US20010019836A1 - L-glutamic acid-producing bacterium and method for producing L-glutamic acid - Google Patents
L-glutamic acid-producing bacterium and method for producing L-glutamic acid Download PDFInfo
- Publication number
- US20010019836A1 US20010019836A1 US09/784,208 US78420801A US2001019836A1 US 20010019836 A1 US20010019836 A1 US 20010019836A1 US 78420801 A US78420801 A US 78420801A US 2001019836 A1 US2001019836 A1 US 2001019836A1
- Authority
- US
- United States
- Prior art keywords
- glutamic acid
- gene
- microorganism
- enterobacter
- activity
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
- C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)
- C12N9/0016—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4) with NAD or NADP as acceptor (1.4.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—Lyases (4.)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/14—Glutamic acid; Glutamine
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/425—Serratia
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/8215—Microorganisms
- Y10S435/822—Microorganisms using bacteria or actinomycetales
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/8215—Microorganisms
- Y10S435/822—Microorganisms using bacteria or actinomycetales
- Y10S435/88—Serratia
Definitions
- the present invention relates to a novel L-glutamic acid-producing bacterium and a method for producing L-glutamic acid by fermentation using the same.
- L-Glutamic acid is an important amino acid as food, drugs and the like.
- L-Glutamic acid has conventionally been produced by fermentation methods utilizing the so-called coryneform L-glutamic acid-producing bacteria which principally belong to the genera Brevibacterium, Corynebacterium , and Microbacterium or variants thereof (“Amino Acid Fermentation”, Gakkai Shuppan Center, pp.195-215, 1986).
- methods for producing L-glutamic acid by fermentation utilizing other bacterial strains there have been known the methods utilizing microorganisms of the genera Bacillus, Streptomyces, Penicillium and the like (U.S. Pat. No.
- the object of the present invention is to find a novel L-glutamic acid-producing bacterium having a high ability to produce L-glutamic acid, thereby developing a more inexpensive and more efficient method for producing L-glutamic acid.
- the present inventors intensively searched for and studied microorganisms having the ability to produce L-glutamic acid that are different from the previously reported microorganisms. As a result, they found that certain strains derived from microorganisms belonging to the genus Enterobacter or Serratia had a high ability to produce L-glutamic acid, and have completed the present invention.
- the present invention provide:
- the microorganism decreases in or is deficient in an activity of an enzyme catalyzing a reaction branching from a pathway for L-glutamic acid biosynthesis and producing a compound other than L-glutamic acid;
- CS citrate synthase
- PEPC phosphoenolpyruvate carboxylase
- GDH glutamate dehydrogenase
- a microorganism of the above (2) wherein the enzyme catalyzing the reaction for the L-glutamic acid biosynthesis includes all of CS, PEPC and GDH;
- ⁇ KGDH ⁇ -ketoglutarate dehydrogenase
- (6) a method for producing L-glutamic acid which comprises culturing the microorganism as defined in any one of the above (1) to (5) in a liquid culture medium to produce and accumulate L-glutamic acid in the culture medium, and collecting the L-glutamic acid from the culture medium.
- the microorganism of the present invention have a high ability to produce L-glutamic acid, it is considered to be possible to impart a further higher production ability to the microorganism by using the breeding techniques previously known for the coryneform L-glutamic acid-producing bacteria and the like, and it is expected to contribute to development of a more inexpensive and more efficient method for producing L-glutamic acid by appropriately selecting culture conditions and the like.
- FIG. 1 shows construction of a plasmid pMWCPG having a gltA gene, a ppc gene and a gdha gene.
- FIG. 2 shows construction of a plasmid pSTVG having the gdhA gene.
- FIG. 3 shows construction of a plasmid RSF-Tet having a replication origin of a wide-host-range plasmid RSF1010 and a tetracycline resistance gene.
- FIG. 4 shows construction of a plasmid RSFCPG having the replication origin of the wide-host-range plasmid RSF1010, the tetracycline resistance gene, the gltA gene, the ppc gene and the gdhA gene.
- FIG. 5 shows construction of a plasmid pMWCB having the gltA gene.
- FIG. 6 shows a restriction map of a DNA fragment of pTWVEK101 derived from Enterobacter agglomerans.
- FIG. 7 shows comparison of an amino acid sequence deduced from a nucleotide sequence of a sucA gene derived from Enterobacter agglomerans with one derived from Escherichia coli .
- FIG. 8 shows comparison of an amino acid sequence deduced from a nucleotide sequence of a sucB gene derived from Enterobacter agglomerans with one derived from Escherichia coli.
- FIG. 9 shows comparison of an amino acid sequence deduced from a nucleotide sequence of a sdhB gene derived from Enterobacter agglomerans with one derived from Escherichia coli.
- FIG. 10 shows comparison of an amino acid sequence deduced from a nucleotide sequence of a sucC gene derived from Enterobacter agglomerans with one derived from Escherichia coli.
- the microorganism of the present invention is a microorganism belonging to the genus Enterobacter or Serratia , and having at least one of the following properties:
- the microorganism decreases in or is deficient in an activity of an enzyme catalyzing a reaction branching from a pathway for L-glutamic acid biosynthesis and producing a compound other than L-glutamic acid.
- Such a microorganism can be obtained by using a microorganism belonging to the genus Enterobacter or the genus Serratia as a parent strain, and imparting the properties of the above (a) and/or (b) to the microorganism.
- Examples of the microorganism belonging to the genus Enterobacter or the genus Serratia that can be used as the parent strain are listed below:
- the Enterobacter agglomerans AJ13355 was deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry on Feb. 19, 1998, and received an accession number of FERM P-16644, and then transferred to an international deposition under the Budapest Treaty on Jan. 11, 1999, and received an accession number of FERM BP-6614.
- the Enterobacter agglomerans ATCC 12287, and the Serratia liquefacience ATCC 14460 are available from ATCC.
- the Enterobacter agglomerans AJ13355 is a strain isolated from soil in Iwata-shi, Shizuoka, Japan.
- Physiological properties of AJ13355 are as follows:
- Citric acid Positive
- AJ13355 is determined to be Enterobacter agglomerans.
- Enterobacter agglomerans ATCC12287 Enterobacter agglomerans ATCC12287, Enterobacter agglomerans AJ13355, and Serratia liquefacience ATCC14460 were used as starting parent strains for obtaining strains which increase in the activity of the enzyme catalyzing the reactions for the L-glutamic acid biosynthesis, or strains which decrease in or are deficient in the activity of the enzyme catalyzing the reaction branching from the pathway for L-glutamic acid biosynthesis and producing the compound other than L-glutamic acid.
- the microorganism of the present invention is a microorganism belonging to the genus Enterobacter or the genus Serratia and having an ability to produce L-glutamic acid.
- the expression “having an ability to produce L-glutamic acid” as herein used means to have an ability to accumulate L-glutamic acid in a culture medium during cultivation.
- the ability to produce L-glutamic acid is imparted by giving either one or both of the following characteristics:
- the microorganism decreases in or is deficient in the activity of the enzyme catalyzing the reaction branching from the pathway for L-glutamic acid biosynthesis and producing the compound other than L-glutamic acid.
- GDH glutamine synthetase
- glutamate synthase glutamate synthase
- isocitrate dehydrogenase aconitate hydratase
- CS CS
- PEPC pyruvate dehydrogenase
- pyruvate kinase enolase
- phosphoglyceromutase phosphoglycerate kinase
- glyceraldehyde-3-phosphate dehydrogenase triosephosphate isomerase
- fructose bisphosphate aldolase phosphofructokinase
- glucose phosphate isomerase and the like.
- CS CS
- PEPC a microorganism of the present invention
- activities of all of the three kinds of enzymes, CS, PEPC and GDH are increased.
- degree of the increase of the activity can be determined by measuring the enzyme activity of a bacterial cell extract or a purified fraction, and comparing it with that of a wild type strain or a parent strain.
- the microorganism of the present invention which belongs to the genus Enterobacter or Serratia , and increases in the activity of the enzyme catalyzing the reaction for L-glutamic acid biosynthesis, can be obtained as, for example, a variant where mutation has been made in a gene encoding the enzyme or a genetic recombinant strain by using any of the microorganisms mentioned above as a starting parent strain.
- a gene encoding CS, PEPC or GDH can be cloned in a suitable plasmid, and the aforementioned starting parent strain as a host can be transformed with the resulting plasmid.
- This can increase the copy number of each of the genes encoding CS, PEPC and GDH (hereinafter abbreviated as “gltA gene”, “ppc gene”, and “gdhA gene”, respectively), and as a result the activities of CS, PEPC and GDH can be increased.
- One or two or three kinds selected from the cloned gltA gene, ppc gene and gdhA gene in any combination are introduced into the starting parent strain mentioned above.
- two or three kinds of the genes are introduced, either the two or three kinds of the genes are cloned in one kind of plasmid, and introduced into the host, or they are separately cloned in two or three kinds of plasmids that can exist in the same host, and introduced into the host.
- the plasmid is not particularly limited so long as it can autonomously replicate in a microorganism belonging to the genus Enterobacter or Serratia .
- Examples of the plasmid include, for example, pUC19, pUC18, pBR322, pHSG299, pHSG298, pHSG399, pHSG398, RSF1010, pMW119, pMW118, pMW219, pMW218 and the like.
- phage DNA vectors can also be utilized.
- Transformation can be achieved by, for example, the method of D. M. Morrison (Methods in Enzymology 68, 326 (1979)), the method by increasing permeability of recipient cells for DNA with calcium chloride (Mandel, M. and Higa, A., J. Mol. Biol., 53, 159 (1970)), or the like.
- the activities of CS, PEPC and GDH can also be increased by using multiple copies of the gltA gene, the ppc gene and/or the gdh gene present on the chromosome DNA of the starting parent strain as a host.
- the ppc gene and/or the gdhA gene into a chromosome DNA of a microorganism belonging to the genus Enterobacter or Serratia
- sequences present on chromosome DNA in a multiple copy number such as repetitive DNA, and inverted repeats present at an end of transposition factors can be utilized.
- multiple copies of the genes can also be introduced into a chromosome DNA by utilizing transposition of transposons carrying the gltA gene, the ppc gene, or the gdhA gene. These techniques can increase the copy number of the gltA gene, the ppc gene, and the gdhA gene in transformant cells, and as a result increase the activities of CS, PEPC and GDH.
- Any organisms can be used as a source of the gltA gene, the ppc gene and the gdhA gene used for increasing copy numbers, so long as the organisms have the CS, PEPC and GDH activities.
- bacteria i.e., prokaryotes, such as those bacteria belonging to the genera Enterobacter, Klebsiella, Erwinia, Pantoea, Serratia, Escherichia, Corynebacterium, Brevibacterium , and Bacillus are preferred.
- Escherichia coli can be mentioned.
- the gltA gene, the ppc gene and the gdhA gene can be obtained from a chromosome DNA of such microorganisms as mentioned above.
- the gltA gene, the ppc gene and the gdhA gene can each be obtained from a chromosome DNA of any of the aforementioned microorganisms by isolating a DNA fragment complementing auxotrophy of a variant strain lacking the CS, PEPC or GDH activity.
- the nucleotide sequences of these genes of bacteria of the genus Escherichia or Corynebacterium have already been elucidated (Biochemistry, Vol. 22, pp.5243-5249, 1983; J. Biochem. Vol. 95, pp.909-916, 1984; Gene, Vol. 27, pp.193-199, 1984; Microbiology, Vol.
- the genes can be obtained by PCR using primers synthesized based on each of the elucidated nucleotide sequences, and the chromosome DNA as a template.
- the activity of CS, PEPC or GDH can also be increased by, other than by the gene amplification mentioned above, enhancing expression of the gltA gene, the ppc gene or the gdhA gene.
- the expression is enhanced by replacing the promoter of the gltA gene, the ppc gene, or the gdhA gene with another stronger promoter.
- a strong promoter include, for example, a lac promoter, a trp promoter, a trc promoter, a tac promoter, a P R promoter and a P L promoter of lambda phage and the like.
- the gltA gene, the ppc gene, or the gdhA gene of which promoter has been substituted is cloned into a plasmid and introduced into a host microorganism, or introduced into a chromosome DNA of host microorganism using a repetitive DNA, inverted repeat, transposon or the like.
- the activities of CS, PEPC or GDH can also be increased by replacing the promoter of the gltA gene, the ppc gene, or the gdhA gene on a chromosome with another stronger promoter (see WO87/03006, and Japanese Patent Application Laid-Open (KOKAI) No. 61-268183(1986)), or inserting a strong promoter at the upstream of each coding sequence of the genes (see Gene, 29, pp. 231-241, 1984).
- these are achieved by homologous recombination between the gltA gene, the ppc gene, or the gdhA gene of which promoter is replaced with a stronger promoter or DNA containing a part of them, and a corresponding gene on the chromosome.
- microorganism belonging to the genus Enterobacter or Serratia of which CS, PEPC or GDH activity is increased include, for example, Enterobacter agglomerans ATCC12287/RSFCPG, Enterobacter agglomerans AJ13355/RSFCPG, and Serratia liquefacience ATCC14460/RSFCPG.
