WO2001005959A1 - Obtention d'une substance cible par un procede de fermentation - Google Patents

Obtention d'une substance cible par un procede de fermentation Download PDF

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Publication number
WO2001005959A1
WO2001005959A1 PCT/JP2000/004773 JP0004773W WO0105959A1 WO 2001005959 A1 WO2001005959 A1 WO 2001005959A1 JP 0004773 W JP0004773 W JP 0004773W WO 0105959 A1 WO0105959 A1 WO 0105959A1
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WO
WIPO (PCT)
Prior art keywords
target substance
gene
microorganism
substrate
strain
Prior art date
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PCT/JP2000/004773
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English (en)
Japanese (ja)
Inventor
Eiichiro Kimura
Hisao Ito
Osamu Kurahashi
Original Assignee
Ajinomoto Co., Inc.
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Ajinomoto Co., Inc. filed Critical Ajinomoto Co., Inc.
Priority to AU60183/00A priority Critical patent/AU6018300A/en
Publication of WO2001005959A1 publication Critical patent/WO2001005959A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
    • C12P1/04—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using bacteria
    • 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
    • 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 a target substance using a microorganism, and more particularly, to a target substance such as L-amino acid, an antibiotic, a vitamin, a growth factor, or a physiologically active substance. It discloses a means for improving the productivity of a substance which is the final target product in a method for producing a microorganism using a microorganism.
  • a target substance such as L-amino acid, an antibiotic, a vitamin, a growth factor, or a physiologically active substance.
  • BACKGROUND ART As a typical method for producing a substance using a microorganism, a method for producing L-amino acid by a fermentation method is known. L-amino acid is used not only as a seasoning and food, but also as a component of various nutritional mixtures for medical purposes.
  • L-amino acids such as L-lysine and L-homoserine by microorganisms.
  • microorganisms capable of producing L-amino acids by fermentation coryneform bacteria, Escherichia bacteria, Bacillus bacteria, Serratia bacteria, and the like are known.
  • the present invention relates to a method for producing a target substance such as an L-amino acid, an antibiotic, a vitamin, a growth factor, or a physiologically active substance using a microorganism. It is an object to provide a method for improvement.
  • the present inventors have conducted intensive studies to solve the above problems, and as a result, a mutant or recombinant strain in which the amount of intermediates or substrates in the biosynthetic system of the target substance has been reduced has a low productivity of the target substance. Was found to be improved, and the present invention was completed.
  • a method for producing a target substance using a microorganism wherein the microorganism is cultured in a medium, the target substance is produced and accumulated in the medium, and the target substance is collected from the culture.
  • microorganism is one in which an intermediate of a biosynthetic system of a target substance or an excretion system of a substrate is defective or weakened.
  • microorganism is a bacterium belonging to the genus Escherichia or a coryneform bacterium;
  • the target substance produced by the present invention is not particularly limited as long as it is a substance that can be produced by a microorganism.
  • L-threonine, L-lysine, L-glutamic acid, L-leucine, L-isoleucine, L-valine, L- Various L-amino acids such as phenylalanine are exemplified.
  • nucleic acids such as guanylic acid and inosinic acid
  • vitamins, antibiotics, growth factors, and bioactive substances the intermediates of biosynthesis or substrates are excreted by substrates. Anything that exists can be used.
  • the present invention can be used for substances which are not currently produced using microorganisms as long as they have an intermediate for biosynthesis or an excretion system for substrates.
  • the target substance and the intermediate or the substrate are relative concepts, and whether the substance is the target substance, the intermediate or the substrate depends on the object to be produced.
  • the amino acid is the target substance, but when producing a peptide antibiotic using the amino acid as a precursor, the target substance is the same antibiotic.
  • Amino acids are intermediates or substrates.
  • the microorganism used in the present invention can be used without any particular limitation as long as it is a microorganism having an ability to produce a target substance, for example, a microorganism conventionally used for producing a useful substance by a fermentation method.
  • the present invention can be applied to microorganisms that have not been conventionally used industrially as long as they have the ability to produce the target substance.
  • the term “ability to produce a target substance” refers to the ability of a microorganism of the present invention to accumulate a significant amount of the target substance in the medium or in the cells when cultured in the medium.
