WO2001005960A1 - Procede de production d'un acide l-amine - Google Patents

Procede de production d'un acide l-amine Download PDF

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Publication number
WO2001005960A1
WO2001005960A1 PCT/JP2000/004772 JP0004772W WO0105960A1 WO 2001005960 A1 WO2001005960 A1 WO 2001005960A1 JP 0004772 W JP0004772 W JP 0004772W WO 0105960 A1 WO0105960 A1 WO 0105960A1
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Prior art keywords
lysine
rrna gene
gene
producing
acid
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PCT/JP2000/004772
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English (en)
Japanese (ja)
Inventor
Eiichiro Kimura
Hisao Ito
Osamu Kurahashi
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Ajinomoto Co Inc
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Ajinomoto Co Inc
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Priority to AU60182/00A priority Critical patent/AU6018200A/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/14—Glutamic acid; Glutamine

Definitions

  • the present invention relates to a method for producing L-amino acid by a fermentation method, particularly to a method for producing L-lysine and L-glutamic acid.
  • L-Lysine is widely used as a feed additive
  • L-glutamic acid is widely used as a seasoning material.
  • L-amino acids such as L-lysine and L-glutamic acid have been fermented using a coryneform bacterium belonging to the genus Brevipacterium or Corynebacterium having the ability to produce these L-amino acids. It is industrially produced by the law. For these coryneform bacteria, strains isolated from the natural world or artificial mutants of the strains are used in order to improve productivity.
  • the L-glutamic acid dehydrogenase gene, the isocitrate dehydrogenase gene, the aconitate hydrase gene, and the citrate synthase gene are enhanced to increase L-glutamic acid.
  • a technique for increasing the productivity of minic acid has been disclosed (JP-A-63-214189).
  • the gene encoding rRNA has already been cloned in Escherichia coli and its nucleotide sequence has been reported (16S RNA gene: Brosius, J. et al., Proc. Natl. Acad. Sci.
  • An object of the present invention is to provide a method for producing an L-amino acid such as L-lysine or L-glutamic acid by a fermentation method which has been further improved, and a strain used therefor.
  • the present inventors have conducted intensive studies in order to solve the above problems, and as a result, introduced an operon encoding rRNA of Escherichia coli into a coryneform bacterium, thereby enhancing the expression level of rRNA.
  • the present inventors have found that the production amount of lysine or L-glucamic acid can be increased, and that the production rate of these L-amino acids can be increased, thereby completing the present invention.
  • the present invention is as follows.
  • a coryneform bacterium with enhanced rRNA gene expression in cells and L-amino acid producing ability (2) The coryneform bacterium according to (1), wherein the L-amino acid is selected from L-lysine and L-glucamic acid.
  • the coryneform bacterium according to any one of (1) to (5) is cultured in a medium, L-amino acids are produced and accumulated in the culture, and L-amino acids are collected from the culture.
  • the 16 S rRNA gene, 23 S rRNA gene or 5 S rRNA gene, or any one, two or three of these, or an operon containing them, is collectively referred to as rRNA gene. There is.
  • the coryneform bacterium of the present invention is a coryneform bacterium having an L-amino acid-producing ability and having enhanced expression of the 16SrRNA gene in cells.
  • the L-amino acid include various L-amino acids such as L-lysine, L-glutamic acid, L-threonine, L-one-isine, L-isodicine, L-parin, and L-phenylalanine. Among these, L-lysine and L-glutamic acid are preferred.
  • the coryneform bacteria referred to in the present invention include: A group of microorganisms defined in Bergey's Manual of Determinative Bacteriology, 8th Edition, p. 599 (1974), aerobic, Gram-positive, nonacid-fast, sporulation It is a bacillus that has no function and includes bacteria that were previously classified into the genus Brevibacterium but are now integrated as Corynebacterium genus (Int. J. Syst. Bacteriol., 41, 55 (1981)). In addition, it includes bacteria of the genus Brevipacterium and those of the genus Microbacterium which are very closely related to the genus Corynebacterium. Examples of the strains of coryneform bacteria suitably used for producing L-lysine or L-glucamic acid include, for example, those shown below. Corynebacterium acetate film ATCC 13870