- Examples of the enzyme catalyzing the reaction branching from the pathway of L-glutamic acid biosynthesis and producing the compound other than L-glutamic acid include, for example, ⁇ KGDH, isocitrate lyase, phosphate acetyltransferase, acetate kinase, acetohydroxy acid synthase, acetolactate synthase, formate acetyltransferase, lactate dehydrogenase, glutamate decarboxylase, 1-pyrroline dehydrogenase and the like.
- ⁇ KGDH is preferred.
- a mutation causing the decrease or deficiency of the enzyme activity can be introduced into a gene encoding the enzyme by a conventional mutagenesis technique or genetic engineering technique.
- Examples of the mutagenesis technique include, for example, the method utilizing irradiation of X-ray or ultraviolet light, the method utilizing treatment with a mutagenic agent such as N-methyl-N′-nitro-N-nitrosoguanidine and the like.
- the site of gene to which a mutation is introduced may be a coding region encoding an enzyme protein, or an expression regulatory region such as a promoter.
- Examples of the genetic engineering technique include, for example, genetic recombination, genetic transduction, cell fusion and the like.
- a drug resistance gene is inserted into a target gene to produce a functionally inactivated gene (defective gene). Then, this defective gene is introduced into a cell of a microorganism belonging to the genus Enterobacter or Serratia , and the target gene on a chromosome is replaced with the defective gene by homologous recombination (gene disruption).
- Whether a microorganism decreases in an activity of a target enzyme or is deficient in the activity, and degree of the decrease of the activity can be determined by measuring the enzyme activity of a bacterial cell extract or a purified fraction of a candidate strain, and comparing it with that of a wild type strain or a parent strain.
- the ⁇ KGDH enzymatic activity can be measured by, for example, the method of Reed et al. (L. J. Reed and B. B. Mukherjee, Methods in Enzymology 1969, 13, p.55-61).
- a target variant can be selected based on a phenotype of the variant.
- a variant which is deficient in the ⁇ KGDH activity or decreases in the activity cannot grow on a minimal medium containing glucose, or a minimal medium containing acetic acid or L-glutamic acid as an exclusive carbon source, or shows markedly reduced growth rate therein under aerobic conditions.
- it can exhibit normal growth by addition of succinic acid or lysine, methionine and diaminopimelate to the minimal medium containing glucose. Based on these phenomena, a variant that is deficient in the ⁇ KGDH activity or decreases in the activity can be selected.
- a method for producing a Brevibacterium lactofermentum strain lacking the ⁇ KGDH gene based on homogenous recombination is detailed in WO95/34672, and a similar method can be used for microorganisms belonging to the genera Enterobacter and Serratia.
- Enterobacter agglomerans AJ13356 An example of the variant strain that is deficient in the ⁇ KGDH activity or decreases in the activity obtained as described above is Enterobacter agglomerans AJ13356.
- the Enterobacter agglomerans AJ13356 was deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry on Feb. 19, 1998, received an accession number of FERM P-16645, and then transferred to an international deposition under the Budapest Treaty on Jan. 11, 1999, and received an accession number of FERM BP-6615.
- microorganism belonging to the genus Enterobacter or Serratia and having at least one of the properties (a) and (b) and an ability to produce L-glutamic acid can be cultured in a liquid medium to produce and accumulate L-glutamic acid in the medium.
- the culture medium may be an ordinary nutrient medium containing a carbon source, a nitrogen source, and inorganic salts, as well as organic trace nutrients such as amino acids, vitamins and the like, as required. It can be a synthetic medium or a natural medium. Any carbon sources and nitrogen sources can be used for the culture medium so long as they can be utilized by the microorganism to be cultured.
- the carbon source may be a saccharide such as glucose, glycerol, fructose, sucrose, maltose, mannose, galactose, starch hydrolysates, molasses and the like.
- an organic acid such as acetic acid and citric acid may also be used alone or in combination with other carbon sources.
- the nitrogen source may be ammonia, ammonium salts such as ammonium sulfate, ammonium carbonate, ammonium chloride, ammonium phosphate, and ammonium acetate, nitrates and the like.
- inorganic salt phosphates, magnesium salts, calcium salts, iron salts, manganese salts and the like are used.
- cultivation may be performed under aerobic conditions at a temperature of 20 to 42° C. and a pH of 4 to 8. The cultivation can be continued for 10 hours to 4 days to accumulate a considerable amount of L-glutamic acid in the liquid culture medium.
- L-glutamic acid accumulated in the culture medium may be collected by a known method.
- it can be isolated by a method comprising concentrating the medium after removing the cells to crystallize the product, ion exchange chromatography or the like.
- a plasmid pBRGDH having a gdhA gene derived from Escherichia coli was digested with HindIII and SphI, and the both ends were blunt-ended by a treatment with T4 DNA polymerase. Then, a DNA fragment containing the gdhA gene was purified and collected.
- a plasmid pMWCP having a gltA gene and a ppc gene derived from Escherichia coli was digested with XbaI, and the both ends were blunt-ended by a treatment with T4 DNA polymerase. This was mixed with the DNA fragment having the gdhA gene purified above, and ligated with T4 ligase, giving a plasmid pMWCPG, which corresponds to the pMWCP further carrying the gdhA gene (FIG. 1).
- a DNA fragment having the gdhA gene obtained by digesting the pBRGDH with HindIII and SalI was purified and collected, and introduced into the HindIII-SalI site of a plasmid pSTV29 (purchased from Takara Shuzo) to obtain a plasmid pSTVG (FIG. 2).
- a product obtained by digesting a plasmid pVIC40 having a replication origin of a wide-host-range plasmid RSF1010 Japanese Patent Application Laid-Open (KOKAI) No. 8-047397(1996)) with NotI, followed by T4 DNA polymerase treatment and PstI digestion, and a product obtained by digesting pBR322 with EcoT141, followed by T4 DNA polymerase treatment and PstI digestion, were mixed and ligated with T4 ligase to obtain a plasmid RSF-Tet having the replication origin of RSF1010 and a tetracycline resistance gene (FIG. 3).
- the pMWCPG was digested with EcoRI and PstI, and a DNA fragment having the gltA gene, the ppc gene and the gdhA gene was purified and collected.
- the RSF-Tet was digested with EcoRI and PstI, and a DNA fragment having the replication origin of RSF1010 was purified and collected.
- Those DNA fragments were mixed and ligated with T4 ligase to obtain a plasmid RSFCPG composed of RSF-Tet carrying the gltA gene, the ppc gene and the gdhA gene (FIG. 4).
- a plasmid having a gltA gene derived from Brevibacterium lactofermentum was constructed as follows. PCR was performed by using primers having the nucleotide sequences represented in SEQ ID NOS: 6 and 7 selected based on the nucleotide sequence of the gltA gene of Corynebacterium glutamicum (Microbiology, 140, 1817-1828, 1994), and a chromosome DNA of Brevibacteriuin lactofermentum ATCC 13869 as a template to obtain a gltA gene fragment of about 3 kb.
- This fragment was inserted into a plasmid pHSG399 (purchased from Takara Shuzo) digested with SmaI to obtain a plasmid pHSGCB (FIG. 5). Then, the pHSGCB was digested with HindIII, and an excised gltA gene fragment of about 3 kb was inserted into a plasmid pMW218 (purchased from Nippon Gene) digested with HindIII to obtain a plasmid pMWCB (FIG. 5). Expression of the gltA gene by the resulting plasmid pMWCB was confirmed by determination of enzyme activity in the Enterobacter agglomerans AJ13355.
- the Enterobacter agglomerans ATCC 12287, the Enterobacter agglomerans AJ13355 and the Serratia liquefacience ATCC 14460 were transformed with the RSFCPG, pMWCB and pSTVG by electroporation (Miller J. H., “A Short Course in Bacterial Genetics; Handbook” Cold Spring Harbor Laboratory Press, USA, 1992) to obtain transformants exhibiting tetracycline resistance.
- Each of the resulting transformants and the parent strains was inoculated into 50 ml-volume large size test tube containing 5 ml of a culture medium comprising 40 g/L glucose, 20 g/L ammonium sulfate, 0.5 g/L magnesium sulfate heptahydrate, 2 g/L potassium dihydrogenphosphate, 0.5 g/L sodium chloride, 0.25 g/L calcium chloride heptahydrate, 0.02 g/L ferrous sulfate heptahydrate, 0.02 g/L manganese sulfate tetrahydrate, 0.72 mg/L zinc sulfate dihydrate, 0.64 mg/L copper sulfate pentahydrate, 0.72 mg/L cobalt chloride hexahydrate, 0.4 mg/L boric acid, 1.2 mg/L sodium molybdate dihydrate, 2 g/L yeast extract, and 30 g/L calcium carbonate, and cultured at 37° C.
- sucAB gene of the Enterobacter agglomerans AJ13355 was cloned by selecting a DNA fragment complementing acetate non-assimilation of an Escherichia coli strain lacking the ⁇ KGDH-E1 subunit gene (referred to as “sucA” hereinafter) from the chromosome DNA of the Enterobacter agglomerans AJ13355.
- the chromosome DNA of the Enterobacter agglomerans AJ13355 strain was isolated by the same method as conventionally used for extracting chromosome DNA from Escherichia coli (Seibutsu Kogaku Jikken-sho (Textbook of Bioengineering Experiments), Ed. by the Society of Fermentation and Bioengineering, Japan, p.97-98, Baifukan, 1992).
- the pTWV228 used as the vector (ampicillin resistant) was a marketed product from Takara Shuzo.
- the Escherichia coli JRG465 carrying the pTWVEK101 recovered the characteristics of acetate non-assimilability as well as auxotrophy for succinic acid or L-lysine and L-methionine. This suggests that the pTWVEK101 contains the sucA gene of Enterobacter agglomerans.
- FIG. 6 A restriction map of Enterobacter agglomerans -derived DNA fragment of pTWVEK101 is shown in FIG. 6.
- SEQ ID NO: 1 The result of nucleotide sequencing of the hatched portion in FIG. 6 is shown in SEQ ID NO: 1.
- SEQ ID NOS: 2 to 5 Amino acid sequences that can be encoded by these ORFs and the partial sequences thereof are shown in SEQ ID NOS: 2 to 5 in order from the 5′ ends.
- nucleotide sequence As a result of homology analysis of these sequences, it was found that the portion of which nucleotide sequence had been determined contained a 3′ partial sequence of succinate dehydrogenase iron-sulfur protein gene (sdhB), full length sucA and ⁇ KGDH-E2 subunit gene (sucB gene), and 5′ partial sequence of succinyl-CoA synthetase ⁇ subunit gene (sucC gene). Comparison of the amino acid sequences deduced from these nucleotide sequences with those of Escherichia coli . (Eur. J. Biochem., 141, 351-359 (1984), Eur. J.
- FIGS. 7 to 9 the amino acid sequences exhibited markedly high homology. It was also found that a cluster of sdhB-sucA-sucB-sucC is formed on the Enterobacter agglomerans chromosome like Escherichia coli (Eur. J. Biochem., 141, 351-359 (1984), Eur. J. Biochem., 141, 361-374 (1984), and Biochemistry, 24, 6245-6252 (1985)).
- pTWVEK101 was digested with BglII to remove the C-terminus region corresponding to about half of the sucA gene and the full length of the sucB gene.
- BglII chloramphenicol resistance gene fragment cut out from the pHSG399 (Takara Shuzo) with AccI was then inserted.
- the region of sdhB- ⁇ sucAB::Cm r -sucC obtained above was cut out with AflII and SacI.
- the resulting DNA fragment was used to transform the Enterobacter agglomerans AJ13355 strain by electroporation to obtain a chloramphenicol resistant strain, and thus a Enterobacter agglomerans AJ13356 strain lacking the sucAB gene where the sucAB gene on the chromosome was replaced by sucAB::Cm r was obtained.
- Each of the AJ13355 and AJ13356 strains was inoculated into a 500 ml-volume flask containing 20 ml of a culture medium comprising 40 g/L glucose, 20 g/L ammonium sulfate, 0.5 g/L magnesium sulfate heptahydrate, 2 g/L potassium dihydrogenphosphate, 0.5 g/L sodium chloride, 0.25 g/L calcium chloride heptahydrate, 0.02 g/L ferrous sulfate heptahydrate, 0.02 g/L manganese sulfate tetrahydrate, 0.72 mg/L zinc sulfate dihydrate, 0.64 mg/L copper sulfate pentahydrate, 0.72 mg/L cobalt chloride hexahydrate, 0.4 mg/L boric acid, 1.2 mg/L sodium molybdate dihydrate, 2 g/L yeast extract, 30 g/L calcium carbonate, 200 mg/L L-ly
- the AJ13356 strain deficient in the ⁇ KGDH activity accumulated 1.5 g/L of L-glutamic acid, and simultaneously accumulated 3.2 g/L of ⁇ KG.