  • the microorganism of the present invention may originally have the ability to produce the target substance, or may have the ability to produce the target substance by breeding using a mutation method, recombinant DNA technology, or the like. You may.
  • bacteria belonging to the genus Escherichia such as Escherichia coli and Brevibacterium Examples include, but are not limited to, coryneform bacteria such as mucolactofermentum, Bacillus bacteria such as Bacillus subtilis, and Serratia bacteria such as Serratia marcescens.
  • the target substance is L-threonine, Escherichia coli VKPM B-3996 (RIA 1867) (see U.S. Pat. No. 5,175,107), Corynebacterium acetoacidophilum AJ12318 (FERM BP-1172) (US Patent No. 5,188,949), and in the case of L-lysine, Escherichia coli E. coli AJ11441 (NRRL B-12185, FERM BP-1543) (see US Pat. No. 4,346,170), Brevibacterium. Amentum AJ3990 1 "( ⁇ 31269) (see U.S. Patent No.
  • the intermediate or substrate in the biosynthesis system of the target substance is a substance involved in the biosynthesis of the target substance, and may be any substance as long as it has an emission system.
  • the intermediate or substrate is not limited to a biosynthetic intermediate or substrate peculiar to the target substance such as a precursor, but may be, for example, a glycolytic intermediate or substrate when the target substance is an L-amino acid.
  • the intermediates or substrates include substances such as protons and electron donors or acceptors as long as they are involved in the biosynthesis reaction of the target substance.
  • an intermediate or a substrate of the biosynthesis system of the above intermediate or substrate is also included.
  • Table 1 shows examples of the target substance, the intermediate or substrate thereof, and the efflux gene in the present invention.
  • Table 1 Target substance Intermediate or substrate Efflux gene Lek, rutamic acid-ketok ', rutalic acid-ketok, rutale-toha. -Mia -se, gene (kgtP)
  • the microorganism used in the present invention is a mutant strain or a recombinant strain in which the amount of intermediates or substrates of the biosynthetic system of the target substance is reduced as described above.
  • Examples of the strain whose efflux is reduced include strains in which the efflux system of the intermediate or the substrate is defective or weakened.
  • the strain whose efflux system is deficient or weakened can be obtained by disrupting or mutating one or more genes related to the efflux system so that the efflux system does not function properly. it can.
  • the mutant strain used in the present invention is obtained by subjecting a wild strain of a microorganism or a mutant strain having a preferable mutation to the production of a target substance to a mutation treatment, and excreting the intermediate or the substrate is smaller than that of the wild strain. Intracellular concentration of intermediate or substrate is equal to or higher than wild type By selecting a strain that is Even if the mutant has a smaller amount of the intermediate or the substrate than the wild-type strain, the mutant having an incomplete biosynthesis system of the target substance is not preferable as the microorganism used in the present invention.
  • mutation treatment examples include irradiation with ultraviolet light or treatment of a microorganism with a mutagen that is commonly used for mutation treatment, such as N-methyl-N, 1,2-nitro-dinitrosoguanidine (NTG) or nitrite. It is possible.
  • a mutagen that is commonly used for mutation treatment, such as N-methyl-N, 1,2-nitro-dinitrosoguanidine (NTG) or nitrite. It is possible.
  • the recombinant strain used in the present invention can be created by gene disruption by homologous recombination.
  • a microorganism containing an excretory gene that has been deleted so that it does not function properly by deleting the 5 'end and / or the 3' end of a gene involved in the excretory system is used.
  • the efflux gene on the chromosome can be destroyed.
  • gene disruption by homologous recombination has already been established, and gene disruption can also be performed by a method using linear DNA or a method using a plasmid containing a temperature-sensitive replication origin. The following describes a method using a plasmid containing a temperature-sensitive replication origin.
  • Microorganisms are transformed with DNA containing a gene (deletion gene) that has been deleted so that it does not function properly by deleting the interior of the efflux gene. By causing the recombination, it is possible to destroy the excretory gene on the chromosome. Such gene disruption by homologous recombination has already been established, and there are a method using linear DNA and a method using a plasmid containing a temperature-sensitive replication origin.
  • a recombinant DNA is prepared by inserting a temperature-sensitive replication origin, a deletion type gene, and a marker gene showing resistance to a drug such as chloramphenicol, and a microorganism is transformed with the recombinant DNA.
