WO2002097086A1 - Micro-organismes utiles a echelle industrielle - Google Patents

Micro-organismes utiles a echelle industrielle Download PDF

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Publication number
WO2002097086A1
WO2002097086A1 PCT/JP2002/005200 JP0205200W WO02097086A1 WO 2002097086 A1 WO2002097086 A1 WO 2002097086A1 JP 0205200 W JP0205200 W JP 0205200W WO 02097086 A1 WO02097086 A1 WO 02097086A1
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WO
WIPO (PCT)
Prior art keywords
microorganism
group
pseudomonas
serratia
useful substance
Prior art date
Application number
PCT/JP2002/005200
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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 JP2003500252A priority Critical patent/JPWO2002097086A1/ja
Publication of WO2002097086A1 publication Critical patent/WO2002097086A1/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
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • 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
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides

Definitions

  • the present invention relates to a microorganism useful for industrial production, and a method for producing a useful substance using the microorganism.
  • Direct view technology
  • An object of the present invention is to provide a microorganism in which a specific gene is inactivated or deleted, and a method for producing a useful substance using the microorganism, which is generally effective for producing various target useful substances. Aim.
  • microorganisms have degradation systems for various primary metabolites, and some of the primary metabolites, amino acids or nucleic acid compounds, are degraded and wasted.
  • the present inventors have found that by exhaustively destroying the degradation system for the primary metabolite, the waste of the primary metabolite is reduced, and the basic metabolites of the microorganism are reduced. Microorganisms with improved efficiency can be produced, and microorganisms without such a degradation system are considered to be ideal as parent strains for producing various useful substances. Various genes involved in the degradation system for metabolites were inactivated or deleted, and the production efficiency of useful substances was compared.
  • the present invention provides the following (1) to (7).
  • the genes involved in amino acid or nucleic acid degradation are: ⁇ , g ⁇ i / i_A ⁇ adiA c «d! A, goflG ⁇ gabD gabT gabP 5 ⁇ _ ⁇ ⁇ sdaB ⁇ sda, tdcA tdcB tdcC tdcD tdcE, tdcF tdcG tdcR yhaP yhaQ tdh, IdcC kbl gcvH gcvP ⁇ gcvT putA ⁇ tnaA ⁇ tnaB tnaL ⁇ aspA ushA add deoD deoAs.
  • the microorganism according to the above (1) which is a gene selected from a group of insect genes. -
  • Microorganisms are Enterobacteriaceae Enterobacteriaceae ⁇ , Clostridiaceae, Corynepacteridae
  • the microorganism according to (1) or (2) which is a microorganism belonging to a family selected from the group consisting of (Nocardiaceae).
  • Microbial activity 5 a group selected from the group consisting of Klebsiella Erwinia Serratia s Salmonella, Escherichia, Proteus Clostridium Coj nebacterium, Pseudomonas ⁇ Xanthomonas j3 ⁇ 4 s Bacillus, Art irobflcter ⁇ and i / io ⁇ cocci ⁇ A microorganism belonging to the genus
  • microorganism according to any one of (1) to (3). (5) microbial force 5, Klebsiella aerogenes Erwinia berbicola Erwinia amylovora ⁇ Serratia marcescens Serratia ficaria, Serratia fonticola Serratia liquefaciens Escherichia coli Salmonella typhimurium Proteus rettgeri Corynebacterium glutamicum Corynebacterium ammoniagenes ⁇ Corynebacterium mycetoides ⁇ Corynebacterium variabilis Clostridium butyricum ⁇ Pseudomonas putida Pseudomonas fluorescens ⁇ Pseudomonas aeruginosa ⁇ Pseudomonas dacunhae Pseudomonas thazdirwphilum,
  • microorganism according to any one of (1) to (4), which is a microorganism selected from the group consisting of Arthrobacter obae and Rhodococcus erythropolis.
  • microorganism according to any one of (1) to (5) above 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.
  • the microorganism may be a wild-type microorganism or a microorganism that has been industrially usefully improved.
  • microorganisms of the present invention may be any of a mutant strain, a cell fusion strain, a transduced strain, or a recombinant strain created using a gene recombination technique of the microorganism.
  • a mutant strain a cell fusion strain, a transduced strain, or a recombinant strain created using a gene 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. After washing the cells with an appropriate buffering agent, for example, 0.05 M Tris-maleic acid buffer (pH 6.0), the cell concentration is adjusted to 10 4 to 10 1 () cells / ml. In the same buffer. Using the suspension, mutagenesis is performed by a conventional method. As a usual method, for example, add N-methyl-N'-nitro-N-nitrosoguanidine (NTG) to the suspension to a final concentration of 600 mg / l, and hold at room temperature for 20 minutes. Mutation processing methods can be given. Apply the mutagenized suspension to the complete medium.
  • NTG N-methyl-N'-nitro-N-nitrosoguanidine
  • Molecular cloning A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989) (hereinafter referred to as Molecular 'Clothing') is a powerful method for deleting or inactivating a gene of interest on a chromosome of a microorganism. 2nd edition), the method of GM Church et al. [Journal of Bacteriology, 179, 6228-6237 (1997)], and the method of BL Wanner et al. [Proc. Natl. Acad. Sci. USA, 97, 6640 (2000)]. )] Can be used. Genes on chromosomes can also be deleted using transposons (Gene, 27, 131-149 (1984) ⁇ ).
  • 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 pK03 and pKD20.
  • suicide genes include « JCJB and the like derived from Bacillus subtilis.
  • the DNA in which two DNA fragments homologous to a region of about 1 to 3 kbp at both ends of the target gene region, are introduced into a temperature-sensitive plasmid containing a suicide gene.
  • the plasmid is inserted on the microbial chromosome under the limiting temperature.
  • the resulting recombinant strain is cultured under conditions where the suicide gene acts, and the grown strain is stained with the plasmid. Acquire as a strain that has dropped off from the body.
  • 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 III, 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. be able to.
  • a PCR is used to produce linear DNA in which a DNA homologous to a region of about 1 to 3 kbp at both ends of the target gene region is added to the drug resistance gene.