- the AJ13356 strain was transformed with the RSFCPG, and the resulting strain introduced with the RSFCPG, AJ13356/RSFCPG, was inoculated into a 500 ml-volume flask containing 20 ml of a culture medium comprising 40 g/L glucose, 20 g/L ammonium sulfate, 0.5 g/L magnesium sulfate heptahydrate, 2 g/L potassium dihydrogenphosphate, 0.5 g/L sodium chloride, 0.25 g/L calcium chloride heptahydrate, 0.02 g/L ferrous sulfate heptahydrate, 0.02 g/L manganese sulfate tetrahydrate, 0.72 mg/L zinc sulfate dihydrate, 0.64 mg/L copper sulfate pentahydrate, 0.72 mg/L cobalt chloride hexahydrate, 0.4 mg/L boric acid, 1.2 mg/L sodium molybdate dihydrate, 2
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
L-Glutamic acid is produced by culturing in a liquid culture medium a microorganism belonging to the genus Enterobacter or Serratia and having an ability to produce L-glutamic acid, which increases in an activity of enzyme catalyzing a reaction for L-glutamic acid biosynthesis, or which decreases in or is deficient in an activity of an enzyme catalyzing a reaction branching from a pathway for L-glutamic acid biosynthesis and producing a compound other than L-glutamic acid, and collecting produced L-glutamic acid from the culture medium.
Description
- The present invention relates to a novel L-glutamic acid-producing bacterium and a method for producing L-glutamic acid by fermentation using the same. L-Glutamic acid is an important amino acid as food, drugs and the like.
- L-Glutamic acid has conventionally been produced by fermentation methods utilizing the so-called coryneform L-glutamic acid-producing bacteria which principally belong to the generaBrevibacterium, Corynebacterium, and Microbacterium or variants thereof (“Amino Acid Fermentation”, Gakkai Shuppan Center, pp.195-215, 1986). As methods for producing L-glutamic acid by fermentation utilizing other bacterial strains, there have been known the methods utilizing microorganisms of the genera Bacillus, Streptomyces, Penicillium and the like (U.S. Pat. No. 3,220,929), the methods utilizing microorganisms of the genera Pseudomonas, Arthrobacter, Serratia, Candida and the like (U.S. Pat. No. 3,563,857), the methods utilizing microorganisms of the genera Bacillus, Pseudomonas, Serratia and the like or Aerobacter aerogenes (currently referred to as Enterobacter aerogenes) (Japanese Patent Publication (KOKOKU) No. 32-9393(1957)), the method utilizing variant strains of Escherichia coli (Japanese Patent Application Laid-Open (KOKAI) No. 5-244970(1993)) and the like.
- Though the productivity of L-glutamic acid has considerably been improved by breeding of such microorganisms as mentioned above or improvements of production methods, it is still desired to develop a more inexpensive and more efficient method for producing L-glutamic acid in order to meet the expected markedly increasing future demand of the amino acid.
- The object of the present invention is to find a novel L-glutamic acid-producing bacterium having a high ability to produce L-glutamic acid, thereby developing a more inexpensive and more efficient method for producing L-glutamic acid.
- To achieve the aforementioned object, the present inventors intensively searched for and studied microorganisms having the ability to produce L-glutamic acid that are different from the previously reported microorganisms. As a result, they found that certain strains derived from microorganisms belonging to the genusEnterobacter or Serratia had a high ability to produce L-glutamic acid, and have completed the present invention.
- Thus, the present invention provide:
- (1) a microorganism belonging to the genusEnterobacter or Serratia and having an ability to produce L-glutamic acid and at least one of the following properties:
- (a) the microorganism increases in an activity of an enzyme catalyzing a reaction for L-glutamic acid biosynthesis; and
- (b) the microorganism decreases in or is deficient in an activity of an enzyme catalyzing a reaction branching from a pathway for L-glutamic acid biosynthesis and producing a compound other than L-glutamic acid;
- (2) a microorganism of the above (1) wherein the enzyme catalyzing the reaction for the L-glutamic acid biosynthesis is at least one selected from the group consisting of citrate synthase (abbreviated as “CS” hereinafter), phosphoenolpyruvate carboxylase (abbreviated as “PEPC” hereinafter), and glutamate dehydrogenase (abbreviated as “GDH” hereinafter);
- (3) a microorganism of the above (2) wherein the enzyme catalyzing the reaction for the L-glutamic acid biosynthesis includes all of CS, PEPC and GDH;
- (4) a microorganism of any one of the above (1) to (3) wherein the enzyme catalyzing the reaction branching from the pathway for L-glutamic acid biosynthesis and producing the compound other than L-glutamic acid is α-ketoglutarate dehydrogenase (abbreviated as “αKGDH” hereinafter);
- (5) a microorganism of any one of the above (1) to (4) which isEnterobacter agglomerans or Serratia liquefacience; and
- (6) a method for producing L-glutamic acid which comprises culturing the microorganism as defined in any one of the above (1) to (5) in a liquid culture medium to produce and accumulate L-glutamic acid in the culture medium, and collecting the L-glutamic acid from the culture medium.
- Because the microorganism of the present invention have a high ability to produce L-glutamic acid, it is considered to be possible to impart a further higher production ability to the microorganism by using the breeding techniques previously known for the coryneform L-glutamic acid-producing bacteria and the like, and it is expected to contribute to development of a more inexpensive and more efficient method for producing L-glutamic acid by appropriately selecting culture conditions and the like.
- FIG. 1 shows construction of a plasmid pMWCPG having a gltA gene, a ppc gene and a gdha gene.
- FIG. 2 shows construction of a plasmid pSTVG having the gdhA gene.
- FIG. 3 shows construction of a plasmid RSF-Tet having a replication origin of a wide-host-range plasmid RSF1010 and a tetracycline resistance gene.
- FIG. 4 shows construction of a plasmid RSFCPG having the replication origin of the wide-host-range plasmid RSF1010, the tetracycline resistance gene, the gltA gene, the ppc gene and the gdhA gene.
- FIG. 5 shows construction of a plasmid pMWCB having the gltA gene.
- FIG. 6 shows a restriction map of a DNA fragment of pTWVEK101 derived fromEnterobacter agglomerans.
- FIG. 7 shows comparison of an amino acid sequence deduced from a nucleotide sequence of a sucA gene derived fromEnterobacter agglomerans with one derived from Escherichia coli. The upper sections: Enterobacter agglomerans, the lower sections: Escherichia coli (the same shall apply hereinafter).
- FIG. 8 shows comparison of an amino acid sequence deduced from a nucleotide sequence of a sucB gene derived fromEnterobacter agglomerans with one derived from Escherichia coli.
- FIG. 9 shows comparison of an amino acid sequence deduced from a nucleotide sequence of a sdhB gene derived fromEnterobacter agglomerans with one derived from Escherichia coli.
- FIG. 10 shows comparison of an amino acid sequence deduced from a nucleotide sequence of a sucC gene derived fromEnterobacter agglomerans with one derived from Escherichia coli.
- The present invention will be explained in detail hereinafter.
- The microorganism of the present invention is a microorganism belonging to the genusEnterobacter or Serratia, and having at least one of the following properties:
- (a) the microorganism increases in an activity of an enzyme catalyzing a reaction for L-glutamic acid biosynthesis; and
- (b) the microorganism decreases in or is deficient in an activity of an enzyme catalyzing a reaction branching from a pathway for L-glutamic acid biosynthesis and producing a compound other than L-glutamic acid.
- Such a microorganism can be obtained by using a microorganism belonging to the genusEnterobacter or the genus Serratia as a parent strain, and imparting the properties of the above (a) and/or (b) to the microorganism. Examples of the microorganism belonging to the genus Enterobacter or the genus Serratia that can be used as the parent strain are listed below:
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
- More preferably, those bacterial strains listed below can be mentioned:
-
-
-
- TheEnterobacter agglomerans AJ13355 was deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry on Feb. 19, 1998, and received an accession number of FERM P-16644, and then transferred to an international deposition under the Budapest Treaty on Jan. 11, 1999, and received an accession number of FERM BP-6614. The Enterobacter agglomerans ATCC 12287, and the Serratia liquefacience ATCC 14460 are available from ATCC.
- TheEnterobacter agglomerans AJ13355 is a strain isolated from soil in Iwata-shi, Shizuoka, Japan.
- Physiological properties of AJ13355 are as follows:
- (1) Gram stain: Negative
- (2) Behavior for oxygen: Facultative anaerobe
- (3) Catalase: Negative
- (4) Oxidase: Positive
- (5) Nitrate reduction: Negative
- (6) voges-Proskauer reaction: Positive
- (7) Methyl Red test: Negative
- (8) Urease: Negative
- (9) Indole production: Positive
- (10) Motility: Present
- (11) Hydrogen sulfide production in TSI culture medium: Slightly active
- (12) β-Galactosidase: Positive
- (13) Sugar assimilability:
- Arabinose: Positive
- Sucrose: Positive
- Lactose: Positive
- Xylose: Positive
- Sorbitol: Positive
- Inositol: Positive
- Trehalose: Positive
- Maltose: Positive
- Melibiose: Positive
- Adonitol: Negative
- Raffinose: Positive
- Salicin: Negative
- Melibiose: Positive
- (14) Glycerose assimilability: Positive
- (15) Organic acid assimilability:
- Citric acid: Positive
- Tartaric acid: Negative
- Gluconic acid: Positive
- Acetic acid: Positive
- Malonic acid: Negative
- (16) Arginine dehydratase: Negative
- (17) Ornithine decarboxylase: Negative
- (18) Lysine decarboxylase: Negative
- (19) Phenylalanine deaminase: Negative
- (20) Pigment formation: Yellow
- (21) Gelatin liquefaction: Positive
- (22) Growth pH: Not good growth at pH 4, good growth at pH 4.5 to 7
- (23) Growth temperature: Good growth at 25° C., good growth at 30° C., good growth at 37° C., growth possible at 42° C., no growth at 45° C.
- From these bacteriological properties, AJ13355 is determined to beEnterobacter agglomerans.
- In the working examples described hereinafter,Enterobacter agglomerans ATCC12287, Enterobacter agglomerans AJ13355, and Serratia liquefacience ATCC14460 were used as starting parent strains for obtaining strains which increase in the activity of the enzyme catalyzing the reactions for the L-glutamic acid biosynthesis, or strains which decrease in or are deficient in the activity of the enzyme catalyzing the reaction branching from the pathway for L-glutamic acid biosynthesis and producing the compound other than L-glutamic acid. However, the sugar metabolism by any of bacteria belonging to the genera Enterobacter and Serratia is achieved via the Embden-Meyerhof pathway, and pyruvate produced in the pathway is oxidized in the tricarboxylic acid cycle under aerobic conditions. L-Glutamic acid is biosynthesized from α-ketoglutaric acid which is an intermediate of the tricarboxylic acid cycle by GDH or glutamine synthetase/glutamate synthase. Thus, these microorganisms share the same biosynthetic pathway for L-glutamic acid, and microorganism belonging to the genera Enterobacter and Serratia are encompassed within a single concept according to the present invention. Therefore, microorganisms belonging to the genera Enterobacter and Serratia other than species and strains specifically mentioned above also fall within the scope of the present invention.
- The microorganism of the present invention is a microorganism belonging to the genusEnterobacter or the genus Serratia and having an ability to produce L-glutamic acid. The expression “having an ability to produce L-glutamic acid” as herein used means to have an ability to accumulate L-glutamic acid in a culture medium during cultivation. According to the present invention, the ability to produce L-glutamic acid is imparted by giving either one or both of the following characteristics:
- (a) the microorganism increases in the activity of the enzyme catalyzing the reaction for the L-glutamic acid biosynthesis; and
- (b) the microorganism decreases in or is deficient in the activity of the enzyme catalyzing the reaction branching from the pathway for L-glutamic acid biosynthesis and producing the compound other than L-glutamic acid.
- As examples of the enzyme catalyzing the reaction for L-glutamic acid biosynthesis of microorganisms of the genusEnterobacter or Serratia, there can be mentioned GDH, glutamine synthetase, glutamate synthase, isocitrate dehydrogenase, aconitate hydratase, CS, PEPC, pyruvate dehydrogenase, pyruvate kinase, enolase, phosphoglyceromutase, phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase, triosephosphate isomerase, fructose bisphosphate aldolase, phosphofructokinase, glucose phosphate isomerase and the like. Among these enzymes, one or two or three kinds of CS, PEPC and GDH are preferred. As for the microorganism of the present invention, it is further preferred that activities of all of the three kinds of enzymes, CS, PEPC and GDH, are increased. Whether a microorganism increases in an activity of a target enzyme, and degree of the increase of the activity can be determined by measuring the enzyme activity of a bacterial cell extract or a purified fraction, and comparing it with that of a wild type strain or a parent strain.
- The microorganism of the present invention, which belongs to the genusEnterobacter or Serratia, and increases in the activity of the enzyme catalyzing the reaction for L-glutamic acid biosynthesis, can be obtained as, for example, a variant where mutation has been made in a gene encoding the enzyme or a genetic recombinant strain by using any of the microorganisms mentioned above as a starting parent strain.
- To enhance the activity of CS, PEPC or GDH, for example, a gene encoding CS, PEPC or GDH can be cloned in a suitable plasmid, and the aforementioned starting parent strain as a host can be transformed with the resulting plasmid. This can increase the copy number of each of the genes encoding CS, PEPC and GDH (hereinafter abbreviated as “gltA gene”, “ppc gene”, and “gdhA gene”, respectively), and as a result the activities of CS, PEPC and GDH can be increased.
