WO2002097089A1 - Micro-organismes utiles pour la production industrielle - Google Patents

Micro-organismes utiles pour la production industrielle Download PDF

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Publication number
WO2002097089A1
WO2002097089A1 PCT/JP2002/005198 JP0205198W WO02097089A1 WO 2002097089 A1 WO2002097089 A1 WO 2002097089A1 JP 0205198 W JP0205198 W JP 0205198W WO 02097089 A1 WO02097089 A1 WO 02097089A1
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WO
WIPO (PCT)
Prior art keywords
microorganism
group
pseudomonas
serratia
gene
Prior art date
Application number
PCT/JP2002/005198
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English (en)
Japanese (ja)
Inventor
Hideo Mori
Tatsuro Fujio
Masao Nishihara
Original Assignee
Kyowa Hakko Kogyo Co., Ltd.
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 Kyowa Hakko Kogyo Co., Ltd. filed Critical Kyowa Hakko Kogyo Co., Ltd.
Priority to JP2003500254A priority Critical patent/JPWO2002097089A1/ja
Publication of WO2002097089A1 publication Critical patent/WO2002097089A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from 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
    • 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

Definitions

  • the present invention relates to a microorganism useful for industrial production, and a method for producing a useful substance using the microorganism.
  • the present invention provides a microorganism having a specific gene inactivated or deleted, and a method for producing a useful compound using the microorganism, which is generally effective for producing various desired useful compounds. With the goal.
  • the present invention provides the following (1) to (7).
  • a microorganism having a flagellum wherein one or more genes selected from the group consisting of genes involved in flagellum configuration and genes involved in flagellar movement are inactivated or deleted.
  • the gene is flgN flgM, flgA, flgB, flgC, flgD, flgE, flgF, flgG, flgH ⁇ flgl ⁇ flgJ ⁇ flgK flgL, flhE ⁇ flhA ⁇ flhB, cheZ ⁇ cheY, cheB ⁇ cheR ⁇ tar ⁇ cheW cheA , MotB ⁇ "wtA, flhC ⁇ flhD ⁇ fUY ⁇ fliZ ⁇ fliA, fliB ⁇ fliC ⁇ fliDs fHS fliT ⁇ fliE fliF ⁇ fliG ⁇ fliH ⁇ flil ⁇ fliJ ⁇ fl
  • the microorganisms are Enterobacteriaceae (Enter bacterioaceae), ku D stridum (Clostridiaceae),
  • microorganism (Pseudomonadaceae), a microorganism according to the above (1) or (2), which is a microorganism belonging to a family selected from the group consisting of Xanthomonas group; Bacillacea).
  • Xanthomonas ovyzae ⁇ microbes selected from the group consisting of Bacillus subtilis and Bacillus licheniformis
  • microorganism according to any one of (1) to (4), which is a product.
  • the microorganism according to any one of the above (1) to (5) is cultured in a medium, a useful substance is produced and accumulated in the culture, and the useful substance is collected. Method for producing useful substances.
  • the useful substance is a useful substance selected from the group consisting of proteins, amino acids, nucleic acids, vitamins, sugars, organic acids, lipids and analogs thereof.
  • microorganism that can be used to construct the microorganism of the present invention any microorganism that can be used industrially can be used.
  • Such microorganisms include Enterobacteriaceae (Enterobacteri cea), Clostridium (Clostridiaceae), and Pseudomonas
  • Pseudomonas putida Pseudomonas fluovescens
  • Pseudomonas aeruginosa Pseudomonas dacunhae
  • Pseudomonas thazdinophilum Xanthomonas ovyzae ⁇ Bacillus subtilis ⁇ Bacillus licheniformis etc. can be opened o
  • the microorganism may be a wild-type microorganism or an industrially useful improved microorganism.
  • microorganisms of the present invention may be a mutant strain, a cell fusion strain, a transduced strain, or a recombinant strain created using the DNA recombination technique of the microorganism.
  • more effective microorganisms of the present invention can be constructed by the following method.
  • the microorganism described in (1) above is cultured according to a conventional method. After culturing, cells are obtained from the obtained culture by centrifugation. The microbial cells, a suitable buffer, e.g., 0.05 M Tris - after washing with maleic acid buffer (pH 6.0) or the like, in the same buffer to cell concentration of 10 4 ⁇ 10 1Q cells / ml Suspend. Mutation treatment is carried out using the suspension by a conventional method.
  • a suitable buffer e.g., 0.05 M Tris - after washing with maleic acid buffer (pH 6.0) or the like
  • N-methyl-N-nitto-N-nitrosguanidine NSG
  • NMG N-methyl-N-nitto-N-nitrosguanidine
  • the mutated suspension is applied to a complete medium and cultured at 15 to 38 ° C for 1 to 4 days. After the culture, the grown and formed colonies are applied to the center of the soft agar medium only and cultured. Motile strains form large colonies due to their motility, whereas non-motile mutants form small colonies only in the center. A non-motile strain is selected as the mutant strain of interest.
  • Molecular cloning A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989) (hereinafter referred to as Molecular 'Cloning 2nd Edition) is a force method for deleting or inactivating a target gene on a chromosome of a microorganism.
  • GM Church et al. Journal of Bacteriology, 179, 6228-6237 (1997)]
  • BL Wanner et al. Pro Natl. Acad. Sci. USA, 97, 6640 (2000)
  • a temperature-sensitive plasmid incorporating a suicide gene is used.