  • Corynebacterium thermoaminogenes AJ12340 (FERM BP-1539) These can be obtained, for example, from the American 'Type' Culture 'collection. That is, a registration number corresponding to each microorganism is assigned, and the microorganism can be ordered by referring to this registration number. The registration number corresponding to each micro-organism is found in the catalog of the American Type 'Kartya I' collection.
  • the AJ12340 strain is a member of the Ministry of International Trade and Industry It has been deposited with the National Institute of Technology under the Budapest Treaty.
  • mutants having L-lysine-producing ability or L-glutamic acid-producing ability derived from these strains can also be used in the present invention.
  • Such human mutants include the following. S— (2-aminoethyl) one cysteine
  • AEC Resistant mutants
  • L-amino acid-producing ability refers to the ability of a coryneform bacterium to accumulate a significant amount of L-amino acid in a medium when cultured in the medium, or the amino acid content in the cells. The ability to increase.
  • the rRNA gene fragment is ligated with a vector, preferably a multicopy type vector, which functions in the bacterium to produce recombinant DNA. Then, this may be introduced into a coryneform bacterium capable of producing L-lysine or L-glutamic acid for transformation.
  • a vector preferably a multicopy type vector
  • the expression of the rRNA gene is enhanced.
  • the rRNA gene to be enhanced includes any one, two or three selected from 16 S rRNA gene, 23 SrRNA gene and 5 SrRNA gene, and all three are enhanced. Is preferably performed.
  • the 16S rRNA gene, the 23S: 3 ⁇ 4 gene and the 53rRNA gene are encoded by the rrnB operon together with a gene encoding glutamate tRNA (Glu-tRNA-2).
  • rRNA gene a gene of a coryneform bacterium or a gene derived from another organism such as a bacterium belonging to the genus Escherichia can be used.
  • Each rRNA gene or rrnB operon can be obtained by the PCR method (PCR: polymerase chain reaction; White, TJ et al; see Trends Genet. 5, 185 (1989)).
  • the rrnB operon can be obtained, for example, using the primers shown in SEQ ID NOs: 1 and 2 in the Sequence Listing.
  • RRNA genes of other microorganisms such as coryneform bacteria can be obtained in a similar manner.
  • the operon may contain Glu-tRNA-2, but it does not have to contain Glu-tRNA-2.
  • Chromosomal DNA is obtained from bacteria that are DNA donors, for example, according to the method of Saito and Miura. (See H. Saito and K. Miura Biochem. Biophys. Acta, 72, 619, (1963), Bioengineering Experiments, edited by The Society of Biotechnology, pages 97-98, Baifukan, 1992), etc. It can be prepared by
  • the rRNA gene amplified by the PCR method is connected to autonomously replicable vector DNA in the cells of Escherichia coli and / or coryneform bacteria to prepare recombinant DNA, which is introduced into Escherichia coli cells. Doing so will make subsequent operations difficult.
  • a vector capable of autonomous replication in Escherichia coli cells a plasmid vector is preferable, and a vector capable of autonomous replication in a host cell is preferable.
  • Examples of vectors capable of autonomous replication in coryneform bacteria cells include PAM330 (see Japanese Patent Publication No. 58-67699) and pHM1519 (see Japanese Patent Application Laid-Open No. 58-77895).
  • a DNA fragment having the ability to enable autonomous replication of plasmid in coryneform bacteria is extracted from one of these vectors, and inserted into the above-mentioned vector for Escherichia coli, whereby both Escherichia coli and coryneform bacteria become autonomous. It can be used as a shuttle vector that cannot be duplicated.
  • Such shuttle vectors include the following. Microorganisms carrying the respective vectors and the accession numbers of the international depository organizations are shown in parentheses.
  • PAJ1844 Escherichia 1 MJ11883 (FERM BP-137)
  • PAJ611 Escherichia AJU884 (FERM BP-138)
  • Ligation is usually performed using a ligase such as ⁇ 4DNA ligase.
  • the transformation may be performed according to the transformation method reported so far. For example, a method of increasing the permeability of DNA by treating recipient cells with calcium chloride, as reported for Escherichia coli K-12 (Mandel, M. and Higa, A., J. Mol. Biol., 53, 159 (1970)), and a method for preparing DNA from transgenic cells and introducing DNA as described in Bacillus subtilis (Duncan, CH, Wilson, GA and Young, FE, Gene, 1, 153 (1977)).