- One or two or three kinds selected from the cloned gltA gene, ppc gene and gdhA gene in any combination are introduced into the starting parent strain mentioned above. When two or three kinds of the genes are introduced, either the two or three kinds of the genes are cloned in one kind of plasmid, and introduced into the host, or they are separately cloned in two or three kinds of plasmids that can exist in the same host, and introduced into the host.
- The plasmid is not particularly limited so long as it can autonomously replicate in a microorganism belonging to the genusEnterobacter or Serratia. Examples of the plasmid include, for example, pUC19, pUC18, pBR322, pHSG299, pHSG298, pHSG399, pHSG398, RSF1010, pMW119, pMW118, pMW219, pMW218 and the like. Other than these plasmids, phage DNA vectors can also be utilized.
- Transformation can be achieved by, for example, the method of D. M. Morrison (Methods in Enzymology 68, 326 (1979)), the method by increasing permeability of recipient cells for DNA with calcium chloride (Mandel, M. and Higa, A., J. Mol. Biol., 53, 159 (1970)), or the like.
- The activities of CS, PEPC and GDH can also be increased by using multiple copies of the gltA gene, the ppc gene and/or the gdh gene present on the chromosome DNA of the starting parent strain as a host. In order to introduce multiple copies of the gltA gene, the ppc gene and/or the gdhA gene into a chromosome DNA of a microorganism belonging to the genusEnterobacter or Serratia, sequences present on chromosome DNA in a multiple copy number such as repetitive DNA, and inverted repeats present at an end of transposition factors can be utilized. Alternatively, multiple copies of the genes can also be introduced into a chromosome DNA by utilizing transposition of transposons carrying the gltA gene, the ppc gene, or the gdhA gene. These techniques can increase the copy number of the gltA gene, the ppc gene, and the gdhA gene in transformant cells, and as a result increase the activities of CS, PEPC and GDH.
- Any organisms can be used as a source of the gltA gene, the ppc gene and the gdhA gene used for increasing copy numbers, so long as the organisms have the CS, PEPC and GDH activities. Among such organisms, bacteria, i.e., prokaryotes, such as those bacteria belonging to the generaEnterobacter, Klebsiella, Erwinia, Pantoea, Serratia, Escherichia, Corynebacterium, Brevibacterium, and Bacillus are preferred. As a specific example, Escherichia coli can be mentioned. The gltA gene, the ppc gene and the gdhA gene can be obtained from a chromosome DNA of such microorganisms as mentioned above.
- The gltA gene, the ppc gene and the gdhA gene can each be obtained from a chromosome DNA of any of the aforementioned microorganisms by isolating a DNA fragment complementing auxotrophy of a variant strain lacking the CS, PEPC or GDH activity. Alternatively, because the nucleotide sequences of these genes of bacteria of the genusEscherichia or Corynebacterium have already been elucidated (Biochemistry, Vol. 22, pp.5243-5249, 1983; J. Biochem. Vol. 95, pp.909-916, 1984; Gene, Vol. 27, pp.193-199, 1984; Microbiology, Vol. 140, pp.1817-1828, 1994; Mol. Gen. Genet. Vol. 218, pp.330-339, 1989; and Molecular Microbiology, Vol. 6, pp.317-326, 1992), the genes can be obtained by PCR using primers synthesized based on each of the elucidated nucleotide sequences, and the chromosome DNA as a template.
- The activity of CS, PEPC or GDH can also be increased by, other than by the gene amplification mentioned above, enhancing expression of the gltA gene, the ppc gene or the gdhA gene. For example, the expression is enhanced by replacing the promoter of the gltA gene, the ppc gene, or the gdhA gene with another stronger promoter. Examples of such a strong promoter include, for example, a lac promoter, a trp promoter, a trc promoter, a tac promoter, a PR promoter and a PL promoter of lambda phage and the like. The gltA gene, the ppc gene, or the gdhA gene of which promoter has been substituted is cloned into a plasmid and introduced into a host microorganism, or introduced into a chromosome DNA of host microorganism using a repetitive DNA, inverted repeat, transposon or the like.
- The activities of CS, PEPC or GDH can also be increased by replacing the promoter of the gltA gene, the ppc gene, or the gdhA gene on a chromosome with another stronger promoter (see WO87/03006, and Japanese Patent Application Laid-Open (KOKAI) No. 61-268183(1986)), or inserting a strong promoter at the upstream of each coding sequence of the genes (see Gene, 29, pp. 231-241, 1984). Specifically, these are achieved by homologous recombination between the gltA gene, the ppc gene, or the gdhA gene of which promoter is replaced with a stronger promoter or DNA containing a part of them, and a corresponding gene on the chromosome.
- Specific examples of the microorganism belonging to the genusEnterobacter or Serratia of which CS, PEPC or GDH activity is increased include, for example, Enterobacter agglomerans ATCC12287/RSFCPG, Enterobacter agglomerans AJ13355/RSFCPG, and Serratia liquefacience ATCC14460/RSFCPG.
- Examples of the enzyme catalyzing the reaction branching from the pathway of L-glutamic acid biosynthesis and producing the compound other than L-glutamic acid include, for example, αKGDH, isocitrate lyase, phosphate acetyltransferase, acetate kinase, acetohydroxy acid synthase, acetolactate synthase, formate acetyltransferase, lactate dehydrogenase, glutamate decarboxylase, 1-pyrroline dehydrogenase and the like. Among these enzymes, αKGDH is preferred.
- In order to obtain such decrease or deficiency of enzyme activity as mentioned above in a microorganism belonging to the genusEnterobacter or Serratia, a mutation causing the decrease or deficiency of the enzyme activity can be introduced into a gene encoding the enzyme by a conventional mutagenesis technique or genetic engineering technique.
- Examples of the mutagenesis technique include, for example, the method utilizing irradiation of X-ray or ultraviolet light, the method utilizing treatment with a mutagenic agent such as N-methyl-N′-nitro-N-nitrosoguanidine and the like. The site of gene to which a mutation is introduced may be a coding region encoding an enzyme protein, or an expression regulatory region such as a promoter.
- Examples of the genetic engineering technique include, for example, genetic recombination, genetic transduction, cell fusion and the like. For example, a drug resistance gene is inserted into a target gene to produce a functionally inactivated gene (defective gene). Then, this defective gene is introduced into a cell of a microorganism belonging to the genusEnterobacter or Serratia, and the target gene on a chromosome is replaced with the defective gene by homologous recombination (gene disruption).
- Whether a microorganism decreases in an activity of a target enzyme or is deficient in the activity, and degree of the decrease of the activity can be determined by measuring the enzyme activity of a bacterial cell extract or a purified fraction of a candidate strain, and comparing it with that of a wild type strain or a parent strain. The αKGDH enzymatic activity can be measured by, for example, the method of Reed et al. (L. J. Reed and B. B. Mukherjee, Methods in Enzymology 1969, 13, p.55-61).
- Depending on the target enzyme, a target variant can be selected based on a phenotype of the variant. For example, a variant which is deficient in the αKGDH activity or decreases in the activity cannot grow on a minimal medium containing glucose, or a minimal medium containing acetic acid or L-glutamic acid as an exclusive carbon source, or shows markedly reduced growth rate therein under aerobic conditions. However, even under the same condition, it can exhibit normal growth by addition of succinic acid or lysine, methionine and diaminopimelate to the minimal medium containing glucose. Based on these phenomena, a variant that is deficient in the αKGDH activity or decreases in the activity can be selected.
- A method for producing aBrevibacterium lactofermentum strain lacking the αKGDH gene based on homogenous recombination is detailed in WO95/34672, and a similar method can be used for microorganisms belonging to the genera Enterobacter and Serratia.
- In addition, procedures of genetic cloning, cleavage and ligation of DNA, transformation and the like are detailed in Molecular Cloning, 2nd edition, Cold Spring Harbor Press (1989) and the like.
- An example of the variant strain that is deficient in the αKGDH activity or decreases in the activity obtained as described above isEnterobacter agglomerans AJ13356. The Enterobacter agglomerans AJ13356 was deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry on Feb. 19, 1998, received an accession number of FERM P-16645, and then transferred to an international deposition under the Budapest Treaty on Jan. 11, 1999, and received an accession number of FERM BP-6615.
- The microorganism belonging to the genusEnterobacter or Serratia, and having at least one of the properties (a) and (b) and an ability to produce L-glutamic acid can be cultured in a liquid medium to produce and accumulate L-glutamic acid in the medium.
- The culture medium may be an ordinary nutrient medium containing a carbon source, a nitrogen source, and inorganic salts, as well as organic trace nutrients such as amino acids, vitamins and the like, as required. It can be a synthetic medium or a natural medium. Any carbon sources and nitrogen sources can be used for the culture medium so long as they can be utilized by the microorganism to be cultured.
- The carbon source may be a saccharide such as glucose, glycerol, fructose, sucrose, maltose, mannose, galactose, starch hydrolysates, molasses and the like. Further, an organic acid such as acetic acid and citric acid may also be used alone or in combination with other carbon sources.
- The nitrogen source may be ammonia, ammonium salts such as ammonium sulfate, ammonium carbonate, ammonium chloride, ammonium phosphate, and ammonium acetate, nitrates and the like.
- As organic trace nutrients, amino acids, vitamins, fatty acids, nucleic acids, materials containing them such as peptone, casamino acid, yeast extract, and soybean protein decomposition products and the like are used, and when an auxotrophic variant which requires an amino acid or the like for its growth is used, it is necessary to complement the nutrient required.
- As the inorganic salt, phosphates, magnesium salts, calcium salts, iron salts, manganese salts and the like are used.
- As for the culture conditions, cultivation may be performed under aerobic conditions at a temperature of 20 to 42° C. and a pH of 4 to 8. The cultivation can be continued for 10 hours to 4 days to accumulate a considerable amount of L-glutamic acid in the liquid culture medium.
- After the completion of the cultivation, L-glutamic acid accumulated in the culture medium may be collected by a known method. For example, it can be isolated by a method comprising concentrating the medium after removing the cells to crystallize the product, ion exchange chromatography or the like.
- Examples
- The present invention will be explained more specifically with reference to the following examples.
- (1) Construction of plasmid having gltA gene, ppc gene and gdhA gene
- Procedure for construction of a plasmid having a gltA gene, a ppc gene and a gdhA gene will be explained by referring to FIG. 1 to FIG. 5.
- A plasmid pBRGDH having a gdhA gene derived fromEscherichia coli (Japanese Patent Application Laid-Open (KOKAI) No. 7-203980(1995)) was digested with HindIII and SphI, and the both ends were blunt-ended by a treatment with T4 DNA polymerase. Then, a DNA fragment containing the gdhA gene was purified and collected. On the other hand, a plasmid pMWCP having a gltA gene and a ppc gene derived from Escherichia coli (WO97/08294) was digested with XbaI, and the both ends were blunt-ended by a treatment with T4 DNA polymerase. This was mixed with the DNA fragment having the gdhA gene purified above, and ligated with T4 ligase, giving a plasmid pMWCPG, which corresponds to the pMWCP further carrying the gdhA gene (FIG. 1).
- A DNA fragment having the gdhA gene obtained by digesting the pBRGDH with HindIII and SalI was purified and collected, and introduced into the HindIII-SalI site of a plasmid pSTV29 (purchased from Takara Shuzo) to obtain a plasmid pSTVG (FIG. 2).
- At the same time, a product obtained by digesting a plasmid pVIC40 having a replication origin of a wide-host-range plasmid RSF1010 (Japanese Patent Application Laid-Open (KOKAI) No. 8-047397(1996)) with NotI, followed by T4 DNA polymerase treatment and PstI digestion, and a product obtained by digesting pBR322 with EcoT141, followed by T4 DNA polymerase treatment and PstI digestion, were mixed and ligated with T4 ligase to obtain a plasmid RSF-Tet having the replication origin of RSF1010 and a tetracycline resistance gene (FIG. 3).
- Then, the pMWCPG was digested with EcoRI and PstI, and a DNA fragment having the gltA gene, the ppc gene and the gdhA gene was purified and collected. Similarly, the RSF-Tet was digested with EcoRI and PstI, and a DNA fragment having the replication origin of RSF1010 was purified and collected. Those DNA fragments were mixed and ligated with T4 ligase to obtain a plasmid RSFCPG composed of RSF-Tet carrying the gltA gene, the ppc gene and the gdhA gene (FIG. 4). Expression of the gitA gene, the ppc gene and the gdhA gene by the resulting plasmid RSFCPG, and expression of the gdhA gene by the pSTVG were confirmed based on complementation of auxotrophy ofEscherichia coli strains lacking the gltA gene, the ppc gene or the gdhA gene, and measurement of each enzyme activity.
