WO2004113550A1 - Procede de production d'acide l-glutamique - Google Patents

Procede de production d'acide l-glutamique 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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protein
seq
amino acid
glutamic acid
activity
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PCT/JP2004/008807
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English (en)
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/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/14—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

L'invention concerne un procédé permettant de produire de l'acide L-glutamique. Ce procédé consiste à mettre en culture une bactérie coryneforme capable de produire de l'acide L-glutamique dans un milieu de culture de manière à provoquer la formation et l'accumulation d'acide L-glutamique dans un produit de culture, et à récupérer cet acide L-glutamique dans le produit de culture. Ce procédé comprend l'utilisation de bactéries coryneformes qui ont subi une modification de manière à augmenter l'activité d'une des protéines suivantes : (A) une protéine présentant la séquence d'acides aminés SEQ ID No. 2, ou (B) une protéine composée de la séquence d'acides aminés SEQ ID No. 2 ayant subi une substitution, une délétion, une insertion ou une addition d'un ou de plusieurs acides aminés. Lorsqu'elle est introduite dans les bactéries coryneformes, ladite protéine a pour effet d'augmenter la capacité de production d'acide L-glutamique.
PCT/JP2004/008807 2003-06-23 2004-06-23 Procede de production d'acide l-glutamique WO2004113550A1 (fr)

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CN102471790B (zh) 2009-07-29 2014-10-29 味之素株式会社 产生l-氨基酸的方法
BR112013027845A2 (pt) 2011-05-18 2017-01-03 Ajinomoto Kk Imunoestimulante, ração, método para produzir um imunoestimulante, e, método para imunoestimulação
PE20150681A1 (es) 2013-05-13 2015-05-15 Ajinomoto Kk Metodo para producir l-aminoacidos
EP3061828B1 (fr) 2013-10-23 2024-08-28 Ajinomoto Co., Inc. Procédé de production d'une substance cible
JP6623690B2 (ja) 2015-10-30 2019-12-25 味の素株式会社 グルタミン酸系ｌ−アミノ酸の製造法
WO2020071538A1 (fr) 2018-10-05 2020-04-09 Ajinomoto Co., Inc. Procédé de production d'une substance cible par fermentation bactérienne
EP4239070A1 (fr) 2020-10-28 2023-09-06 Ajinomoto Co., Inc. Procédé de production d'acide l-aminé

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