  • a recombinant DNA is prepared by inserting a temperature-sensitive replication origin, a deletion type gene, and a marker gene showing resistance to a drug such as chloramphenicol, and a microorganism is transformed with the recombinant DNA.
  • the strain in which the recombinant DNA has been integrated into the chromosome undergoes recombination with the excretory gene sequence originally present on the chromosome, and the excretory gene on the chromosome and the deletion type gene are deleted.
  • Two gene fusion genes have been inserted into the chromosome with the other part of the recombinant DNA (vector part, temperature-sensitive replication origin and drug resistance marker) in between. Therefore, in this state, the normal efflux gene is dominant, so that the transformed strain has an efflux system.
  • a target gene-disrupted strain can be selected using a mutant that requires an intermediate or a substrate in the biosynthetic system of the target substance and using complementation of the requirement of the mutant as an index.
  • the efflux gene-disrupted strain obtained as described above is cultured at a temperature at which the temperature-sensitive replication origin functions (for example, low temperature), the efflux gene is retained in the cells, and the temperature at which the temperature-sensitive replication origin does not function ( If the cells are cultured at high temperatures, for example, the efflux gene is deleted.
  • a temperature at which the temperature-sensitive replication origin functions for example, low temperature
  • the microorganism used in the present invention is constructed and transformed into a rec A- strain, it is possible to prevent the excretory system gene on the plasmid from being integrated into the chromosome during cultivation at a low temperature, and to ensure the loss of the gene. It is preferable because it can be performed.
  • the efflux system gene examples include the peroxidase gene.
  • the gene is known as kgtP in Escherichia coli, and its nucleotide sequence has also been reported (eg, Seol, W. and Shatkin, A, J., Pro Natl. Acad. Sci. USA, 88 , 3802-3806 (1991)).
  • the nucleotide sequence of this gene and the amino acid sequence that can be encoded by this nucleotide sequence are shown in SEQ ID NOs: 1 and 2 in the sequence listing. You.
  • the nucleotide sequence of this gene is included in the nucleotide sequence registered as accession D90886 in DDBJ / EMBL / GenBank.
  • the kgtP gene of Escherichia coli is, for example, a polymerase using the chromosomal DNA of Escherichia coli as a type III and an oligonucleotide having the nucleotide sequence shown in SEQ ID NO: 3 or 4 as a primer. It can be obtained by the one-shot method (PCR: polymerase chain reaction; see White, TJ et al., Trends Genet., 5, 185 (1989)).
  • PHSG415 and pHSG422 which function in bacteria belonging to the genus Escherichia such as Escherichia coli, and Brevi Examples of those that function in coryneform bacteria such as bacterial lactate fermentum include pHS4, pHS22, and pHS23.
  • a plasmid pHSC4 obtained by connecting a DNA fragment containing a replication origin derived from a coryneform bacterium excised from pHS4 to PHSG398, a vector for Escherichia coli, also has a temperature-sensitive plasmid. Can be used in the present invention.
  • PHSC4 grows autonomously in coryneform bacteria and Escherichia coli and confers chloramphenicol resistance on the host.
  • Escherichia coli K. AJ12571 which carries pHSC4, was established on October 11, 1990 at the Institute of Biotechnology and Industrial Technology, Institute of Industrial Science and Technology, Ministry of International Trade and Industry (zip code 305-8566, 1-3 1-3 Tsukuba East, Ibaraki, Japan).
  • Deposited under accession number FERM P-11763 transferred to an international deposit under the Budapest Treaty on August 26, 1991, and deposited under accession number FERM BP-3524.
  • temperature-sensitive plasmids can grow autonomously in coryneform bacterial cells at about 10-32 ° C, but not at about 34 or more.
  • a DNA fragment having a temperature-sensitive replication origin can be obtained, for example, by excising the above pHSC4 with BamHI and KpnI.
  • Microorganisms in which the efflux system gene is disrupted obtained as described above reduce the amount of intermediates or substrates of the biosynthesis system of the target substance that are extracellularly reduced, and therefore the intermediates that are supplied to the biosynthesis of the target substance
  • the amount of body or substrate increases, resulting in an increase in the production of the target substance.
  • the microorganism of the present invention has a reduced biosynthetic enzyme of the target substance in addition to a reduced amount of intermediates or substrates of the biosynthetic system of the target substance.
  • the biosynthetic enzymes of the target substance include, for example, when the target substance is L-glutamic acid, glutamate dehydrogenase, glutamine synthetase, glutamate synthase, and isoquenate dehydrogenase.
  • the microorganism of the present invention may have a reduced or defective activity of an enzyme that catalyzes a reaction that produces a compound other than the target substance by branching off from the target substance biosynthetic pathway.