- A plasmid having a gltA gene derived fromBrevibacterium lactofermentum was constructed as follows. PCR was performed by using primers having the nucleotide sequences represented in SEQ ID NOS: 6 and 7 selected based on the nucleotide sequence of the gltA gene of Corynebacterium glutamicum (Microbiology, 140, 1817-1828, 1994), and a chromosome DNA of Brevibacteriuin lactofermentum ATCC 13869 as a template to obtain a gltA gene fragment of about 3 kb. This fragment was inserted into a plasmid pHSG399 (purchased from Takara Shuzo) digested with SmaI to obtain a plasmid pHSGCB (FIG. 5). Then, the pHSGCB was digested with HindIII, and an excised gltA gene fragment of about 3 kb was inserted into a plasmid pMW218 (purchased from Nippon Gene) digested with HindIII to obtain a plasmid pMWCB (FIG. 5). Expression of the gltA gene by the resulting plasmid pMWCB was confirmed by determination of enzyme activity in the Enterobacter agglomerans AJ13355.
- (2) Introduction of RSFCPG, pMWCB and pSTVG intoEnterobacter agglomerans or Serratia liquefacience, and evaluation of L-glutamic acid productivity
- TheEnterobacter agglomerans ATCC 12287, the Enterobacter agglomerans AJ13355 and the Serratia liquefacience ATCC 14460 were transformed with the RSFCPG, pMWCB and pSTVG by electroporation (Miller J. H., “A Short Course in Bacterial Genetics; Handbook” Cold Spring Harbor Laboratory Press, USA, 1992) to obtain transformants exhibiting tetracycline resistance.
- Each of the resulting transformants and the parent strains was inoculated into 50 ml-volume large size test tube containing 5 ml of a culture medium comprising 40 g/L glucose, 20 g/L ammonium sulfate, 0.5 g/L magnesium sulfate heptahydrate, 2 g/L potassium dihydrogenphosphate, 0.5 g/L sodium chloride, 0.25 g/L calcium chloride heptahydrate, 0.02 g/L ferrous sulfate heptahydrate, 0.02 g/L manganese sulfate tetrahydrate, 0.72 mg/L zinc sulfate dihydrate, 0.64 mg/L copper sulfate pentahydrate, 0.72 mg/L cobalt chloride hexahydrate, 0.4 mg/L boric acid, 1.2 mg/L sodium molybdate dihydrate, 2 g/L yeast extract, and 30 g/L calcium carbonate, and cultured at 37° C. with shaking until the glucose contained in the culture medium was consumed. However, as for the AJ13355/pMWCB strain and the AJ13355/pSTVG strain, the cultivation was stopped when about 10 g/L of glucose was consumed, i.e., cultivated for 15 hours like the parent strain AJ13355, because their glucose consumption rates were low. To the culture medium of the transformants, 25 mg/L of tetracycline was added. After the cultivation was completed, L-glutamic acid accumulated in the culture medium was measured. The results are shown in Table 1.
TABLE 1 Accumulated amount of L-glutamic acid Accumulated amount of L- Bacterial strain glutamic acid ATCC12287 0.0 g/L ATCC12287/RSFCPG 6.1 AJ13355 0.0 AJ13355/RSFCPG 3.3 AJ13355/pMWCB 0.8 AJ13355/pSTVG 0.8 ATCC14460 0.0 ATCC14460/RSFCPG 2.8 Culture medium alone 0.2 - While theEnterobacter agglomerans ATCC12287, the Enterobacter agglomerans AJ13355 and the Serratia liquefacience ATCC14460 did not accumulate L-glutamic acid, the strains whose CS, PEPC and GDH activities were amplified by introducing RSFCPG accumulated 6.1 g/L, 3.3 g/L, and 2.8 g/L of L-glutamic acid, respectively. The AJ13355 strain of which CS activity alone was amplified accumulated 0.8 g/L of L-glutamic acid, and the strain of which GDH activity alone was amplified also accumulated 0.8 g/L of L-glutamic acid.
- (3) Cloning of αKGDH gene (referred to as “sucAB” hereinafter) ofEnterobacter agglomerans AJ13355
- The sucAB gene of the Enterobacter agglomerans AJ13355 was cloned by selecting a DNA fragment complementing acetate non-assimilation of anEscherichia coli strain lacking the αKGDH-E1 subunit gene (referred to as “sucA” hereinafter) from the chromosome DNA of the Enterobacter agglomerans AJ13355.
- The chromosome DNA of theEnterobacter agglomerans AJ13355 strain was isolated by the same method as conventionally used for extracting chromosome DNA from Escherichia coli (Seibutsu Kogaku Jikken-sho (Textbook of Bioengineering Experiments), Ed. by the Society of Fermentation and Bioengineering, Japan, p.97-98, Baifukan, 1992). The pTWV228 used as the vector (ampicillin resistant) was a marketed product from Takara Shuzo.
- Products obtained by digesting the chromosome DNA of the AJ13355 strain with EcoT221 and products obtained by digesting the pTWV228 with PstI were ligated by T4 ligase, and theEscherichia coli JRG465 lacking sucA (Herbert J. et al., Mol. Gen. Genetics, 1969, 105, p.182) was transformed with them. Strains grown on the acetic acid minimal medium were selected from the transformants obtained as described above, and a plasmid extracted from them was designated as pTWVEK101. The Escherichia coli JRG465 carrying the pTWVEK101 recovered the characteristics of acetate non-assimilability as well as auxotrophy for succinic acid or L-lysine and L-methionine. This suggests that the pTWVEK101 contains the sucA gene of Enterobacter agglomerans.
- A restriction map ofEnterobacter agglomerans-derived DNA fragment of pTWVEK101 is shown in FIG. 6. The result of nucleotide sequencing of the hatched portion in FIG. 6 is shown in SEQ ID NO: 1. In this sequence, two full length ORFs and two nucleotides sequences considered as partial sequences of ORFs were found. Amino acid sequences that can be encoded by these ORFs and the partial sequences thereof are shown in SEQ ID NOS: 2 to 5 in order from the 5′ ends. As a result of homology analysis of these sequences, it was found that the portion of which nucleotide sequence had been determined contained a 3′ partial sequence of succinate dehydrogenase iron-sulfur protein gene (sdhB), full length sucA and αKGDH-E2 subunit gene (sucB gene), and 5′ partial sequence of succinyl-CoA synthetase β subunit gene (sucC gene). Comparison of the amino acid sequences deduced from these nucleotide sequences with those of Escherichia coli. (Eur. J. Biochem., 141, 351-359 (1984), Eur. J. Biochem., 141, 361-374 (1984), and Biochemistry, 24, 6245-6252 (1985)) is shown in FIGS. 7 to 9. As shown by these results, the amino acid sequences exhibited markedly high homology. It was also found that a cluster of sdhB-sucA-sucB-sucC is formed on the Enterobacter agglomerans chromosome like Escherichia coli (Eur. J. Biochem., 141, 351-359 (1984), Eur. J. Biochem., 141, 361-374 (1984), and Biochemistry, 24, 6245-6252 (1985)).
- (4) Acquisition of strain deficient in αKGDH derived fromEnterobacter agglomerans AJ13355
- Using the sucAB gene ofEnterobacter agglomerans obtained as described above, a strain lacking αKGDH of Enterobacter agglomerans was obtained by homologous recombination.
- First, pTWVEK101 was digested with BglII to remove the C-terminus region corresponding to about half of the sucA gene and the full length of the sucB gene. To this site, a chloramphenicol resistance gene fragment cut out from the pHSG399 (Takara Shuzo) with AccI was then inserted. The region of sdhB-ΔsucAB::Cmr-sucC obtained above was cut out with AflII and SacI. The resulting DNA fragment was used to transform the Enterobacter agglomerans AJ13355 strain by electroporation to obtain a chloramphenicol resistant strain, and thus a Enterobacter agglomerans AJ13356 strain lacking the sucAB gene where the sucAB gene on the chromosome was replaced by sucAB::Cmr was obtained.
- To confirm that the AJ13356 strain obtained as described above was deficient in the αKGDH activity, its enzymatic activity was determined by the method of Reed (L. J. Reed and B. B. Mukherjee, Methods in Enzymology 1969, 13, p.55-61). As a result, the αKGDH activity could not be detected in the AJ13356 strain as shown in Table 2, and thus it was confirmed that the strain lacked the sucAB as desired.
TABLE 2 αKGDH activity αKGDH activity (ΔABS/min/mg Bacterial strain protein) AJ13355 0.481 AJ13356 <0.0001 - (5) Evaluation of L-glutamic acid productivity ofEnterobacter agglomerans strain deficient in αKGDH
- Each of the AJ13355 and AJ13356 strains was inoculated into a 500 ml-volume flask containing 20 ml of a culture medium comprising 40 g/L glucose, 20 g/L ammonium sulfate, 0.5 g/L magnesium sulfate heptahydrate, 2 g/L potassium dihydrogenphosphate, 0.5 g/L sodium chloride, 0.25 g/L calcium chloride heptahydrate, 0.02 g/L ferrous sulfate heptahydrate, 0.02 g/L manganese sulfate tetrahydrate, 0.72 mg/L zinc sulfate dihydrate, 0.64 mg/L copper sulfate pentahydrate, 0.72 mg/L cobalt chloride hexahydrate, 0.4 mg/L boric acid, 1.2 mg/L sodium molybdate dihydrate, 2 g/L yeast extract, 30 g/L calcium carbonate, 200 mg/L L-lysine monohydrochloride, 200 mg/L L-methionine and 200 mg/L DL-α,ε-diaminopimelic acid (DAP), and cultured at 37° C. with shaking until the glucose contained in the culture medium was consumed. After the cultivation was completed, L-glutamic acid and α-ketoglutaric acid (abbreviated as “αKG” hereinafter) accumulated in the culture medium were measured. The results are shown in Table 3.
TABLE 3 Accumulated amounts of L-glutamic acid and αKG Accumulated Bacterial amount of L- Accumulated strain glutamic acid amount of αKG AJ13355 0.0 g/L 0.0 g/L AJ13356 1.5 3.2 - The AJ13356 strain deficient in the αKGDH activity accumulated 1.5 g/L of L-glutamic acid, and simultaneously accumulated 3.2 g/L of αKG.
- (6) Introduction of RSFCPG intoEnterobacter agglomerans strain lacking αKGDH and evaluation of L-glutamic acid productivity
- The AJ13356 strain was transformed with the RSFCPG, and the resulting strain introduced with the RSFCPG, AJ13356/RSFCPG, was inoculated into a 500 ml-volume flask containing 20 ml of a culture medium comprising 40 g/L glucose, 20 g/L ammonium sulfate, 0.5 g/L magnesium sulfate heptahydrate, 2 g/L potassium dihydrogenphosphate, 0.5 g/L sodium chloride, 0.25 g/L calcium chloride heptahydrate, 0.02 g/L ferrous sulfate heptahydrate, 0.02 g/L manganese sulfate tetrahydrate, 0.72 mg/L zinc sulfate dihydrate, 0.64 mg/L copper sulfate pentahydrate, 0.72 mg/L cobalt chloride hexahydrate, 0.4 mg/L boric acid, 1.2 mg/L sodium molybdate dihydrate, 2 g/L yeast extract, 25 mg/L tetracycline, 30 g/L calcium carbonate, 200 mg/L L-lysine monohydrochloride, 200 mg/L L-methionine and 200 mg/L DL-α,ε-DAP, and cultured at 37° C. with shaking until the glucose contained in the culture medium was consumed. After the cultivation was completed, L-glutamic acid and αKG accumulated in the culture medium were measured. The results are shown in Table 4.
TABLE 4 Accumulated amounts of L-glutamic acid and αKG Accumulated Bacterial amount of L- Accumulated strain glutamic acid amount of αKG AJ13356 1.4 g/L 2.9 g/L AJ13356/RSFCPG 5.1 0.0 - In the strain of which CS, PEPC and GDH activities were amplified by the introduction of RSFCPG, the accumulated amount of αKG was reduced, and the accumulated amount of L-glutamic acid was further improved.
Claims (6)
1. A microorganism belonging to the genus Enterobacter or Serratia and having an ability to produce L-glutamic acid and at least one of the following properties:
(a) the microorganism increases in an activity of an enzyme catalyzing a reaction for the L-glutamic acid biosynthesis; and
(b) the microorganism decreases in or is deficient in an activity of an enzyme catalyzing a reaction branching from a pathway for L-glutamic acid biosynthesis and producing a compound other than L-glutamic acid.
