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WO2022210236A1 - Vanillic-acid-producing transformed microorganism and use of same - Google Patents

Vanillic-acid-producing transformed microorganism and use of same Download PDF

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WO2022210236A1
WO2022210236A1 PCT/JP2022/013826 JP2022013826W WO2022210236A1 WO 2022210236 A1 WO2022210236 A1 WO 2022210236A1 JP 2022013826 W JP2022013826 W JP 2022013826W WO 2022210236 A1 WO2022210236 A1 WO 2022210236A1
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gene
strain
lignin
ngc7
acid
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PCT/JP2022/013826
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French (fr)
Japanese (ja)
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和典 園木
雄大 樋口
英司 政井
直史 上村
曉弘 吉田
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国立大学法人弘前大学
国立大学法人長岡技術科学大学
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Publication of WO2022210236A1 publication Critical patent/WO2022210236A1/en

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    • 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, e.g. protozoa; 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
    • 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
    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • 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
    • C12N9/0004—Oxidoreductases (1.)
    • 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
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/42—Hydroxy-carboxylic acids

Definitions

  • the present invention relates to a transformed microorganism capable of producing vanillic acid and a method for producing vanillic acid using the transformed microorganism.
  • the present invention relates to transformed microorganisms capable of selectively producing vanillic acid from a mixture of aromatic compounds derived from p -hydroxyphenyllignin, guaiacyllignin and syringyllignin.
  • Lignin is an amorphous macromolecular substance that exists as a component of the vascular cell wall of plants. It is a complex condensation of phenylpropane-based structural units, and its chemical structure is characterized by the presence of methoxy groups. ing. Lignin has the function of binding lignified plant cells to each other and strengthening the tissue, and is present in wood at approximately 18% to 36% and in herbs at approximately 15% to 25%. Therefore, attempts have been made to decompose lignin to obtain useful compounds in order to effectively utilize wood.
  • lignin derived from biomass such as wood: p -hydroxyphenyl lignin (H-type lignin), guaiacyl lignin (G-type lignin) and syringyl lignin (S-type lignin). .
  • H-type lignin p -hydroxyphenyl lignin
  • G-type lignin guaiacyl lignin
  • S-type lignin syringyl lignin
  • Vanillic acid is one of the candidates for lignin-derived aromatic monomers.
  • a polymer obtained by polymerizing vanillic acid is characterized by a very high melting point.
  • a highly functional polymer obtained using a monomer obtained by modifying vanillic acid as a raw material has a high melting point and high functionality (see, for example, Patent Documents 1 and 2 below).
  • SYK-6 strain (hereinafter also referred to as SYK-6 strain) is known as a microorganism that decomposes lignin or lignin-derived aromatic compounds.
  • the SYK-6 strain can degrade vanillic acid derived from G-type lignin and syringic acid derived from S-type lignin, and can also degrade p -hydroxybenzoic acid derived from H-type lignin in the presence of methionine.
  • a transformed microorganism transformed from the SYK-6 strain to delete the ligM gene (SYK-6 ⁇ ligM strain) or a transformed microorganism transformed from the SYK-6 strain to delete the ligM gene and desA gene ( SYK-6 ⁇ ligM ⁇ desA strain) is reported to lack some or all of the vanillic acid degradability (see, for example, Non-Patent Document 1 below).
  • NGC7 strain (accession number: NITE BP-03043) is capable of degrading p -hydroxybenzoic acid, vanillic acid and syringic acid, which are lignin-derived aromatic compounds. ; hereinafter also referred to as NGC7 strain), and transformed the NGC7 strain to successfully obtain muconic acid from the lignin-derived aromatic compound (see, for example, Patent Document 3 below).
  • Non-Patent Document 1 Although the SYK-6 ⁇ ligM ⁇ desA strain described in Non-Patent Document 1 does not degrade vanillic acid, it also loses the ability to decompose syringic acid, an analog of vanillic acid, and in the absence of methionine, aromatics derived from H-type lignin The compound cannot be grown as a carbon source. Therefore, when attempting to produce vanillic acid using the SYK-6 ⁇ ligM ⁇ desA strain, there is a problem that the types of biomass that can be used are limited because broad-leaved trees containing a large amount of S-type lignin cannot be used.
  • Non-Patent Document 1 degrades syringic acid, but also degrades vanillic acid, and therefore cannot accumulate vanillic acid. Therefore, even if the SYK-6 ⁇ ligM strain is used, there is a problem that vanillic acid cannot be selectively produced.
  • Non-Patent Document 1 can specifically decompose either vanillic acid or its analog syringic acid.
  • the NGC7 strain and the NGC7 transformant described in Patent Document 3 cannot accumulate vanillic acid because they degrade vanillic acid. Further, according to investigations by the present inventors, no genes corresponding to the ligM gene and desA gene of the SYK-6 strain have been found in the NGC7 strain by genome analysis so far.
  • the present invention decomposes not only H-type lignin-derived aromatic compounds but also S-type lignin-derived aromatic compounds, and selectively produces and / or selectively accumulates vanillic acid while using it as a carbon source. It is an object of the present invention to provide a microorganism that can be used to produce vanillic acid and a method for producing vanillic acid using the microorganism.
  • vanillate O 2 -demethylase consists of a vanillate O 2 -demethylase oxygenase component (VanA) and a vanillate O 2 -demethylase oxidoreductase component (VanB).
  • the present inventors found that the NGC7 strain is closely related to Pseudomonas putida .
  • the NGC7 strain has 4 genes corresponding to the vanA gene and 6 genes corresponding to the vanB gene. Microorganisms having multiple types of vanA genes and vanB genes have thus far been scarcely known.