  • the DNA is integrated into the chromosome of the microorganism by homologous recombination using the incoming 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 gene of interest may be any gene as long as it is involved in the degradation of primary metabolites.
  • gacM, gadB, gdhA, adiA ⁇ cadA ⁇ goaG ⁇ gabD ⁇ gabT ⁇ gabP ⁇ sdaA ⁇ sdaB ⁇ sdaC ⁇ tacA ⁇ tdcB ⁇ tdcC tdcD tdcE ⁇ tdcF ⁇ tdcG ⁇ tdcR ⁇ yhaP ⁇ yha Q tdh, ldc , Kbl ⁇ gcvH, gcvP g vT putA s tnaA ⁇ tnaB ⁇ tnaL ⁇ aspA ⁇ ushA ⁇ add ⁇ deoD ⁇ deoA yicP amn ⁇ ygfP ⁇ dgt ⁇ rihA ⁇
  • Production of useful substances using the microorganism of the present invention created in I Can be carried out using the microorganism culturing method described above.
  • Examples of the useful substance include proteins, amino acids, nucleic acids, vitamins, sugars, organic acids, lipids, and analogs thereof.
  • a synthetic medium or a natural medium can be used as long as it contains a sufficient amount of carbon sources, nitrogen sources, inorganic salts, and other trace nutrients required by the microorganism used. It is.
  • the carbon source may be any one that can be assimilated by each microorganism, such as glucose, fructose, sucrose, molasses containing them, carbohydrates such as starch or starch hydrolyzate, acetic acid, propionic acid, etc. Alcohols such as organic acids, ethanol, and propanol are used.
  • Nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate and other inorganic acids and ammonium salts of organic acids, other nitrogen-containing compounds, peptone, meat extract, yeast extract and corn. Chip liquor, casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented cells and digests thereof are used.
  • potassium (II) phosphate potassium (II) phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate, etc. are used.
  • potassium (II) phosphate potassium (II) phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate, etc.
  • the culture is performed under aerobic conditions such as shaking culture or submerged agitation culture.
  • the culture temperature is preferably 15 to 40 ° C, and the culture time is usually 16 hours to 7 days.
  • the pH is maintained at 3.0 to 9.0. Adjustment of the pH is performed using an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia or the like.
  • an antibacterial substance such as ampicillin tetracycline may be added to the medium during the culture.
  • an inducer may be added to the medium as needed when culturing.
  • lac promotion when culturing a microorganism transformed with an expression vector using Yuichi, when culturing a microorganism transformed with an expression vector using a tvp motor, such as isopropyl-1- / D-thiogalactopyranoside (IPTG). May be added to the medium with indoleacrylic acid (IAA).
  • IAA indoleacrylic acid
  • the target useful substance can be isolated and purified from the culture solution.
  • the cells are collected from the culture solution and then crushed by an appropriate method such as a mechanical or chemical method.
  • the target useful substance can be isolated and purified from the lysate of the cells by using an ion exchange treatment, a concentration method, a salting-out method and the like together. ⁇ ⁇ Shame bear to give
  • K-12 strain Escherichia coli MG 1655 (parent strain), a group of genes (ii / cA, tdcB, tdcC, tdcD, tdcE, tdcF, tdcG) that are involved in the degradation of serine and threonine and exist continuously on the chromosome And two DNA fragments homologous to a region of about 2 kbp adjacent to both ends of tdcR) are introduced into a temperature-sensitive plasmid pK03 having a suicide gene, sacB. According to the method of GM Church et al.
  • the plasmid was introduced into the chromosome of K-12 strain E. coli MG 1655 to obtain a recombinant strain I do.
  • the recombinant strain is cultured in a medium containing sucrose, and the grown strain is obtained as a recombinant strain in which the plasmid region has been removed from the chromosome.
  • the target genes (tdcA, tdcB ⁇ tdcC, tdcD, Strains lacking tdcE, tdcF, trfcG and ticR) (deficient strains) were selected.
  • the genotype is OCL-32, which has the genotype: OL412, MyqjA-yha O-. -.Km ⁇ ] Junichi Kato Available from the professor.
  • the parent strain and the defective strain were each liquid-cultured overnight at 30 ° C.
  • Each of the obtained culture solutions was inoculated at 1% into 8 ml of a complete medium, cultured at 30 ° C, and sampled with time.
  • the obtained sample was diluted so that the absorbance at 660 nm was about 0.03 to 0.3, and the turbidity was measured using a spectrophotometer.
  • the amount of protein derived from the bacterial cells at 23 hours after the culture was measured by the following method.
  • washed cells After centrifuging sample 200 ⁇ 1 at 16,000 X g for 6 minutes, the supernatant was carefully discarded. 1 ml of physiological saline was added to the obtained cells (precipitated fraction), and the cells were washed by stirring. After washing, the mixture was centrifuged at 16,000 X g for 5 minutes, and the supernatant was carefully discarded. Further, the mixture was centrifuged at 16,000 X g for 4 minutes, the supernatant was carefully discarded, and the washing solution adhering to the walls and the like was removed to obtain washed cells. Since the washed cells could be stored at ⁇ 30 ° C., when measuring the amount of evening protein, the washed cells frozen and stored were used.
  • a 200% 1% SDS solution is added to the washed cells or the cells obtained by thawing the cryopreserved cells, the cells are suspended, and then the cells are heated at 100 ° C for 5 minutes. Was dissolved.
  • the resulting lysate was serially diluted using 1% SDS.
  • the amount of evening protein in the diluted solution was measured using a DC protein atskit 1 manufactured by Biorad in accordance with the instructions attached to the kit, and the evening protein amount per 1 ml of the culture solution was calculated.
  • the defective strain showed higher turbidity than the parent strain, indicating that the growth was improved compared to the parent strain.
  • the defective strain significantly increased the amount of bacterial cells per medium volume as compared with the parent strain.
  • the present invention it is possible to provide a microorganism useful for producing a useful substance while suppressing waste of a primary metabolite, 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)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