2. A microorganism according to wherein the enzyme catalyzing the reaction for the L-glutamic acid biosynthesis is at least one selected from the group consisting of citrate synthase, phosphoenolpyruvate carboxylase, and glutamate dehydrogenase.
claim 1
3. A microorganism according to wherein the enzyme catalyzing the reaction for the L-glutamic acid biosynthesis includes all of citrate synthase, phosphoenolpyruvate carboxylase, and glutamate dehydrogenase.
claim 2
4. A microorganism according to any one of to wherein the enzyme catalyzing the reaction branching from the pathway for L-glutamic acid biosynthesis and producing the compound other than L-glutamic acid is α-ketoglutarate dehydrogenase.
claims 1
3
5. A microorganism according to any one of to which is Enterobacter agglomerans or Serratia liquefacience.
claims 1
4
6. A method for producing L-glutamic acid which comprises culturing the microorganism as defined in any one of to in a liquid culture medium to produce and accumulate L-glutamic acid in the culture medium, and collecting the L-glutamic acid from the culture medium.
claims 1
5
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US09/784,208 US20010019836A1 (en) | 1998-03-18 | 2001-02-16 | L-glutamic acid-producing bacterium and method for producing L-glutamic acid |
US10/315,023 US20030119153A1 (en) | 1998-03-18 | 2002-12-10 | L-glutamic acid-producing bacterium and method for producing L-glutamic acid |
US11/624,080 US8129151B2 (en) | 1998-03-18 | 2007-01-17 | L-glutamic acid-producing bacterium and method for producing L-glutamic acid |
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP6906898 | 1998-03-18 | ||
JP10-69068 | 1998-10-19 | ||
JP29712998 | 1998-10-19 | ||
US09/271,438 US6331419B1 (en) | 1998-03-18 | 1999-03-18 | L-glutamic acid-producing bacterium and method for producing L-glutamic acid |
US09/784,208 US20010019836A1 (en) | 1998-03-18 | 2001-02-16 | L-glutamic acid-producing bacterium and method for producing L-glutamic acid |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US09/271,438 Continuation US6331419B1 (en) | 1998-03-18 | 1999-03-18 | L-glutamic acid-producing bacterium and method for producing L-glutamic acid |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US10/315,023 Continuation US20030119153A1 (en) | 1998-03-18 | 2002-12-10 | L-glutamic acid-producing bacterium and method for producing L-glutamic acid |
Publications (1)
Publication Number | Publication Date |
---|---|
US20010019836A1 true US20010019836A1 (en) | 2001-09-06 |
Family
ID=26410250
Family Applications (4)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US09/271,438 Expired - Lifetime US6331419B1 (en) | 1998-03-18 | 1999-03-18 | L-glutamic acid-producing bacterium and method for producing L-glutamic acid |
US09/784,208 Abandoned US20010019836A1 (en) | 1998-03-18 | 2001-02-16 | L-glutamic acid-producing bacterium and method for producing L-glutamic acid |
US10/315,023 Abandoned US20030119153A1 (en) | 1998-03-18 | 2002-12-10 | L-glutamic acid-producing bacterium and method for producing L-glutamic acid |
US11/624,080 Expired - Fee Related US8129151B2 (en) | 1998-03-18 | 2007-01-17 | L-glutamic acid-producing bacterium and method for producing L-glutamic acid |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US09/271,438 Expired - Lifetime US6331419B1 (en) | 1998-03-18 | 1999-03-18 | L-glutamic acid-producing bacterium and method for producing L-glutamic acid |
Family Applications After (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US10/315,023 Abandoned US20030119153A1 (en) | 1998-03-18 | 2002-12-10 | L-glutamic acid-producing bacterium and method for producing L-glutamic acid |
US11/624,080 Expired - Fee Related US8129151B2 (en) | 1998-03-18 | 2007-01-17 | L-glutamic acid-producing bacterium and method for producing L-glutamic acid |
Country Status (13)
Country | Link |
---|---|
US (4) | US6331419B1 (en) |
EP (2) | EP0952221B1 (en) |
KR (2) | KR100626102B1 (en) |
CN (1) | CN100402657C (en) |
AU (1) | AU756507B2 (en) |
BR (1) | BR9901173B1 (en) |
DE (2) | DE69921881T2 (en) |
ES (2) | ES2327838T3 (en) |
ID (1) | ID22246A (en) |
MY (1) | MY126522A (en) |
PE (1) | PE20000342A1 (en) |
PL (1) | PL196781B1 (en) |
TW (1) | TWI235179B (en) |
Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1233070A2 (en) * | 2001-02-20 | 2002-08-21 | Ajinomoto Co., Ltd. | Method for producing L-glutamic acid |
US20030119153A1 (en) * | 1998-03-18 | 2003-06-26 | Ajinomoto Co. Inc. | L-glutamic acid-producing bacterium and method for producing L-glutamic acid |
US20060084151A1 (en) * | 2003-05-07 | 2006-04-20 | Hiroshi Ueda | Method for producing L-glutamic acid |
US7208296B2 (en) | 1999-08-20 | 2007-04-24 | Ajinomoto Co., Inc. | Method for producing L-glutamic acid by fermentation accompanied by precipitation |
US20070092953A1 (en) * | 2001-02-05 | 2007-04-26 | Ajinomoto Co. Inc | Method for producing l-glutamine by fermentation and l-glutamine producing bacterium |
US20090286290A1 (en) * | 2006-12-19 | 2009-11-19 | Yoshihiko Hara | Method for producing an l-amino acid |
USRE41800E1 (en) | 2001-02-20 | 2010-10-05 | Ajinomoto Co., Inc. | Method for producing l-glutamic acid |
US8222007B2 (en) | 2006-08-18 | 2012-07-17 | Ajinomoto Co., Inc. | L-glutamic acid producing bacterium and a method for producing L-glutamic acid |
Families Citing this family (100)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
PE20001128A1 (en) * | 1998-10-19 | 2000-11-14 | Ajinomoto Kk | L-GLUTAMIC ACID PRODUCING BACTERIA AND PROCEDURE TO OBTAIN L-GLUTAMIC ACID |
AU2005200716B2 (en) * | 1999-08-20 | 2008-02-14 | Ajinomoto Co., Inc. | Method for producing L-glutamic acid by fermentation accompanied by precipitation |
JP2002238592A (en) | 2001-02-20 | 2002-08-27 | Ajinomoto Co Inc | Method for producing l-glutamic acid |
JP4292724B2 (en) | 2001-02-20 | 2009-07-08 | 味の素株式会社 | Organic nitrogen-containing composition and fertilizer containing the same |
US7052883B2 (en) | 2001-04-03 | 2006-05-30 | Degussa Ag | Process for the production of L-amino acids using strains of the family Enterobacteriaceae that contain an attenuated fruR gene |
DE10116518A1 (en) * | 2001-04-03 | 2002-10-17 | Degussa | Process for the fermentative production of L-amino acids using strains of the Enterobacteriaceae family |
EP1407022B1 (en) * | 2001-07-18 | 2009-09-02 | Evonik Degussa GmbH | Process for the preparation of l-amino acids using strains of the enterobacteriaceae family which contain an enhanced rsea gene |
US20050130256A1 (en) * | 2001-09-26 | 2005-06-16 | Northeastern University | Isolation and cultivation of microorganisms from natural environments and drug discovery based thereon |
US20030059866A1 (en) * | 2001-09-26 | 2003-03-27 | Kim Lewis | Isolation and cultivation of microorganisms from natural environments and drug discovery based thereon |
US7011957B2 (en) * | 2001-09-26 | 2006-03-14 | Northeastern University | Isolation and cultivation of microorganisms from natural environments and drug discovery based thereon |
JP3932945B2 (en) | 2002-03-27 | 2007-06-20 | 味の素株式会社 | Method for producing L-amino acid |
EP1655374B1 (en) * | 2003-06-10 | 2014-10-15 | Ajinomoto Co., Inc. | Process for producing l-glutamic acid |
US7344874B2 (en) * | 2004-03-04 | 2008-03-18 | Ajinomoto Co., Inc. | L-glutamic acid-producing microorganism and a method for producing L-glutamic acid |
US7501282B2 (en) * | 2005-02-25 | 2009-03-10 | Ajinomoto Co., Inc. | Plasmid autonomously replicable in Enterobacteriaceae family |
JP2009118740A (en) | 2006-03-03 | 2009-06-04 | Ajinomoto Co Inc | Method for producing l-amino acid |
EP2055771A3 (en) | 2006-03-23 | 2009-07-08 | Ajinomoto Co., Inc. | A method for producing an L-amino acid using bacterium of the Enterobacteriaceae family with attenuated expression of a gene coding for small RNA |
EP2007873B1 (en) | 2006-04-18 | 2015-11-18 | Ajinomoto Co., Inc. | A METHOD FOR PRODUCING AN L-AMINO ACID USING A BACTERIUM OF THE ENTEROBACTERIACEAE FAMILY WITH ATTENUATED EXPRESSION OF THE sfmACDFH-fimZ CLUSTER OR THE fimZ GENE |
JP2009165355A (en) | 2006-04-28 | 2009-07-30 | Ajinomoto Co Inc | L-amino acid-producing microorganism and method for producing l-amino acid |
JP2010017082A (en) | 2006-10-10 | 2010-01-28 | Ajinomoto Co Inc | Method for producing l-amino acid |
RU2006143864A (en) | 2006-12-12 | 2008-06-20 | Закрытое акционерное общество "Научно-исследовательский институт Аджиномото-Генетика" (ЗАО АГРИ) (RU) | METHOD FOR PRODUCING L-AMINO ACIDS USING THE BACTERIA OF THE ENTEROBACTERIACEAE FAMILY IN WHICH THE EXPRESSION OF GENES cynT, cynS, cynX, OR cynR, OR THEIR COMBINATION IS DECREASED |
ES2631360T3 (en) | 2007-01-22 | 2017-08-30 | Ajinomoto Co., Inc. | Microorganism that produces an L-amino acid and procedure to produce an L-amino acid |
JP2010088301A (en) | 2007-02-01 | 2010-04-22 | Ajinomoto Co Inc | Method for production of l-amino acid |
JP2010110216A (en) | 2007-02-20 | 2010-05-20 | Ajinomoto Co Inc | Method for producing l-amino acid or nucleic acid |
JP2010110217A (en) | 2007-02-22 | 2010-05-20 | Ajinomoto Co Inc | L-amino acid-producing microorganism and method for producing l-amino acid |
JP2010130899A (en) | 2007-03-14 | 2010-06-17 | Ajinomoto Co Inc | Microorganism producing l-glutamic acid-based amino acid, and method for producing amino acid |
EP2149608A4 (en) | 2007-04-16 | 2013-02-20 | Ajinomoto Kk | Method for production of organic acid |
EP2147970B1 (en) | 2007-04-17 | 2014-12-31 | Ajinomoto Co., Inc. | A method for producing an acidic substance having a carboxyl group |
EP2192170B1 (en) | 2007-09-04 | 2017-02-15 | Ajinomoto Co., Inc. | Amino acid-producing microorganism and method of producing amino acid |
JP5644108B2 (en) | 2007-12-06 | 2014-12-24 | 味の素株式会社 | Method for producing organic acid |
US7953493B2 (en) | 2007-12-27 | 2011-05-31 | Ebr Systems, Inc. | Optimizing size of implantable medical devices by isolating the power source |
JP2011067095A (en) | 2008-01-10 | 2011-04-07 | Ajinomoto Co Inc | Method for producing target substance by fermentation process |
WO2009093703A1 (en) | 2008-01-23 | 2009-07-30 | Ajinomoto Co., Inc. | Method of producing l-amino acid |
RU2008105793A (en) | 2008-02-19 | 2009-08-27 | Закрытое акционерное общество "Научно-исследовательский институт Аджиномото-Генетика" (ЗАО АГРИ) (RU) | METHOD FOR DESIGNING OPERONS CONTAINING TRANSLATION-CONJUGATED GENES, BACTERIA CONTAINING SUCH OPERON, METHOD FOR PRODUCING USEFUL METABOLITIS AND METHOD FOR EXPRESS MONITORING |
JP5476545B2 (en) | 2008-02-21 | 2014-04-23 | 味の素株式会社 | L-cysteine producing bacterium and method for producing L-cysteine |
JP5332237B2 (en) | 2008-03-06 | 2013-11-06 | 味の素株式会社 | L-cysteine producing bacterium and method for producing L-cysteine |
KR100955884B1 (en) | 2008-04-07 | 2010-05-06 | 고려대학교 산학협력단 | Method for detection of Enterobacter sakazakii using salicin and medium for detection of Enterobacter sakazakii |