  • the inventors of the present invention conducted trial and error to set pairs of genes for multiple types of vanA genes and multiple types of vanB genes, and came to select sets of four pairs of vanA genes and vanB genes. Then, when NGC7 transformant strains lacking each of these four pairs of gene sets were produced, some transformant strains were able to degrade syringic acid and proliferate, while others were inferior in assimilation of syringic acid. , some transformants degrade vanillic acid, and some transformants hardly degrade vanillic acid.
  • an NGC7 transformant strain lacking a specific set of vanA and vanB genes degrades syringic acid to a level equal to or greater than that of the wild-type NGC7 strain and proliferates, while producing vanillic acid. almost did not decompose.
  • vanillic acid can be produced from aromatic compounds derived from G-type lignin while growing using aromatic compounds derived from S-type lignin.
  • acetovanillone which is a major aromatic compound contained in kraft cooking liquor and soda cooking liquor, which are lignin decomposition methods currently used at the industrial level, is also used in the NGC7 transformant to convert acetovanillone to vanillic acid.
  • a gene group that enables conversion vanillic acid could be produced.
  • the present inventors have finally found that the problems of the present invention are solved by finding G-type lignin-derived aromatic compounds such as H-type lignin-derived aromatic compounds and S-type lignin-derived aromatic compounds.
  • G-type lignin-derived aromatic compounds such as H-type lignin-derived aromatic compounds and S-type lignin-derived aromatic compounds.
  • the present invention is an invention completed based on the findings and successful examples obtained for the first time by these inventors.
  • the host microorganism is Pseudomonas sp. NGC7 strain (accession number: NITE BP-03043), A transformed microorganism lacking the vanA4 gene (SEQ ID NO: 1) or the vanA4 gene and the vanB4 gene (SEQ ID NO: 2) on the chromosome.
  • the host microorganism is Pseudomonas sp.
  • NGC7 strain accession number: NITE BP-03043
  • the vanA4 gene (SEQ ID NO: 1) or the vanA4 gene and the vanB4 gene (SEQ ID NO: 2) on the chromosome are deleted, and A transformed microorganism expressing the inserted acvA gene, acvB gene, acvC gene, acvD gene, acvE gene, acvF gene, vceA gene and vceB gene.
  • a method for producing vanillic acid comprising a step of obtaining vanillic acid by allowing an aromatic compound derived from guaiacyllignin to act on the transformed microorganism of [1] or [2].
  • a mixture of an aromatic compound derived from guaiacyl lignin and an aromatic compound derived from p -hydroxyphenyl lignin and/or an aromatic compound derived from syringyl lignin is added to the trait of [1] or [2].
  • a method for producing vanillic acid comprising a step of obtaining vanillic acid by allowing the transformed microorganism to act.
  • a method for producing vanillic acid comprising a step of obtaining vanillic acid by allowing a mixture of aromatic compounds derived from guaiacyl lignin containing acetovanillone to act on the transformed microorganism of [2].
  • H-type lignin-derived aromatic compounds and / or S-type lignin-derived aromatic compounds are decomposed and used as a carbon source, while microbiologically vanillic acid is produced from G-type lignin-derived aromatic compounds. It can be selectively produced and/or accumulated.
  • lignin since it is possible to use either of H-type lignin and S-type lignin as a lignin decomposition product that serves as a carbon source, lignin can be obtained without using a carbon source other than lignin such as glucose.
  • a decomposition product can also be used as a carbon source, and vanillic acid can be produced from an aromatic compound derived from G-type lignin inexpensively and independently of the type of lignin. Therefore, according to the present invention, production of vanillic acid on an industrial scale is expected as part of effective utilization of biomass containing lignin.
  • FIG. 1A is a copy of the receipt of accession number NITE BP-03043 (Pseudomonas sp. NGC7 strain).
  • FIG. 1B is a copy of the certificate of survival of accession number NITE BP-03043 (Pseudomonas sp. NGC7 strain).
  • FIG. 2A shows the OD over time when the NGC7 strain, NGC7 ⁇ vanA1B1 strain, NGC7 ⁇ vanA2B2 strain, NGC7 ⁇ vanA3B3 strain, and NGC7 ⁇ vanA4B4 strain were cultured using vanillic acid (VA) as a carbon source, as described in Examples below.
  • VA vanillic acid
  • FIG. 2B shows the OD over time when the NGC7 strain, NGC7 ⁇ vanA1B1 strain, NGC7 ⁇ vanA2B2 strain, NGC7 ⁇ vanA3B3 strain, and NGC7 ⁇ vanA4B4 strain were cultured using syringic acid (SA) as a carbon source, as described in Examples below.
  • SA syringic acid
  • 6 is a diagram showing the results of measuring 600 ;
  • FIG. FIG. 3A is a diagram showing the results of evaluating the degradability of VA by the NGC7 strain, the NGC7 ⁇ vanA1B1 strain, the NGC7 ⁇ vanA2B2 strain, the NGC7 ⁇ vanA3B3 strain, and the NGC7 ⁇ vanA4B4 strain, as described in Examples below.
  • FIG. 3B is a diagram showing the results of evaluating the degradability of SA by the NGC7 strain, the NGC7 ⁇ vanA1B1 strain, the NGC7 ⁇ vanA2B2 strain, the NGC7 ⁇ vanA3B3 strain, and the NGC7 ⁇ vanA4B4 strain, as described in Examples below.
  • FIG. 4A is a diagram showing the results of measuring the SA concentration and OD 600 over time when the NGC7 ⁇ vanA4B4 strain was cultured using SA as a carbon source, as described in Examples below.
  • FIG. 4B shows the results of measuring the VA concentration and OD 600 over time when the NGC7 ⁇ vanA4B4 strain was cultured using VA as a carbon source, as described in Examples below.
  • FIG. 4C shows the results of measuring the HBA concentration and OD 600 over time when the NGC7 ⁇ vanA4B4 strain was cultured using p -hydroxybenzoic acid (HBA) as a carbon source, as described in Examples below. It is a diagram.