  • the DNA of the recipient DNA is transformed into a protoplast or spheroplast that readily incorporates the recombinant DNA, as is known for Bacillus subtilis, actinomycetes, and yeast.
  • a protoplast or spheroplast that readily incorporates the recombinant DNA, as is known for Bacillus subtilis, actinomycetes, and yeast.
  • the transformation method used in Examples of the present invention is the electric pulse method (see Japanese Patent Application Laid-Open No. 2-207791).
  • Enhancement of the rRNA gene can also be achieved by causing multiple copies of the rRNA gene to be present on the chromosome DNA of the host.
  • homologous recombination is performed by using a sequence present in multiple copies on the chromosomal DNA as a target.
  • a sequence present in multiple copies on the chromosome DNA it is possible to use reactive DNA, or an integrated repeat present at the end of a transposable element.
  • Enhancement of the expression of the rRNA gene can be achieved by replacing the expression regulatory sequence such as the promoter of the rRNA gene on the chromosomal DNA or plasmid with a strong one, in addition to the gene amplification described above.
  • a strong one in addition to the gene amplification described above.
  • lac promoter one, trp promoter, trc promoter Isseki one, tac promoter evening one, P R promoter of lambda phages, the P L promoter, and the like have been known as a powerful promoter Isseki scratch. The substitution of these promoters enhances the expression of the rRNA gene.
  • the coryneform bacterium of the present invention has enhanced enzymatic activities of the L-amino acid biosynthetic pathway or glycolytic pathway, in addition to enhancing the expression of the rRNA gene, thereby enhancing their enzymatic activities.
  • genes that can be used for the production of L-lysine include aspartokinase subunit protein and / or the synergistic feedback inhibition by L-lysine and L-threonine has been substantially released.
  • Subunit protein-encoding gene W094 / 25605 international publication pamphlet
  • Coryneform bacterium-derived wild-type phosphoenolpyruvate carboxylase gene Japanese Patent Publication No. 60-87788
  • Coryneform bacterium-derived wild-type Genes encoding dihydrodibicolinate synthase Japanese Patent Publication No. 6-55149
  • Homoserine dehydrogenase is an enzyme that catalyzes a reaction that diverges from the L-glucamic acid biosynthetic pathway to produce a compound other than L-glucamic acid (see WO95 / 23864).
  • genes that can be used for the production of L-glutamic acid include glycosylated phosphofructokinase (PFK, JP-A-63-102692), and phosphoenolpyruvate carboxylase of the anapretictic pathway.
  • an enzyme that catalyzes a reaction that branches off from the biosynthetic pathway of L-glucaminic acid to produce a compound other than L-glucaminic acid may be reduced or defective.
  • Enzymes that catalyze the reaction that diverges from the biosynthetic pathway of L-glucaminic acid to produce compounds other than L-glucaminic acid include ketoglutarate dehydrogenase (Hg).
  • KGDH isoquinate lyase, acetyl phosphate transferase, acetate kinase, acetate hydroxysynthesis, acetate lactate synthase, formate acetyl transferase, lactate dehydrogenase, glutamate decarboxylase , 1-pyrroline dehydrogenase, and the like.
  • a biotin action inhibitor such as a surfactant to a coryneform bacterium capable of producing L-glutamic acid
  • the biotin action inhibitor in a medium containing an excessive amount of biotin can be added.
  • L-glutamic acid can be produced in the absence of E. coli (see W096 / 06180).
  • Examples of such coryneform bacteria include Brevipacterium lactofermentum AJ13029 described in W096 / 06180. AJ13029 strain was registered on September 2, 1994 with the Institute of Biotechnology, Industrial Science and Technology (Postal Code 305-8566, Tsukuba East 1-3-1, Ibaraki, Japan) under the accession number FERM P-14501. Deposited and transferred to an international deposit under the Budapest Convention on August 1, 1995, and given accession number FERM BP-5189.
  • a coryneform bacterium having the ability to produce L-lysine and L-glutamic acid is subjected to a temperature-sensitive mutation against a biotin-inhibiting substance, so that the biotin-inhibiting substance can be expressed in a medium containing an excessive amount of biotin.
  • L-lysine and L-glutamic acid can be produced simultaneously in the absence (see W096 / 06180).
