WO2020100888A1 - Micro-organisme appartenant au genre cupriavidus - Google Patents

Micro-organisme appartenant au genre cupriavidus Download PDF

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Publication number
WO2020100888A1
WO2020100888A1 PCT/JP2019/044316 JP2019044316W WO2020100888A1 WO 2020100888 A1 WO2020100888 A1 WO 2020100888A1 JP 2019044316 W JP2019044316 W JP 2019044316W WO 2020100888 A1 WO2020100888 A1 WO 2020100888A1
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Prior art keywords
starch
cupriavidus
microorganism
genus
amylase
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PCT/JP2019/044316
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English (en)
Japanese (ja)
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俊輔 佐藤
尚志 有川
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株式会社カネカ
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Priority to JP2020555716A priority Critical patent/JPWO2020100888A1/ja
Publication of WO2020100888A1 publication Critical patent/WO2020100888A1/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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/62—Carboxylic acid esters

Definitions

  • the present invention relates to a microorganism belonging to the genus Cupriavidus.
  • a carbon source that can be suitably assimilated by microorganisms (for example, a carbon source for cultivation and / or fermentation) is required.
  • a carbon source for cultivation and / or fermentation for example, a carbon source for cultivation and / or fermentation
  • sugars eg starch
  • oils and fats eg vegetable oils
  • fatty acids eg plant-derived fatty acids
  • Starch which is a sugar raw material, is an inexpensive fermentation raw material that can be produced from potato, corn, rice, cassava and the like, and may be considered as one of suitable carbon source candidates.
  • ⁇ It is easy to use as a raw material a carbon source that has a structure that is easily taken up by cells and catabolized and assimilated by microorganisms, such as low molecular weight sugar raw materials such as glucose and sucrose.
  • starch is a polymer in which a large number of glucose are bound, and in particular raw starch has a micelle structure (crystal structure) in which chains of amylose and amylopectin are regularly assembled by hydrogen bonds, and thus is not decomposed by an enzyme. It is difficult to receive and cannot be assimilated by microorganisms as it is.
  • the microorganisms responsible for producing the substance can secrete amylase capable of decomposing the raw starch and produce the substance directly from the raw starch.
  • a microorganism belonging to the genus Cupriavidus can be mentioned as a host for substance production using microorganisms.
  • Examples of substance production using a microorganism belonging to the genus Cupriavidus include aliphatic polyesters such as polyhydroxyalkanoate (PHA), butanol and isopropanol.
  • Non-Patent Document 1 There is an example of producing acetone and butanol using a cupriavidus necator, which is a microorganism belonging to the genus Cupriavidus (Non-Patent Document 1). There is an example of producing isopropanol from fructose, which is a sugar raw material, using Cupriavidus necator (Non-patent Document 2). Also, an example of producing polyhydroxyalkanoate (PHA) from sucrose using Cupriavidus necator has been reported (Non-Patent Document 3). However, neither uses starch as a raw material.
  • PHA polyhydroxyalkanoate
  • Non-patent document 4 Since Cupriavidus necator does not have a gene encoding ⁇ -amylase, it cannot express a starch-degrading enzyme and cannot properly use starch, so an example of producing PHA using saccharified potato-derived starch has been reported. (Non-patent document 4).
  • Non-patent Document 5 an ⁇ -amylase derived from Streptococcus bovis (Non-patent Document 5), which is reported to be capable of degrading non-solubilized starch (also referred to as raw starch), into the Cupriavidus necator H16 strain, and The starch degrading activity was confirmed, but it did not show the degrading activity. Thus, it is not easy to suitably use starch, especially raw starch, as a carbon source.
  • Non-Patent Document 6 a Rhizopus-derived secretory signal sequence is used to cause yeast Saccharomyces cerevisiae to express Streptococcus bovis-derived ⁇ -amylase on the surface (a type of secretory expression).
  • a Rhizopus-derived secretory signal sequence is used to cause yeast Saccharomyces cerevisiae to express Streptococcus bovis-derived ⁇ -amylase on the surface (a type of secretory expression).
  • it is essential to introduce a gene sequence in which a secretory signal and a gene encoding the protein are linked.
  • the target microorganism will be It is difficult to find out if it exhibits degrading activity. Secreting the enzyme extracellularly is considered more difficult than simply expressing the heterologous gene intracellularly. Further research is necessary to industrially enable efficient production of substances by microorganisms belonging to the genus Cupriavidus that can produce aliphatic polyesters such as polyhydroxyalkanoate (PHA) using