  • FIG. 4D shows the results of measuring the SA concentration, VA concentration, and OD 600 over time when the NGC7 ⁇ vanA4B4 strain was cultured using a mixture of SA and VA as the carbon source, as described in Examples below. It is a diagram.
  • FIG. 4E shows the results of measuring the SA concentration, HBA concentration, and OD 600 over time when the NGC7 ⁇ vanA4B4 strain was cultured using a mixture of SA and HBA as the carbon source, as described in Examples below. It is a diagram.
  • FIG. 4C shows the results of measuring the HBA concentration and OD 600 over time when the NGC7 ⁇ vanA4B4 strain was cultured using p -hydroxybenzoic acid (HBA) as a carbon source, as described
  • FIG. 4F shows the results of measuring the VA concentration, HBA concentration, and OD 600 over time when the NGC7 ⁇ vanA4B4 strain was cultured using a mixture of VA and HBA as the carbon source, as described in Examples below. It is a diagram.
  • FIG. 4G shows the SA concentration, VA concentration, HBA concentration, and OD 600 measured over time when the NGC7 ⁇ vanA4B4 strain was cultured using a mixture of SA, VA, and HBA as the carbon source, as described in Examples below.
  • FIG. 10 is a diagram showing the results of the experiment.
  • FIG. 5A shows the degradability of acetovanilone (AV) by the NGC7 [pSEVA241_P lac -acv, pTS093_vceA-B] strain as described in the Examples below, and the AV concentration, vanilloyl acetic acid (VAA) concentration, and VA concentration were evaluated. It is the figure which showed the result of having measured with time.
  • FIG. 5B shows the results of evaluating the degradability of AV by the NGC7 ⁇ vanA4B4 [pSEVA241_P lac -acv, pTS093_vceA-B] strain and measuring the AV concentration, VAA concentration, and VA concentration over time, as described in Examples below. It is a figure showing.
  • FIG. 5B shows the degradability of acetovanilone (AV) by the NGC7 [pSEVA241_P lac -acv, pTS093_vceA-B] strain as described in the Examples below, and the AV concentration
  • FIG. 6 shows the degradability of the lignin degradation product model by the NGC7 ⁇ vanA4B4 [pSEVA241_P lac -acv, pTS093_vceA-B] strain, as described in the Examples described later.
  • FIG. 4 is a diagram showing the results of measuring OD 600 over time;
  • FIG. 7 shows the evaluation of the degradability of lignin degradation products by the NGC7 ⁇ vanA4B4 [pSEVA241_P lac -acv, pTS093_vceA-B] strain, as described in the Examples below, and the AV concentration, VN concentration, VA concentration, glucose concentration, and OD 600 is a diagram showing the results of measuring 600 over time.
  • FIG. 4 is a diagram showing the results of measuring OD 600 over time
  • FIG. 7 shows the evaluation of the degradability of lignin degradation products by the NGC7 ⁇ vanA4B4 [pSEVA241_P lac -acv, pTS09
  • NGC7 ⁇ vanA1B1 ⁇ vanA4B4 ⁇ aph ⁇ vanA2B2 ⁇ vanA3B3 [pSEVA241_P lac -acv, pTS093_vceA-B] strain to evaluate the degradability of lignin degradation products, AV concentration, VN concentration , VA concentration, glucose concentration, and OD 600 over time.
  • Gene deletion means that the gene does not function normally and the expression of the gene is prevented, such as the gene not being transcribed normally or the protein that should be produced by the expression of the gene not being translated normally. means that Gene deletion can occur, for example, by disruption, deletion, substitution, insertion, or the like of all or part of a gene, resulting in a change in the structure of the gene. However, gene deletion can also occur by suppressing gene expression by, for example, blocking the regulatory region of the gene without altering the structure of the gene. "Expression of a gene” means that a protein encoded by a gene is produced through transcription, translation, etc. so as to have the original structure and activity.
  • a transformed microorganism of one aspect of the present invention is a microorganism transformed from a host microorganism such that a specific gene on the chromosome of the host microorganism is deleted.
  • the transformed microorganism that is one aspect of the present invention preferably transforms the host microorganism so as to express the acetovanilone-degrading enzyme gene group inserted as a foreign gene.
  • the host microorganism Pseudomonas sp. NGC7 strain (accession number: NITE BP-03043), has vanA4 gene (SEQ ID NO: 1) and vanB4 gene (SEQ ID NO: 2) on the chromosome.
  • vanA4 gene which is an oxygenase component, directly acts on vanillic acid. Therefore, the transformed microorganism of one aspect of the present invention lacks the vanA4 gene among these genes on the chromosome.
  • the transformed microorganism of one aspect of the present invention is not particularly limited as long as it lacks the vanA4 gene that the NGC7 strain originally has on the chromosome. Preferably both vanB4 genes are deleted.
  • the NGC7 strain has 4 types of presumed vanA genes and 6 types of presumed vanB genes. Microorganisms that have multiple types of presumed vanA and vanB genes , such as the NGC7 strain, are extremely rare.
  • the vanA gene is the gene that expresses the vanillate O - demethylase oxygenase component.
  • the vanillate O -demethylase oxygenase component (EC 1.14.13.82) utilizes electrons from NADH or NADPH supplied via the oxygenductase component and oxygen atoms supplied from molecular oxygen to convert vanillin Cleavage the methyl ether bond of the acid to produce protocatechuic acid, formaldehyde and water.
  • vanillate O -demethylase oxygenase component has a Rieske [2Fe-2S] iron-sulfur domain (W7-V107, PROSITE entry no. PS51296) in its amino acid sequence, and C and H (C47, H49, C66, H69) are involved in Fe—S bonding.
  • the vanB gene is the gene that expresses the vanillate O - demethylase oxidoreductase component.