  • Such strains include Brevipacterium 'Lactofamentum AJ12993 strain described in W096 / 06180.
  • the strain was granted to the Institute of Biotechnology and Industrial Technology, Institute of Industrial Science and Technology (June 3, 1904, Tsukuba 1-chome 1-3, Ibaraki, Japan 305-8566, Japan) on June 3, 1994, with accession number FERM. Deposited under P-14348, transferred to an international deposit under the Budapest Treaty on August 1, 1995, and given accession number FERM BP-5188.
  • L-lysine and L-glutamic acid When a coryneform bacterium capable of producing an acid is cultured in a medium, L-lysine and L-glutamic acid accumulate in the medium. When L-lysine and L-glutamic acid are simultaneously produced by fermentation, the L-lysine-producing bacterium may be cultured under conditions for producing L-glutamic acid, or a coryneform strain having L-lysine-producing ability may be used. Bacteria and coryneform bacteria having an ability to produce L-glucamic acid may be mixed and cultured (Japanese Patent Application Laid-Open No. 5-37993).
  • the medium used for producing L-lysine and / or L-glucamic acid using the microorganism of the present invention may be a conventional medium containing a carbon source, a nitrogen source, inorganic ions, and if necessary, other organic micronutrients.
  • Carbon sources include carbohydrates such as glucose, lactose, galactose, fructose, sucrose, molasses, starch hydrolysates, alcohols such as ethanol and inositol, acetic acid, fumaric acid, and quen.
  • Organic acids such as acid and succinic acid can be used.
  • Nitrogen sources include inorganic ammonium salts such as ammonium sulfate, ammonium nitrate, ammonium chloride, ammonium phosphate, and ammonium acetate, ammonia, peptone, meat extract, yeast extract, yeast extract, corn 'Stib' liquor, and soybean hydrolysis. Organic nitrogen, ammonia gas, aqueous ammonia, etc., can be used.
  • inorganic ammonium salts such as ammonium sulfate, ammonium nitrate, ammonium chloride, ammonium phosphate, and ammonium acetate, ammonia, peptone, meat extract, yeast extract, yeast extract, corn 'Stib' liquor, and soybean hydrolysis.
  • Organic nitrogen, ammonia gas, aqueous ammonia, etc. can be used.
  • inorganic ions small amounts of potassium phosphate, magnesium sulfate, iron ions, manganese ions and the like are added.
  • organic trace nutrients it is desirable to include a required substance such as a vitamin or a yeast extract in an appropriate amount as necessary.
  • Culture is preferably carried out for 16 to 72 hours under aerobic conditions such as shaking culture, aeration and agitation culture, and the culture temperature is 30 X; Control to 5-9.
  • aerobic conditions such as shaking culture, aeration and agitation culture, and the culture temperature is 30 X; Control to 5-9.
  • pH adjustment an inorganic or organic acidic or alkaline substance, ammonia gas or the like can be used.
  • the collection of L-lysine from the fermentation broth can usually be carried out by a combination of an ion exchange resin method, a precipitation method and other known methods.
  • the method for collecting L-glutamic acid may be a conventional method, for example, an ion exchange resin method, a crystallization method, or the like. Specifically, L-glutamic acid may be adsorbed and separated by an anion exchange resin, or may be neutralized and crystallized. When producing both L-lysine and L-glutamic acid, when these are used as a mixture, these aminos It is not necessary to separate the acids from each other.
  • BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described more specifically with reference to examples.
  • SEQ ID NO: 1 corresponds to the sequence from 251st to 270th base of the rrnB gene base sequence registered in EMBL / GenBank / DDBJ accession No. J01695
  • SEQ ID NO: 2 corresponds to the sequence from base 7488 to base 7508.
  • the recognition sequence of the restriction enzyme Kpnl is inserted into the nucleotide sequences of SEQ ID NOS: 1 and 2.
  • the chromosome D ⁇ of Escherichia coli W3110 was prepared by a conventional method (Bioengineering Experiments, edited by The Biotechnology Society of Japan, pages 97-98, Baifukan, 1992).
  • the standard reaction conditions described on page 185 of the PCR method forefront were used.
  • the resulting PCR product was purified by a conventional method, reacted with a restriction enzyme Kpnl, and ligated with a plasmid pVK7 cut with the restriction enzyme Kpnl using a ligation kit (Takara Shuzo).
  • PVK7 Said, as follows, is a vector one for Eshierihia 'coli pHSG 299 (Km r;. Takeshita , S. et al, Gene, 61, 63-74, (1987) see) Burebiba Kuteriumu-in It was constructed by combining pAM330, a lactofamentum cribtic plasmid. PAM330 was prepared from Brevipacterium 'lactofermentum ATCC13869 strain. pHSG299 is cut with Aval I (Takara Shuzo Co., Ltd.) which is a one-site cleavage enzyme, blunt-ended with T4 DNA polymerase, and then cut with Hind III (Takara Shuzo Co., Ltd.). PAM blunt-ended with T4 DNA polymerase 7
  • pVK6 The two kinds of generated plasmids were named pVK6 and pVK7 according to the insertion direction of pAM330 into pHSG299, and pVK7 was used in the following experiments.
  • pVK7 is capable of autonomous replication in E. coli and Brevibacterium lactofermentum cells and has a multiple cloning site derived from pHSG299, lacZ 'and a kanamycin resistance gene as a marker. ing.
  • Topiranoside 10 ⁇ g / ml, X-Gal (5-bromo 4- 4-cloth 3-indolyl /?-D-galactoside) 40 ⁇ g / ml and kanamycin 25 ⁇ g / ml Applied to L medium containing 10 ml / ml (Pacto Tributon 10 g / L, Pactoistractose tract 5 g / L, NaCl 5 g / L, Agar 15 g / L, pH 7.2), appeared after overnight culture The isolated white colonies were picked and separated into single colonies to obtain a transformant.
  • Plasmid was prepared from the transformant using the alkaline method (Bioengineering Experiments, edited by The Biotechnology Society of Japan, p. 105, Baifukan, 1992), and the DNA fragment inserted into the vector was prepared. A restriction map was constructed and compared with the reported restriction map of the rrnB operon. The plasmid into which the DNA fragment having the same restriction map had been inserted was named pVKRRN.
  • Brevibacterium lactofermentum AJ13029 was transformed with the plasmid pVKRRN by the electric pulse method (see Japanese Patent Application Laid-Open No. 2-207791) to obtain the resulting transformant.
  • culture for producing L-glucamic acid was performed as follows.
  • AJ13029 / pVKRRN cells obtained by culturing on a CM2B plate medium containing 25 ⁇ g / ml kanamycin were added to a seed culture medium containing 25 ⁇ g / ml kanamycin and having the composition shown in Table 1. Inoculation was performed and shaking culture was performed at 31.5 ° C for 24 hours to obtain a seed culture.
  • the main culture medium having the composition shown in Table 1 was dispensed into a 500-ml glass jar fermenter in an amount of 300 ml each, sterilized by heating, and then inoculated with 40 ml of the above seed culture.
  • the culture was started at a culture temperature of 31.5 ° C with a stirring speed of 800 to 130 Orpin and an aeration rate of 1/2 to 1/1 vvm.
  • the pH of the culture was maintained at 7.5 with ammonia gas.
  • Eight hours after the start of the culture the culture temperature was shifted to 37.
  • a strain obtained by transforming pVK7 into a corynebacterium bacterium AJ13029 by an electric pulse method was cultured in the same manner as described above. Table 1 Concentration
  • AJ11082 / pVKRRN strain obtained by culturing on a CM2B plate medium containing 25 ⁇ g / ml kanamycin is inoculated into an L-lysine production medium containing 25 ⁇ g / ml kanamycin and having the following composition Then, shaking culture was performed at 31.5 ° C until the sugar in the medium was consumed.
  • a strain in which pVK7 was transformed into a corynebacterium bacterium AJ11082 by an electric pulse method was cultured in the same manner as described above.
  • Protein hydrolyzate (bean concentrate) 30 ml
  • Brevipacterium lactofermentum AJ12993 was transformed with plasmid pVKRRN by the electric pulse method (see Japanese Patent Application Laid-Open No. 2-207791) to obtain the obtained transformant.
  • cultivation for producing L-lysine and L-glutamic acid was performed as follows.
  • Cells of the AJ12993 / pVKRRN strain obtained by culturing on a CM2B plate medium containing 25 ⁇ g / ml kanamycin were inoculated into the L-lysine production medium containing 25 ⁇ g / ml kanamycin. The cells were cultured at 31.5 ° C.
  • the culture temperature was shifted to 34 ° C, and the culture was performed with shaking until the sugar in the medium was consumed.
  • a strain obtained by transforming pVK7 into a corynebacterium bacterium AJ12993 by an electric pulse method was cultured in the same manner as described above.
  • L-amino acids such as L-lysine, L-glutamic acid, L-threonine, L-leucine, L-isoleucine, L-valine, and L-phenylalanine of coryneform bacteria Performance can be improved.
  • the production rate of these L-amino acids can be improved.

