WO2020100888A1 - Microorganism belonging to genus cupriavidus - Google Patents

Microorganism belonging to genus cupriavidus Download PDF

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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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starch
cupriavidus
microorganism
genus
amylase
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PCT/JP2019/044316
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French (fr)
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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Publication of WO2020100888A1 publication Critical patent/WO2020100888A1/en

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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.

Abstract

The present invention makes it possible to provide a microorganism belonging to the genus Cupriavidus that is capable of degrading and effectively assimilating starch.　A microorganism belonging to the genus Cupriavidus into which a gene encoding α-amylase, to which a secretion signal derived from a microorganism belonging to the genus Cupriavidus is linked, has been introduced.

Description

Ｃｕｐｒｉａｖｉｄｕｓ属に属する微生物Microorganisms belonging to the genus Cupriavidus
　本発明は、Cupriavidus属に属する微生物に関する。 The present invention relates to a microorganism belonging to the genus Cupriavidus.
　環境問題、食糧問題、健康及び安全に対する意識の高まり；並びに天然及び自然志向の高まり等を背景に、微生物の生産、及び微生物による物質製造（例えば、発酵生産、及びバイオ変換等）の意義及び重要性が益々高まっている。 Significance and importance of microbial production and substance production by microorganisms (for example, fermentative production, bioconversion, etc.) against the backdrop of heightened awareness of environmental issues, food issues, health and safety; Sex is increasing.
　微生物の生産、及び微生物による物質の製造においては、微生物によって好適に資化される炭素源（例えば、培養及び／又は発酵等のための炭素源）が必要である。その炭素源の代表的なものとして、糖質（例えば、澱粉）、油脂（例えば、植物油脂）、及び脂肪酸（例えば、植物由来の脂肪酸）等の利用が益々望まれてきている。糖質原料である澱粉は、ポテト、コーン、米及びキャッサバ等から生産され得る安価な発酵原料であり、好適な炭素源の候補の一つとして考えられるかもしれない。 In the production of microorganisms and the production of substances by microorganisms, a carbon source that can be suitably assimilated by microorganisms (for example, a carbon source for cultivation and / or fermentation) is required. As typical carbon sources, utilization of sugars (eg starch), oils and fats (eg vegetable oils), and fatty acids (eg plant-derived fatty acids) has been increasingly desired. 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.
　グルコースやシュークロース等の低分子量の糖質原料のように、一般に微生物が細胞内に取り込み、異化及び同化されやすい構造の炭素源を原料として利用することは容易である。しかし、澱粉は、グルコースが多数結合した高分子であり、特に生澱粉は、アミロースとアミロペクチンの鎖が水素結合によって規則的に集合したミセル構造（結晶構造）を有しており、酵素による分解を受けにくく、そのままでは微生物によって資化できない問題がある。 ▽ 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. However, 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.
　生澱粉をグルコース等の低分子量の糖質原料に分解するためには、熱や物理的破壊等のエネルギー投入により結晶化度を低下させることが必要であり、通常、加熱糊化（可溶化、又はα化とも言う）が行われる（特許文献１）。そして、その後、分解酵素であるアミラーゼを作用させて糖化する。しかし、このような方法では糊化工程でエネルギーを消費してしまう。このため、澱粉、特に生澱粉は有効に産業利用されているとはいえない。 In order to decompose raw starch into low-molecular-weight sugar raw materials such as glucose, it is necessary to reduce the crystallinity by inputting heat or energy such as physical destruction, and usually heat gelatinization (solubilization, solubilization, Alternatively, it is also referred to as alpha conversion) (Patent Document 1). Then, after that, amylase which is a degrading enzyme is caused to act on the saccharification. However, this method consumes energy in the gelatinization process. Therefore, it cannot be said that starch, particularly raw starch, is effectively used industrially.
　生澱粉の利用に伴うエネルギー消費を少なくするため、物質生産を担う微生物が生澱粉を分解可能なアミラーゼを分泌でき、生澱粉を原料として物質を直接生産させる事ができれば意義深い。 To reduce the energy consumption associated with the use of raw starch, it is significant if 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.
　微生物を用いた物質生産の宿主としてCupriavidus属に属する微生物が挙げられる。また、Cupriavidus属に属する微生物を用いた物質生産の例として、例えば、ポリヒドロキシアルカノエート（ＰＨＡ）等の脂肪族ポリエステル、並びにブタノール及びイソプロパノール等が挙げられる。 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.
　Cupriavidus属に属する微生物であるCupriavidus necatorを用いて、アセトンとブタノールを生産した例がある（非特許文献１）。Cupriavidus necatorを用いて、糖質原料であるフルクトースからイソプロパノールを生産した例がある（非特許文献２）。また、Cupriavidus necatorを用いて、スクロースからポリヒドロキシアルカノエート（ＰＨＡ）を生産した例が報告されている（非特許文献３）。しかし、いずれも原料として澱粉は利用していない。 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.