  • the temperature-sensitive plasmid those in which the protein essential for plasmid replication has become temperature-sensitive can be used, and specific examples include pKL03 and pKD20.
  • Suicide genes include ⁇ from Bacillus subtilis. DNA, which is obtained by joining two DNA fragments homologous to a region of about 1 to 3 kbp at both ends of the target gene region, is introduced into a temperature-sensitive plasmid containing a suicide gene. The plasmid is introduced onto the microbial chromosome under the limiting temperature.
  • the obtained recombinant strain is cultured under conditions in which a suicide gene acts, and a grown strain is obtained as a strain in which the plasmid has dropped off from the chromosome.
  • the culture conditions under which the suicide gene acts include conditions for culturing in a medium containing sucrose.
  • the chromosome structure of the obtained strain is analyzed, and a strain lacking the target gene region is selected. Chromosome structure analysis can be performed according to a conventional method, for example, a method in which the chromosome of the strain is type II, the sequence around the gene region to be disrupted is used as a primer, and the structure of the peripheral region is analyzed by PCR. Can be given.
  • the method of B.L. Wanner et al. Is described in detail below.
  • a linear DNA is prepared by PCR using a drug-resistant gene and DNA homologous to a region of about 1 to 3 kbp at both ends of the target gene region.
  • the DNA is integrated into the chromosome of the microorganism by homologous recombination using the z Red recombination system.
  • a strain in which the target gene region has been replaced with a drug resistance gene can be selected as a drug resistant strain.
  • the structural analysis of the chromosome can be performed according to the above method.
  • the gene of interest may be any gene that constitutes flagella or is involved in flagellar movement.
  • Production of a useful substance using the microorganism of the present invention created in the above item I can be carried out using a usual microorganism culturing method.
  • Examples of the useful substance include proteins, amino acids, nucleic acids, vitamins, sugars, organic acids, lipids, and analogs thereof.
  • any synthetic medium or natural medium can be used as long as it contains a carbon source, a nitrogen source, an inorganic salt, and a trace amount of nutrients required by the microorganism used. is there.
  • Any carbon source can be used as long as each microorganism can assimilate it.
  • Alcohols such as organic acids, ethanol, and propafur are used.
  • Nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate, etc., ammonium salts of various inorganic and organic acids, other nitrogen-containing compounds, peptone, meat extract, yeast extract, corn steep liquor. , Casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented cells and digests thereof.
  • potassium (I) phosphate potassium (II) phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate, and the like are used.
  • the culture is performed under aerobic conditions such as shaking culture or deep aeration stirring culture (the culture temperature is preferably 15 to 40 ° C, and the culture time is usually 16 hours to 7 days.
  • the pH is 3.0.
  • the pH is adjusted to 9.0 by adjusting the pH with an inorganic or organic acid, alkali solution, urea, calcium carbonate, ammonia, or the like.
  • an antibiotic such as ampicillin-tetracycline may be added to the medium during the culturing.
  • an inducer may be added to the medium as needed when culturing.
  • an inducer may be added to the medium as needed when culturing.
  • culturing a microorganism transformed with an expression vector using the Zac promoter isopropyl-1D-thiogalactobyranoside (IPTG) or the like is transformed with an expression vector using Promo-Yuichi.
  • IPTG isopropyl-1D-thiogalactobyranoside
  • ⁇ indoleacrylic acid
  • the objective useful substance can be isolated and purified from the culture solution.
  • the cells are recovered from the culture solution and then disrupted by an appropriate method such as mechanical or chemical method.
  • the target useful substance can be isolated and purified from the cell lysate by subjecting the cell lysate to an ion exchange treatment, a concentration method, a salting-out method and the like.
  • the present invention it is possible to provide a microorganism useful for producing a useful substance, and a method for efficiently producing a useful substance using the microorganism.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Biophysics (AREA)
  • Biotechnology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicinal Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne des micro-organismes flagellés dans lesquels un ou plusieurs gènes sélectionnés dans le groupe constitué par les gènes intervenant dans la formation des flagelles les gènes intervenant dans mouvements des flagelles ont été inactivés ou délétés, et un procédé permettant de produire des composants utiles tels que protéines, acides aminés, acides nucléiques, vitamines, saccharides, acides organiques, lipides ou des analogues de ceux-ci au moyen de ces micro-organismes.
PCT/JP2002/005198 2001-05-29 2002-05-29 Micro-organismes utiles pour la production industrielle WO2002097089A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2003500254A JPWO2002097089A1 (ja) 2001-05-29 2002-05-29 工業的生産に有用な微生物