  • an enzyme that catalyzes a reaction that produces a compound other than the target substance by branching off from the target substance biosynthetic pathway.
  • the enzyme may be: And acetolactate synthase, acetyl formate transferase, lactate dehydrogenase, L-glucaminic acid decarboxylase, and 11-pyrroline dehydrogenase.
  • the microorganism of the present invention may be provided with other properties that are favorable for production of the target substance.
  • the target substance is L-glutamic acid and the microorganism is a coryneform bacterium
  • a temperature-sensitive mutation for a biotin-inhibiting substance such as a surfactant is imparted to a medium containing an excessive amount of biotin.
  • Piotin at L-glutamic acid can be produced in the absence of an action inhibitor (see W096 / 06180).
  • the target substance is produced by culturing a microorganism having an improved target substance production ability in a medium as described above, producing and accumulating the target substance in the medium, and collecting the target substance from the culture.
  • the culture medium and culture conditions used for the culture may be appropriately selected depending on the host used.
  • the target substance produced as described above can be used as necessary to purify the target substance from a cell extract or culture medium using standard methods such as ion exchange chromatography, gel filtration chromatography, adsorption chromatography, and solvent precipitation. It can be purified using BEST MODE FOR CARRYING OUT THE INVENTION
  • BEST MODE FOR CARRYING OUT THE INVENTION will be described more specifically with reference to examples.
  • the whole genome DNA of E. coli K12 strain was prepared by the method of Saito and Miura (Biochem. Biophys. Acta., 72, 619 (1963)).
  • two primers having the sequences shown in SEQ ID NOs: 3 and 4 were prepared based on the known nucleotide sequence of the kgtP gene. Using these, a PCR reaction was performed to amplify the kgtP gene.
  • the obtained DNA was inserted into the EcoRI site of Vector-1 PHSG399 (manufactured by Takara Shuzo Co., Ltd.) to obtain brassmid p399KGTP.
  • the above-mentioned plasmid P399KGTP is cut with restriction enzymes Sphl and Nhel, blunt-ended with T4 DNA polymerase, and subjected to self-ligation using T4 DNA ligase to delete the inside of the kgtP gene.
  • P399AKGTP was obtained.
  • a deletion-type kgtP gene of ⁇ 399 ⁇ was obtained from a plasmid capable of autonomously replicating in Escherichia coli, and a self-replicating ability with a temperature-sensitive mutant plasmid PHSG415 (Hashimoto-Gotoh, T. et al, Gene, 16, 227-235 (1981)).
  • p399AKGTP was digested with EcoRI, and the obtained fragment containing the deleted kgtP gene was introduced into the EsoRI site of brassmid PHSG415 to produce brassmid p415AKGTP.
  • Escherichia coli AJ13199 strain an L-glucamic acid-producing bacterium of Escherichia coli, was transformed using this plasmid, and the kgtP gene on the chromosome was replaced with a deletion type.
  • AJ13199 / p415AKGTP into which plasmid was introduced was shaken for 6 hours at 25 ° C in an LB medium (containing 10 g of pak tributone, 5 g of bacteriose tract, and 5 g of NaCl in 1 L of water). After culturing, the cells were spread on an LB agar medium containing 25 mg / ml of kanamycin, and cultured at 42 ° C to obtain colonies formed as plasmid-incorporated strains. Next, a strain that became susceptible to kanamycin at 42 ° C was obtained from this strain by the Levri force method. The nucleotide sequence of the kgtP gene on the chromosome was examined from this susceptible strain, and it was confirmed that the gene was replaced with a deletion type. This was named AkgtP strain.
  • Escherichia coli AJ13199 strain is a DL-aspartic acid-hydroxamate-resistant strain, No. 305-8566, No. 1-3, Tsukuba, Higashi 1-chome, Ibaraki Pref., Japan, under the accession number FERM P-15573. BP-5807 has been granted.
  • AJ13199 strain and mumm kgtP strain were added to a glumic acid production medium (composition: glucose 40.0 g / L, magnesium sulfate (separately sterilized) 1.0 g / L, ammonium sulfate 20.0 g / L, phosphorus Potassium dihydrogen acid 1.0 g / L, ferrous sulfate heptahydrate 10.0 mg / Ls Manganese sulfate pentahydrate 1 0.O mg / L, Park toast extract 2.0 g / L, thiamine hydrochloride 10 0.
  • a glumic acid production medium composition: glucose 40.0 g / L, magnesium sulfate (separately sterilized) 1.0 g / L, ammonium sulfate 20.0 g / L, phosphorus Potassium dihydrogen acid 1.0 g / L, ferrous sulfate heptahydrate 10.0 mg / Ls Manganese
  • the AJ13199 strain and the AkgtP strain were cultured in a glutamic acid producing medium for 37 to 40 hours, and the amount of L-glutamic acid in the medium was measured using a Biotech Analyzer AS-210 manufactured by Asahi Kasei Corporation. Table 3 shows the results. Table 3 Strain L-glutamic acid (g / L)
  • the present invention it is possible to reduce the amount of an intermediate or a substrate of a biosynthetic system of a target substance that is released into cells, and as a result, it is possible to improve the productivity of the target substance. .