La présente invention concerne des micro-organismes dans lesquels un ou plusieurs gènes participant à la décomposition des acides aminés ou des acides nucléiques ont été inactivés ou supprimés. Cette invention concerne aussi un processus de production de substances qui conviennent pour une utilisation avec les micro-organismes susmentionnés, tels que des protéines des acides aminés, des acides nucléiques, des vitamines des saccharides, des acides organiques, des lipides ou des analogues de ceux-ci
PCT/JP2002/005200 2001-05-29 2002-05-29 Micro-organismes utiles a echelle industrielle WO2002097086A1 (fr)

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JP2003500252A JPWO2002097086A1 (ja) 2001-05-29 2002-05-29 工業的生産に有用な微生物

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005304320A (ja) * 2004-04-19 2005-11-04 Mitsukan Group Honsha:Kk γ−アミノ酪酸資化能低下納豆菌、及びγ−アミノ酪酸含有納豆
JP2006211934A (ja) * 2005-02-02 2006-08-17 Kao Corp 組換え微生物
WO2011080301A3 (fr) * 2009-12-30 2012-04-12 Metabolic Explorer Souches et procédé pour la production de méthionine
WO2015142021A1 (fr) * 2014-03-21 2015-09-24 Cj Cheiljedang Corp. Micro-organisme ayant une productivité améliorée en acides l-aminés et procédé de production d'acides l-aminés les utilisant
JP2016534741A (ja) * 2013-10-28 2016-11-10 味の素株式会社 プトレシン分解経路が破壊された腸内細菌科の細菌を使用するｌ−アミノ酸の製造方法
CN106536724A (zh) * 2014-04-30 2017-03-22 赢创德固赛有限公司 使用甘氨酸裂解系统在棒杆菌中生产l‑氨基酸的方法
CN108753669A (zh) * 2018-05-25 2018-11-06 苏州引航生物科技有限公司 一种腺嘌呤生产菌株及其构建方法和应用
CN110951797A (zh) * 2019-12-05 2020-04-03 湖北大学 消化链球菌谷氨酸脱氢酶GdhA在提高地衣芽胞杆菌聚γ-谷氨酸产量中的应用
CN112322561A (zh) * 2020-12-23 2021-02-05 新疆农业大学 一株克雷伯氏菌及其在果树梨火疫病防治中的应用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH10248588A (ja) * 1997-03-14 1998-09-22 Ajinomoto Co Inc 発酵法によるｌ−セリンの製造法

Patent Citations (1)

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Publication number Priority date Publication date Assignee Title
JPH10248588A (ja) * 1997-03-14 1998-09-22 Ajinomoto Co Inc 発酵法によるｌ−セリンの製造法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SAWERS G.: "The anaerobic degradation of L-serine and L-threonine in enterobacteria: networks of pathways and regulatory signals", ARCH. MICROBIOL., vol. 171, no. 1, 1998, pages 1 - 5, XP002953871 *

Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005304320A (ja) * 2004-04-19 2005-11-04 Mitsukan Group Honsha:Kk γ−アミノ酪酸資化能低下納豆菌、及びγ−アミノ酪酸含有納豆
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
US9382561B2 (en) 2009-12-30 2016-07-05 Metabolic Explorer Strains and method for the production of methionine
WO2011080301A3 (fr) * 2009-12-30 2012-04-12 Metabolic Explorer Souches et procédé pour la production de méthionine
US9840725B2 (en) 2013-10-28 2017-12-12 Ajinomoto Co., Inc. Method for producing an L-amino acid using a bacterium of the family Enterobacteriaceae having a disrupted putrescine degradation pathway
JP2016534741A (ja) * 2013-10-28 2016-11-10 味の素株式会社 プトレシン分解経路が破壊された腸内細菌科の細菌を使用するｌ−アミノ酸の製造方法
JP2017504349A (ja) * 2014-03-21 2017-02-09 シージェイ チェイルジェダン コーポレーション Ｌ−アミノ酸の生産能が向上された微生物、及びそれを利用してｌ−アミノ酸を生産する方法
CN105980545B (zh) * 2014-03-21 2019-12-24 Cj第一制糖株式会社 具有增强的l-氨基酸生产能力的微生物和使用该微生物生产l-氨基酸的方法
KR101599800B1 (ko) * 2014-03-21 2016-03-04 씨제이제일제당 주식회사 Ｌ-아미노산의 생산능이 향상된 미생물 및 이를 이용하여 ｌ-아미노산을 생산하는 방법
KR20150109990A (ko) * 2014-03-21 2015-10-02 씨제이제일제당 (주) Ｌ-아미노산의 생산능이 향상된 미생물 및 이를 이용하여 ｌ-아미노산을 생산하는 방법
CN105980545A (zh) * 2014-03-21 2016-09-28 Cj第制糖株式会社 具有增强的l-氨基酸生产能力的微生物和使用该微生物生产l-氨基酸的方法
WO2015142021A1 (fr) * 2014-03-21 2015-09-24 Cj Cheiljedang Corp. Micro-organisme ayant une productivité améliorée en acides l-aminés et procédé de production d'acides l-aminés les utilisant
US10113191B2 (en) 2014-03-21 2018-10-30 Cj Cheiljedang Corporation Microorganisms having enhanced L-amino acids productivity and process for producing L-amino acids using the same
TWI639698B (zh) * 2014-03-21 2018-11-01 Ｃｊ第一製糖股份有限公司 具有提高的ｌ－胺基酸生產力的微生物和使用其的ｌ－胺基酸的生產方法
CN106536724A (zh) * 2014-04-30 2017-03-22 赢创德固赛有限公司 使用甘氨酸裂解系统在棒杆菌中生产l‑氨基酸的方法
CN108753669A (zh) * 2018-05-25 2018-11-06 苏州引航生物科技有限公司 一种腺嘌呤生产菌株及其构建方法和应用
CN110951797A (zh) * 2019-12-05 2020-04-03 湖北大学 消化链球菌谷氨酸脱氢酶GdhA在提高地衣芽胞杆菌聚γ-谷氨酸产量中的应用
CN110951797B (zh) * 2019-12-05 2020-08-25 湖北大学 消化链球菌谷氨酸脱氢酶GdhA在提高地衣芽胞杆菌聚γ-谷氨酸产量中的应用
CN112322561A (zh) * 2020-12-23 2021-02-05 新疆农业大学 一株克雷伯氏菌及其在果树梨火疫病防治中的应用
CN112322561B (zh) * 2020-12-23 2023-05-02 新疆农业大学 一株克雷伯氏菌及其在果树梨火疫病防治中的应用

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