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2001159843 2001-05-29
JP2001-159843 2001-05-29

Publications (1)

Publication Number Publication Date
WO2002097089A1 true WO2002097089A1 (fr) 2002-12-05

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PCT/JP2002/005198 WO2002097089A1 (fr) 2001-05-29 2002-05-29 Micro-organismes utiles pour la production industrielle

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JP (1) JPWO2002097089A1 (fr)
WO (1) WO2002097089A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006211934A (ja) * 2005-02-02 2006-08-17 Kao Corp 組換え微生物
EP2559754A2 (fr) 2011-08-18 2013-02-20 Ajinomoto Co., Inc. Procédé de production d'un acide L-aminé utilisant une bactérie de la famille des entérobactéries á expression améliorée des gènes de formation des flagelles et de cascade motilitée
WO2018117168A1 (fr) * 2016-12-20 2018-06-28 株式会社カネカ Procédé de production de poly(acide hydroxyalcanoïque) et microbes

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BALLADO T. ET AL.: "The hook gene (flgE) is expressed from the flgBCDEF operon in rhodobactor spheroides: study of an flgE mutant", J. BACTERIOL., vol. 183, no. 5, March 2001 (2001-03-01), pages 1680 - 1687, XP002956652 *
DAILEY F.E. ET AL.: "Mutants in disulfide bond formation that discrupt flagellar assembly in escherichia coli", PROC. NATL. ACAD. SCI. USA, vol. 90, 1993, pages 1043 - 1047, XP002956653 *
KARLINSEY J.E. ET AL.: "The flk gene of salmonella typhimurium couples flagellar P-and L-ring assembly to flagellar morphogenesis", J. BACTERIOL., vol. 179, no. 7, 1997, pages 2389 - 2400, XP002956655 *
KAWAGISHI I. ET AL.: "Characterization of the flagellar hook length control protein FliK of salmonella typhimurium and escherichia coli", J. BACTERIOL., vol. 178, no. 10, 1996, pages 2954 - 2959, XP002956654 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006211934A (ja) * 2005-02-02 2006-08-17 Kao Corp 組換え微生物
JP4643999B2 (ja) * 2005-02-02 2011-03-02 花王株式会社 組換え微生物
EP2559754A2 (fr) 2011-08-18 2013-02-20 Ajinomoto Co., Inc. Procédé de production d'un acide L-aminé utilisant une bactérie de la famille des entérobactéries á expression améliorée des gènes de formation des flagelles et de cascade motilitée
WO2013024904A1 (fr) 2011-08-18 2013-02-21 Ajinomoto Co.,Inc. Procédé de production d'un acide aminé l au moyen d'une bactérie de la famille des enterobacteriaceae présentant une expression accrue de gènes impliqués dans la cascade de médiation de la motilité et la formation de flagelles
EP2559754A3 (fr) * 2011-08-18 2013-03-20 Ajinomoto Co., Inc. Procédé de production d'un acide L-aminé utilisant une bactérie de la famille des entérobactéries á expression améliorée des gènes de formation des flagelles et de cascade motilitée
US9284584B2 (en) 2011-08-18 2016-03-15 Ajinomoto Co., Inc. Method for producing an L-amino acid using a bacterium of the family Enterobacteriaceae having enhanced expression of the flagella formation and motility cascade genes
WO2018117168A1 (fr) * 2016-12-20 2018-06-28 株式会社カネカ Procédé de production de poly(acide hydroxyalcanoïque) et microbes

Also Published As

Publication number Publication date
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