WO2010027022A1 (en) | 2008-09-05 | 2010-03-11 | 味の素株式会社 | Bacterium capable of producing l-amino acid, and method for producing l-amino acid |
BRPI0918299B1 (en) * | 2008-09-08 | 2018-08-14 | Ajinomoto Co., Inc. | METHOD FOR PRODUCING L-AMINO ACID |
RU2411289C2 (en) * | 2008-09-30 | 2011-02-10 | Закрытое акционерное общество "Научно-исследовательский институт Аджиномото-Генетика" (ЗАО АГРИ) | Pantoea genus bacteria, producer of l-aspartate or metabolite, which is derivative of l-aspartate, and method of producing l-aspartate or metabolite which is derivative of l-aspartate |
JP2010142200A (en) | 2008-12-22 | 2010-07-01 | Ajinomoto Co Inc | Method for producing l-lysine |
WO2010084995A2 (en) | 2009-01-23 | 2010-07-29 | Ajinomoto Co.,Inc. | A method for producing an l-amino acid |
JP5359409B2 (en) | 2009-03-12 | 2013-12-04 | 味の素株式会社 | L-cysteine producing bacterium and method for producing L-cysteine |
WO2011013707A1 (en) | 2009-07-29 | 2011-02-03 | 味の素株式会社 | Method for producing l-amino acid |
WO2011021717A2 (en) | 2009-08-21 | 2011-02-24 | Ajinomoto Co.,Inc. | Method for producing hydroxylated amino acids |
CN102639691B (en) | 2009-11-30 | 2014-04-16 | 味之素株式会社 | L-cysteine-producing bacterium, and process for production of L-cysteine |
RU2010101135A (en) | 2010-01-15 | 2011-07-20 | Закрытое акционерное общество "Научно-исследовательский институт "Аджиномото-Генетика" (ЗАО АГРИ) (RU) | BACTERIA OF THE ENTEROBACTERIACEAE FAMILY - PRODUCER OF L-ASAPPARATE OR METABOLITES, L-ASPARATE DERIVATIVES, AND METHOD OF PRODUCING L-ASAPPARATE OR METABOLITES, PRODUCED L-ASAPPARATE |
RU2460793C2 (en) | 2010-01-15 | 2012-09-10 | Закрытое акционерное общество "Научно-исследовательский институт "Аджиномото-Генетика" (ЗАО АГРИ) | Method for producing l-amino acids with use of bacteria of enterobacteriaceae family |
JP2013074795A (en) | 2010-02-08 | 2013-04-25 | Ajinomoto Co Inc | MUTANT rpsA GENE AND METHOD FOR PRODUCING L-AMINO ACID |
RU2471868C2 (en) | 2010-02-18 | 2013-01-10 | Закрытое акционерное общество "Научно-исследовательский институт "Аджиномото-Генетика" (ЗАО АГРИ) | Mutant adenylate cyclase, dna coding it, bacteria of enterobacteriaceae family containing said dan and method for preparing l-amino acids |
RU2501858C2 (en) | 2010-07-21 | 2013-12-20 | Закрытое акционерное общество "Научно-исследовательский институт "Аджиномото-Генетика" (ЗАО АГРИ) | METHOD FOR OBTAINING L-AMINOACID USING BACTERIUM OF Enterobacteriaceae FAMILY |
RU2482188C2 (en) | 2010-07-21 | 2013-05-20 | Закрытое акционерное общество "Научно-исследовательский институт "Аджиномото-Генетика" (ЗАО АГРИ) | METHOD FOR PREPARING L-ARGININE WITH USE OF BACTERIA OF GENUS Escherichia WHEREIN astCADBE OPERON IS INACTIVATED |
WO2012036151A1 (en) | 2010-09-14 | 2012-03-22 | 味の素株式会社 | Sulfur amino acid-producing bacteria and method for producing sulfur amino acids |
JP2014036576A (en) | 2010-12-10 | 2014-02-27 | Ajinomoto Co Inc | Method for producing l-amino acids |
JP2014087259A (en) | 2011-02-22 | 2014-05-15 | Ajinomoto Co Inc | L-cysteine-producing bacterium, and production method of l-cysteine |
JP6020443B2 (en) | 2011-04-01 | 2016-11-02 | 味の素株式会社 | Method for producing L-cysteine |
JP2014131487A (en) | 2011-04-18 | 2014-07-17 | Ajinomoto Co Inc | Method for producing l-cysteine |
RU2496867C2 (en) | 2011-04-25 | 2013-10-27 | Закрытое акционерное общество "Научно-исследовательский институт Аджиномото-Генетика" (ЗАО "АГРИ") | Method to produce l-amino acid of glutamate family using coryneformic bacterium |
EP2711013A4 (en) | 2011-05-18 | 2015-03-04 | Ajinomoto Kk | Immunostimulant for animals, feed containing same, and method for manufacturing same |
EP2738247B1 (en) | 2011-07-29 | 2016-09-14 | Mitsui Chemicals, Inc. | Microorganism having carbon dioxide fixation cycle introduced thereinto |
RU2011134436A (en) | 2011-08-18 | 2013-10-27 | Закрытое акционерное общество "Научно-исследовательский институт Аджиномото-Генетика" (ЗАО "АГРИ") | METHOD FOR PRODUCING L-AMINO ACID USING THE ENTEROBACTERIACEAE FAMILY POSSESSING AN INCREASED EXPRESSION OF GENES OF THE CASCADE OF THE FORMATION OF FLAGELLS AND CELL MOBILITY |
WO2013069634A1 (en) | 2011-11-11 | 2013-05-16 | 味の素株式会社 | Method for producing target substance by fermentation |
WO2013179711A1 (en) | 2012-05-29 | 2013-12-05 | 味の素株式会社 | Method for producing 3-acetylamino-4-hydroxybenzoic acid |
JP6169073B2 (en) | 2012-05-30 | 2017-07-26 | 株式会社ブリヂストン | Isoprene synthase, polynucleotide encoding the same, and method for producing isoprene monomer |
WO2014115896A1 (en) | 2013-01-24 | 2014-07-31 | Mitsui Chemicals, Inc. | Microorganism for production of chemicals derived from acetyl-coa |
MY172023A (en) | 2013-01-24 | 2019-11-12 | Mitsui Chemicals Inc | Microorganism having carbon dioxide fixation cycle introduced thereinto |
RU2013118637A (en) | 2013-04-23 | 2014-10-27 | Закрытое акционерное общество "Научно-исследовательский институт Аджиномото-Генетика" (ЗАО "АГРИ") | METHOD FOR PRODUCING L-AMINO ACIDS USING THE BACTERIA OF THE ENTEROBACTERIACEAE FAMILY IN WHICH THE yjjK GENE IS ADJUSTABLE |
JP2016165225A (en) | 2013-07-09 | 2016-09-15 | 味の素株式会社 | Method for producing useful substance |
RU2013140115A (en) | 2013-08-30 | 2015-03-10 | Закрытое акционерное общество "Научно-исследовательский институт Аджиномото-Генетика" (ЗАО "АГРИ") | METHOD FOR PRODUCING L-AMINO ACIDS USING THE BACTERIA OF THE Enterobacteriaceae FAMILY IN WHICH THE EXPRESSION OF THE znuACB GENERAL CLUSTER IS DISORDERED |
JP2016192903A (en) | 2013-09-17 | 2016-11-17 | 味の素株式会社 | Method for manufacturing l-amino acid from biomass derived from seaweed |
RU2013144250A (en) | 2013-10-02 | 2015-04-10 | Закрытое акционерное общество "Научно-исследовательский институт Аджиномото-Генетика" (ЗАО "АГРИ") | METHOD FOR PRODUCING L-AMINO ACIDS USING THE BACTERIA OF THE Enterobacteriaceae FAMILY IN WHICH THE EXPRESSION OF A GENE CODING A PHOSPHATE TRANSPORTER IS DECREASED |
WO2015050234A1 (en) | 2013-10-02 | 2015-04-09 | 味の素株式会社 | Ammonia control apparatus and ammonia control method |
JP6459962B2 (en) | 2013-10-21 | 2019-01-30 | 味の素株式会社 | Method for producing L-amino acid |
JPWO2015076392A1 (en) | 2013-11-22 | 2017-03-16 | 味の素株式会社 | Modified isoprene synthase |
JPWO2015080273A1 (en) | 2013-11-28 | 2017-03-16 | 味の素株式会社 | Method for producing isoprene monomer |
RU2014105547A (en) | 2014-02-14 | 2015-08-20 | Адзиномото Ко., Инк. | METHOD FOR PRODUCING L-AMINO ACIDS USING THE ENTEROBACTERIACEAE FAMILY HAVING A SUPER EXPRESSED GENE yajL |
RU2014112066A (en) | 2014-03-28 | 2015-10-10 | Адзиномото Ко., Инк. | METHOD FOR PRODUCING MONOMERIC ISOPRENE |
JP2017216881A (en) | 2014-12-26 | 2017-12-14 | 味の素株式会社 | Method for producing dicarboxylate |
RU2015120052A (en) | 2015-05-28 | 2016-12-20 | Аджиномото Ко., Инк. | A method for producing an L-amino acid using a bacterium of the Enterobacteriaceae family in which gshA gene expression is weakened |
BR112018017227A2 (en) | 2016-02-25 | 2019-02-05 | Ajinomoto Kk | method to produce an l-amino acid |
US11248207B2 (en) * | 2016-05-31 | 2022-02-15 | Toray Industries, Inc. | Method for producing alpha-hydromuconic acid |
US11078503B2 (en) * | 2016-05-31 | 2021-08-03 | Toray Industries, Inc. | Method for producing 3-hydroxyadipic acid |
WO2018079683A1 (en) | 2016-10-26 | 2018-05-03 | Ajinomoto Co., Inc. | Method for producing objective substance |
JP7074133B2 (en) | 2016-10-26 | 2022-05-24 | 味の素株式会社 | Manufacturing method of target substance |
EP3532627A1 (en) | 2016-10-26 | 2019-09-04 | Ajinomoto Co., Inc. | Method for producing objective substance |
EP3532628B1 (en) | 2016-10-26 | 2024-02-28 | Ajinomoto Co., Inc. | Method for producing objective substance |
WO2018079686A1 (en) | 2016-10-26 | 2018-05-03 | Ajinomoto Co., Inc. | Method for producing l-methionine or metabolites requiring s-adenosylmethionine for synthesis |
WO2018079705A1 (en) | 2016-10-27 | 2018-05-03 | Ajinomoto Co., Inc. | Method for producing aldehyde |
JP7066977B2 (en) | 2017-04-03 | 2022-05-16 | 味の素株式会社 | Manufacturing method of L-amino acid |
US10858676B2 (en) | 2017-05-22 | 2020-12-08 | Ajinomoto Co., Inc. | Method for producing objective substance |
US11680279B2 (en) | 2017-11-29 | 2023-06-20 | Ajinomoto Co., Inc. | Method for producing objective substance |
WO2019163827A1 (en) | 2018-02-20 | 2019-08-29 | 味の素株式会社 | Method for inducing rna silencing |
WO2020027251A1 (en) | 2018-08-03 | 2020-02-06 | Ajinomoto Co., Inc. | Method for producing objective substance |
WO2020071538A1 (en) | 2018-10-05 | 2020-04-09 | Ajinomoto Co., Inc. | Method for producing target substance by bacterial fermentation |
BR112021014194A2 (en) | 2019-02-22 | 2021-12-28 | Ajinomoto Kk | Method for producing an l-amino acid |
EP3960870A4 (en) | 2019-03-29 | 2023-06-07 | Ajinomoto Co., Inc. | Method for producing allolactose |
WO2020204179A1 (en) | 2019-04-05 | 2020-10-08 | Ajinomoto Co., Inc. | Method of producing l-amino acids |
WO2020226087A1 (en) | 2019-05-08 | 2020-11-12 | 味の素株式会社 | Vanillin production method |
WO2021060337A1 (en) | 2019-09-25 | 2021-04-01 | Ajinomoto Co., Inc. | Method for producing 2-methyl-butyric acid by bacterial fermentation |
WO2021060438A1 (en) | 2019-09-25 | 2021-04-01 | Ajinomoto Co., Inc. | Method for producing l-amino acids by bacterial fermentation |
US20240117393A1 (en) | 2022-09-30 | 2024-04-11 | Ajinomoto Co., Inc. | Method for producing l-amino acid |
Family Cites Families (47)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS329393B1 (en) | 1954-12-25 | 1957-11-07 | ||
GB864562A (en) | 1957-08-16 | 1961-04-06 | Ajinomoto Kk | A process for the production of glutamic acid or a salt thereof |
US3220929A (en) | 1964-02-10 | 1965-11-30 | Kyowa Hakko Kogyo Kk | Microbiological production of amino acid by reductive amination |
US3563857A (en) * | 1967-03-20 | 1971-02-16 | Sanraku Ocean Co | Process for producing l-glutamic acid by fermentation |
WO1980002433A1 (en) * | 1979-05-02 | 1980-11-13 | Nat Res Dev | The identification of bacteria |
GB2076853B (en) * | 1980-04-17 | 1983-12-21 | Ajinomoto Kk | L-glutamic acid producing microorganisms |
JPS589693A (en) * | 1981-07-03 | 1983-01-20 | Tanabe Seiyaku Co Ltd | Production of l-glutamic acid through fermentation process |
JPH0783714B2 (en) | 1983-08-29 | 1995-09-13 | 味の素株式会社 | Fermentation method for producing L-amino acid |
JPH0746994B2 (en) * | 1984-10-04 | 1995-05-24 | 味の素株式会社 | Method for producing L-amino acid by fermentation method |
JPS61268185A (en) | 1984-12-27 | 1986-11-27 | Asahi Chem Ind Co Ltd | Dna fragment containing glutamic acid dehydrogenase-producing gene |
FR2581653B1 (en) | 1985-05-10 | 1989-06-30 | Asahi Chemical Ind | DNA FRAGMENT CONTAINING A GENE ENCODING PHOSPHOENOLPYRUVATE CARBOXYLASE, RECOMBINANT DNA CONTAINING THE SAME, AND MICROORGANISM CONTAINING THE SAME |