  • Vanillate O -demethylase oxidoreductase component (EC 1.14.13.82) is known as one of the oxidoreductases that extract electrons from NADH or NADPH and transfer them to oxygenases.
  • the vanillate O 2 -demethylase oxidoreductase component transfers electrons from NADH or NADPH to the vanillate O 2 -demethylase oxygenase component, VanA.
  • the vanillate O -demethylase oxidoreductase component has a 2Fe-2S Ferredoxin type iron-sulfer binding domain (G229-I316, PROSITE entry no. PS51085) in its amino acid sequence, and C (C265, C270 , C273, C303) are involved in the bonding of Fe—S.
  • the vanillate O -demethylase oxidoreductase component has in its amino acid sequence the NAD-binding domain (L109-D201, Pfam entry no. PF00175) and the Ferredoxin reductase type FAD-binding domain (M1-A101, PROSITE entry no. PS514). ).
  • VanAB vanylate O -demethylase
  • VanAB which is responsible for the demethylation of vanillate, can convert syringate to 3- O-methylgallic acid and 3-O-methylgallic acid to gallic acid, respectively, thus syringate O - demethylase , It can also function as a 3- O -methylgallate demethylase.
  • Sphingobium sp.
  • the SYK-6 strain has the desA gene as a gene encoding syringate O -demethylase and the ligM gene as a gene encoding vanillate/3-O-methylgallate O -demethylase.
  • Enzymes encoded by the desA gene and the ligM gene of the SYK-6 strain catalyze a methyl transfer reaction to tetrahydrofolic acid, and require tetrahydrofolic acid for the progress of the reaction. Since regeneration of tetrahydrofolate requires C1 metabolism to supply C1 compounds required for methionine, thymidine, purine biosynthesis, etc., these enzymes must function in conjunction with C1 metabolism.
  • the vanillate O -demethylase expressed by the NGC7 strain is oxygenated and consists of an oxygenase component and an oxidoreductase component.
  • vanA gene and vanB gene are considered to function as a set of genes. Therefore, when the present inventors searched for the vanA gene and vanB gene possessed by the NGC7 strain using the genomic DNA of the NGC7 strain, they unexpectedly found vanA1 gene (SEQ ID NO: 11), vanA2 gene (SEQ ID NO: 12), Having four types of vanA genes, vanA3 gene (SEQ ID NO: 13) and vanA4 gene (SEQ ID NO: 1), and vanB1 gene (SEQ ID NO: 14), vanB2 gene (SEQ ID NO: 15), vanB3 gene (SEQ ID NO: 16), It was found to have six types of vanB genes, vanB4 gene (SEQ ID NO: 2), vanB5 gene (SEQ ID NO: 17) and vanB6 gene (SEQ ID NO: 18).
  • the present inventors proceeded with further studies, and based on these gene information, selected genes to be paired with each other, and assumed a total of four gene sets of vanA genes and vanB genes. These four pairs of gene sets were defined as a gene set of vanA1 gene and vanB1 gene, a gene set of vanA2 gene and vanB2 gene, a gene set of vanA3 gene and vanB3 gene, and a gene set of vanA4 gene and vanB4 gene.
  • the present inventors produced strains lacking the above-mentioned four pairs of gene sets and tested the vanillic acid degradability. The discovery led to the completion of the present invention.
  • the transformed microorganism of one embodiment of the present invention inhibits the expression of vanillate O -demethylase, which is deeply involved in the decomposition of vanillic acid, among a plurality of vanillate O -demethylases that are assumed to exist. Therefore, it is preferably an NGC7 transformant strain lacking both the vanA4 gene and the vanB4 gene (hereinafter also referred to as NGC7 ⁇ vanA4B4 strain).
  • the transformed microorganism of one aspect of the present invention may lack other vanA and/or vanB genes in addition to the vanA4 and vanB4 genes.
  • the NGC7 transformant lacking the vanA1 and vanB1 genes and the NGC7 transformant lacking the vanA2 and vanB2 genes are inferior in syringic acid assimilation compared to the NGC7 and NGC7 ⁇ vanA4B4 strains. The result is obtained. Therefore, when an aromatic compound derived from S-type lignin such as syringic acid is used as a carbon source, the transformed microorganism of one aspect of the present invention is selected from the group consisting of vanA1 gene, vanB1 gene, vanA2 gene and vanB2 gene.
  • the transformed microorganism of one aspect of the present invention preferably lacks another vanA gene and/or another vanB gene.
  • the transformed microorganism of one aspect of the present invention lacks the vanA4 gene and the vanB4 gene, and vanA1 preferably retains the vanA4 and vanB4 genes; and preferably retains the vanA2 and vanB2 genes; or lacks the vanA4 and vanB4 genes ; Moreover, it is preferable to retain the vanA1 gene, the vanB1 gene, and the vanA2 gene and the vanB2 gene.
  • the purpose is to selectively produce and / or selectively accumulate vanillic acid while decomposing G-type lignin-derived aromatic compounds and / or H-type lignin-derived aromatic compounds and using them as carbon sources. or when using an aromatic compound derived from p -hydroxyphenyl lignin or a carbon source such as glucose, in order to increase the recovery rate of vanillic acid, the transformed microorganism of one embodiment of the present invention comprises vanA4 gene and vanB4 gene.
  • the feature of being able to produce vanillic acid at a high yield is the transformed microorganism lacking the vanA1 gene and the vanB1 gene, the transformed microorganism lacking the vanA2 gene and the vanB2 gene, and the vanillin gene lacking the vanA3 gene and the vanB3 gene. It is a surprising feature that cannot be expected from a transformed microorganism that is missing.
  • the SYK-6 strain is capable of degrading acetovanilone, one of the aromatic compounds derived from lignin.