  • An object of the present invention is to provide a microorganism belonging to the genus Cupriavidus that can decompose starch and effectively assimilate it.
  • the present invention relates to a microorganism belonging to the genus Cupriavidus, into which a gene encoding an ⁇ -amylase, into which a secretory signal derived from a microorganism belonging to the genus Cupriavidus has been introduced.
  • the ⁇ -amylase may be an ⁇ -amylase derived from a microorganism belonging to the genus Streptococcus.
  • a microorganism belonging to the genus Cupriavidus may synthesize polyhydroxyalkanoate.
  • the microorganism belonging to the genus Cupriavidus may be Cupriavidus necator.
  • the polyhydroxyalkanoate may be poly (3-hydroxybutyrate-co-3-hydroxyhexanoate).
  • the present invention it is possible to provide a microorganism belonging to the genus Cupriavidus that can decompose starch and effectively assimilate it.
  • the microorganism of the present disclosure is a microorganism belonging to the genus Cupriavidus, into which a gene encoding an ⁇ -amylase, into which a secretion signal derived from a microorganism belonging to the genus Cupriavidus is linked, has been introduced.
  • the present inventors have found a gene encoding a protein secreted extracellularly (secretory protein) from genes possessed by microorganisms belonging to the genus Cupriavidus, and present the signal present in the gene.
  • a gene in which a similar sequence (secretion signal) is linked to the ⁇ -amylase gene was introduced into a microorganism belonging to the genus Cupriavidus.
  • ⁇ -amylase which is a starch-degrading enzyme having a starch-degrading activity, can be extracellularly secreted, raw starch can be assimilated without being converted to ⁇ and can be effectively used.
  • the substance production using the microorganism of the present disclosure is also suitable for industrial substance production.
  • the microorganism of the present disclosure is not limited as long as it is a microorganism belonging to the genus Cupriavidus, but it is preferable to synthesize polyhydroxyalkanoate, that is, to have PHA synthesizing ability.
  • Cupriavidus necator is particularly preferable because it has high PHA synthesizing ability.
  • microorganism belonging to the genus Cupriavidus having PHA synthesizing ability not only a wild strain originally having a PHA synthase gene but also a mutant strain obtained by artificially mutating such a wild strain, or PHA synthesis It may be a recombinant strain into which an exogenous PHA synthase gene has been introduced by a genetic engineering technique into a microorganism that does not originally have the enzyme gene.
  • the microorganism belonging to the genus Cupriavidus having PHA synthesizing ability is a recombinant strain into which an exogenous PHA synthase gene has been introduced by a genetic engineering method
  • the exogenous PHA synthase gene for example, the Cupriavidus necator H16 strain is A PHA synthase gene encoding the amino acid sequence described in SEQ ID NO: 2 or a PHA encoding a polypeptide having 85% or more sequence identity to the amino acid sequence and having PHA synthesis activity.
  • Synthetic enzyme gene and PHA synthase gene encoding the amino acid sequence of SEQ ID NO: 3 held by Aeromonas caviae, or having 85% or more sequence identity to the amino acid sequence, and having PHA synthetic activity
  • the PHA synthase gene include a PHA synthase gene that encodes a polypeptide having: and other PHA synthase genes can be preferably used.
  • the sequence identity is preferably 90% or more, more preferably 95% or more, and particularly preferably 99% or more.
  • a PHA synthase gene having a synthetic activity of poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) (sometimes referred to as PHBH) as PHA is preferable.
  • PHBH may be synthesized by a transformant in which the PHA synthase gene derived from Aeromonas caviae is introduced into Cupriavidus necator.
  • the above-mentioned gene introduced into the microorganism of the present disclosure encodes an ⁇ -amylase linked with a secretory signal derived from a microorganism belonging to the genus Cupriavidus.
  • Secretory signal is a DNA sequence encoding a secretory signal peptide.
  • the secretory signal peptide is a signal peptide necessary for secreting a protein synthesized inside the cell, outside the intracellular membrane or outside the cell.
  • the secretory signal is not particularly limited as long as it is a secretory signal derived from a microorganism belonging to the genus Cupriavidus.
  • secretion signals derived from microorganisms belonging to the genus Cupriavidus such as Cupriavidus necator, Cupriavidus oxalaticus, Cupriavidus pauculus, Cupriavidus respiraculi, Cupriavidus taiwanensis and Cupriavidus metallidurans can be used.
  • ⁇ -amylase is not limited as long as it is an ⁇ -amylase having a starch-degrading activity, and the gene introduced into the microorganism of the present disclosure is not limited as long as it encodes such ⁇ -amylase. is not.
  • Examples of the gene encoding ⁇ -amylase include genes encoding ⁇ -amylase derived from microorganisms of Bacillus genus, Streptococcus genus, Panibacillus genus, and Rhyzopus genus. Among these, a gene encoding an ⁇ -amylase derived from a microorganism of the genus Streptococcus is preferable. This is because the microorganism of the present disclosure can suitably decompose raw starch.
  • genes encoding ⁇ -amylase derived from microorganisms belonging to the genus Bacillus include genes encoding ⁇ -amylase derived from microorganisms such as Bacillus stearothermophilus and Bacillus amyloliquefaciens.
  • genes encoding ⁇ -amylase derived from microorganisms belonging to the genus Streptococcus include genes encoding ⁇ -amylase derived from microorganisms such as Streptococcus bovis.
  • genes encoding ⁇ -amylase derived from microorganisms belonging to the genus Paenibacillus include genes encoding ⁇ -amylase derived from microorganisms such as Paenibacillus sp.
  • the “ligation” between the secretory signal derived from a microorganism belonging to the genus Cupriavidus and the gene encoding ⁇ -amylase may be a direct binding between the secretory signal and the part encoding ⁇ -amylase, and the secretory signal peptide and ⁇ -amylase It may be an indirect bond containing one or more bases with the coding portion.
  • a method for introducing into a microorganism belonging to the genus Cupriavidus or a method of introducing into a genomic DNA is used. be able to.
  • Introduction into a microorganism belonging to the genus Cupriavidus using a plasmid vector can be performed using any method commonly used, for example, an electric introduction method, a calcium method and the like.
  • the introduction into the genomic DNA can be carried out using any method commonly used, for example, homologous recombination, a method using a transposon, a method using a phage, or the like.
  • the plasmid vector may be prepared by ligating a DNA fragment having a nucleotide sequence encoding ⁇ -amylase to which a secretion signal derived from a microorganism belonging to the genus Cupriavidus is ligated with a plasmid vector such as pCUP2.
  • the culture condition of the microorganism belonging to the genus Cupriavidus of the present disclosure may be any condition that allows the microorganism to grow.
  • the temperature condition is 20 ° C. or higher and 50 ° C. or lower
  • the culture time is 10 hours or longer and 100 hours. It may be about the following.
  • substances accumulated in microorganisms belonging to the genus Cupriavidus can be collected by a known method.
  • the accumulated substance is PHA
  • it can be recovered by the following method, for example.
  • the cells are separated from the culture solution by a centrifuge or the like, and the cells are washed with methanol or the like and dried.
  • PHA is extracted from the dried cells using an organic solvent such as chloroform.
  • bacterial components are removed by filtration or the like, and a poor solvent such as hexane is added to the filtrate to precipitate PHA. Further, the supernatant may be removed by filtration or centrifugation, and dried to recover PHA.
  • ⁇ -amylase having a starch-degrading activity can be secreted extracellularly, and thus starch can be decomposed and effectively assimilated.
  • the microorganism of the present disclosure is a microorganism belonging to the genus Cupriavidus that synthesizes PHA, PHA can be directly synthesized from starch, and thus it is expected that PHA can be produced at low cost and energy saving.
  • PHA is a thermoplastic polyester that microorganisms produce and accumulate in the microbial cells, has biodegradability, and is incorporated into the natural carbon cycle process, so it enters the ecosystem. It is expected that the adverse effect of PHA will be small, and the practical application of PHA is earnestly desired. Therefore, PHA is one of the good examples of the substance produced using the microorganism of the present disclosure.
  • the microorganism belonging to the genus Cupriavidus of the present disclosure can be used for the production of alcohols such as ethanol, butanol and propanol; acids such as lactic acid, acetic acid, amino acids and nucleic acids, lipids, and fats and oils as well as PHA. ..
  • PHA production examples using Cupriavidus malaysiensis (Non-patent document 7) and Cupriavidus sp.USMAA1020 (Non-patent document 8) have been reported.
  • KNK005dZG strain The KNK005 ⁇ phaZ1,2,6 / nagEG793C, dR strain (hereinafter referred to as “KNK005dZG strain”) used in the following Production Examples, Examples, and Comparative Examples is Aeromonas caviae-derived on the chromosome of the Cupriavidus necator H16 strain.
  • a PHA synthase gene (a gene encoding a PHA synthase having the amino acid sequence set forth in SEQ ID NO: 3) was introduced, and the phaZ1,2,6 gene, which is a PHA-degrading enzyme gene on the chromosome, was deleted to relate to sugar uptake
  • G which is the 793th base of the nagE structural gene, is replaced with C, and the transformed microorganism has a deletion from the start codon to the stop codon of the nagR gene.
  • This transformed microorganism can be produced according to the method described in WO 2017/104722.
  • a plasmid vector was prepared.
  • the manufacturing method is as follows. A nucleotide encoding the trc promoter and the amino acid sequence (SEQ ID NO: 1) of a secretory ⁇ -amylase derived from Streptococcus bovis 148 strain (NRIC 1535) (hereinafter referred to as “amyA1535”) by PCR using a synthetic oligo DNA. A DNA fragment having a sequence (SEQ ID NO: 4) was obtained. The obtained DNA fragment was digested with restriction enzymes EcoRI and SpeI.
  • the secretory ⁇ -amylase is an ⁇ -amylase having a secretory signal peptide.
  • the plasmid vector pCUP2-trc-amyA1535 was introduced into the KNK005dZG strain to obtain a transformant pCUP2-trc-amyA1535 / KNK005dZG strain.
  • the introduction of the plasmid vector into the cells was carried out by electric introduction as follows.
  • a gene pulser manufactured by Biorad was used as the gene introduction device, and a gap 0.2 cm also manufactured by Biorad was used as the cuvette.
  • 400 ⁇ l of competent cells and 20 ⁇ l of expression vector were injected into a cuvette and set in a pulse device, and an electric pulse was applied under the conditions of a capacitance of 25 ⁇ F, a voltage of 1.5 kV and a resistance value of 800 ⁇ .
  • the bacterial solution in the cuvette was cultivated with shaking in Nutrient Broth medium (manufactured by DIFCO) at 30 ° C. for 3 hours, and then at 30 ° C.
  • a plasmid vector was prepared. The production was performed as follows. By PCR using a synthetic oligo DNA, a trc promoter and a DNA fragment having a nucleotide sequence encoding amyA1535 (SPfliCamyA1535) in which the secretory signal peptide is replaced with the secretory signal peptide derived from the Cupriavidus necator H16 strain fliC gene (SEQ ID NO: 5) Got The obtained DNA fragment was digested with restriction enzymes EcoRI and SpeI.
  • This DNA fragment was ligated with the plasmid vector pCUP2 described in WO 2007/049716 cleaved with restriction enzymes MunI and SpeI to obtain a plasmid vector pCUP2-trc-SPfliCamyA1535.
  • the plasmid vector pCUP2-trc-SPfliCamyA1535 was introduced into the KNK005dZG strain in the same manner as in Production Example 1 to obtain a transformant pCUP2-trc-SPfliCamyA1535 / KNK005dZG strain.
  • This DNA fragment was ligated with a plasmid vector pCUP2 described in WO 2007/049716 cut with restriction enzymes MunI and SpeI to obtain a plasmid vector pCUP2-trc-SPplcN4amyA1535.
  • the plasmid vector pCUP2-trc-SPplcN4amyA1535 was introduced into the KNK005dZG strain in the same manner as in Production Example 1 to obtain a transformant pCUP2-trc-SPplcN4amyA1535 / KNK005dZG strain.
  • the composition of the plate medium containing starch was the nutrient agar medium (Difco) to which 1 w / v% corn starch was added.
  • a glycerol stock (50 ⁇ L) of the KNK005dZG strain was inoculated into a seed medium (5 mL) and shake-cultured at a culture temperature of 30 ° C. for 24 hours, and the obtained culture solution was used as a seed mother.
  • Comparative Example 2 Starch-degrading test using pCUP2-trc-amyA1535 / KNK005dZG strain
  • the composition of the seed mother medium and the starch-containing plate medium was the same as that described in Comparative Example 1.
  • the pCUP2-trc-amyA1535 / KNK005dZG strain prepared in Production Example 1 was subjected to culture and starch degrading test in the same manner as in Comparative Example 1. However, kanamycin was added to the seed mother medium and the starch-containing plate medium so that the final concentration was 100 ⁇ g / ml.
  • Example 1 Starch-degrading test using pCUP2-trc-SPfliCamyA1535 / KNK005dZG strain
  • the composition of the seed mother medium and the starch-containing plate medium was the same as that described in Comparative Example 1.
  • the pCUP2-trc-SPfliCamyA1535 / KNK005dZG strain prepared in Production Example 2 was subjected to culture and starch degrading test in the same manner as in Comparative Example 1. However, kanamycin was added to the seed mother medium and the starch-containing plate medium so that the final concentration was 100 ⁇ g / ml.
  • Example 2 Starch-degrading test using pCUP2-trc-SPplcN4amyA1535 / KNK005dZG strain
  • the composition of the seed mother medium and the starch-containing plate medium was the same as that described in Comparative Example 1.
  • the pCUP2-trc-SPplcN4amyA1535 / KNK005dZG strain prepared in Production Example 3 was subjected to culture and starch degrading test in the same manner as in Comparative Example 1. However, kanamycin was added to the seed mother medium and the starch-containing plate medium so that the final concentration was 100 ⁇ g / ml.

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Abstract

Micro-organisme appartenant au genre Cupriavidus capable de dégrader et d'assimiler efficacement l'amidon.　Plus particulièrement, l'invention porte sur un micro-organisme appartenant au genre Cupriavidus auquel un gène codant pour l'alpha-amylase a été introduit; ledit gêne étant lié à un signal de sécrétion dérivé dudit micro-organisme.
PCT/JP2019/044316 2018-11-15 2019-11-12 Micro-organisme appartenant au genre cupriavidus WO2020100888A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114804371A (zh) * 2022-04-01 2022-07-29 安徽农业大学 重金属污染环境的生物修复方法
KR102609658B1 (ko) * 2023-05-17 2023-12-06 코스맥스 주식회사 쿠프리아비더스 메탈리듀란스 균주 및 그의 피부 항당화, 피부 상태 개선 용도

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WO2008010296A1 (fr) * 2006-07-21 2008-01-24 Kaneka Corporation Micro-organisme doté d'un gène remplacé et procédé de production de polyester à l'aide dudit micro-organisme
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JP2015171340A (ja) * 2014-03-12 2015-10-01 東ソー株式会社 組換え水素酸化細菌およびそれを用いたタンパク質製造方法

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WO2008010296A1 (fr) * 2006-07-21 2008-01-24 Kaneka Corporation Micro-organisme doté d'un gène remplacé et procédé de production de polyester à l'aide dudit micro-organisme
TW200918669A (en) * 2007-04-20 2009-05-01 Univ Yuan Ze Method for producing polyhydroxyalkanonate (PHAs) by microorganisms utilizing different carbon sources
JP2015171340A (ja) * 2014-03-12 2015-10-01 東ソー株式会社 組換え水素酸化細菌およびそれを用いたタンパク質製造方法

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KAZUYOSHI SUNAHARA: "Analysis of biopolyester-producing bacterium of ralstonia eutropha that is amylase transgenic strain", PROCEEDINGS OF THE ENZYME ENGINEERING WORKSHOP; NOVEMBER 16, 2018, vol. 80, 16 November 2018 (2018-11-16), pages 61, XP009521223 *
SHASHI KANT BHATIA ET AL: "Starch based polyhydroxybutyrate production in engineered Escherichia coli", BIOPROCESS AND BIOSYSTEMS ENGINEERING, vol. 38, no. 8, 28 March 2015 (2015-03-28), pages 1479 - 1484, XP035524018, ISSN: 1615-7591, DOI: 10.1007/s00449-015-1390-y *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114804371A (zh) * 2022-04-01 2022-07-29 安徽农业大学 重金属污染环境的生物修复方法
KR102609658B1 (ko) * 2023-05-17 2023-12-06 코스맥스 주식회사 쿠프리아비더스 메탈리듀란스 균주 및 그의 피부 항당화, 피부 상태 개선 용도

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