　Cupriavidus necatorは、αアミラーゼをコードする遺伝子を有さないため、澱粉分解酵素を発現できず、澱粉を好適に利用できないため、糖化したポテト由来の澱粉を用いてＰＨＡを生産した例が報告されている（非特許文献４）。 Since Cupriavidus necator does not have a gene encoding α-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).
特許第４７６７１２８号公報Japanese Patent No. 4767128
　本発明者らは、まず、非可溶化澱粉（生澱粉とも言う）を分解可能であると報告されているStreptococcus bovis由来のαアミラーゼ（非特許文献５）をCupriavidus necator H16株に導入し、その澱粉分解活性を確認したが、分解活性を示さなかった。このように、澱粉、特に生澱粉を炭素源として好適に利用することは容易ではない。 The present inventors first introduced an α-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.
　例えば、非特許文献６ではRhizopus由来の分泌シグナル配列を利用し、酵母Saccharomyces cerevisiaeにStreptococcus bovis由来αアミラーゼを表層提示発現（分泌発現の一種）させている。タンパク質を分泌発現させるためには、分泌シグナルとタンパク質をコードする遺伝子を連結させた遺伝子配列の導入が必須であるが、分泌タンパク質と分泌シグナルをどのように組み合わせれば、目的とする微生物が澱粉分解活性を示すのかを見出すのは困難である。細胞外に酵素を分泌させることは、細胞内で単に異種遺伝子を発現させるよりも困難とされている。澱粉を原料として、ポリヒドロキシアルカノエート（ＰＨＡ）等の脂肪族ポリエステル等を生産可能なCupriavidus属に属する微生物による効率的な物質製造を工業的に可能にするためには、更なる研究が必要である。 For example, in Non-Patent Document 6, a Rhizopus-derived secretory signal sequence is used to cause yeast Saccharomyces cerevisiae to express Streptococcus bovis-derived α-amylase on the surface (a type of secretory expression). In order to secrete and express a protein, it is essential to introduce a gene sequence in which a secretory signal and a gene encoding the protein are linked. However, if the secretory protein and the secretory signal are combined, 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 starch as a raw material. is there.
　本発明は、澱粉を分解し、有効に資化し得るCupriavidus属に属する微生物を提供することを目的とする。 An object of the present invention is to provide a microorganism belonging to the genus Cupriavidus that can decompose starch and effectively assimilate it.
　本発明は、Cupriavidus属に属する微生物由来の分泌シグナルが連結した、αアミラーゼをコードする遺伝子を導入した、Cupriavidus属に属する微生物に関する。 The present invention relates to a microorganism belonging to the genus Cupriavidus, into which a gene encoding an α-amylase, into which a secretory signal derived from a microorganism belonging to the genus Cupriavidus has been introduced.
　前記微生物において、前記αアミラーゼが、Streptococcus属に属する微生物由来のαアミラーゼであってよい。 In the microorganism, the α-amylase may be an α-amylase derived from a microorganism belonging to the genus Streptococcus.
　前記微生物において、前記Cupriavidus属に属する微生物が、ポリヒドロキシアルカノエートを合成してよい。 In the above microorganism, a microorganism belonging to the genus Cupriavidus may synthesize polyhydroxyalkanoate.
　前記微生物において、前記Cupriavidus属に属する微生物が、Cupriavidus necatorであってよい。 Among the microorganisms, the microorganism belonging to the genus Cupriavidus may be Cupriavidus necator.
　前記ポリヒドロキシアルカノエートが、ポリ（３－ヒドロキシブチレート－コ－３－ヒドロキシヘキサノエート）であってよい。 The polyhydroxyalkanoate may be poly (3-hydroxybutyrate-co-3-hydroxyhexanoate).
　本発明によれば、澱粉を分解し、有効に資化し得るCupriavidus属に属する微生物を提供することができる。 According to the present invention, it is possible to provide a microorganism belonging to the genus Cupriavidus that can decompose starch and effectively assimilate it.
　本開示の微生物は、Cupriavidus属に属する微生物由来の分泌シグナルが連結した、αアミラーゼをコードする遺伝子を導入した、Cupriavidus属に属する微生物である。 The microorganism of the present disclosure is a microorganism belonging to the genus Cupriavidus, into which a gene encoding an α-amylase, into which a secretion signal derived from a microorganism belonging to the genus Cupriavidus is linked, has been introduced.
　本開示の微生物は、本発明者らが、Cupriavidus属に属する微生物が保有する遺伝子の中から、細胞外に分泌されるタンパク質（分泌タンパク質）をコードする遺伝子を見出し、該遺伝子中に存在するシグナル様配列（分泌シグナル）とαアミラーゼ遺伝子とを連結させた遺伝子をCupriavidus属に属する微生物に導入したものである。当該微生物によれば、澱粉分解活性を有する澱粉分解酵素であるαアミラーゼを細胞外に分泌させることが可能であるため、生澱粉をα化することなく資化でき、有効利用できる。本開示の微生物を用いた物質生産は、工業的な物質生産としても適している。 As for the microorganism of the present disclosure, 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 α-amylase gene was introduced into a microorganism belonging to the genus Cupriavidus. According to the microorganism, since α-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 α and can be effectively used. The substance production using the microorganism of the present disclosure is also suitable for industrial substance production.