  • the acetovanilone degradability of the SYK-6 strain includes acvA gene (SEQ ID NO: 3), acvB gene (SEQ ID NO: 4), acvC gene (SEQ ID NO: 5), acvD gene (SEQ ID NO: 6), acvE gene (SEQ ID NO: 7 ), acvF gene (SEQ ID NO: 8), vceA gene (SEQ ID NO: 9) and vceB gene (SEQ ID NO: 10) are involved.
  • the transformed microorganism of one embodiment of the present invention has the following foreign genes: acvA gene (SEQ ID NO: 3), acvB gene (SEQ ID NO: 4), acvC gene (SEQ ID NO: 5), acvD
  • the gene (SEQ ID NO: 6), acvE gene (SEQ ID NO: 7), acvF gene (SEQ ID NO: 8), vceA gene (SEQ ID NO: 9) and vceB gene (SEQ ID NO: 10) are preferably inserted and expressed.
  • the transformed microorganism of one aspect of the present invention is capable of degrading acetovanillone and producing and accumulating vanillic acid by inserting and expressing the acetovanillone-degrading enzyme gene group as a foreign gene.
  • the inserted gene may not be completely identical to the gene originally possessed by the SYK-6 strain (that is, the wild-type gene), but at least is identical to the protein that the wild-type gene expresses (that is, the wild-type protein). or a nucleotide sequence that hybridizes under stringent conditions with a nucleotide sequence complementary to that of the wild-type gene, as long as the gene expresses a protein with similar enzymatic properties.
  • a "nucleotide sequence that hybridizes under stringent conditions” is obtained by colony hybridization, plaque hybridization, Southern blot hybridization, etc. using DNA having the nucleotide sequence of a wild-type gene as a probe. It refers to the nucleotide sequence of the resulting DNA.
  • “Stringent conditions” are conditions under which a specific hybrid signal is clearly distinguished from a non-specific hybrid signal, and vary depending on the hybridization system used and the type, sequence and length of the probe. . Such conditions can be determined by varying the temperature of hybridization, varying the temperature and salt concentration of washing. For example, when even non-specific hybrid signals are strongly detected, the specificity can be increased by raising the temperature of hybridization and washing and, if necessary, lowering the salt concentration of washing. If no specific hybrid signal is detected, hybrids can be stabilized by lowering the hybridization and washing temperatures and, if necessary, increasing the washing salt concentration.
  • a DNA probe is used as a probe, and hybridization is performed using 5 ⁇ SSC, 1.0% (w/v) blocking reagent for nucleic acid hybridization (Roche Diagnostics). , 0.1% (w/v) N-lauroyl sarcosine, 0.02% (w/v) SDS, overnight (8 to 16 hours). Wash twice for 15 minutes using 0.1-0.5 ⁇ SSC, 0.1% (w/v) SDS, preferably 0.1 ⁇ SSC, 0.1% (w/v) SDS conduct. The temperature for hybridization and washing is 65°C or higher, preferably 68°C or higher.
  • DNA having a nucleotide sequence that hybridizes under stringent conditions for example, DNA having the nucleotide sequence of a wild-type gene derived from colonies or plaques, or a filter immobilized with a fragment of the DNA, DNA obtained by hybridization under stringent conditions, or in the presence of 0.5 M to 2.0 M NaCl, after hybridization at 40 ° C. to 75 ° C., preferably 0.7 M to 1 After performing hybridization at 65°C in the presence of 0 M NaCl, 0.1 to 1 x SSC solution (1 x SSC solution is 150 mM sodium chloride and 15 mM sodium citrate) was used to perform hybridization at 65°C. Examples include DNA that can be identified by washing the filter with .
  • Probe preparation and hybridization methods are described in Molecular Cloning: A Laboratory Manual, 2nd-Ed. , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. , 1989, Current Protocols in Molecular Biology, Supplement 1-38, John Wiley & Sons, 1987-1997 (hereinafter, these documents may be referred to as reference technical documents), etc. can be done. It should be noted that those skilled in the art would be able to determine the nucleotide sequence of the wild-type gene by taking into account various conditions such as probe concentration, probe length, reaction time, etc., in addition to conditions such as buffer salt concentration and temperature. Conditions for obtaining a DNA having a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence complementary to , can be appropriately set.
  • DNAs containing nucleotide sequences that hybridize under stringent conditions include DNAs having a certain or more sequence identity with the nucleotide sequence of the DNA having the nucleotide sequence of the wild-type gene used as a probe. 80% or more, preferably 85% or more, more preferably 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more of the nucleotide sequence of the type gene , 98% or more, or 99% or more, more preferably 99.5% or more of sequence identity.
  • the upper limit of the sequence identity is not particularly limited and is typically 100%.
  • nucleotide sequence that hybridizes under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence of the wild-type gene for example, if 100 nucleotides in the nucleotide sequence are one unit, the nucleotide sequence of the wild-type gene , 1 to several, preferably 1 to 20, more preferably 1 to 15, still more preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 per unit It includes nucleotide sequences with nucleotide deletions, substitutions, additions, and the like.
  • nucleotide deletion means that there is a missing or missing nucleotide in the sequence
  • nucleotide substitution means that a nucleotide in the sequence is replaced with another nucleotide
  • Additional of a nucleotide means that a new nucleotide is added to insert.
  • a protein encoded by a nucleotide sequence that hybridizes under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence of the wild-type gene has one to several amino acids in the protein encoded by the nucleotide sequence of the wild-type gene. It is likely that the protein has an amino acid sequence with single amino acid deletions, substitutions, additions, etc., but has the same enzymatic activity as the protein encoded by the nucleotide sequence of the wild-type gene.
  • a protein having the same or similar enzymatic properties as a wild-type protein is an amino acid sequence in which one to several amino acids are deleted, substituted, added, etc. in the amino acid sequence of the wild-type protein. It may consist of Here, the range of "1 to several" in the "deletion, substitution or addition of one to several amino acids" of the amino acid sequence is not particularly limited, but for example, 100 amino acids in the amino acid sequence may be regarded as one unit. For example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 per unit, preferably means about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, more preferably about 1, 2, 3, 4 or 5.