Abstract

Cette invention concerne un procédé d'obtention d'une substance cible à partir d'un micro-organisme cultivé dans un milieu. Dans ce milieu, on produit et l'on fait se développer la substance cible, puis l'on recueille ladite substance. Comme micro-organisme, on utilise une variante ou une version recombinante présentant une moindre décharge d'intermédiaire ou de substrat dans le système de biosynthèse de la substance cible de manière à accroître le taux de production de ladite substance.
PCT/JP2000/004773 1999-07-19 2000-07-14 Obtention d'une substance cible par un procede de fermentation WO2001005959A1 (fr)

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Application Number Priority Date Filing Date Title
AU60183/00A AU6018300A (en) 1999-07-19 2000-07-14 Process for producing target substance by fermentation method

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JP20526699A JP2003159065A (ja) 1999-07-19 1999-07-19 発酵法による目的物質の製造法
JP11/205266 1999-07-19

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7335496B2 (en) 2003-06-05 2008-02-26 Ajinomoto Co., Inc. Method for producing target substance
EP3165608A1 (fr) * 2015-10-30 2017-05-10 Ajinomoto Co., Inc. Procédé de production d'acide aminé l de la famille glutamate
US9822385B2 (en) 2007-04-17 2017-11-21 Ajinomoto Co., Inc. Method for producing an L-glutamic acid and L-aspartic acid using a recombinant microorganism having enhanced expression of a ybjL protein
WO2018091525A1 (fr) 2016-11-15 2018-05-24 Danmarks Tekniske Universitet Cellules bactériennes à tolérance améliorée aux diacides

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2010130899A (ja) * 2007-03-14 2010-06-17 Ajinomoto Co Inc Ｌ−グルタミン酸系アミノ酸生産微生物及びアミノ酸の製造法
JP5445453B2 (ja) * 2008-07-09 2014-03-19 味の素株式会社 アミノヒドロキシ安息香酸類の製造方法
JP2016165225A (ja) 2013-07-09 2016-09-15 味の素株式会社 有用物質の製造方法

Citations (3)

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Publication number Priority date Publication date Assignee Title
JPH01296994A (ja) * 1987-01-20 1989-11-30 Kyowa Hakko Kogyo Co Ltd Ｌ−グルタミン酸の製造法
WO1994014955A1 (fr) * 1992-12-21 1994-07-07 Purdue Research Foundation Deblocage de la voie commune de la synthese d'aminoacide aromatique
EP0955368A2 (fr) * 1998-03-18 1999-11-10 Ajinomoto Co., Ltd. Bactérie produisant de l'acide glutamique et procédé de préparation de l'acide glutamique

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH01296994A (ja) * 1987-01-20 1989-11-30 Kyowa Hakko Kogyo Co Ltd Ｌ−グルタミン酸の製造法
WO1994014955A1 (fr) * 1992-12-21 1994-07-07 Purdue Research Foundation Deblocage de la voie commune de la synthese d'aminoacide aromatique
EP0955368A2 (fr) * 1998-03-18 1999-11-10 Ajinomoto Co., Ltd. Bactérie produisant de l'acide glutamique et procédé de préparation de l'acide glutamique

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7335496B2 (en) 2003-06-05 2008-02-26 Ajinomoto Co., Inc. Method for producing target substance
US9822385B2 (en) 2007-04-17 2017-11-21 Ajinomoto Co., Inc. Method for producing an L-glutamic acid and L-aspartic acid using a recombinant microorganism having enhanced expression of a ybjL protein
EP3165608A1 (fr) * 2015-10-30 2017-05-10 Ajinomoto Co., Inc. Procédé de production d'acide aminé l de la famille glutamate
CN107034250A (zh) * 2015-10-30 2017-08-11 味之素株式会社 谷氨酸类l‑氨基酸的制造方法
US9932614B2 (en) 2015-10-30 2018-04-03 Ajinomoto Co., Inc. Method for producing L-amino acid of glutamate family
CN107034250B (zh) * 2015-10-30 2021-09-21 味之素株式会社 谷氨酸类l-氨基酸的制造方法
WO2018091525A1 (fr) 2016-11-15 2018-05-24 Danmarks Tekniske Universitet Cellules bactériennes à tolérance améliorée aux diacides

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