　本開示の微生物は、Cupriavidus属に属する微生物であれば、限定されるものではないが、ポリヒドロキシアルカノエートを合成すること、つまり、ＰＨＡ合成能を有することが好ましい。Cupriavidus属に属する微生物の中でも、高いＰＨＡ合成能を有するため、Cupriavidus necatorが特に好ましい。 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. Among microorganisms belonging to the genus Cupriavidus, Cupriavidus necator is particularly preferable because it has high PHA synthesizing ability.
　ＰＨＡ合成能を有するCupriavidus属に属する微生物としては、ＰＨＡ合成酵素遺伝子を本来的に有する野生株だけではなく、そのような野生株を人工的に突然変異処理して得られる変異株や、ＰＨＡ合成酵素遺伝子を本来的に有しない微生物に対して遺伝子工学的手法により外来のＰＨＡ合成酵素遺伝子が導入された組み換え菌株であってもよい。 As a 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.
　ＰＨＡ合成能を有するCupriavidus属に属する微生物が、遺伝子工学的手法により外来のＰＨＡ合成酵素遺伝子が導入された組み換え菌株である場合、前記外来のＰＨＡ合成酵素遺伝子としては、例えば、Cupriavidus necator H16株が保有する配列番号２に記載するアミノ酸配列をコードするＰＨＡ合成酵素遺伝子、又は、該アミノ酸配列に対して８５％以上の配列同一性を有し、且つ、ＰＨＡ合成活性を有するポリペプチドをコードするＰＨＡ合成酵素遺伝子；並びにAeromonas caviaeが保有する配列番号３に記載するアミノ酸配列をコードするＰＨＡ合成酵素遺伝子、又は、該アミノ酸配列に対して８５％以上の配列同一性を有し、且つ、ＰＨＡ合成活性を有するポリペプチドをコードするＰＨＡ合成酵素遺伝子等が挙げられるが、これらに限定されず、その他のＰＨＡ合成酵素遺伝子も好適に利用出来る。なお、上記配列同一性は好ましくは９０％以上であり、より好ましくは９５％以上、特に好ましくは９９％以上である。これらの中でも、ＰＨＡとしてポリ（３－ヒドロキシブチレート－コ－３－ヒドロキシヘキサノエート）（ＰＨＢＨと称することがある）を合成活性を有するＰＨＡ合成酵素遺伝子が好ましい。ＰＨＢＨは、Cupriavidus necatorに、Aeromonas caviae由来のＰＨＡ合成酵素遺伝子が導入された形質転換体により合成すればよい。 When 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, as 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 Examples of 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. Among these, 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.
　本開示の微生物に導入された上記遺伝子は、Cupriavidus属に属する微生物由来の分泌シグナルが連結したαアミラーゼをコードするものである。 The above-mentioned gene introduced into the microorganism of the present disclosure encodes an α-amylase linked with a secretory signal derived from a microorganism belonging to the genus Cupriavidus.
　分泌シグナルとは、分泌シグナルペプチドをコードするＤＮＡ配列である。また、分泌シグナルペプチドとは、細胞内で合成されたタンパク質を細胞内膜外又は細胞外へ分泌させるために必要なシグナルペプチドである。 Secretory signal is a DNA sequence encoding a secretory signal peptide. In addition, 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.
　分泌シグナルは、Cupriavidus属に属する微生物由来の分泌シグナルであれば特に制限されない。例えばCupriavidus necator、Cupriavidus oxalaticus、Cupriavidus pauculus、Cupriavidus respiraculi、Cupriavidus taiwanensis及びCupriavidus metallidurans等のCupriavidus属に属する微生物由来の分泌シグナルが利用出来る。 The secretory signal is not particularly limited as long as it is a secretory signal derived from a microorganism belonging to the genus Cupriavidus. For example, 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.
　αアミラーゼは、澱粉分解活性を有するαアミラーゼであればなんら制限されるものではなく、本開示の微生物に導入された上記遺伝子は、このようなαアミラーゼをコードしていればなんら制限されるものではない。 α-amylase is not limited as long as it is an α-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 α-amylase. is not.
　αアミラーゼをコードする遺伝子としては、例えばBacillus属、Streptococcus属、Panibacillus属、及びRhyzopus属の微生物由来のαアミラーゼをコードする遺伝子が挙げられる。これらの中でも、Streptococcus属の微生物由来のαアミラーゼをコードする遺伝子が好ましい。本開示の微生物が生澱粉を好適に分解可能となるためである。 Examples of the gene encoding α-amylase include genes encoding α-amylase derived from microorganisms of Bacillus genus, Streptococcus genus, Panibacillus genus, and Rhyzopus genus. Among these, a gene encoding an α-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.
　Bacillus属に属する微生物由来のαアミラーゼをコードする遺伝子として、Bacillus stearothermophilus、及びBacillus amyloliquefaciens等の微生物由来のαアミラーゼをコードする遺伝子が挙げられる。 Examples of genes encoding α-amylase derived from microorganisms belonging to the genus Bacillus include genes encoding α-amylase derived from microorganisms such as Bacillus stearothermophilus and Bacillus amyloliquefaciens.
　Streptococcus属に属する微生物由来のαアミラーゼをコードする遺伝子として、Streptococcus bovis等の微生物由来のαアミラーゼをコードする遺伝子が挙げられる。 Examples of genes encoding α-amylase derived from microorganisms belonging to the genus Streptococcus include genes encoding α-amylase derived from microorganisms such as Streptococcus bovis.
　Paenibacillus属に属する微生物由来のαアミラーゼをコードする遺伝子として、Paenibacillus sp. 等の微生物由来のαアミラーゼをコードする遺伝子が挙げられる。 Examples of genes encoding α-amylase derived from microorganisms belonging to the genus Paenibacillus include genes encoding α-amylase derived from microorganisms such as Paenibacillus sp.
　Cupriavidus属に属する微生物由来の分泌シグナルとαアミラーゼをコードする遺伝子との「連結」は、分泌シグナルとαアミラーゼをコードする部分との直接的な結合であってよく、分泌シグナルペプチドとαアミラーゼをコードする部分との間に１又は２以上の塩基が含まれる間接的な結合であってよい。 The “ligation” between the secretory signal derived from a microorganism belonging to the genus Cupriavidus and the gene encoding α-amylase may be a direct binding between the secretory signal and the part encoding α-amylase, and the secretory signal peptide and α-amylase It may be an indirect bond containing one or more bases with the coding portion.
　Cupriavidus属に属する微生物由来の分泌シグナルが連結したαアミラーゼをコードする遺伝子を導入する方法としては、プラスミドベクターを用いてCupriavidus属に属する微生物に導入する方法や、ゲノムＤＮＡに導入する方法などを用いることができる。プラスミドベクターを用いたCupriavidus属に属する微生物への導入は、一般的に行われている方法、例えば、電気導入法、カルシウム法等の任意の方法を用いて行うことができる。ゲノムＤＮＡへの導入は、一般的に行われている方法、例えば、相同性組み換え、トランスポゾンを利用する方法、及びファージを利用する方法等の任意の方法を用いて行うことができる。 As a method for introducing a gene encoding an α-amylase to which a secretion signal derived from a microorganism belonging to the genus Cupriavidus is linked, a method of 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.
　プラスミドベクターは、Cupriavidus属に属する微生物由来の分泌シグナルが連結したαアミラーゼをコードする塩基配列を有するＤＮＡ断片を、ｐＣＵＰ２等のプラスミドベクターと連結することにより作製すればよい。 The plasmid vector may be prepared by ligating a DNA fragment having a nucleotide sequence encoding α-amylase to which a secretion signal derived from a microorganism belonging to the genus Cupriavidus is ligated with a plasmid vector such as pCUP2.
　本開示のCupriavidus属に属する微生物の培養条件は、当該微生物が育成可能な条件であればよいが、例えば、温度条件としては、２０℃以上５０℃以下、培養時間としては、１０時間以上１００時間以下程度であってよい。 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. For example, the temperature condition is 20 ° C. or higher and 50 ° C. or lower, and the culture time is 10 hours or longer and 100 hours. It may be about the following.
　また、Cupriavidus属に属する微生物内に蓄積された物質は公知の方法により回収することができる。蓄積された物質がＰＨＡである場合、例えば、次のような方法により回収することができる。Cupriavidus属に属する微生物の培養終了後、培養液から遠心分離器等で菌体を分離し、その菌体をメタノール等により洗浄し、乾燥させる。この乾燥菌体から、クロロホルム等の有機溶剤を用いてＰＨＡを抽出する。このＰＨＡを含んだ有機溶剤の溶液から、濾過等によって菌体成分を除去し、そのろ液にヘキサン等の貧溶媒を加えてＰＨＡを沈殿させる。さらに、濾過や遠心分離によって上澄み液を除去し、乾燥させてＰＨＡを回収すればよい。 Also, substances accumulated in microorganisms belonging to the genus Cupriavidus can be collected by a known method. When the accumulated substance is PHA, it can be recovered by the following method, for example. After the culture of the microorganism belonging to the genus Cupriavidus is completed, 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. From the solution of the organic solvent containing PHA, 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.
　本開示の微生物によれば、澱粉分解活性を有するαアミラーゼを細胞外に分泌させることが可能であるため、澱粉を分解し、有効に資化し得る。 According to the microorganism of the present disclosure, α-amylase having a starch-degrading activity can be secreted extracellularly, and thus starch can be decomposed and effectively assimilated.
　さらに、本開示の微生物がＰＨＡを合成するCupriavidus属に属する微生物であれば、澱粉から直接ＰＨＡを合成できるため、省エネルギー、低コストでのＰＨＡ生産が可能になることが期待される。 Furthermore, if 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.
　現在、環境への意識の高まりから非石油由来のプラスチックが注目されている。非石油来のプラスチックのうち、特にＰＨＡは微生物が菌体内に産生及び蓄積される熱可塑性ポリエステルであって、生分解性を有しており、自然界の炭素循環プロセスに取り込まれることから生態系への悪影響が小さいと予想され、ＰＨＡの実用化が切望されている。そのため、ＰＨＡは、本開示の微生物を用いて生産される物質の好例の一つである。 Currently, non-petroleum-derived plastics are attracting attention due to increasing environmental awareness. Among non-petroleum-derived plastics, PHA, in particular, 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.
　本開示のCupriavidus属に属する微生物は、ＰＨＡの他、エタノール、ブタノール及びプロパノール等のアルコール類；乳酸、酢酸、アミノ酸及び核酸等の酸類、脂質類、並びに油脂類等の生産にも用いることができる。例えば、Cupriavidus malaysiensis（非特許文献７）やCupriavidus sp. USMAA1020（非特許文献８）を用いたＰＨＡの生産例が報告されている。 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. .. For example, PHA production examples using Cupriavidus malaysiensis (Non-patent document 7) and Cupriavidus sp.USMAA1020 (Non-patent document 8) have been reported.
　以下に実施例で本発明を詳細に説明するが、本発明はこれら実施例によって何ら制限されるものではない。 The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.
　全体的な遺伝子操作は、Molecular Cloning （Cold Spring Harbor Laboratory Press (1989又は2001)）の記載に従い行うことができる。また、遺伝子操作に使用する酵素、及びクローニング宿主等は、市場の供給者から購入し、その説明に従い使用することができる。なお、酵素としては、遺伝子操作に使用できるものであれば特に限定されない。 ∙ Overall genetic manipulation can be performed as described in Molecular Cloning (Cold Spring Harbor Laboratory Press (1989 or 2001)). The enzyme used for genetic manipulation, the cloning host and the like can be purchased from suppliers on the market and used according to the instructions. The enzyme is not particularly limited as long as it can be used for gene manipulation.
　以下の製造例、実施例、及び比較例で使用されるＫＮＫ００５ΔｐｈａＺ１，２，６／ｎａｇＥＧ７９３Ｃ，ｄＲ株（以下、「ＫＮＫ００５ｄＺＧ株」と記載する）は、Cupriavidus necator H16株の染色体上にAeromonas caviae由来のＰＨＡ合成酵素遺伝子（配列番号３に記載のアミノ酸配列を有するＰＨＡ合成酵素をコードする遺伝子）が導入され、染色体上のＰＨＡ分解酵素遺伝子であるｐｈａＺ１，２，６遺伝子が欠失し、糖取込みに関するｎａｇオペロン遺伝子のうち、ｎａｇＥ構造遺伝子の７９３番目の塩基であるＧがＣに置換され、さらにｎａｇＲ遺伝子の開始コドンから終止コドンまでを欠失した形質転換微生物である。この形質転換微生物は、国際公開第２０１７／１０４７２２号に記載の方法に準じて作製することができる。 The KNK005ΔphaZ1,2,6 / 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 In the nag operon gene, 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.
　（製造例１）ｐＣＵＰ２－ｔｒｃ－ａｍｙＡ１５３５／ＫＮＫ００５ｄＺＧ株の作製
　まず、プラスミドベクターの作製を行った。作製方法は以下のとおりである。合成オリゴＤＮＡを用いたＰＣＲにより、ｔｒｃプロモーター、及びStreptococcus bovis 148株（NRIC 1535）に由来する分泌型αアミラーゼ（以下、「ａｍｙＡ１５３５」と記載する）のアミノ酸配列（配列番号１）をコードする塩基配列を有するＤＮＡ断片（配列番号４）を得た。得られたＤＮＡ断片を制限酵素ＥｃｏＲＩ及びＳｐｅＩで消化した。このＤＮＡ断片を、国際公開公報２００７／０４９７１６号公報に記載のプラスミドベクターｐＣＵＰ２を制限酵素ＭｕｎＩ及びＳｐｅＩで切断したものと連結し、プラスミドベクターｐＣＵＰ２－ｔｒｃ－ａｍｙＡ１５３５を得た。なお、当該分泌型αアミラーゼとは、分泌シグナルペプチドを有するαアミラーゼである。 (Production Example 1) Preparation of pCUP2-trc-amyA1535 / KNK005dZG strain First, 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 α-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. This DNA fragment was ligated with the plasmid vector pCUP2 described in WO2007 / 049716 cleaved with restriction enzymes MunI and SpeI to obtain a plasmid vector pCUP2-trc-amyA1535. The secretory α-amylase is an α-amylase having a secretory signal peptide.
　次に、プラスミドベクターｐＣＵＰ２－ｔｒｃ－ａｍｙＡ１５３５をＫＮＫ００５ｄＺＧ株へ導入し、形質転換体ｐＣＵＰ２－ｔｒｃ－ａｍｙＡ１５３５／ＫＮＫ００５ｄＺＧ株を得た。 Next, the plasmid vector pCUP2-trc-amyA1535 was introduced into the KNK005dZG strain to obtain a transformant pCUP2-trc-amyA1535 / KNK005dZG strain.
　プラスミドベクターの細胞への導入は以下のように電気導入によって行った。遺伝子導入装置はＢｉｏｒａｄ社製のジーンパルサーを用い、キュベットは同じくＢｉｏｒａｄ社製のｇａｐ０．２ｃｍを用いた。キュベットに、コンピテント細胞４００μｌと発現ベクター２０μｌを注入してパルス装置にセットし、静電容量２５μＦ、電圧１．５ｋＶ、抵抗値８００Ωの条件で電気パルスをかけた。パルス後、キュベット内の菌液をＮｕｔｒｉｅｎｔＢｒｏｔｈ培地（ＤＩＦＣＯ社製）で３０℃、３時間振とう培養し、選択プレート（ＮｕｔｒｉｅｎｔＡｇａｒ培地（ＤＩＦＣＯ社製）、カナマイシン１００ｍｇ／Ｌ）で、３０℃にて２日間培養して、生育してきた形質転換体ｐＣＵＰ２－ｔｒｃ－ａｍｙＡ１５３５／ＫＮＫ００５ｄＺＧ株を取得した。 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 μl of competent cells and 20 μl 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 μF, a voltage of 1.5 kV and a resistance value of 800Ω. After the pulse, 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. with a selection plate (Nutrient Agar medium (manufactured by DIFCO), 100 mg / L) at 30 ° C. After culturing for one day, the transformant pCUP2-trc-amyA1535 / KNK005dZG strain that had grown was obtained.
　（製造例２）ｐＣＵＰ２－ｔｒｃ－ＳＰｆｌｉＣａｍｙＡ１５３５／ＫＮＫ００５ｄＺＧ株の作製
　まず、プラスミドベクターの作製を行った。作製は以下のように行った。合成オリゴＤＮＡを用いたＰＣＲにより、ｔｒｃプロモーター、及び分泌シグナルペプチドがCupriavidus necator H16株ｆｌｉＣ遺伝子由来の分泌シグナルペプチドに置換されたａｍｙＡ１５３５（ＳＰｆｌｉＣａｍｙＡ１５３５）をコードする塩基配列を有するＤＮＡ断片（配列番号５）を得た。得られたＤＮＡ断片を制限酵素ＥｃｏＲＩ及びＳｐｅＩで消化した。このＤＮＡ断片を、国際公開公報第２００７／０４９７１６号公報に記載のプラスミドベクターｐＣＵＰ２を制限酵素ＭｕｎＩ及びＳｐｅＩで切断したものと連結し、プラスミドベクターｐＣＵＰ２－ｔｒｃ－ＳＰｆｌｉＣａｍｙＡ１５３５を得た。 (Production Example 2) Preparation of pCUP2-trc-SPfliCamyA1535 / KNK005dZG strain First, 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.
　次に、プラスミドベクターｐＣＵＰ２－ｔｒｃ－ＳＰｆｌｉＣａｍｙＡ１５３５を、製造例１と同様の方法で、ＫＮＫ００５ｄＺＧ株へ導入し、形質転換体ｐＣＵＰ２－ｔｒｃ－ＳＰｆｌｉＣａｍｙＡ１５３５／ＫＮＫ００５ｄＺＧ株を得た。 Next, 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.
　（製造例３）ｐＣＵＰ２－ｔｒｃ－ＳＰｐｌｃＮ４ａｍｙＡ１５３５／ＫＮＫ００５ｄＺＧ株の作製
　まず、プラスミドベクターの作製を行った。作製は以下のように行った。合成オリゴＤＮＡを用いたＰＣＲにより、ｔｒｃプロモーター、及び分泌シグナルペプチドがCupriavidus necator H16株ｐｌｃＮ４遺伝子由来の分泌シグナルペプチドに置換されたａｍｙＡ１５３５（ＳＰｐｌｃＮ４ａｍｙＡ１５３５）をコードする塩基配列を有するＤＮＡ断片（配列番号６）を得た。得られたＤＮＡ断片を制限酵素ＥｃｏＲＩ及びＳｐｅＩで消化した。このＤＮＡ断片を、国際公開公報第２００７／０４９７１６号公報に記載のプラスミドベクターｐＣＵＰ２を制限酵素ＭｕｎＩ及びＳｐｅＩで切断したものと連結し、プラスミドベクターｐＣＵＰ２－ｔｒｃ－ＳＰｐｌｃＮ４ａｍｙＡ１５３５を得た。 (Production Example 3) Preparation of pCUP2-trc-SPplcN4amyA1535 / KNK005dZG strain First, a plasmid vector was prepared. The production was performed as follows. A DNA fragment having a nucleotide sequence encoding amyA1535 (SPplcN4amyA1535) in which the secretion signal peptide derived from the cupriavidus necator H16 strain plcN4 gene has been replaced by PCR by using a synthetic oligo DNA (PCR sequence number 6) Got The obtained DNA fragment was digested with restriction enzymes EcoRI and SpeI. 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.
　次に、プラスミドベクターｐＣＵＰ２－ｔｒｃ－ＳＰｐｌｃＮ４ａｍｙＡ１５３５を、製造例１と同様の方法で、ＫＮＫ００５ｄＺＧ株へ導入し、形質転換体ｐＣＵＰ２－ｔｒｃ－ＳＰｐｌｃＮ４ａｍｙＡ１５３５／ＫＮＫ００５ｄＺＧ株を得た。 Next, 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.
　（比較例１）ＫＮＫ００５ｄＺＧ株による澱粉分解能試験
　種母培地の組成は、１ｗ／ｖ％　Ｍｅａｔ－ｅｘｔｒａｃｔ、１ｗ／ｖ％　Ｂａｃｔｏ－Ｔｒｙｐｔｏｎ、０．２ｗ／ｖ％　Ｙｅａｓｔ－ｅｘｔｒａｃｔ、０．９ｗ／ｖ％　Ｎａ２ＨＰＯ４・１２Ｈ２Ｏ、０．１５ｗ／ｖ％　ＫＨ２ＰＯ４とした。 (Comparative Example 1) Starch-degrading test using KNK005dZG strain The composition of the seed mother medium was 1 w / v% Meat-extract, 1 w / v% Bacto-Trypton, 0.2 w / v% Yeast-extract, 0.9 w / v%. Na 2 HPO 4 · 12H 2 O and 0.15 w / v% KH 2 PO 4 .
　澱粉含有プレート培地の組成は、ニュートリエント寒天培地（Ｄｉｆｃｏ）に１ｗ／ｖ％　コーンスターチを加えたものとした。 The composition of the plate medium containing starch was the nutrient agar medium (Difco) to which 1 w / v% corn starch was added.
　ＫＮＫ００５ｄＺＧ株のグリセロールストック（５０μＬ）を種母培地（５ｍＬ）に接種して培養温度３０℃で２４時間振とう培養し、得られた培養液を種母とした。 A glycerol stock (50 μL) 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.
　澱粉分解能試験は以下のように行った。滅菌水を用いてＯＤ６００＝２．０に調整した種母培養液１０μｌを澱粉含有プレート培地（ニュートリエント寒天培地（Ｄｉｆｃｏ）に１ｗ／ｖ％　コーンスターチ）にスポットし、３０℃で３日間生育させた。生育したコロニーの周囲に、澱粉が分解されたことによるクリアゾーン（ハローとも称する）が観察されるか否かを判定した。また、クリアゾーンの直径が大きい方が、澱粉分解能が高く優れる。 The starch resolution test was performed as follows. 10 μl of seed culture broth adjusted to OD600 = 2.0 with sterilized water was spotted on a starch-containing plate medium (1 w / v% corn starch on nutrient agar medium (Difco)) and grown at 30 ° C. for 3 days .. It was determined whether or not a clear zone (also referred to as a halo) due to the decomposition of starch was observed around the grown colonies. In addition, the larger the diameter of the clear zone, the better the starch decomposing ability.
　その結果、本比較例において、クリアゾーンは観察されず、ＫＮＫ００５ｄＺＧ株は澱粉分解能を示さなかった。結果は表１に示す。 As a result, in this comparative example, no clear zone was observed, and the KNK005dZG strain did not show starch decomposing ability. The results are shown in Table 1.
　（比較例２）ｐＣＵＰ２－ｔｒｃ－ａｍｙＡ１５３５／ＫＮＫ００５ｄＺＧ株による澱粉分解能試験
　種母培地及び澱粉含有プレート培地の組成は、比較例１に記載のものと同様とした。製造例１で作製したｐＣＵＰ２－ｔｒｃ－ａｍｙＡ１５３５／ＫＮＫ００５ｄＺＧ株について、比較例１と同様の方法で培養及び澱粉分解能試験を行った。ただし、種母培地及び澱粉含有プレート培地は、カナマイシンを最終濃度１００μｇ／ｍｌとなるように添加した。 (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 μg / ml.
　本比較例において、クリアゾーンは観察されず、ｐＣＵＰ２－ｔｒｃ－ａｍｙＡ１５３５／ＫＮＫ００５ｄＺＧ株は澱粉分解能を示さなかった。結果は表１に示す。 In this comparative example, no clear zone was observed, and the pCUP2-trc-amyA1535 / KNK005dZG strain did not show starch degrading ability. The results are shown in Table 1.
　（実施例１）ｐＣＵＰ２－ｔｒｃ－ＳＰｆｌｉＣａｍｙＡ１５３５／ＫＮＫ００５ｄＺＧ株による澱粉分解能試験
　種母培地及び澱粉含有プレート培地の組成は、比較例１に記載のものと同様とした。製造例２で作製したｐＣＵＰ２－ｔｒｃ－ＳＰｆｌｉＣａｍｙＡ１５３５／ＫＮＫ００５ｄＺＧ株について、比較例１と同様の方法で培養及び澱粉分解能試験を行った。ただし、種母培地及び澱粉含有プレート培地は、カナマイシンを最終濃度１００μｇ／ｍｌとなるように添加した。 (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 μg / ml.
　本実施例においては、クリアゾーンが観察され、ｐＣＵＰ２－ｔｒｃ－ＳＰｆｌｉＣａｍｙＡ１５３５／ＫＮＫ００５ｄＺＧ株は澱粉分解能を示した。クリアゾーンの直径を測定した。結果は表１に示す。 In this example, a clear zone was observed, and the pCUP2-trc-SPfliCamyA1535 / KNK005dZG strain showed starch degrading ability. The diameter of the clear zone was measured. The results are shown in Table 1.
　（実施例２）ｐＣＵＰ２－ｔｒｃ－ＳＰｐｌｃＮ４ａｍｙＡ１５３５／ＫＮＫ００５ｄＺＧ株による澱粉分解能試験
　種母培地及び澱粉含有プレート培地の組成は、比較例１に記載のものと同様とした。製造例３で作製したｐＣＵＰ２－ｔｒｃ－ＳＰｐｌｃＮ４ａｍｙＡ１５３５／ＫＮＫ００５ｄＺＧ株について、比較例１と同様の方法で培養及び澱粉分解能試験を行った。ただし、種母培地及び澱粉含有プレート培地は、カナマイシンを最終濃度１００μｇ／ｍｌとなるように添加した。 (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 μg / ml.
　本実施例においては、クリアゾーンが観察され、ｐＣＵＰ２－ｔｒｃ－ＳＰｐｌｃＮ４ａｍｙＡ１５３５／ＫＮＫ００５ｄＺＧ株は澱粉分解能を示した。クリアゾーンの直径を測定した。結果は表１に示す。 In this example, a clear zone was observed, and the pCUP2-trc-SPplcN4amyA1535 / KNK005dZG strain showed starch degrading ability. The diameter of the clear zone was measured. The results are shown in Table 1.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　Cupriavidus属に属する微生物由来の分泌シグナルが連結した、αアミラーゼをコードする遺伝子を導入した実施例の微生物は、澱粉を分解できたため、澱粉を有効に資化し得ることが示された。 It was shown that the microorganism of the example into which the gene encoding α-amylase, in which a secretory signal derived from a microorganism belonging to the genus Cupriavidus was linked, was able to decompose starch, and thus can effectively assimilate starch.

Claims (5)

  1. 　Cupriavidus属に属する微生物由来の分泌シグナルが連結した、αアミラーゼをコードする遺伝子を導入した、Cupriavidus属に属する微生物。 A microorganism belonging to the genus Cupriavidus, into which a gene encoding α-amylase, which is linked with a secretion signal derived from a microorganism belonging to the genus Cupriavidus, has been introduced.
  2. 　前記αアミラーゼが、Streptococcus属に属する微生物由来のαアミラーゼである、請求項１に記載の微生物。 The microorganism according to claim 1, wherein the α-amylase is an α-amylase derived from a microorganism belonging to the genus Streptococcus.
  3. 　前記Cupriavidus属に属する微生物が、ポリヒドロキシアルカノエートを合成する、請求項１又は２に記載の微生物。 The microorganism according to claim 1 or 2, wherein the microorganism belonging to the genus Cupriavidus synthesizes polyhydroxyalkanoate.
  4. 　前記Cupriavidus属に属する微生物が、Cupriavidus necatorである、請求項１～３の何れか一項に記載の微生物。 The microorganism according to any one of claims 1 to 3, wherein the microorganism belonging to the genus Cupriavidus is Cupriavidus necator.
  5. 　前記ポリヒドロキシアルカノエートが、ポリ（３－ヒドロキシブチレート－コ－３－ヒドロキシヘキサノエート）である、請求項３又は４に記載の微生物。 The microorganism according to claim 3 or 4, wherein the polyhydroxyalkanoate is poly (3-hydroxybutyrate-co-3-hydroxyhexanoate).
PCT/JP2019/044316 2018-11-15 2019-11-12 Microorganism belonging to genus cupriavidus WO2020100888A1 (en)

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