  • amino acid deletion means deletion or disappearance of an amino acid residue in a sequence
  • amino acid substitution means that an amino acid residue in a sequence is replaced with another amino acid residue.
  • addition of an amino acid means that a new amino acid residue is added to insert into the sequence.
  • “deletion, substitution, addition of one to several amino acids” include embodiments in which one to several amino acids are replaced with other chemically similar amino acids.
  • replacement of a hydrophobic amino acid with another hydrophobic amino acid replacement of a polar amino acid with another polar amino acid having the same charge, and the like can be mentioned.
  • Such chemically similar amino acids are known in the art for each amino acid.
  • nonpolar (hydrophobic) amino acids include alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, methionine, and the like.
  • Polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, cysteine, and the like.
  • positively charged basic amino acids include arginine, histidine, and lysine.
  • aspartic acid, glutamic acid, etc. are mentioned as an acidic amino acid with a negative charge.
  • Amino acid sequences having one to several amino acid deletions, substitutions, additions, etc. in the amino acid sequence of the wild-type protein include amino acid sequences having a certain degree or more of sequence identity with the amino acid sequence of the wild-type protein. , for example, 80% or more, preferably 85% or more, more preferably 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% of the amino acid sequence of the wild-type protein As mentioned above, amino acid sequences having sequence identity of 97% or more, 98% or more, or 99% or more, more preferably 99.5% or more can be mentioned.
  • the upper limit of the sequence identity is not particularly limited and is typically 100%.
  • the method for determining the sequence identity of nucleotide sequences and amino acid sequences is not particularly limited. It is determined by using a program for aligning the nucleotide sequence and amino acid sequence and calculating the rate of identity between the two sequences.
  • Examples of programs for calculating the rate of identity between two nucleotide sequences and amino acid sequences include the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87:2264-2268, 1990; Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993), and a BLAST program using this algorithm has been developed by Altschul et al. (J. Mol. Biol. 215:403-410, 1990). Furthermore, Gapped BLAST, which is a program for determining sequence identity with higher sensitivity than BLAST, is also known (Nucleic Acids Res. 25:3389-3402, 1997).
  • a person skilled in the art can, for example, use the programs described above to search the database for sequences showing high sequence identity to a given sequence. These are available, for example, on the Internet website of the US National Center for Biotechnology Information (http://blast.ncbi.nlm.nih.gov/Blast.cgi).
  • the acetovanilone degrading enzyme gene group to be inserted is Sphingobium sp.
  • the acetovanilone-degrading enzyme gene group is inserted. and belonging to the gamma proteobacteria network, the order Pseudomodales, the family Pseudomonadaceae, the genus Pseudomonas , and having the ability to degrade acetovanilone and analogues thereof. are also not excluded.
  • the host microorganism is Pseudomonas sp. strain NGC7 (accession number: NITE BP-03043).
  • the genome of the NGC7 strain contains the aph gene (SEQ ID NO: 37), which is thought to be a genetic factor that causes the NGC7 strain to exhibit kanamycin resistance. Therefore, when drug selection using kanamycin is used for selection of transformants, it is preferable to delete the aph gene on the genome of the NGC7 strain. Thus, in order to increase the selection efficiency of transformants, genes on the genome of the NGC7 strain may be deleted depending on the selection conditions.
  • Genes to be deleted and inserted can be inserted into various known appropriate vectors. Furthermore, this vector can be introduced into the NGC7 strain, which is a host microorganism, to produce a transformant (transformed microorganism) in which the gene has been deleted or the gene has been inserted.
  • the gene to be deleted is preferably a wild-type gene whose structure has been altered by disruption, deletion, substitution, insertion, or the like, in whole or in part.
  • the gene to be inserted is preferably a gene that expresses the same or similar protein as the wild-type gene.
  • transformation and transformants include transduction and transductants, respectively.
  • chromosomal DNA and mRNA can be extracted from derived organisms and various microorganisms that have wild-type genes related to the gene to be deleted or inserted by a conventional method, for example, the method described in the reference technical document.
  • cDNA can be synthesized using the extracted mRNA as a template.
  • a library of chromosomal DNA and cDNA can be constructed.
  • the gene to be inserted can be obtained by cloning using, as templates, the chromosomal DNA and cDNA of the derived organism containing the wild-type gene related to the gene.
  • Wild-type genes are derived from the SYK-6 strain and its relatives as described above.
  • the SYK-6 strain is cultured, water is removed from the obtained cells, and the cells are ground physically using a mortar or the like while cooling in liquid nitrogen to obtain fine powdery cells.
  • a chromosomal DNA fraction is extracted from the cell piece by a conventional method.
  • a commercially available chromosomal DNA extraction kit such as DNeasy Blood & Tissue Kit (Qiagen) can be used for the chromosomal DNA extraction procedure.
  • chromosomal DNA and genomic DNA are synonymous.
  • DNA is then amplified by polymerase chain reaction (PCR) using the chromosomal DNA as a template and synthetic primers complementary to the 5' and 3' terminal sequences.
  • the primer is not particularly limited as long as it can amplify a DNA fragment containing the gene to be inserted. Examples thereof include primers represented by SEQ ID NOs: 35 and 36 designed with reference to the genome sequence of the NGC7 strain for amplifying the vanA4 gene.
  • the full-length target gene can be amplified using such primers.
  • screening of target gene clones from a shotgun library, or appropriate PCR such as Inverse PCR, Nested PCR, 5'RACE method, 3'RACE method, etc. is used to amplify DNA containing the target gene fragment. , and ligating them to obtain a DNA containing the full-length target gene.
  • the method of obtaining the gene to be deleted or inserted is not particularly limited as described above, and it is possible to construct the gene using, for example, a chemical synthesis method without using genetic engineering techniques.