JPS62201585A (en) | 1985-11-26 | 1987-09-05 | Asahi Chem Ind Co Ltd | Dna fragment containing citric acid synthase-producing gene |
JPH07121228B2 (en) | 1986-11-07 | 1995-12-25 | 協和醗酵工業株式会社 | Process for producing L-glutamic acid and L-proline |
JP2520895B2 (en) * | 1987-03-04 | 1996-07-31 | 旭化成工業株式会社 | Method for producing L-glutamic acid |
FR2680178B1 (en) * | 1991-08-07 | 1994-10-14 | Ajinomoto Kk | PROCESS FOR PRODUCING L-GLUTAMIC ACID BY FERMENTATION. |
HU219600B (en) * | 1993-08-24 | 2001-05-28 | Ajinomoto Co., Inc. | Variant phosphoenolpyruvate carboxylase, gene thereof, and process for producing amino acid |
HU221120B1 (en) | 1993-10-28 | 2002-08-28 | Ajinomoto Kk | Process for producing different substances with use of microorganism |
JP3880636B2 (en) * | 1994-01-10 | 2007-02-14 | 味の素株式会社 | Method for producing L-glutamic acid by fermentation |
RU2084520C1 (en) | 1994-01-19 | 1997-07-20 | Всесоюзный научно-исследовательский институт генетики и селекции промышленных микроорганизмов | Strain of bacterium brevibacterium flavum - a producer of l-glutamine (variants) |
JP2781954B2 (en) | 1994-03-04 | 1998-07-30 | メック株式会社 | Copper and copper alloy surface treatment agent |
US5821351A (en) | 1994-06-10 | 1998-10-13 | Dnx Biotherapeutics | Production of hemoglobin having a delta-like globin |
CN1177926C (en) * | 1994-06-14 | 2004-12-01 | 味之素株式会社 | Method for stick bacteria capable of producing L-glutaminic acid and producing 1-glutaminc acid |
CA2153561A1 (en) * | 1994-07-11 | 1996-01-12 | Ryoichi Katsumata | Process for producing an optically active .gamma.-hydroxy-l-glutamic acid |
EP0780477B1 (en) * | 1994-08-19 | 2003-04-09 | Ajinomoto Co., Inc. | Process for producing l-lysine and l-glutamic acid by fermentation |
US6110714A (en) * | 1995-08-23 | 2000-08-29 | Ajinomoto Co., Inc. | Process for producing L-glutamic acid by fermentation |
JPH09285294A (en) * | 1996-04-23 | 1997-11-04 | Ajinomoto Co Inc | Production of l-glutamic acid by fermentation |
AU746542B2 (en) * | 1998-03-18 | 2002-05-02 | Ajinomoto Co., Inc. | L-glutamic acid-producing bacterium and method for producing L-glutamic acid |
AU756507B2 (en) * | 1998-03-18 | 2003-01-16 | Ajinomoto Co., Inc. | L-glutamic acid-producing bacterium and method for producing L-glutamic acid |
PE20001128A1 (en) | 1998-10-19 | 2000-11-14 | Ajinomoto Kk | L-GLUTAMIC ACID PRODUCING BACTERIA AND PROCEDURE TO OBTAIN L-GLUTAMIC ACID |
JP4427878B2 (en) * | 1999-08-20 | 2010-03-10 | 味の素株式会社 | Method for producing L-glutamic acid by fermentation method with precipitation |
TW200734459A (en) * | 1999-10-04 | 2007-09-16 | Ajinomoto Kk | Genes for heat resistant enzymes of amino acid biosynthetic pathway derived from thermophilic coryneform bacteria |
JP4304837B2 (en) * | 2000-07-05 | 2009-07-29 | 味の素株式会社 | L-glutamic acid-producing bacterium and method for producing L-glutamic acid |
JP4560998B2 (en) * | 2001-02-05 | 2010-10-13 | 味の素株式会社 | Method for producing L-glutamine by fermentation and L-glutamine producing bacteria |
JP4599726B2 (en) * | 2001-02-20 | 2010-12-15 | 味の素株式会社 | Method for producing L-glutamic acid |
JP4292724B2 (en) * | 2001-02-20 | 2009-07-08 | 味の素株式会社 | Organic nitrogen-containing composition and fertilizer containing the same |
JP2002238592A (en) * | 2001-02-20 | 2002-08-27 | Ajinomoto Co Inc | Method for producing l-glutamic acid |
JP4599725B2 (en) * | 2001-02-20 | 2010-12-15 | 味の素株式会社 | Method for producing L-glutamic acid |
RU2230114C2 (en) * | 2001-11-30 | 2004-06-10 | Закрытое акционерное общество "Научно-исследовательский институт Аджиномото-Генетика" | Mutant glutamine synthetase, dna fragment, strain of escherichia coli as p roducer of l-glutamine and method for preparing l-amino acids |
JP3932945B2 (en) * | 2002-03-27 | 2007-06-20 | 味の素株式会社 | Method for producing L-amino acid |
US7344874B2 (en) * | 2004-03-04 | 2008-03-18 | Ajinomoto Co., Inc. | L-glutamic acid-producing microorganism and a method for producing L-glutamic acid |
US7205132B2 (en) * | 2004-09-10 | 2007-04-17 | Ajinomoto Co., Inc. | L-glutamic acid-producing microorganism and a method for producing L-glutamic acid |
US7794989B2 (en) * | 2004-12-28 | 2010-09-14 | Ajinomoto Co., Inc. | L-glutamic acid-producing microorganism and a method for producing L-glutamic acid |
US7501282B2 (en) * | 2005-02-25 | 2009-03-10 | Ajinomoto Co., Inc. | Plasmid autonomously replicable in Enterobacteriaceae family |
JP5011675B2 (en) * | 2005-08-09 | 2012-08-29 | 味の素株式会社 | Method for simulating material production process |
WO2007024010A1 (en) * | 2005-08-26 | 2007-03-01 | Ajinomoto Co., Inc. | L-glutamic acid-producing bacterium and method for production of l-glutamic acid |
JP2007117082A (en) * | 2005-09-27 | 2007-05-17 | Ajinomoto Co Inc | L-amino acid producing bacteria and preparation process of l-amino acid |
EP2054500B1 (en) * | 2006-08-18 | 2016-11-23 | Ajinomoto Co., Inc. | An l-glutamic acid producing bacterium and a method for producing l-glutamic acid |
-
1999
- 1999-03-16 AU AU21223/99A patent/AU756507B2/en not_active Ceased
- 1999-03-17 DE DE69921881T patent/DE69921881T2/en not_active Expired - Lifetime
- 1999-03-17 DE DE69941137T patent/DE69941137D1/en not_active Expired - Lifetime
- 1999-03-17 KR KR1019990008985A patent/KR100626102B1/en not_active IP Right Cessation
- 1999-03-17 BR BRPI9901173-5A patent/BR9901173B1/en not_active IP Right Cessation
- 1999-03-17 ID IDP990233D patent/ID22246A/en unknown
- 1999-03-17 ES ES04013665T patent/ES2327838T3/en not_active Expired - Lifetime
- 1999-03-17 ES ES99105508T patent/ES2232984T3/en not_active Expired - Lifetime
- 1999-03-17 EP EP99105508A patent/EP0952221B1/en not_active Expired - Lifetime
- 1999-03-17 EP EP04013665A patent/EP1462523B1/en not_active Expired - Lifetime
- 1999-03-17 MY MYPI99001009A patent/MY126522A/en unknown
- 1999-03-18 TW TW088104255A patent/TWI235179B/en not_active IP Right Cessation
- 1999-03-18 CN CNB991055497A patent/CN100402657C/en not_active Expired - Lifetime
- 1999-03-18 PL PL332072A patent/PL196781B1/en unknown
- 1999-03-18 PE PE1999000227A patent/PE20000342A1/en not_active IP Right Cessation
- 1999-03-18 US US09/271,438 patent/US6331419B1/en not_active Expired - Lifetime
-
2001
- 2001-02-16 US US09/784,208 patent/US20010019836A1/en not_active Abandoned
-
2002
- 2002-12-10 US US10/315,023 patent/US20030119153A1/en not_active Abandoned
-
2006
- 2006-05-19 KR KR1020060045355A patent/KR100626568B1/en not_active IP Right Cessation
-
2007
- 2007-01-17 US US11/624,080 patent/US8129151B2/en not_active Expired - Fee Related
Cited By (17)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20030119153A1 (en) * | 1998-03-18 | 2003-06-26 | Ajinomoto Co. Inc. | L-glutamic acid-producing bacterium and method for producing L-glutamic acid |
US8129151B2 (en) | 1998-03-18 | 2012-03-06 | Ajinomoto Co., Inc. | L-glutamic acid-producing bacterium and method for producing L-glutamic acid |
US20090263874A1 (en) * | 1998-03-18 | 2009-10-22 | Ajinomoto Co. Inc | L-glutamic acid-producing bacterium and method for producing l-glutamic acid |
US7208296B2 (en) | 1999-08-20 | 2007-04-24 | Ajinomoto Co., Inc. | Method for producing L-glutamic acid by fermentation accompanied by precipitation |
US20070134773A1 (en) * | 1999-08-20 | 2007-06-14 | Ajinomoto Co., Inc. | Method for producing l-glutamic acid by fermentation accompanied by precipitation |
US20090162907A1 (en) * | 1999-08-20 | 2009-06-25 | Ajinomoto Co., Inc. | Method for producing l-glutamic acid by fermentation accompanied by precipitation |
USRE42350E1 (en) | 1999-08-20 | 2011-05-10 | Ajinomoto Co., Inc. | Method for producing L-glutamic acid by fermentation accompanied by precipitation |
US20070092953A1 (en) * | 2001-02-05 | 2007-04-26 | Ajinomoto Co. Inc | Method for producing l-glutamine by fermentation and l-glutamine producing bacterium |
USRE41800E1 (en) | 2001-02-20 | 2010-10-05 | Ajinomoto Co., Inc. | Method for producing l-glutamic acid |
EP1233070A3 (en) * | 2001-02-20 | 2002-11-27 | Ajinomoto Co., Inc. | Method for producing L-glutamic acid |
US6653110B2 (en) | 2001-02-20 | 2003-11-25 | Ajinomoto Co., Inc. | Method for producing L-glutamic acid |
EP1233070A2 (en) * | 2001-02-20 | 2002-08-21 | Ajinomoto Co., Ltd. | Method for producing L-glutamic acid |
US20060084151A1 (en) * | 2003-05-07 | 2006-04-20 | Hiroshi Ueda | Method for producing L-glutamic acid |
US7294491B2 (en) | 2003-05-07 | 2007-11-13 | Ajinomoto Co., Inc. | Method for producing L-glutamic acid |
US8222007B2 (en) | 2006-08-18 | 2012-07-17 | Ajinomoto Co., Inc. | L-glutamic acid producing bacterium and a method for producing L-glutamic acid |
US20090286290A1 (en) * | 2006-12-19 | 2009-11-19 | Yoshihiko Hara | Method for producing an l-amino acid |
US8058035B2 (en) | 2006-12-19 | 2011-11-15 | Ajinomoto Co., Inc. | Method for producing an L-amino acid |
Also Published As
Publication number | Publication date |
---|---|
PE20000342A1 (en) | 2000-04-24 |
EP0952221A2 (en) | 1999-10-27 |
US20030119153A1 (en) | 2003-06-26 |
PL332072A1 (en) | 1999-09-27 |
DE69921881D1 (en) | 2004-12-23 |
DE69941137D1 (en) | 2009-08-27 |
US20090263874A1 (en) | 2009-10-22 |
KR100626102B1 (en) | 2006-09-20 |
MY126522A (en) | 2006-10-31 |
DE69921881T2 (en) | 2005-07-21 |
KR100626568B1 (en) | 2006-09-25 |
KR19990077973A (en) | 1999-10-25 |
PL196781B1 (en) | 2008-01-31 |
BR9901173A (en) | 2000-03-28 |
ES2327838T3 (en) | 2009-11-04 |
EP1462523A3 (en) | 2004-11-17 |
US6331419B1 (en) | 2001-12-18 |
EP0952221B1 (en) | 2004-11-17 |
EP1462523A2 (en) | 2004-09-29 |
ES2232984T3 (en) | 2005-06-01 |
US8129151B2 (en) | 2012-03-06 |
ID22246A (en) | 1999-09-23 |
KR20060061785A (en) | 2006-06-08 |
TWI235179B (en) | 2005-07-01 |
CN1233660A (en) | 1999-11-03 |
CN100402657C (en) | 2008-07-16 |
EP0952221A3 (en) | 2001-09-12 |
BR9901173B1 (en) | 2010-11-30 |
AU2122399A (en) | 1999-09-30 |
AU756507B2 (en) | 2003-01-16 |
EP1462523B1 (en) | 2009-07-15 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US8129151B2 (en) | L-glutamic acid-producing bacterium and method for producing L-glutamic acid | |
US6197559B1 (en) | L-glutamic acid-producing bacterium and method for producing L-glutamic acid | |
US6596517B2 (en) | Method for producing L-glutamic acid | |
US7208296B2 (en) | Method for producing L-glutamic acid by fermentation accompanied by precipitation | |
USRE41800E1 (en) | Method for producing l-glutamic acid | |
KR100674401B1 (en) | L-Glutamic acid producing bacterium and process for producing L-glutamic acid | |
JP4144098B2 (en) | L-glutamic acid-producing bacterium and method for producing L-glutamic acid | |
US6653110B2 (en) | Method for producing L-glutamic acid |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
STCB | Information on status: application discontinuation |
Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION |