WO2021049207A1 - 形質転換微生物、及び当該微生物を用いたポリヒドロキシアルカン酸の製造方法 - Google Patents

形質転換微生物、及び当該微生物を用いたポリヒドロキシアルカン酸の製造方法 Download PDF

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Publication number
WO2021049207A1
WO2021049207A1 PCT/JP2020/029763 JP2020029763W WO2021049207A1 WO 2021049207 A1 WO2021049207 A1 WO 2021049207A1 JP 2020029763 W JP2020029763 W JP 2020029763W WO 2021049207 A1 WO2021049207 A1 WO 2021049207A1
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gene
strain
pha
amino acid
expression
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English (en)
French (fr)
Japanese (ja)
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佳弘 毛利
尚志 有川
俊輔 佐藤
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Kaneka Corp
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Kaneka Corp
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Priority to JP2021545163A priority Critical patent/JP7535527B2/ja
Priority to CN202080062790.XA priority patent/CN114616320B/zh
Priority to US17/641,358 priority patent/US12351854B2/en
Priority to EP20862422.1A priority patent/EP4029946A4/en
Publication of WO2021049207A1 publication Critical patent/WO2021049207A1/ja
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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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/62—Carboxylic acid esters
    • C12P7/625—Polyesters of hydroxy carboxylic acids
    • 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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms; 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • 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
    • 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
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/101—Plasmid DNA for bacteria
    • 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
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • the present invention relates to a transformed microorganism and a method for producing polyhydroxyalkanoic acid using the microorganism.
  • PHA synthase gene The PHA synthase gene is not particularly limited, but PHA synthase genes derived from organisms similar to Ralstonia, Cupriavidus, Woutercia, Alcaligenes, Aeromonas, Pseudomonas, Norcadia, and Chromobacterium, and their PHA synthase genes. Variants and the like can be mentioned. As the variant, a base sequence encoding a PHA synthase in which one or more amino acid residues are deleted, added, inserted, or substituted can be used.
  • P (3HB) which is a homopolymer of 3-hydroxybutyrate (abbreviation: 3HB), and P (3HB-co-3HV), a copolymer of 3HB and 3-hydroxyvaleric acid (abbreviation: 3HV), 3HB and 3-.
  • Examples include, but are not limited to, PHA containing lactic acid (abbreviation: LA) as a constituent component, for example, a copolymer P (LA-co-3HB) of 3HB and LA.
  • PHBH is preferable from the viewpoint of having a wide range of applications as a polymer.
  • the type of PHA produced depends on the type of PHA synthase gene possessed or separately introduced by the microorganism to be used, the type of metabolic gene involved in the synthesis, the culture conditions, etc., depending on the purpose. It can be selected as appropriate.
  • the minC gene in the present disclosure is a polypeptide represented by the amino acid sequence shown in SEQ ID NO: 3 (UniProtKB ID Q0KFI3), or a polypeptide represented by an amino acid sequence having 85% or more sequence homology with respect to the amino acid sequence. It is a gene having a base sequence encoding.
  • the sequence homology is preferably 90% or more, more preferably 95% or more, still more preferably 97% or more, and particularly preferably 99% or more.
  • the enhancement of gene expression in the present disclosure refers to a state in which the transcription amount of the target gene or the expression level of the polypeptide encoded by the target gene is increased as compared with the strain in which the expression of the target gene is not enhanced.
  • the amount of increase is not particularly limited, but it may be more than 1 time as compared with the strain in which the expression of the target gene is not enhanced, preferably 1.1 times or more, more preferably 1.2 times or more, still more preferable. Is an increase of 1.5 times or more, and more preferably 2 times or more.
  • a method in which a sacB gene derived from Bacillus subtilis coexists and a microbial strain in which the gene has been lost by homologous recombination in the second stage is easily isolated as a sucrose-resistant strain can also be used.
  • a genome editing technique Y. Wang et al., ACS Synth Biol. 2016, 5 (7): 721-732 by the CRISPR / Cas9 system for modifying the target DNA can also be used. ..
  • the guide RNA gRNA
  • the guide RNA has a sequence that can bind to a part of the base sequence of the genomic DNA to be modified, and has a role of transporting Cas9 to the target.
  • an A0597 deletion disruption strain was prepared as follows. Escherichia coli S17-1 strain (ATCC47055) was transformed with the plasmid vector pNS2X-sacB + A0597UD for gene insertion and disruption, and the transformed microorganisms obtained thereby were mixed and cultured on KNK-005 strain and Nutrient Agar medium (manufactured by Difco). Joint transmission was performed.
  • the cell diameter of the A2405 insertion-disrupted strain measured under the above conditions increased by 20% or more as compared with the parent strain KNK-005.
  • the productivity of PHA was also equivalent to that of the KNK-005 strain.
  • Example 3 PHA production using the A2405 deletion-disrupted strain
  • Table 1 shows the measurement results of the ratio of PHA accumulation amount and cell diameter. Moreover, the microscopic observation photograph of the cell performed as described above is shown in FIG.
  • Example 5 PHA production using a minCD-expressing A1386-disrupted strain
  • a culture study using a minCD-expressing A1386-disrupted strain was carried out under the same conditions as in Comparative Example 1.
  • Table 1 shows the measurement results of the ratio of PHA accumulation amount and cell diameter. Moreover, the microscopic observation photograph of the cell performed as described above is shown in FIG.

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  • Chemical & Material Sciences (AREA)
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  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
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  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
PCT/JP2020/029763 2019-09-09 2020-08-04 形質転換微生物、及び当該微生物を用いたポリヒドロキシアルカン酸の製造方法 Ceased WO2021049207A1 (ja)

Priority Applications (4)

Application Number Priority Date Filing Date Title
JP2021545163A JP7535527B2 (ja) 2019-09-09 2020-08-04 形質転換微生物、及び当該微生物を用いたポリヒドロキシアルカン酸の製造方法
CN202080062790.XA CN114616320B (zh) 2019-09-09 2020-08-04 转化微生物、以及使用了该微生物的聚羟基烷酸酯的制造方法
US17/641,358 US12351854B2 (en) 2019-09-09 2020-08-04 Transformed microorganism and method of producing polyhydroxyalkanoate using the microorganism
EP20862422.1A EP4029946A4 (en) 2019-09-09 2020-08-04 PROCESSED MICRO-ORGANISM AND PROCESS FOR PRODUCTION OF POLYHYDROXYALCANOIC ACID USING SAID MICRO-ORGANISM

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JP2019-163743 2019-09-09
JP2019163743 2019-09-09

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WO2023085374A1 (ja) * 2021-11-11 2023-05-19 株式会社カネカ ポリヒドロキシアルカン酸の製造方法

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JP7554177B2 (ja) * 2019-02-28 2024-09-19 株式会社カネカ 形質転換微生物、及びポリヒドロキシアルカン酸の製造方法

Citations (5)

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Publication number Priority date Publication date Assignee Title
JP2007259708A (ja) 2006-03-27 2007-10-11 Kaneka Corp 新規生分解性ポリエステルの製造方法
US7384766B2 (en) 2006-07-26 2008-06-10 Kaneka Corporation Gene-substituted microorganisms, and production method of polyesters using the same
CN105779488A (zh) * 2016-03-23 2016-07-20 清华大学 一种诱导外源基因在革兰氏阴性菌中表达的系统及其应用
WO2016194771A1 (ja) * 2015-05-29 2016-12-08 国立大学法人北海道大学 ｍｔｇＡ遺伝子の欠損した微生物
WO2019142845A1 (ja) * 2018-01-17 2019-07-25 株式会社カネカ 高組成比率の３ｈｈモノマー単位を含む共重合ｐｈａを生産する形質転換微生物およびそれによるｐｈａの製造方法

Patent Citations (5)

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Publication number Priority date Publication date Assignee Title
JP2007259708A (ja) 2006-03-27 2007-10-11 Kaneka Corp 新規生分解性ポリエステルの製造方法
US7384766B2 (en) 2006-07-26 2008-06-10 Kaneka Corporation Gene-substituted microorganisms, and production method of polyesters using the same
WO2016194771A1 (ja) * 2015-05-29 2016-12-08 国立大学法人北海道大学 ｍｔｇＡ遺伝子の欠損した微生物
CN105779488A (zh) * 2016-03-23 2016-07-20 清华大学 一种诱导外源基因在革兰氏阴性菌中表达的系统及其应用
WO2019142845A1 (ja) * 2018-01-17 2019-07-25 株式会社カネカ 高組成比率の３ｈｈモノマー単位を含む共重合ｐｈａを生産する形質転換微生物およびそれによるｐｈａの製造方法

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SHEN, RUI ET AL.: "Manipulation of polyhydroxyalkanoate granular sizes in Halomonas bluephagenesis", METABLIC ENGINEERING, vol. 54, 6 April 2019 (2019-04-06), pages 117 - 126, XP085706711, DOI: 10.1016/j.ymben.2019.03.011 *
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023085374A1 (ja) * 2021-11-11 2023-05-19 株式会社カネカ ポリヒドロキシアルカン酸の製造方法

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JP7535527B2 (ja) 2024-08-16
CN114616320A (zh) 2022-06-10
US20220315958A1 (en) 2022-10-06
JPWO2021049207A1 (https=) 2021-03-18
EP4029946A1 (en) 2022-07-20
US12351854B2 (en) 2025-07-08
EP4029946A4 (en) 2023-08-30
CN114616320B (zh) 2025-02-07

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