルタミカム ATCC15806 Specifically, the following strains can be exemplified. Corynebacterium 'Acetoglutamicum ATCC15806
コリネバタテリゥム 'アル力ノリティカム ATCC21511 Corynebacterium 'Al Force Noricam Cam ATCC21511
コリネバタテリゥム 'カルナェ ATCC15991 Corynebacterium 'Karnae ATCC15991
コリネバタテリゥム 'グルタミカム ATCC13020, ATCC13032, ATCC13060 コリネバタテリゥム 'リリウム ATCC15990 Corynebacterium Glutamicum ATCC13020, ATCC13032, ATCC13060 Corynebacterium Lilium ATCC15990
コリネバタテリゥム 'メラセコーラ ATCC17965 Corynebacterium 'Merase Cola ATCC17965
コリネバタテリゥム.サーモアミノゲネス AJ12340(FERM BP-1539) Corynebacterium thermoaminogenes AJ12340 (FERM BP-1539)
コリネバタテリゥム.ハーキユリス ATCC13868 Corynebate territory Herkiyurisu ATCC13868
ブレビバタテリゥム.ディバリカタム ATCC14020 Brevi Batterium. Divaricatam ATCC14020
ブレビバタテリゥム.フラバム ATCC13826, ATCC14067, AJ12418(FERM BP-2205) ブレビバタテリゥム.インマリオフィラム ATCC14068 Brevi Batteretium Flavum ATCC13826, ATCC14067, AJ12418 (FERM BP-2205) Brevi Batteretium Inmario Filum ATCC14068
ブレビバタテリゥム 'ラタトフアーメンタム ATCC13869 ブレビバタテリゥム'ロゼゥム ATCC13825 Brevi Batteries' Ratato Armament ATCC13869 Brevi Batteries' Rosem ATCC13825
ブレビバタテリゥム 'サッカロリティカム ATCC14066  Brevi Batteries' Saccharity Cam ATCC14066
ブレビバタテリゥム.チォゲ二タリス ATCC19240  Brevi Batteries. Chogeitaris ATCC19240
コリネバタテリゥム'アンモニアゲネス ATCC6871、 ATCC6872  Corynebacterium Ammonia Genes ATCC6871, ATCC6872
ブレビバタテリゥム.アルバム ATCC15111  Brevi Batteries Album ATCC15111
ブレビバタテリゥム 'セリヌム ATCC15112  Brevi Batteries' Selinum ATCC15112
ミクロバタテリゥム.アンモニアフィラム ATCC15354  Micro Batteries Ammonia Filum ATCC15354
[0014] これらを入手するには、例えばアメリカン'タイプ'カルチャー 'コレクションより分譲を 受けることができる。すなわち、各菌株毎に対応する登録番号が付与されており、こ の登録番号を利用して分譲を受けることができる。各菌株に対応する登録番号はァメ リカン 'タイプ'カルチャー 'コレクションのカタログに記載されている。また、 AJ12340株 は、 1987年 10月 27日付けで通商産業省工業技術院生命工学工業技術研究所 (現 独立行政法人産業技術総合研究所特許微生物寄託センター） ( τ 305-5466 日本 国茨城県つくば巿東 1丁目 1番地 1 中央第 6)に FERM BP-1539の受託番号でブダ ペスト条約に基づいて寄託されている。また、 AJ12418株は、 1989年 1月 5日付けで通 商産業省工業技術院生命工学工業技術研究所に FERM ΒΡ-2205の受託番号でブ ダペスト条約に基づいて寄託されている。 [0014] These can be obtained from, for example, the American 'Type' Culture 'collection. That is, a corresponding registration number is assigned to each strain, and the strain can be ordered using this registration number. The registration numbers for each strain are listed in the catalog of the American 'Type' Culture 'collection. In addition, AJ12340 strain was established on October 27, 1987 by the Ministry of International Trade and Industry, National Institute of Advanced Industrial Science and Technology, Biotechnology and Industrial Technology Research Institute (currently the National Institute of Advanced Industrial Science and Technology, Patent Microorganisms Depositary) (τ 305-5466 Japan It has been deposited at Tsukuba East 1-chome 1-chome 1 Central No. 6) under the Budapest Treaty under the accession number of FERM BP-1539. AJ12418 strain was deposited on January 5, 1989 with the Institute of Biotechnology, Industrial Technology Institute of the Ministry of International Trade and Industry under the accession number FERM II-2205 under the Budapest Treaty.
[0015] 本発明において、「L_グルタミン酸生産能」とは、本発明のコリネ型細菌を培養した ときに、培地中に Lーグノレタミン酸を蓄積する能力をいう。この L一グルタミン酸生産能 は、コリネ型細菌の野生株の性質として有するものであってもよぐ育種によって付与 または増強された性質であってもよい。 [0015] In the present invention, "L_glutamic acid-producing ability" refers to the ability to accumulate L-gnoretamic acid in a medium when the coryneform bacterium of the present invention is cultured. This L-glutamic acid-producing ability may be a property of a wild type coryneform bacterium or a property imparted or enhanced by breeding.
[0016] 育種によって L一グルタミン酸生産能を付与または増強するには、 6-ジァゾ -5-ォキ ソ -ノルロイシン耐性を付与する方法（特開平 3-232497)、プリンアナログ耐性および /またはメチォニンスルホキサイド耐性を付与する方法（特開昭 61-202694)、 a -ケト マロン酸耐性を付与する方法（特開昭 56-151495)、グルタミン酸を含有するペプチド に耐性を付与する方法 (特開平 2-186994)などが挙げられる。 [0016] In order to impart or enhance the ability to produce L-glutamic acid by breeding, a method of imparting 6-diazo-5-oxo-norleucine resistance (Japanese Patent Laid-Open No. 3-232497), purine analog resistance and / or A method for imparting ninsulfoxide resistance (JP-A-61-202694), a method for imparting a-ketomalonic acid resistance (JP-A-56-151495), and a method for imparting resistance to glutamic acid-containing peptides Kaihei 2-186994).
[0017] L一グルタミン酸生産能を有するコリネ型細菌の具体例としては、下記のような菌株 が挙げられる。 ブレビバタテリゥム 'フラバム AJ11573(FERM P-5492)特開昭 56-151495公報参照 ブレビバタテリゥム 'フラバム AJ12210(FERM P-8123)特開昭 61-202694公報参照 ブレビバタテリゥム 'フラバム AJ12212(FERM P-8123)特開昭 61-202694公報参照 ブレビバタテリゥム.フラバム AJ12418(FERM-BP2205)特開平 2-186994公報参照 ブレビバタテリゥム.フラバム DH18(FERM P-11116)特開平 3-232497公報参照 コリネバタテリゥム 'メラセコラ DH344(FERM P-11117)特開平 3-232497公報参照 コリネバタテリゥム.グルタミカム AJ11574(FERM P-5493)特開昭 56-151495公報参照[0017] Specific examples of the coryneform bacterium having the ability to produce L-glutamic acid include the following strains. Brevibataterum 'Flavum AJ11573 (FERM P-5492) See Japanese Unexamined Patent Publication No. 56-151495 Brevibataterum' Flavam AJ12210 (FERM P-8123) Japanese Patent Application Laid-Open No. 61-202694) Brevibataterim 'Flavam AJ12212 ( FERM P-8123) JP-A-61-202694 See Brevibataterum.Flavam AJ12418 (FERM-BP2205) JP-A-2-186994 See JP-B2-186994 JP Brevibataterite.Flavum DH18 (FERM P-11116) JP-A-3-232497 Refer to the gazette Corynebatadium 'Merasecora DH344 (FERM P-11117) See Japanese Unexamined Patent Publication (Kokai) No. 3-232497 Corynebata.
[0018] 「細胞内の ORF1554産物の活性が上昇するように改変された」とは、細胞当たりの 前記活性が非改変株、例えば野生型のコリネ型細菌のそれよりも高くなつたことをい う。例えば、細胞当たりの ORF1554産物分子の数が増加した場合や、 ORF1554産物 分子当たりの活性が上昇した場合などが該当する。また、比較対象となる野生型のコ リネ型細菌とは、例えばブレビバタテリゥム 'ラタトフアーメンタム ATCC13869である。コ リネ型細菌の ORF1554産物の活性が上昇すると、同コリネ型細菌の L一グルタミン酸 生産能が向上する。本発明において、「コリネ型細菌の Lーグノレタミン酸生産能を向上 させる活性」とは、このような ORF1554産物が持つ活性をいう。具体的には、 ORF1554 産物を野生株又は非改変株よりも過剰に発現するように改変されたコリネ型細菌の 菌株を培地で培養したときに、野生株又は非改変株よりも、 L-グノレタミン酸の培地中 の蓄積量が多いか、又は、 L一グルタミン酸の生産速度が高ければ、前記改変株は L 一グルタミン酸生産能が向上しているといえる。 “Modified to increase the activity of an ORF1554 product in a cell” means that the activity per cell is higher than that of a non-modified strain, for example, a wild-type coryneform bacterium. U. For example, the case where the number of ORF1554 product molecules per cell increases or the case where the activity per ORF1554 product molecule increases is applicable. The wild-type coryneform bacterium to be compared is, for example, Brevibataterium 'ratatophamentum ATCC13869. When the activity of the ORF1554 product of a coryneform bacterium increases, the ability of the coryneform bacterium to produce L-glutamic acid increases. In the present invention, the “activity for improving the ability of coryneform bacterium to produce L-gnoretamic acid” refers to the activity of such an ORF1554 product. Specifically, when a strain of a coryneform bacterium that has been modified to overexpress the ORF1554 product in excess of the wild-type or non-modified strain is cultured in a culture medium, L-gnoretamine is higher than the wild-type or non-modified strain. If the amount of acid accumulated in the medium is high or the production rate of L-glutamic acid is high, it can be said that the modified strain has improved L-glutamic acid-producing ability.