  • Nucleotide sequences of amplification products amplified by PCR and chemically synthesized genes can be confirmed, for example, as follows.
  • a recombinant DNA is prepared by inserting a DNA whose sequence is to be confirmed into an appropriate vector according to a conventional method.
  • known or commercially available kits such as In-Fusion HD Cloning Kit (Takara Bio) and TA Cloning Kit (Invitrogen); pUC4K (see Gene, vol.19, p259-268, 1982).
  • pEX18Amp see Gene, vol.212, p77-86, 1998), pPS858 (see Gene, vol.212, p77-86, 1998), pUC118 (Takara Bio), pJB866 (Plasmid, vol.38, p35-51, 1997), pMCL200 (Gene, vol.162, p157-158, 1995), pQE30 (Qiagen), pUC119 (Takara Bio), pUC18 (Takara Bio), pBR322 (Takara Bio ), pAK405 (see Andreas Kaczmarczyk et al., Applied and Environmental Microbiology, 2012, vol.78(10), p3774-3777), pK18 mobsacB (see Andreas Schafer et al., Gene, 1994, vol.6-9-3777) ) or other known or commercially available plasmid vectors; ⁇ EMBL3 (Stratagene) or
  • the recombinant DNA is introduced into, for example, Escherichia coli , preferably Escherichia coli JM109 strain (Takara Bio Inc.) and Escherichia coli DH5 ⁇ strain (Takara Bio Inc.). Then, the recombinant DNA contained in the resulting transformant can be purified using QIAGEN Plasmid Mini Kit (Qiagen) or the like.
  • the nucleotide sequence of each gene inserted into the recombinant DNA is determined by the dideoxy method (see Methods in Enzymology, 101, p20-78, 1983, etc.).
  • the sequence analyzer used for nucleotide sequence determination is not particularly limited, but examples include Li-COR MODEL 4200L Sequencer (Aloka), 370 DNA Sequence System (PerkinElmer), CEQ2000XL DNA Analysis System (Beckman), etc. mentioned. Then, based on the determined nucleotide sequence, the amino acid sequence of the protein to be translated can be known.
  • Recombinant vectors (recombinant DNA) containing genes to be deleted or inserted should combine PCR amplification products containing genes to be deleted or inserted with various vectors in a form that allows gene deletion or expression.
  • the recombinant vector is introduced into the host microorganism, and the gene in the recombinant vector is replaced with the gene in the host microorganism by homologous recombination, so the recombinant vector is deleted. It preferably includes upstream and downstream regions of the gene.
  • a method for producing a recombinant vector containing a gene to be inserted is, for example, excising a DNA fragment containing any of the genes to be inserted with an appropriate restriction enzyme, and cleaving the DNA fragment with an appropriate restriction enzyme. It can be constructed by ligating the resulting plasmid vector using a commercially available recombinant vector preparation kit such as In-Fusion HD Cloning Kit (Takara Bio).
  • a DNA fragment containing a gene with sequences homologous to the plasmid vector added to both ends and a plasmid-derived DNA fragment amplified by inverse PCR are combined with a commercially available product such as the In-Fusion HD Cloning Kit (Takara Bio Inc.). It can be obtained by ligation using a recombinant vector preparation kit.
  • a recombinant vector containing a gene to be deleted or inserted contains at least the gene to be deleted or inserted and a gene (nucleotide sequence) derived from a plasmid vector.
  • recombinant vectors include recombinant vectors containing vanA4 gene and/or vanB4 gene; recombinant vectors containing acetovanilone degrading enzyme gene group;
  • the recombinant vector may contain genes other than the genes described above as long as they do not interfere with the solution of the problems of the present invention.
  • a recombinant vector may contain a heterologous gene or heterologous nucleotide sequence.
  • the heterologous gene is not particularly limited as long as it is a gene not naturally occurring in the NGC7 strain. Examples include genes derived from NGC7 strain, genes derived from organisms such as other microorganisms different from the NGC7 strain, plants, animals, and viruses. Specific examples of heterologous genes include, but are not limited to, DNA fragments derived from pUC118, such as the lactose promoter region (P lac ).
  • the method for producing the transformed microorganism is not particularly limited, and includes, for example, a method of inserting into the NGC7 strain in such a manner that gene deletion or insertion is achieved according to a conventional method.
  • a DNA construct is prepared by inserting any of the genes to be inserted between an expression-inducing promoter and a terminator, and then the NGC7 strain is transformed with the DNA construct to express the gene to be inserted.
  • a transformed microorganism is obtained.
  • a transformed microorganism lacking the gene can be obtained by preparing a DNA construct containing the gene to be deleted and the upstream and downstream regions of the gene, and then transforming the host microorganism with the DNA construct.
  • Recombinant vectors constructed to transform strain NGC7 are collectively referred to herein as DNA constructs.
  • the method of introducing the DNA construct into the NGC7 strain is not particularly limited.
  • the introduced DNA construct is introduced into the NGC7 strain so as to autonomously proliferate and express the gene.
  • Method a method of directly inserting a DNA construct into the chromosome of the NGC7 strain by utilizing homologous recombination.
  • the DNA construct is ligated between sequences homologous to the upstream and downstream regions of the recombination site on the chromosome. , can be inserted into the genome of strain NGC7.
  • the vector-host system used for the preparation of the NGC7 strain is not particularly limited as long as it is a system in which the inserted gene can be expressed in the NGC7 strain or a system in which the gene on the chromosome can be deleted. , vol.38(1), p35-51, 1997)-Pseudomonas microorganism system, pKT230 (Gene, vol.16, p237-247, 1981)-Pseudomonas microorganism system, pSEVA (Nucleic Acids Research, vol. 48(D1), pD1164-D1170, 2020)-Pseudomonas microorganism system.