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Abstract

Selon le procédé de l'invention, on transfère un ARNr de codage de gène dans un Corynebacterium capable de produire un acide L-aminé, tel que la L-lysine ou l'acide L-glutamique, afin d'accroître l'expression du gène ARNr, ce qui permet d'augmenter le rendement de l'acide L-aminé et la vitesse de production.
PCT/JP2000/004772 1999-07-19 2000-07-14 Procede de production d'un acide l-amine Ceased WO2001005960A1 (fr)

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AU60182/00A AU6018200A (en) 1999-07-19 2000-07-14 Process for producing l-amino acid

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JP20526899A JP2003159066A (ja) 1999-07-19 1999-07-19 Ｌ−アミノ酸の製造法
JP11/205268 1999-07-19

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JP2006340603A (ja) * 2003-06-23 2006-12-21 Ajinomoto Co Inc Ｌ−グルタミン酸の製造法

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JPH10229881A (ja) * 1997-02-19 1998-09-02 Mitsubishi Chem Corp コリネ型細菌内で蛋白質を効率よく翻訳させる配列を有するｄｎａ

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH10229881A (ja) * 1997-02-19 1998-09-02 Mitsubishi Chem Corp コリネ型細菌内で蛋白質を効率よく翻訳させる配列を有するｄｎａ

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LIEBL W. ET AL.: "Transfer of brevibacterium divaricatum DSM 2029T, 'Brevibacterim flavum' DSM 20411, 'Brevibacterium lactofermentun' DSM20412 and DSM 1412 and corynebacterium lilium DSM 20137T to corynebacterium glutamicum and their distinction by rRNA gene restriction patterns", JOURNAL OF SYSTEMATIC BACTERIOLOGY, vol. 41, no. 2, 1991, pages 255 - 260, XP002932537 *
WIRTH R. ET AL.: "Factors modulating transcription and translation in vitro of ribosomal protein S20 and isoleucyl-tRNA synthetase from escherichia coli", EUR. J. BIOCHEM., vol. 114, 1981, pages 429 - 437, XP002932536 *

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