[0019] コリネ型細菌細胞内の ORF1554産物活性の増強は、 ORF1554の発現を増強するこ とによって達成される。同遺伝子の発現量の増強は、 ORF1554のコピー数を高めるこ とによって達成される。例えば、 ORF1554断片を、該細菌で機能するベクター、好まし くはマルチコピー型のベクターと連結して組換え DNAを作製し、これを L—グルタミン 酸生産能を有する宿主に導入して形質転換すればよい。また、野生型のコリネ型細 菌に上記組換え DNAを導入して形質転換株を得、その後当該形質転換株に Lーグノレ タミン酸生産能を付与してもよレ、。  [0019] Enhancement of the activity of the ORF1554 product in coryneform bacterium cells is achieved by enhancing the expression of ORF1554. Enhancement of the expression level of the gene can be achieved by increasing the copy number of ORF1554. For example, the ORF1554 fragment is ligated to a vector that functions in the bacterium, preferably a multicopy vector, to produce a recombinant DNA, which is then introduced into a host capable of producing L-glutamic acid to transform the DNA. do it. Alternatively, a transformant may be obtained by introducing the recombinant DNA into a wild-type coryneform bacterium, and then imparting L-gnoretamic acid-producing ability to the transformant.
[0020] ORF1554は、コリネ型細菌由来の遺伝子およびェシエリヒア属細菌等の他の生物 由来の遺伝子のいずれも使用することができる。このうち、発現の容易さの観点から は、コリネ型細菌由来の遺伝子が好ましい。 As ORF1554, any of genes derived from coryneform bacteria and genes derived from other organisms such as bacteria belonging to the genus Escherichia can be used. Of these, from the viewpoint of ease of expression Is preferably a gene derived from a coryneform bacterium.
[0021] ブレビバタテリゥム 'ラタトフアーメンタムの ORF1554の配列は、本発明によって明ら かになつており（配列番号 1)、コリネバタテリゥム.グルタミカムの ORF1554のホモログ と推定される遺伝子も、既に配列が明らかにされているので（DDBJ/EMBL/GenBank accession # AP005277-302)、それらの塩基配列に基づいて作製したプライマー、例 えば配列番号 9及び 10に示すプライマーを用いて、コリネ型細菌の染色体 DNAを铸 型とする Pし R法 (PCR : polymerase chain reaction；
Figure imgf000009_0001
et al, Trends Genet. 5， 185 (1989)参照）によって、 ORF1554とその隣接領域を取得することができる。他の 微生物の ORF1554のホモログも、同様にして取得され得る。 [0021] The sequence of ORF1554 of Brevibataterum 'ratatophamentum' has been clarified according to the present invention (SEQ ID NO: 1), and a gene putatively homologous to ORF1554 of Corynebacterium glutamicum also includes Since the sequence has already been determined (DDBJ / EMBL / GenBank accession # AP005277-302), a coryneform bacterium was prepared using primers prepared based on those nucleotide sequences, for example, the primers shown in SEQ ID NOS: 9 and 10. P method using chromosomal DNA of type III (PCR: polymerase chain reaction;
Figure imgf000009_0001
et al, Trends Genet. 5, 185 (1989)) to obtain ORF1554 and its adjacent regions. Homologs of ORF1554 of other microorganisms can be obtained in a similar manner.
[0022] 染色体 DNAは、 DNA供与体である細菌から、例えば、斎藤、三浦の方法（H.  [0022] Chromosomal DNA can be obtained from a DNA donor bacterium by, for example, the method of Saito and Miura (H.
Saito and K.Miura, Biochem.B iophys. Acta, 72, 619 (1963)、生物工学実験書、 日本 生物工学会編、 97— 98頁、培風館、 1992年参照）等により調製することができる。  Saito and K. Miura, Biochem. Biophys. Acta, 72, 619 (1963), Bioengineering Experiments, edited by Biotechnology Society of Japan, pages 97-98, Baifukan, 1992) and the like.
[0023] PCR法により増幅された ORF1554は、ェシエリヒア 'コリ及び/またはコリネ型細菌の 細胞内において自律複製可能なベクター DNAに接続して組換え DNAを調製し、これ をェシエリヒア'コリに導入しておくと、後の操作がしゃすくなる。ェシエリヒア'コリ細胞 内において自律複製可能なベクターとしては、 pUC19、 pUC18、 pHSG299,  The ORF1554 amplified by the PCR method is connected to a vector DNA capable of autonomous replication in cells of Escherichia coli and / or coryneform bacteria to prepare a recombinant DNA, which is introduced into Escherichia coli. If you keep it, later operations will be slow. Vectors capable of autonomous replication in Escherichia coli cells include pUC19, pUC18, pHSG299,
PHSG399, PHSG398, RSF1010, pBR322, pACYC184, pMW219等が挙げられる。 PHSG399, P HSG398, RSF1010, pBR322 , pACYC184, pMW219 , and the like.
[0024] コリネ型細菌で機能するベクターとは、例えばコリネ型細菌で自律複製できるプラス ミドである。具体的に例示すれば、以下のものが挙げられる。  [0024] A vector that functions in a coryneform bacterium is, for example, a plasmid that can autonomously replicate in a coryneform bacterium. Specific examples include the following.
PAM330 特開昭 58-67699号公報参照  PAM330 See JP-A-58-67699
PHM1519 特開昭 58-77895号公報参照  PHM1519 See JP-A-58-77895.
[0025] また、これらのベクターからコリネ型細菌中でプラスミドを自律複製可能にする能力 を持つ DNA断片を取り出し、前記ェシエリヒア'コリ用のベクターに揷入すると、ェシ エリヒア'コリ及びコリネ型細菌の両方で自律複製可能ないわゆるシャトルベクターとし て使用すること力 Sできる。  [0025] From these vectors, a DNA fragment having the ability to autonomously replicate a plasmid in a coryneform bacterium is taken out, and inserted into the above-mentioned vector for Escherichia coli. Can be used as a so-called shuttle vector that can replicate autonomously in both cases.
[0026] このようなシャトルベクターとしては、以下のものが挙げられる。尚、それぞれのベタ ターを保持する微生物及び国際寄託機関の受託番号をかっこ内に示した。  [0026] Examples of such shuttle vectors include the following. In addition, the microorganisms holding each solid and the accession number of the international depositary organization are shown in parentheses.
PAJ655 ェシエリヒア'コリ AJ11882(FERM BP- 136) コリネハ、、クテリゥム'ダルタミクム SR8201(ATCC39135) PAJ655 Escherichia Cori AJ11882 (FERM BP-136) Coryneha, Cterium 'Dartamicum SR8201 (ATCC39135)
PAJ1844ェシエリヒア 'コリ AJ11883(FERM BP-137)  PAJ1844 Escherichia 'Kori AJ11883 (FERM BP-137)
コリネハ'、クテリゥム ·グルタミクム SR8202(ATCC39136)  Coryneha ', Cterium Glutamicum SR8202 (ATCC39136)
pAJ611 ェシエリヒア'コリ AJ11884(FERM BP— 138)  pAJ611 Escherichia's coli AJ11884 (FERM BP— 138)
PAJ3148コリネハ、、クテリゥム.ダルタミクム SR8203(ATCC39137)  PAJ3148 Coryneha, Cterium.Daltamikum SR8203 (ATCC39137)
pAJ440 ハ'、チルス'ス'、フ'、チリス AJ11901(FERM BP- 140)  pAJ440 C ', Chill's', F', Chillis AJ11901 (FERM BP-140)
pHC4 ェシエリヒア.コリ AJ12617(FERM BP— 3532)  pHC4 Escherichia coli AJ12617 (FERM BP— 3532)
[0027] これらのベクターは、寄託微生物から次のようにして得られる。対数増殖期に集めら れた細胞をリゾチーム及び SDSを用いて溶菌し、 30000 X gで遠心分離して溶解物 力 得た上澄液にポリエチレングリコールを添カ卩し、セシウムクロライド一ェチジゥムブ ロマイド平衡密度勾配遠心分離により分別精製する。 [0027] These vectors are obtained from the deposited microorganism as follows. The cells collected during the logarithmic growth phase were lysed using lysozyme and SDS, centrifuged at 30,000 X g, and the lysate was obtained.The supernatant obtained was added with polyethylene glycol, and the cesium chloride-etidium bromide equilibrium was added. Separate and purify by density gradient centrifugation.
[0028] また、後記実施例に示すプラスミド pSFK6 (特開 2000-262288号公報、米国特許第 [0028] Further, the plasmid pSFK6 shown in Examples described later (Japanese Patent Application Laid-Open No. 2000-262288,
6,303,383号）及び pSAC4 (米国特許第 5, 846, 790号）も、シャトルベクターとして用いる こと力 Sできる。  6,303,383) and pSAC4 (US Pat. No. 5,846,790) can also be used as shuttle vectors.
[0029] ORF1554とコリネ型細菌で機能するベクターを連結して組換え DNAを調製するには 、 ORF1554の末端に合うような制限酵素でベクターを切断する。連結は T4DNAリガ一 ゼ等のリガーゼを用いて行うのが普通である。  [0029] In order to prepare a recombinant DNA by ligating ORF1554 and a vector that functions in coryneform bacteria, the vector is cut with a restriction enzyme that matches the end of ORF1554. Ligation is usually performed using a ligase such as T4 DNA ligase.
[0030] 上記のように調製した組換え DNAをコリネ型細菌に導入するには、これまでに報告 されている形質転換法に従って行えばよい。例えば、ェシエリヒア'コリ K一 12につい て報告されてレ、るような、受容菌細胞を塩ィヒカルシウムで処理して DNAの透過性を 増す方法（Mandel，M.and Higa'A.J. Mol. Biol. , 53, 159 (1970))があり、バチルス'ズ プチリスについて報告されているような、増殖段階の細胞からコンビテントセルを調製 して DNAを導入する方法（ Duncan,C.H.，Wilson,G.A.and Young,F.E.， Gene, 1, 153 (1977))がある。あるいは、バチルス'ズブチリス、放線菌類及び酵母について知られ ているような、 DNA受容菌の細胞を、組換え DNAを容易に取り込むプロトプラストま たはスフヱ口プラストの状態にして組換え DNAを DNA受容菌に導入する方法（ uhang,S.and uhoen,S.N.,Molec. uen. enet. , 168, 1丄丄  [0030] The recombinant DNA prepared as described above may be introduced into coryneform bacteria according to a transformation method that has been reported so far. For example, as described in Escherichia coli K-12, a method of increasing the permeability of DNA by treating recipient cells with calcium chloride (Mandel, M. and Higa'AJ Mol. Biol., 53, 159 (1970)), and a method for preparing DNA and introducing competent cells from cells in the growth stage (Duncan, CH, Wilson, GA and Young, FE, Gene, 1, 153 (1977)). Alternatively, the recombinant DNA can be transformed into protoplasts or sperm plasts that readily incorporate the recombinant DNA, as is known for Bacillus subtilis, actinomycetes and yeast. (Uhang, S. and uhoen, SN, Molec. Uen. Enet., 168, 1 丄 丄
(1979);Bibb,M.J.,WardJ.M.and Hopwood,O.A., Nature, 274, 398 (1978); Hinnen'A. , Hicks J.B. and Fink,G.R.,Proc. Natl. Acad. Sci. USA, 75 1929 (1978))も 応用できる。また、電気パルス法（特開平 2-207791号公報）によっても、コリネ型細菌 の形質転換を行うことができる。 (1979); Bibb, MJ, Ward J. M. and Hopwood, OA, Nature, 274, 398 (1978); Hinnen'A., Hicks JB and Fink, GR, Proc. Natl. Acad. Sci. USA, 75 1929 (1978)). Also, coryneform bacteria can be transformed by the electric pulse method (Japanese Patent Application Laid-Open No. 2-207791).
[0031] ORF1554のコピー数を高めることは、 ORF1554をコリネ型細菌の染色体 DNA上に多 コピー存在させることによつても達成できる。コリネ型細菌の染色体 DNA上に [0031] Increasing the copy number of ORF1554 can also be achieved by causing ORF1554 to exist in multiple copies on the chromosomal DNA of a coryneform bacterium. On the chromosomal DNA of coryneform bacteria
◦RF1554を多コピーで導入するには、染色体 DNA上に多コピー存在する配列を標 的に利用して相同組換えにより行う。染色体 DNA上に多コピー存在する配列としては 、レペティティブ DNA、転移因子の端部に存在するインバーテッド'リピートが利用で きる。あるいは、特開平 2-109985号公報に開示されているように、 ORF1554をトランス ポゾンに搭載してこれを転移させて染色体 DNA上に多コピー導入することも可能であ る。  • In order to introduce RF1554 in multiple copies, homologous recombination is performed using a sequence present in multiple copies on chromosomal DNA. As a sequence present in multiple copies on chromosomal DNA, repetitive DNA and inverted 'repeat existing at the end of a transposable element can be used. Alternatively, as disclosed in Japanese Patent Application Laid-Open No. 2-109985, ORF1554 can be mounted on a transposon, transferred, and introduced into multiple copies on chromosomal DNA.
[0032] ORF1554産物活性の増強は、上記の遺伝子増幅による以外に、染色体 DNA上ま たはプラスミド上の ORF1554のプロモーター等の発現調節配列を強力なものに置換 することによつても達成される。例えば、 lacプロモーター、 t卬プロモーター、 trcプロモ 一ター等が強力なプロモーターとして知られている。また、国際公開 WO00/18935に 開示されているように、 ORF1554のプロモーター領域に数塩基の塩基置換を導入し 、より強力なものに改変することも可能である。これらのプロモーター置換または改変 により ORF1554の発現が強化され、 ORF1554産物活性が増強される。これら発現調 節配列の改変は、 ORF1554のコピー数を高めることと組み合わせてもよい。  [0032] In addition to the gene amplification described above, enhancement of the ORF1554 product activity can also be achieved by replacing an expression regulatory sequence such as the promoter of ORF1554 on chromosomal DNA or a plasmid with a strong one. . For example, the lac promoter, the t 、 promoter, the trc promoter and the like are known as strong promoters. In addition, as disclosed in International Publication WO00 / 18935, it is also possible to introduce a base substitution of several bases into the promoter region of ORF1554 to modify the promoter region to a stronger one. These promoter substitutions or modifications enhance the expression of ORF1554 and enhance the activity of the ORF1554 product. These alterations in the expression control sequence may be combined with increasing the copy number of ORF1554.
[0033] 発現調節配列の置換は、例えば、温度感受性プラスミドを用いた遺伝子置換と同 様にして行うことができる。コリネ型酸菌の温度感受性プラスミドとしては、 p48K及び pSFKT2 (以上、特開 2000-262288号公報参照）、 pHSC4 (フランス特許公開 1992年 2667875号公報、特開平 5-7491号公報参照）等が挙げられる。  [0033] The replacement of the expression control sequence can be performed, for example, in the same manner as the gene replacement using a temperature-sensitive plasmid. Examples of temperature-sensitive plasmids of coryneform acid bacteria include p48K and pSFKT2 (see JP-A-2000-262288), pHSC4 (see French Patent Publication No. 196676767, and JP-A-5-7491). Can be
[0034] 本発明に用いる ORF1554は、コードされるタンパク質の ORF1554産物活性が損な われない限り、 1若しくは複数の位置での 1若しくは数個のアミノ酸の置換、欠失、揷 入、又は付加を含む ORF1554産物をコードするものであってもよレ、。ここで、「数個」と は、アミノ酸残基のタンパク質の立体構造における位置や種類によっても異なるが、 具体的には 2から 30個、好ましくは、 2から 20個、より好ましくは 2から 10個である。 [0035] 上記の ORF1554産物の変異は、 ORF1554産物の活性が維持されるような保存的変 異である。置換は、アミノ酸配列中の少なくとも 1残基が除去され、そこに他の残基が 挿入される変化である。 ORF1554産物の元々のアミノ酸を置換し、かつ、保存的置換 とみなされるアミノ酸としては、 Alaから ser又は thrへの置換、 argから gln、 his又は へ の ：、 asn力、ら glu、 gln、 lys、 his又は aspへの置換、 asp力ら asn、 glu又は ginへの置換 、 cys力、り ser又 f alaへの置換、 gin力、ら asn、 glu、 lys、 his、 asp又 largへの置換、 glu力 ら gly、 asn、 gln、 lys又 iaspへの置換、 giy ら proへの置換、 his力、ら asn、 lys、 gln、 arg 又は tyrへの置換、 ile力ら leu、 met, val又は pheへの置換、 leu力、ら ile、 met, val又は pheへの置換、 lys力ら asn、 glu、 gln、 his又は argへの置換、 met力ら ile、 leu、 val又は pheへの置換、 phe力ら t卬、 tyr, met, ile又は leuへの置換、 serから thr又は alaへの置 換、 thrから ser又は alaへの置換、 t卬から phe又は tyrへの置換、 tyr力、ら his、 phe又は trpへの置換、及び、 valから met、 ile又は leuへの置換が挙げられる。 [0034] The ORF1554 used in the present invention has one or several amino acid substitutions, deletions, insertions, or additions at one or more positions, as long as the ORF1554 product activity of the encoded protein is not impaired. Including those encoding the ORF1554 product. Here, the term “several” differs depending on the position and type of the amino acid residue in the three-dimensional structure of the protein, but specifically 2 to 30, preferably 2 to 20, and more preferably 2 to 10 Individual. [0035] The mutation of the ORF1554 product described above is a conservative mutation that maintains the activity of the ORF1554 product. Substitutions are changes in which at least one residue in the amino acid sequence has been removed and another residue inserted therein. Amino acids that replace the original amino acids of the ORF1554 product and are considered conservative substitutions include Ala to ser or thr substitutions, arg to gln, his or to: asn force, et al. Glu, gln, lys , His or asp, asp power, asn, glu or gin, cys power, ser or fala, gin power, asn, glu, lys, his, asp or larg Replacement of glu force with gly, asn, gln, lys or iasp, replacement of giy with pro, his force, replacement of asn, lys, gln, arg or tyr, replacement of ile force with leu, met, val or substitution with phe, substitution with leu force, ile, met, val or phe, substitution of lys force with asn, glu, gln, his or arg, substitution of met force with ile, leu, val or phe, phe force et al. substitution of t 卬, tyr, met, ile or leu, substitution of ser for thr or ala, substitution of thr for ser or ala, substitution of t 卬 for phe or tyr, tyr force, etc. his, phe or trp And val to met, ile or leu.
[0036] 上記のようなアミノ酸の置換を有する ORF1554産物として具体的には、例えば、配 列番号 2の 81位のグノレタミン酸残基がグリシン残基に置換されたアミノ酸配列を有す る ORF1554産物が挙げられる。  [0036] Specific examples of the ORF1554 product having the amino acid substitution as described above include, for example, an ORF1554 product having an amino acid sequence in which the gnoletamic acid residue at position 81 of SEQ ID NO: 2 is substituted with a glycine residue. Is mentioned.
[0037] 上記のような ORF1554産物と実質的に同一のタンパク質をコードする DNAは、例 えば部位特異的変異法によって、特定の部位のアミノ酸残基が置換、欠失、挿入、 付カロ、又は逆位を含むように、 ORF1554の塩基配列を改変することによって得られる 。また、上記のような改変された DNAは、従来知られている変異処理によっても取得 され得る。変異処理としては、変異処理前の DNAをヒドロキシルァミン等でインビトロ 処理する方法、及び変異処理前の DNAを保持する微生物、例えばェシエリヒア属細 菌を、紫外線照射または N—メチル _Nしニトロ—N—ニトロソグァ二ジン（NTG)もしくは 亜硝酸等の通常変異処理に用いられている変異剤によって処理する方法が挙げら れる。  [0037] The DNA encoding the protein substantially the same as the ORF1554 product as described above may be obtained by substituting, deleting, inserting, attaching, or removing amino acid residues at a specific site by, for example, site-directed mutagenesis. It can be obtained by modifying the nucleotide sequence of ORF1554 so as to include an inversion. The modified DNA as described above can also be obtained by a conventionally known mutation treatment. Examples of the mutation treatment include a method in which DNA before the mutation treatment is treated in vitro with hydroxylamine or the like; — A method of treating with a mutagen commonly used in mutagenesis treatment such as nitrosoguanidine (NTG) or nitrous acid.
[0038] 上記のような変異を有する DNAを、適当な細胞で発現させ、発現産物の活性を調 ベることにより、 ORF1554産物と実質的に同一のタンパク質をコードする DNAが得ら れる。また、変異を有する ORF1554産物をコードする DNAまたはこれを保持する細 胞から、例えば配列表の配列番号 1に記載の塩基配列のうち塩基番号 749— 1414か らなる塩基配列又はその一部を有するプローブとストリンジェントな条件下でハイプリ ダイズし、かつ、 ORF1554産物活性を有するタンパク質をコードする DNAが得られる。 ここでいう「ストリンジェントな条件」とは、いわゆる特異的なハイブリッドが形成され、非 特異的なハイブリッドが形成されない条件をいう。この条件を明確に数値化すること は困難であるが、一例を示せば、相同性が高い DNA同士、例えば 50%以上、好ま しくは 70%以上、より好ましくは 80%以上、特に好ましくは 90。/o以上、最も好ましくは 95%以上の相同性を有する DNA同士がハイブリダィズし、それより相同性が低い D NA同士がハイブリダィズしない条件、あるいは通常のサザンハイブリダィゼーシヨン の洗レヽの条件である 60。C、 1 X SSC, 0. 1%SDS、好ましくは、 0. 1 X SSC、 0. 1 %SDSに相当する塩濃度でハイブリダィズする条件が挙げられる。 [0038] DNA having the above mutation is expressed in a suitable cell, and the activity of the expression product is examined, whereby a DNA encoding a protein substantially identical to the ORF1554 product can be obtained. In addition, DNA encoding the ORF1554 product having a mutation or a cell carrying the same can be obtained, for example, from the nucleotide sequence of nucleotides 749 to 1414 in the nucleotide sequence of SEQ ID NO: 1 in the sequence listing. A DNA encoding a protein having an ORF1554 product activity that hybridizes with a probe having the base sequence or a part thereof under stringent conditions is obtained. The term "stringent conditions" as used herein refers to conditions under which a so-called specific hybrid is formed and a non-specific hybrid is not formed. Although it is difficult to quantify these conditions clearly, as an example, DNAs with high homology, for example, 50% or more, preferably 70% or more, more preferably 80% or more, and particularly preferably 90% or more . / o or more, and most preferably 95% or more DNAs having homology are hybridized with each other, and DNA having lower homology is not hybridized with DNA, or under conditions of washing of ordinary Southern hybridizations. There are 60. Conditions for hybridization at a salt concentration corresponding to C, 1 × SSC, 0.1% SDS, preferably 0.1 × SSC, 0.1% SDS.
[0039] プローブとして、配列番号 1の塩基配列の一部の配列を用いることもできる。そのよ うなプローブは、配列番号 1の塩基配列に基づいて作製したオリゴヌクレオチドをブラ イマ一とし、配列番号 1の塩基配列を含む DNA断片を铸型とする PCRによって作製 すること力 Sできる。プローブとして、 300bp程度の長さの DNA断片を用いる場合には 、ハイブリダィゼーシヨンの洗いの条件は、 50°C、 2 X SSC、 0. 1 %SDSが挙げられ る。 [0039] A partial sequence of the nucleotide sequence of SEQ ID NO: 1 can also be used as a probe. Such a probe can be prepared by PCR using an oligonucleotide prepared based on the nucleotide sequence of SEQ ID NO: 1 as a primer and a DNA fragment containing the nucleotide sequence of SEQ ID NO: 1 as a type II. When a DNA fragment having a length of about 300 bp is used as a probe, the conditions for washing the hybridization include 50 ° C., 2 × SSC, and 0.1% SDS.
[0040] ORF1554産物と実質的に同一のタンパク質をコードする DNAとして具体的には、 配列番号 2に示すアミノ酸配列と、好ましくは 50%以上、より好ましくは 70%以上、さ らに好ましくは 80%、特に好ましくは 90%以上、最も好ましくは 95%以上の相同性を 有し、かつ ORF1554産物活性を有するタンパク質をコードする DNAが挙げられる。  [0040] Specifically, the DNA encoding the protein substantially identical to the ORF1554 product may be, for example, an amino acid sequence represented by SEQ ID NO: 2, preferably 50% or more, more preferably 70% or more, and even more preferably 80% or more. %, Particularly preferably 90% or more, most preferably 95% or more, and DNA encoding a protein having ORF1554 product activity.
[0041] また、本発明のコリネ型細菌は、 ORF1554産物の活性が上昇するように改変された こと以外に、 L一グルタミン酸生合成を触媒する酵素の活性が増強されてレ、てもよレ、。  In addition, the coryneform bacterium of the present invention has been modified so that the activity of the ORF1554 product is increased, and further, the activity of an enzyme that catalyzes L-glutamic acid biosynthesis is enhanced. ,.
L一グルタミン酸生合成を触媒する酵素としては、グノレタミン酸デヒドロゲナーゼ、グノレ タミンシンテターゼ、グルタミン酸シンターゼ、イソクェン酸デヒドロゲナーゼ、アコニッ ト酸ヒドラターゼ、クェン酸シンターゼ、ホスホェノールピルビン酸カルボキシラーゼ、 ホスホェノールピルビン酸シンターゼ、エノラーゼ、ホスホグリセロムターゼ、ホスホグ リセリン酸キナーゼ、グリセルアルデヒド _3_リン酸デヒドロゲナーゼ、トリオースリン酸 イソメラーゼ、フルトースビスリン酸アルドラーゼ、ホスホフルクトキナーゼ、グルコース リン酸イソメラーゼ等がある。 Examples of enzymes that catalyze L-glutamic acid biosynthesis include gnoretamic acid dehydrogenase, gnoretamine synthetase, glutamate synthase, isoquenate dehydrogenase, aconitate hydratase, citrate synthase, phosphoenolpyruvate carboxylase, phosphoenolpyruvate synthase, enolase, and the like. Phosphoglyceromutase, phosphoglycerate kinase, glyceraldehyde_3_phosphate dehydrogenase, triosephosphate isomerase, fructosebisphosphate aldolase, phosphofructokinase, glucose Phosphate isomerase and the like.
[0042] さらに、 L一グルタミン酸の生合成経路から分岐して L一グルタミン酸以外の化合物を 生成する反応を触媒する酵素の活性が低下または欠損してレ、てもよレ、。 L一ダルタミ ン酸の生合成経路から分岐して L -グルタミン酸以外の化合物を生成する反応を触 媒する酵素としては、 ひ—ケトグルタール酸デヒドロゲナーゼ（ひ KGDH)、イソクェン 酸リアーゼ、リン酸ァセチルトランスフェラーゼ、酢酸キナーゼ、ァセトヒドロキシ酸シ ンターゼ、ァセト乳酸シンターゼ、ギ酸ァセチルトランスフェラーゼ、乳酸デヒドロゲナ ーゼ、グルタミン酸デカルボキシラーゼ、 1_ピロリンデヒドロゲナーゼ、等がある。これ らの酵素の中では、 ひ KGDHが好ましい。  [0042] Furthermore, the activity of an enzyme that catalyzes a reaction that diverges from the L-glutamic acid biosynthetic pathway to produce a compound other than L-glutamic acid is reduced or lost. Enzymes that catalyze the reaction that diverges from the biosynthetic pathway of L-daltamate to produce compounds other than L-glutamic acid include polyketoglutarate dehydrogenase (KKGDH), isocitrate lyase, and acetyl phosphate transferase. Acetic acid kinase, acetohydroxyacid synthase, acetolactate synthase, acetyl formate transferase, lactate dehydrogenase, glutamate decarboxylase, 1-pyrroline dehydrogenase, and the like. Of these enzymes, KGDH is preferred.
[0043] 前記ひ KGDHは、 sucA遺伝子によってコードされている。 sucA遺伝子を破壊された コリネ型細菌は、 W095/34672号国際公開パンフレット、及び特開平 7-834672号公 報に詳述されている。  [0043] The KGDH is encoded by the sucA gene. Coryneform bacteria in which the sucA gene has been disrupted are described in detail in W095 / 34672 International Publication Pamphlet and JP-A-7-834672.
[0044] 尚、 sucA遺伝子破壊株は、親株に比べて生育が悪くなる場合があるので、適当な 培地を用いて生育が良好な株を選択することが好ましい。前記培地としては、例えば 、 CM2Bプレート（10g/Lポリペプトン、 10g/Lイーストエキストラタト、 5g/L NaCl、 10 /i g/Lピオチン、 20g/L寒天、 pH7.0)が挙げられる。  Since the growth of the sucA gene-disrupted strain may be worse than that of the parent strain, it is preferable to select a strain that has good growth using an appropriate medium. Examples of the medium include a CM2B plate (10 g / L polypeptone, 10 g / L yeast extratato, 5 g / L NaCl, 10 / ig / L biotin, 20 g / L agar, pH 7.0).
[0045] く 2〉L—グルタミン酸の製造法  [0045] 2> Method for producing L-glutamic acid
上記のようにして得られるコリネ型細菌を培地で培養し、該培地中に L一グルタミン 酸を生成蓄積せしめ、該培地から Lーグノレタミン酸を採取することにより、 Lーグノレタミ ン酸を効率よく製造することができる。  The coryneform bacterium obtained as described above is cultured in a medium, L-glutamic acid is produced and accumulated in the medium, and L-gnoretamic acid is collected from the medium to efficiently produce L-gnoretamic acid. be able to.
[0046] 本発明のコリネ型細菌を用いて L一グルタミン酸を生産するには、炭素源、窒素源、 無機塩類、その他必要に応じてアミノ酸、ビタミン等の有機微量栄養素を含有する通 常の培地を用いて常法により行うことができる。合成培地または天然培地のいずれも 使用可能である。培地に使用される炭素源および窒素源は培養する菌株の利用可 能であるものならばいずれの種類を用いてもよい。  In order to produce L-glutamic acid using the coryneform bacterium of the present invention, a conventional medium containing a carbon source, a nitrogen source, inorganic salts, and, if necessary, organic trace nutrients such as amino acids and vitamins is used. Can be carried out by a conventional method. Either a synthetic medium or a natural medium can be used. As the carbon source and nitrogen source used in the medium, any type may be used as long as the strain to be cultured can be used.
[0047] 炭素源としては、グルコース、グリセロール、フラクトース、スクロース、マルトース、マ ンノース、ガラクトース、澱粉加水分解物、糖蜜等の糖類が使用され、その他、酢酸、 クェン酸等の有機酸、エタノール等のアルコール類も単独あるいは他の炭素源と併 用して用いられる。 [0047] As the carbon source, sugars such as glucose, glycerol, fructose, sucrose, maltose, mannose, galactose, starch hydrolyzate and molasses are used. In addition, organic acids such as acetic acid and citric acid, ethanol and the like are used. Alcohols may be used alone or in combination with other carbon sources. Used for
[0048] 窒素源としては、アンモニア、硫酸アンモニゥム、炭酸アンモニゥム、塩化アンモニ ゥム、りん酸アンモニゥム、酢酸アンモニゥム等のアンモニゥム塩または硝酸塩等が 使用される。  [0048] As the nitrogen source, ammonia, ammonium salts such as ammonium sulfate, ammonium carbonate, ammonium chloride, ammonium phosphate, and ammonium acetate, or nitrates are used.
[0049] 有機微量栄養素としては、アミノ酸、ビタミン、脂肪酸、核酸、更にこれらのものを含 有するペプトン、カザミノ酸、酵母エキス、大豆たん白分解物等が使用され、生育に アミノ酸などを要求する栄養要求性変異株を使用する場合には要求される栄養素を 補添することが好ましい。  [0049] Examples of the organic trace nutrients include amino acids, vitamins, fatty acids, nucleic acids, and peptones, casamino acids, yeast extracts, and soybean protein decomposed products containing these, and nutrients that require amino acids for growth. When an auxotrophic mutant is used, it is preferable to supplement the required nutrients.
[0050] 無機塩類としてはりん酸塩、マグネシウム塩、カルシウム塩、鉄塩、マンガン塩等が 使用される。  [0050] As the inorganic salts, phosphates, magnesium salts, calcium salts, iron salts, manganese salts and the like are used.
[0051] 培養は、発酵温度 20 45°C、 pHを 5 9に制御し、通気培養を行う。培養中に pHが 下がる場合には、炭酸カルシウムを加える力 \アンモニアガス等のアルカリで中和す る。力べして 10時間一 120時間程度培養することにより、培養液中に著量の L一グルタ ミン酸が蓄積される。  [0051] In the culture, aeration culture is performed while controlling the fermentation temperature to 20 45 ° C and the pH to 59. If the pH drops during cultivation, neutralize with an alkali such as ammonia gas for adding calcium carbonate. By vigorously culturing for 10 hours to 120 hours, a significant amount of L-glutamic acid is accumulated in the culture solution.
[0052] 培養終了後の培養液から L -グルタミン酸を採取する方法は、公知の回収方法に 従って行えばよい。例えば、培養液から菌体を除去した後に、濃縮晶析することによ つて採取される。  [0052] The method of collecting L-glutamic acid from the culture solution after completion of the culture may be performed according to a known recovery method. For example, it is collected by removing the cells from the culture solution and then concentrating and crystallization.
実施例  Example
[0053] 以下、本発明を実施例によりさらに具体的に説明する。  Hereinafter, the present invention will be described more specifically with reference to examples.
〔実施例 1〕ブレビバタテリゥム 'ラタトフアーメンタム野生株からの耐酸性変異株の取 得  Example 1 Acquisition of Acid-Resistant Mutant from Wild Strain of Brevibatatium
ブレビバタテリゥム 'ラタトフアーメンタムの野生株 ATCC13869株を、以下の方法で 変異剤 N—メチル _Nしニトロ—N—ニトロソグァ二ジン（NTG)を用いて変異処理した。 ATCC13869株を、 CM2B培地（10g/Lポリペプトン、 10g/Lイーストエキストラタト、 5g/L NaCl、 10 /i g/Lビォチン、 ρΗ7·0) 8mlを入れた 4ml試験管 2本で、 660nmにおける吸 光度（OD )が約 0.7になるまで振とう培養した。培養後の菌体を、 50mMリン酸緩衝  The wild strain ATCC13869 strain of Brevibata terium 'ratatophamentum' was mutated with the following mutations using the mutation agents N-methyl_N and nitro-N-nitrosoguanidine (NTG). Absorbance at 660 nm in two 4 ml test tubes containing 8 ml of ATCC13869 strain in 8 ml of CM2B medium (10 g / L polypeptone, 10 g / L yeast extratato, 5 g / L NaCl, 10 / ig / L biotin, ρΗ7.0) The cells were shake-cultured until (OD) was about 0.7. After culturing the cells, use 50 mM phosphate buffer
660  660
液（pH7.0)で 2回洗浄後、 500 β 1の 50mMリン酸緩衝液（pH7.0)に懸濁した。この菌体 懸濁液に、最終濃度が 0.5 /i g/ /i 1となるように NTGを添加し、 31.5°Cで 10分間インキ ュベート後、 50mMリン酸緩衝液（pH7.0) 3回で洗浄、 200 μ 1の 50mMリン酸緩衝液（ PH7.0)に懸濁した。この菌体を、 8ml CM2B培地に接種し、 31.5°Cでー晚培養し、変 異処理菌体を調製した。 After two washes with liquid (pH 7.0), and suspended in 500 beta 1 of 50mM phosphate buffer (pH 7.0). To this cell suspension, add NTG to a final concentration of 0.5 / ig // i1 and incubate at 31.5 ° C for 10 minutes. After the incubation, the cells were washed three times with 50 mM phosphate buffer (pH 7.0) and suspended in 200 μl of 50 mM phosphate buffer (PH 7.0). The cells were inoculated into 8 ml of a CM2B medium and cultured at 31.5 ° C. to prepare mutated cells.
[0054] 上記の変異処理菌体から、耐酸性変異株を濃縮するため、 S型ジャーフアーメンタ 一を用いて、酸性条件下での連続培養を行った。方法は、以下のとおりである。 300mlの培地（60g/Lグルコース、 1.4g/L H PO、 750mg/L MgSO · 7Η 0、 15mg/L [0054] In order to concentrate the acid-resistant mutant strain from the above-mentioned mutant-treated cells, continuous culture was performed under acidic conditions using an S-type jar armamenter. The method is as follows. 300 ml of medium (60 g / L glucose, 1.4 g / L HPO, 750 mg / L MgSO · 7Η0, 15 mg / L
3 4 4 2  3 4 4 2
FeSO · 7Η〇、 1.35g/L (N量として）大豆加水分解物（「豆濃」（mameno) (味の素 (株)）  FeSO · 7Η〇, 1.35g / L (as N content) Soybean hydrolyzate (“Mameno” (Ajinomoto Co., Inc.)
4 2  4 2
、 450 μ g/Lビタミン B— HC1、 450 μ g/Lピオチン、 3 μ g/Lビタミン Β 、 7.5mg/L PABA  , 450 μg / L vitamin B—HC1, 450 μg / L biotin, 3 μg / L vitamin Β, 7.5 mg / L PABA
1 12  1 12
(パラァミノ安息香酸）、 7.5mg/Lビタミン C、 500mg/L DL_メチォニン、 lml/L消泡剤 AZ-20R (日本油脂社製)）に、上記の変異処理菌体を接種して 31.5°Cで培養した。グ ルコースが完全消費された後からは、フィード液（上記培地と同じ培地）を添加し、培 地の液量が 450mlで一定に保たれるように培養液の引き抜きをフィードと同時に行い 、 6日間の連続培養を行った。 pH7.0で培養を開始し、 6日間培養を行った。培地の pHは、培養時間の経過とともに自然に徐々に低下し、 pH4.9になるまで培養した。  (Paraaminobenzoic acid), 7.5 mg / L vitamin C, 500 mg / L DL_methionine, lml / L defoamer AZ-20R (manufactured by NOF Corporation) C. After the glucose is completely consumed, a feed solution (the same medium as the above medium) is added, and the culture medium is withdrawn at the same time as the feed so that the volume of the medium is kept constant at 450 ml. A continuous culture was performed for a day. The culture was started at pH 7.0 and cultivated for 6 days. The pH of the medium gradually decreased naturally with the passage of the culture time, and the culture was continued until pH 4.9 was reached.
[0055] 上記の連続培養菌体から、耐酸性変異株のスクリーニングを行った。スクリーニング の方法は以下のとおりである。連続培養菌体を、 pH5.7 (KOHで調整）の MM/MES培 地（20g/Lグルコース、 10g/L (NH ) SO、 lg/L KH PO、 0.4g/L MgSO - 7H 0、 [0055] From the above continuously cultured cells, an acid-resistant mutant strain was screened. The screening method is as follows. The continuously cultured cells were cultured in MM / MES medium (pH: 5.7 (adjusted with KOH)) (20 g / L glucose, 10 g / L (NH) SO, lg / L KHPO, 0.4 g / L MgSO-7H0,
4 2 4 2 4 4 2  4 2 4 2 4 4 2
10mg/L FeSO - 7H 0、 lOmg/L MnSO ·4_5Η 0、 200 μ g/Lビタミン B _HC1、 50 μ  10mg / L FeSO-7H 0, lOmg / L MnSO4_5Η 0, 200 μg / L Vitamin B _HC1, 50 μ
4 2 4 2 1  4 2 4 2 1
g/Lピオチン、 5mg/Lニコチンアミド、 lg/L NaCl、 lg/Lカザミノ酸、 50mg/L L_トリプト ファン、 30mg/L L—システィン、 lOOmM MES (2_(N_モルホリノ)エタンスルホン酸）、 20g/L寒天）に、 1プレートあたり約 104個細胞の菌体密度となるように均一に塗り広げ 、 31.5°C、 6日間の培養の後形成されたコロニーを取得した。野生株は pH5.7ではコ ロニー形成ができないため、この操作によって取得される株は、酸性下でコロニー形 成が可能な耐酸性変異株と考えられる。こうして、耐酸性変異株 16-1株及び 16-20株 を取得した。また、 pH5.9の培地を使用したことと、 3日間の培養を行ったこと以外は、 全て同じ方法によって、耐酸性変異株 15-11株を取得した。 g / L biotin, 5mg / L nicotinamide, lg / L NaCl, lg / L casamino acid, 50mg / L L_tryptophan, 30mg / LL—cysteine, lOOmM MES (2_ (N_morpholino) ethanesulfonic acid), (20 g / L agar) was spread evenly to a cell density of about 10 4 cells per plate, and colonies formed after culturing at 31.5 ° C. for 6 days were obtained. Since wild-type strains cannot form colonies at pH 5.7, the strains obtained by this procedure are considered to be acid-resistant mutants that can form colonies under acidic conditions. Thus, the acid-resistant mutants 16-1 and 16-20 were obtained. In addition, acid-resistant mutant strains 15-11 were obtained by the same method, except that a culture medium of pH 5.9 was used and culture was performed for 3 days.
[0056] 〔実施例 2〕ブレビバタテリゥム 'ラタトフアーメンタム 16-1株、 16-20株、及び 15-11株に 特有の遺伝子の単離 く 1〉ゲノムライブラリーの作製 [Example 2] Isolation of genes specific to Brevibata terium 'ratatofumentum 16-1, 16-20, and 15-11 strains <1> Preparation of genomic library
上記のようにして取得したブレビバタテリゥム 'ラタトフアーメンタム 16-1株、 16-20株 、及び 15-11株の各菌株から染色体 DNAを調製し、制限酵素 Sau3AIにより部分分解 し、 4一 6kbpの DNA断片を精製した。これらの DNA断片を、ェシエリヒア.コリとコリネバ クテリゥム属細菌の双方の菌体内で自律複製可能なプラスミドベクター（pSFK6)の BamHIサイトに揷入した。 16-1株では約 14000クローン、 16-20株では約 7000クローン 、 15-11株では約 14000クローン力もなるゲノムライブラリーを得た。  Chromosomal DNA was prepared from each of the strains of Brevibataterium 'ratatophamentum 16-1, 16-20, and 15-11 obtained as described above, and partially degraded with the restriction enzyme Sau3AI. A 6 kbp DNA fragment was purified. These DNA fragments were introduced into the BamHI site of a plasmid vector (pSFK6) capable of autonomous replication in both Escherichia coli and Corynebacterium bacteria. Genomic libraries with about 14,000 clones for the 16-1 strain, about 7,000 clones for the 16-20 strain, and about 14,000 clones for the 15-11 strain were obtained.
[0057] 前記 pSFK6は、ェシエリヒア'コリ用のベクター pHSG399 (S. Takeshita et al： Gene 61,63-74(1987)参照、宝酒造 (株）から購入できる）とストレプトコッカス 'フヱカリスの力 ナマイシン耐性遺伝子から作製されたプラスミド pKlと、ブレビバタテリゥム 'ラタトファ ーメンタム ATCC13869より抽出したプラスミド pAM330 (米国特許第 4,427, 773号、特 開昭 58-67699号公報参照）から構築されたプラスミドである（特開 2000-262288号公 報、米国特許第 6,303,383号)。  [0057] The pSFK6 was obtained from a vector for Escherichia coli pHSG399 (see S. Takeshita et al: Gene 61, 63-74 (1987), which can be purchased from Takara Shuzo Co., Ltd.) and the force of Streptococcus fucaris namycin resistance gene. This is a plasmid constructed from the prepared plasmid pKl and the plasmid pAM330 (see US Pat. No. 4,427,773, and Japanese Patent Publication No. 58-67699) extracted from Brevibatadium 'ratatofamentum ATCC13869 (Japanese Patent Laid-Open No. 2000-2000). -262288, U.S. Patent No. 6,303,383).
[0058] く 2〉耐酸性変異株特有の遺伝子の取得  [0058] (2) Acquisition of genes specific to acid-resistant mutants
16-1株、 16-20株、及び 15-11株の各々のゲノムライブラリーを、ブレビバタテリゥム. ラタトフアーメンタム ATCC13869に導入した。プラスミドの導入には、電気パルス法（ 特開平 2-207791)を用いた。形質転換体を pH5.7の MM/MESプレートに蒔き、 31.5°C 、 7— 9日間の培養の後、コロニーを形成したクローンを取得した。各変異株ゲノムライ ブラリーについて数十万クローンの検索を行った。ブレビバタテリゥム'ラクトファーメ ンタム ATCC13869は、 ρΗ5·7以下の MM/MESプレートでのコロニー形成ができないこ と力ら、取得されたクローンはプラスミド上の遺伝子によって耐酸性が付与されたもの であると考えられる。このようなクローン力 4種類（クローン #D5、クローン #F1、クローン #F2、クローン #H87)取得された。  The genomic libraries of each of the 16-1, 16-20, and 15-11 strains were introduced into Brevibataterium ratatofamentum ATCC13869. The plasmid was introduced by the electric pulse method (Japanese Patent Laid-Open No. 2-207791). The transformant was plated on an MM / MES plate at pH 5.7, and after culturing at 31.5 ° C. for 7 to 9 days, a clone having formed a colony was obtained. Hundreds of thousands of clones were searched for each mutant genomic library. Brevibataterium lactofermentum ATCC13869 is not capable of forming colonies on MM / MES plates with ρΗ5.7 or less. it is conceivable that. Four such clone strengths (clone # D5, clone # F1, clone # F2, clone # H87) were obtained.
[0059] 上記各クローン #D5、 #F1、 #F2、及び #H87が持つプラスミド上のゲノム DNA断片の 塩基配列の一部を、各々順に配列番号 1、 3、 5、 7に示す。これらの配列中には、各 々オープンリーディングフレーム（ORF) (ORF1554 :配列番号 1の塩基番号 749— 1414、 ORF1249 :配列番号 3の塩基番号 250 2748、 ORF39：配列番号 5の塩基番号 55— 1212、 ORF1059 :配列 7の塩基番号 595 1644)が含まれている。各 ORFがコー ドするアミノ酸配列を、配列番号 2、 4、 6及び 8に示す。尚、配列番号 1の塩基番号 1449一 1455、配列番号 3の塩基番号 3317— 3326、配列番号 5の塩基番号 1一 7は、 PHSG399に由来する配列である。 A part of the base sequence of the genomic DNA fragment on the plasmid of each of the clones # D5, # F1, # F2, and # H87 is shown in SEQ ID NOS: 1, 3, 5, and 7, respectively. Each of these sequences contains an open reading frame (ORF) (ORF1554: nucleotides 749 to 1414 of SEQ ID NO: 1, ORF1249: nucleotides 250 to 2748 of SEQ ID NO: 3, ORF39: nucleotides 55 to 1212 of SEQ ID NO: 5). ORF1059: base number 595 1644 of sequence 7). Each ORF is The amino acid sequences to be assigned are shown in SEQ ID NOs: 2, 4, 6, and 8. In addition, nucleotide numbers 1449 to 1455 of SEQ ID NO: 1, nucleotide numbers 3317 to 3326 of SEQ ID NO: 3, and nucleotide numbers 17 to 17 of SEQ ID NO: 5 are sequences derived from PHSG399.
[0060] 配列番号 1、 3、 5及び 7の配列を、ブレビバタテリゥム 'ラタトフアーメンタム  [0060] The sequence of SEQ ID NOs: 1, 3, 5, and 7 was replaced with Brevibatadium 'ratatofamentum
ATCC13869のゲノム配列上の相当する配列と比較したところ、 ORF1249、 ORF39、 ◦RF1059には耐酸性変異株特有の変異点は認められなかった力 ORF1554には、 耐酸性変異株 16-1に特有の変異点が認められた。配列番号 1に示す変異型 ◦RF1554では、 990位の塩基が「A」であるのに対し、野生型では「G」である。その結 果、変異型 ORF1554産物の予想アミノ酸配列では、配列番号 2の 81位が Gluであるの に対し、野生型では Glyである。以下、両者を区別するために、野生型を〇RF1554、 変異型を ORF1554*と表記することがある。  When compared with the corresponding sequence on the genome sequence of ATCC13869, ORF1249, ORF39, and RF1059 did not show any acid-resistant mutant-specific mutation points. Mutation points were observed. Mutant shown in SEQ ID NO: 1 In RF1554, the base at position 990 is “A”, whereas in the wild type, it is “G”. As a result, in the predicted amino acid sequence of the mutant ORF1554 product, position 81 of SEQ ID NO: 2 is Glu, whereas that of the wild type is Gly. Hereinafter, in order to distinguish between the two, the wild type may be referred to as ΔRF1554, and the mutant type may be referred to as ORF1554 *.
[0061] く 3〉〇RF1554、 ORF1249, ORF39、 ORF1059のブレビバタテリゥム'ラタトフアーメンタ ム野生株への導入  [0061] <3> Introduction of RF1554, ORF1249, ORF39, and ORF1059 into a wild strain of Brevibataterium 'ratatofu amentum
配列番号 1に示す塩基配列を有する DNA断片（両末端に Xbal認識配列を有して いる）を、ェシエリヒア'コリとコリネバタテリゥム属細菌の双方の菌体内で自律複製可 能なプラスミドベクター pSAC4の Xbalサイトに挿入し、プラスミド pD5_2Aを作製した。 なお pSAC4は、コリネ型細菌で自律複製可能なプラスミド pHM1519 (Miwa， k. et al., Agric. Biol. Chem., 48 (1984) 2901-2903)を制限酵素 BamHIおよび Kpnlで消化して 得られる複製起点（特開平 5-7491号公報）を、平滑末端化した後、 Sailリンカ一（宝酒 造 (株)製）を用いて、ェシエリヒア'コリ用ベクター pHSG399の Sailサイトに挿入すること により得られたプラスミドである。  A DNA fragment having the nucleotide sequence shown in SEQ ID NO: 1 (having an Xbal recognition sequence at both ends) was transformed into a plasmid vector pSAC4 capable of autonomous replication in both Escherichia coli and Corynebacterium bacteria. And the plasmid pD5_2A was prepared. In addition, pSAC4 is obtained by digesting plasmid pHM1519 (Miwa, k. Et al., Agric. Biol. Chem., 48 (1984) 2901-2903) that can autonomously replicate in coryneform bacteria with restriction enzymes BamHI and Kpnl. The replication origin (Japanese Patent Application Laid-Open No. 5-7491) is obtained by blunt-ending and then inserting it into the Sail site of the Escherichia coli vector pHSG399 using Sail linker (manufactured by Takara Shuzo Co., Ltd.). Plasmid.
[0062] 配列番号 2に示す塩基配列を有する DNA断片（3'末端に BglII、 5'末端に Kpnlの 認識配列を有している）を、 pSAC4の BamHIサイトと Kpnlサイトに連結し、プラスミド pFl_lBを作製した。 [0062] A DNA fragment having the nucleotide sequence shown in SEQ ID NO: 2 (having BglII at the 3 'end and a recognition sequence for Kpnl at the 5' end) was ligated to the BamHI and Kpnl sites of pSAC4, and the plasmid pFl_lB Was prepared.
[0063] 配列番号 3に示す塩基配列を有する DNA断片（両末端に Xbal認識配列を有して いる）を、 pSAC4の Xbalサイトに揷入し、プラスミド pF2_2Aを作製した。  [0063] A DNA fragment having the nucleotide sequence shown in SEQ ID NO: 3 (having an Xbal recognition sequence at both ends) was inserted into the Xbal site of pSAC4 to prepare a plasmid pF2_2A.
[0064] 配列番号 4に示す塩基配列を有する DNA断片（両末端に Nael認識配列を有して いる）を、 pSAC4の Smalサイトに揷入し、プラスミド pH87_4Aを作製した。 [0065] 以上の 4種のプラスミドを、各々ブレビバタテリゥム 'ラタトフアーメンタム ATCC13869 に導入した。各形質転換株を、酸性 pHに調整した MM/MESプレートで、 31.5°C、 5日 間培養し、コロニー形成を調べた。結果を表 1に示す。その結果、ブレビバタテリゥム 'ラタトフアーメンタム ATCC13869に pSAC4を導入した ATCC13869/pSAC4株（対照 株）では、 pH5.7ではコロニー形成ができなレ、が、上記プラスミド pD5_2A、 pFl_lB、 pF2- 2Aがそれぞれ導入された ATCC13869/pD5_2A株、 ATCC13869/pFl_lB株、 ATCC13869/pF2-2A株では、コロニー形成が可能であった。また、 pH6.0においては 、 ATCC13869/pSAC4株（対照株）に比べ ATCC13869/pH87- 4Aで大きなコロニーを 形成した。このように、 ORF1554, ORF1249, ORF39、 ORF1059を導入することで、耐 酸性を付与することができることがわかった。 [0064] A DNA fragment having the nucleotide sequence shown in SEQ ID NO: 4 (having a Nael recognition sequence at both ends) was inserted into the Smal site of pSAC4 to prepare a plasmid pH87_4A. [0065] The above four types of plasmids were each introduced into Brevibataterium 'ratatophamentum ATCC13869. Each transformant was cultured at 31.5 ° C for 5 days on MM / MES plates adjusted to acidic pH, and colony formation was examined. Table 1 shows the results. As a result, in the ATCC13869 / pSAC4 strain in which pSAC4 was introduced into Brevibataterium 'ratatophamentum ATCC13869 (control strain), colony formation was not possible at pH 5.7, but the plasmids pD5_2A, pFl_lB, and pF2-2A were The introduced ATCC13869 / pD5_2A, ATCC13869 / pFl_lB, and ATCC13869 / pF2-2A strains were able to form colonies. At pH 6.0, a larger colony was formed with ATCC13869 / pH87-4A as compared with ATCC13869 / pSAC4 strain (control strain). Thus, it was found that acid resistance can be imparted by introducing ORF1554, ORF1249, ORF39 and ORF1059.
また、野生型 ORF1554を搭載したプラスミド pD5WT_l (後述する）を、 ATCC13869 に導入した場合にも、 ORF1554*とほぼ同等の耐酸性を示した (表 1)。  Moreover, when the plasmid pD5WT_l (described later) carrying the wild-type ORF1554 was introduced into ATCC13869, it exhibited almost the same acid resistance as ORF1554 * (Table 1).
[0066] [表 1] コロニー形成  [Table 1] Colony formation
菌株  Strain
pHB . 0 PH5.  pHB .0 PH5.
ATCC 1 3869/pSAC4 + ―  ATCC 1 3869 / pSAC4 +-
ATCC 1 3869/pD5-2A (0RF 1 554*) + + + + ATCC 1 3869 / pD5-2A (0RF 1 554 *) + + + +
ATCC 1 3869/pD5WT- 1 (0RF1 554*) + + + +ATCC 1 3869 / pD5WT-1 (0RF1 554 *) + + + +
ATCC 1 3869/pF l - l B (0RF 1 249) + + + ATCC 1 3869 / pF l-l B (0RF 1 249) + + +
ATCC 1 3869/pF 2-2A (0RF39) + + + + ATCC 1 3869 / pF 2-2A (0RF39) + + + +
ATCC 1 3869/pH87-4A (0RF1 059) + + 一 一 ： コロニー形成なし ATCC 13869 / pH87-4A (0RF1 059) ++ No colony formation
十、 + + ： コロニー形成あり（相対的なコロニーサイズ ： + < + + ) く 4〉野生型 ORF1554を搭載したプラスミドの作製  10. ++: colony formation (relative colony size: + <+ +) 4) Preparation of plasmid carrying wild-type ORF1554
配列番号 1の塩基配列を参考に、配列番号 9及び 10に示すプライマーを設計し、 ブレビバタテリゥム'ラタトフアーメンタム ATCC13869から、野生型 ORF1554の上流約 750bpと下流約 30bpを含む領域を、 PCRにより取得した。 PCRは、 pyrobest DNA polymerase (宝酒造 (株)）を用レ、、 94°C 5分の後、 98°C 5秒、 65°C 10秒、 72°C 60秒を 30サイクル行った。取得した約 1.5kbの PCR産物を、両プライマー上に設計しておい た Xbalサイトで切断し、プラスミドベクター pSAC4の Xbalサイトに揷入し、 pD5WT- 1を 作製した。 With reference to the nucleotide sequence of SEQ ID NO: 1, the primers shown in SEQ ID NOs: 9 and 10 were designed, and a region containing about 750 bp upstream and about 30 bp downstream of the wild-type ORF1554 from Brevibatatellium 'ratatophamentum ATCC13869 was subjected to PCR. Acquired by For PCR, pyrobest DNA polymerase (Takara Shuzo Co., Ltd.) was used. After 94 ° C for 5 minutes, 30 cycles of 98 ° C for 5 seconds, 65 ° C for 10 seconds, and 72 ° C for 60 seconds were performed. The obtained 1.5 kb PCR product is cut at the Xbal site designed on both primers, inserted into the Xbal site of the plasmid vector pSAC4, and pD5WT-1 is Produced.
[0068] く 5〉ORF1554、 ORF1554*遺伝子増幅株による L一グルタミン酸生産  [0068] <5> L-Glutamate production by ORF1554 and ORF1554 * gene amplified strains
ORF1554, ORF1554*につレ、て、遺伝子増幅による L一グルタミン酸生産への効果 を調べるため、プラスミド pD5WT-l (ORF1554)及び pD5- 2A (〇RF1554*)をブレビバ クテリゥム 'ラタトフアーメンタム ATCC13869由来の sucA遺伝子欠損株 L30-2株に導 入し、 ORF1554の増幅株 L30_2/pD5WT-l株、及び ORF1554*の増幅株  In order to examine the effect of gene amplification on L-glutamate production by ORF1554 and ORF1554 *, plasmids pD5WT-l (ORF1554) and pD5-2A (〇RF1554 *) were added to Brevibacterium 'ratatofarmentum ATCC13869-derived Introduced into the sucA gene-deficient strain L30-2, and amplified ORF1554 L30_2 / pD5WT-l and amplified ORF1554 *
L30-2/pD5_2A株を作製した。プラスミドの導入は電気パルス法（特開平 2-207791号 公報）によって行った。なお、 L30-2株は、ブレビバタテリゥム 'ラタトフアーメンタム ATCC13869の sucA遺伝子欠損株 Δ S株（W095/34672号国際公開パンフレット）か ら、 CM2Bプレート（10g/Lポリペプトン、 10g/Lイーストエキストラタト、 5g/L NaCl、 10 μ g/Lピオチン、 20g/L寒天、 pH7.0)上で良好なコロニー形成をする株として単離した。  L30-2 / pD5_2A strain was produced. The plasmid was introduced by the electric pulse method (Japanese Patent Application Laid-Open No. 2-207791). The L30-2 strain was obtained from a CM2B plate (10 g / L polypeptone, 10 g / L yeast extra) from the sucA gene-deficient strain ΔS (B095 / 34672 international publication pamphlet) of Brevibataterium 'ratatophamentum ATCC13869. Tato, 5 g / L NaCl, 10 μg / L biotin, 20 g / L agar, pH 7.0).
[0069] 取得した〇RF1554、 ORF1554*増幅株を CMDXプレート（5g/Lグルコース、 10g/Lぺ プトン、 10g/Lイーストエキストラタト、 lg/L KH PO 、 0.4g/L MgSO · 7Η 0、 10mg/L  [0069] The obtained {RF1554, ORF1554 * amplified strain was transferred to a CMDX plate (5 g / L glucose, 10 g / L peptone, 10 g / L yeast extratato, lg / L KHPO, 0.4 g / L MgSO7, 0, 10 mg / L
2 4 4 2  2 4 4 2
FeSO · 7Η 0、 lOmg/L MnSO ·4— 5H 0、 3g/L尿素、 2g/L (N量として)「豆濃」、 FeSO7Η0, lOmg / L MnSO4-5H0, 3g / L urea, 2g / L (as N amount)
4 2 4 2 4 2 4 2
20g/L寒天、 5 /i g/Lクロラムフエ二コール、 pH7.5)で 30°C、ー晚培養後、 1/6プレート 分の菌体を 20mlの培地（30g/Lグルコース、 15g/L (NH ) SO 、 0.4g/L MgSO · 7Η O  After culturing at 20 ° C on 20 g / L agar, 5 / ig / L chloramphenicol, pH 7.5) at 30 ° C, 1/6 plate of cells is cultured in 20 ml of medium (30 g / L glucose, 15 g / L ( NH) SO, 0.4g / L MgSO
4 2 4 4 2 4 2 4 4 2
、 lmg/L FeSO - 7H 0、 lmg/L MnSO ·4_5Η 0、 200 μ g/Lビタミン Bl、 200 μ g/Lビ , Lmg / L FeSO-7H0, lmg / L MnSO4_5Η0, 200 μg / L Vitamin Bl, 200 μg / L
4 2 4 2  4 2 4 2
ォチン、 0.48g/L (N量として）「豆濃」、 lg/フラスコ CaCO、 5 /i g/Lクロラムフエニコー  Votin, 0.48 g / L (as N content) "Tono", lg / flask CaCO, 5 / ig / L chloram fenico
3  Three
ノレ、 pH8.0)の入ったフラスコに接種し、 30°C、 20時間の振とう培養の後、バイオテック アナライザー（サクラ精機）を使用して、培地中のグルコース及び L一グルタミン酸の濃 度を測定した。図 1に、消費グルコースに対する L一グルタミン酸の収率と最終 OD ( 620nm、培養液を 51倍に希釈して測定）を示した。 ORF1554及び ORF1554*の増幅 は、いずれもブレビバタテリゥム 'ラタトフアーメンタムのグルタミン酸収率を約 4%向上さ せることがわかった。  No., pH 8.0), inoculate the flask with shaking at 30 ° C for 20 hours, and use a Biotech Analyzer (Sakura Seiki) to determine the concentration of glucose and L-glutamic acid in the medium. Was measured. FIG. 1 shows the yield of L-glutamic acid and the final OD (620 nm, measured by diluting the culture solution 51-fold) with respect to the consumed glucose. It was found that amplification of ORF1554 and ORF1554 * both increased the glutamic acid yield of Brevibataterium 'ratatofamentum by about 4%.
産業上の利用の可能性  Industrial potential
[0070] 本発明により、ブレビバタテリゥム 'ラタトフアーメンタムの L—グルタミン酸生産能を向 上させることができる。 [0070] According to the present invention, the L-glutamic acid-producing ability of Brevibataterium 'ratatofamentum can be improved.

Claims

請求の範囲 The scope of the claims
[1] L-グルタミン酸生産能を有するコリネ型細菌を培地に培養し、 L-グノレタミン酸を培 養物中に生成蓄積させ、該培養物より L-グルタミン酸を採取する、 L-グノレタミン酸 の製造法において、前記細菌は、下記 (A)又は（B)に示すタンパク質の活性が上昇 するように改変されてレヽることを特徴とする方法。  [1] A method for producing L-gunoletamic acid, comprising culturing a coryneform bacterium capable of producing L-glutamic acid in a medium, producing and accumulating L-gunoletamic acid in a culture, and collecting L-glutamic acid from the culture. In the method, the bacterium is modified so that the activity of the protein shown in the following (A) or (B) is increased, and the bacterium is modified.
(A)配列番号 2に記載のアミノ酸配列を有するタンパク質。  (A) a protein having the amino acid sequence of SEQ ID NO: 2;
(B)配列番号 2に記載のアミノ酸配列において、 1若しくは数個のアミノ酸置換、欠失 、挿入又は付加を含むアミノ酸配列からなり、かつ、コリネ型細菌の Lーグノレタミン酸生 産能を向上させる活性を有するタンパク質。  (B) the amino acid sequence of SEQ ID NO: 2, comprising an amino acid sequence containing one or several amino acid substitutions, deletions, insertions, or additions, and having an activity of improving L-gnoretamic acid productivity of a coryneform bacterium; Protein.
[2] 前記タンパク質が、下記（a)又は (b)に示す DNAによりコードされる請求項 1に記 載の方法。  [2] The method according to claim 1, wherein the protein is encoded by DNA shown in the following (a) or (b).
(a)配列番号 1の塩基番号 749— 1414からなる塩基配列を有する DNA。  (a) DNA having a base sequence consisting of base numbers 749-1414 of SEQ ID NO: 1.
(b)配列番号 1の塩基番号 749— 1414からなる塩基配列とストリンジェントな条件下 でハイブリダィズし、かつ、コリネ型細菌の Lーグノレタミン酸生産能を向上させる活性を 有するタンパク質をコードする DNA。  (b) DNA encoding a protein that hybridizes with a base sequence consisting of base numbers 749 to 1414 of SEQ ID NO: 1 under stringent conditions and has an activity of improving the ability of coryneform bacteria to produce L-gnoretamic acid.
[3] 前記細菌は、前記 (A)又は（B)に示すタンパク質をコードする遺伝子のコピー数を 高めること、又は前記細菌細胞内の前記 (A)又は（B)に示すタンパク質をコードする 遺伝子の発現が増強するように同遺伝子の発現調節配列を改変することにより、細 胞内の前記タンパク質の活性が上昇したことを特徴とする請求項 1又は 2に記載の方 法。  [3] The bacterium may have an increased copy number of a gene encoding the protein shown in (A) or (B), or a gene encoding the protein shown in (A) or (B) in the bacterial cell. 3. The method according to claim 1, wherein the activity of the protein in a cell is increased by modifying an expression control sequence of the gene so as to enhance the expression of the gene.
[4] 前記タンパク質は、配列番号 2の 81位のグルタミン酸残基がグリシン残基に置換さ れたアミノ酸配列を有する請求項 1一 3のいずれか一項に記載の方法。  [4] The method according to any one of claims 13 to 13, wherein the protein has an amino acid sequence in which the glutamic acid residue at position 81 of SEQ ID NO: 2 has been substituted with a glycine residue.
[5] 前記細菌は、 ひーケトグルタル酸デヒドロゲナーゼを欠損していることを特徴とする 請求項 1一 4のいずれか一項に記載の方法。  [5] The method according to any one of [14] to [14], wherein the bacterium is deficient in ketoglutarate dehydrogenase.
[6] L-グルタミン酸生産能を有し、かつ、下記 (A)又は（B)に示すタンパク質の活性が 上昇するように改変されたコリネ型細菌。  [6] A coryneform bacterium which has an ability to produce L-glutamic acid and is modified so that the activity of the protein shown in the following (A) or (B) is increased.
(A)配列番号 2に記載のアミノ酸配列を有するタンパク質。  (A) a protein having the amino acid sequence of SEQ ID NO: 2;
(B)配列番号 2に記載のアミノ酸配列において、 1若しくは数個のアミノ酸の置換、欠 失、挿入又は付加を含むアミノ酸配列からなり、かつ、コリネ型細菌の Lーグノレタミン酸 生産能を向上させる活性を有するタンパク質。 (B) In the amino acid sequence of SEQ ID NO: 2, substitution or deletion of one or several amino acids. A protein consisting of an amino acid sequence containing a deletion, insertion or addition, and having an activity of improving the ability of coryneform bacteria to produce L-gnoretamic acid.
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