  • the DNA construct containing the gene to be inserted is amplified autonomously without being introduced into the chromosome of the NGC7 strain, and even if the gene to be inserted is expressed, the gene to be inserted into the chromosome of the NGC7 strain is expressed. or either.
  • the DNA construct may contain a marker gene to enable selection of transformed cells.
  • the marker gene is not particularly limited, and examples thereof include drug resistance genes for drugs such as gentamicin, kanamycin, tetracycline, ampicillin and carbenicillin.
  • a marker gene may be included in the middle of the deleted gene or to replace the deleted gene.
  • the DNA construct containing the gene to be inserted may contain promoters and terminators that enable expression of the gene in the NGC7 strain, as well as other regulatory sequences (e.g., transcription control sequences such as operators). arrays, etc.).
  • One embodiment of the DNA construct includes, for example, pvanA1B1del plasmid DNA, pvanA2B2del plasmid DNA, pvanA3B3del plasmid DNA, pvanA4B4del plasmid DNA, pSEVA241_P lac -acv plasmid DNA, pTS093_vceA-B plasmid DNA, etc., which are described in Examples below. but not limited to these.
  • a medium suitable for the growth of the NGC7 strain should be used as the medium for selecting and growing transformed microorganisms.
  • selection and growth of transformed microorganisms can be carried out by, for example, culturing the transformed microorganisms in LB medium containing these drugs.
  • the transformed microorganism is placed under conditions in which only the transformed microorganism lacking the gene can survive or under conditions in which only the transformed microorganism expressing the gene to be inserted can survive. It can be achieved by culturing and the like. Alternatively, by culturing the transformed microorganism and then confirming that the amount of vanillic acid in the culture obtained after culturing is greater than the amount of vanillic acid in the culture of the NGC7 strain cultured under the same conditions. , it is possible to confirm that a transformed microorganism has been produced.
  • chromosomal DNA is extracted from the transformed microorganism, PCR is performed using this as a template, and if transformation occurs, a PCR product that can be amplified is generated or the PCR product This may be done, for example, by confirming the properties or the nucleotide sequence.
  • PCR For example, perform PCR using a combination of a forward primer for the nucleotide sequence of the promoter of the gene to be deleted or inserted and a reverse primer for the nucleotide sequence of the marker gene, and confirm that a product of the expected length is generated.
  • PCR is performed with a combination of a forward primer located upstream from the used upstream homologous region and a reverse primer located downstream from the used downstream homologous region, It is preferable to confirm that a product of the expected length is produced when homologous recombination occurs.
  • the acetovanillone degrading enzyme gene cluster is a gene optimized for codons, secondary structure, GC content, etc. for expression in the NGC7 strain. There may be.
  • a specific embodiment of the transformed microorganism is a transformed microorganism in which the host microorganism is strain NGC7 and the vanA4 gene (SEQ ID NO: 1) on the chromosome is deleted.
  • a specific embodiment of the transformed microorganism is a transformed microorganism in which the host microorganism is the NGC7 strain and lacks the vanB4 gene (SEQ ID NO: 2) in addition to the vanA4 gene (SEQ ID NO: 1) on the chromosome. is.
  • the host microorganism is the NGC7 strain, and in addition to the vanA4 gene (SEQ ID NO: 1) and vanB4 gene on the chromosome, vanA1 gene (SEQ ID NO: 11), vanA2 gene (SEQ ID NO: 12), vanA3 gene (SEQ ID NO: 13), vanB1 gene (SEQ ID NO: 14), vanB2 gene (SEQ ID NO: 15), vanB3 gene (SEQ ID NO: 16), vanB5 gene (SEQ ID NO: 17) and vanB6 gene (SEQ ID NO: 18)
  • a transformed microorganism lacking one or more genes selected from the group consisting of These transformed microorganisms are also collectively referred to as transformed microorganisms (1).
  • a specific embodiment of the transformed microorganism (hereinafter, also referred to as transformed microorganism (2)) has, in addition to deletion of the above genes, the inserted acvA gene, acvB gene, acvC gene, acvD gene, acvE gene, It is a transformed microorganism expressing an acetovanilone-degrading enzyme gene group consisting of the acvF gene, the vceA gene and the vceB gene.
  • the transformed microorganism (1) lacks at least the vanA4 gene (SEQ ID NO: 1) or the vanA4 gene and the vanB4 gene (SEQ ID NO: 2) on the chromosome, thereby degrading vanillic acid. is deleted, but by maintaining the assimilation of syringic acid, aromatic compounds derived from H-type lignin such as p -hydroxybenzoic acid, which is impossible with the transformed microorganism described in Patent Document 3, and / Or, it is possible to produce vanillic acid from aromatic compounds derived from G-type lignin such as ferulic acid and vanillin while growing using aromatic compounds derived from S-type lignin such as syringic acid as a carbon source.
  • the inserted acetovanilone degrading enzyme gene group is expressed at least, so that aromatic aromatics derived from H-type lignin such as p -hydroxybenzoic acid G-type lignin-derived aromatic compounds such as ferulic acid and vanillin and G-type lignin-derived aromatic compounds such as ferulic acid and vanillin and acetovanilone or G-type lignin-derived aromatics containing acetovanillone It is possible to produce vanillic acid from mixtures of family compounds.
  • H-type lignin such as p -hydroxybenzoic acid
  • G-type lignin-derived aromatic compounds such as ferulic acid and vanillin
  • G-type lignin-derived aromatic compounds such as ferulic acid and vanillin and acetovanilone or G-type lignin-derived aromatics containing acetovanillone
  • transformed microorganisms (1) and (2) include, but are not limited to, NGC7 ⁇ vanA4B4 strain and NGC7 ⁇ vanA4B4 [pSEVA241_P lac -acv, pTS093_vceA-B] strains, which are described in Examples below. .
  • a production method of one embodiment of the present invention (hereinafter referred to as production method (1)) comprises an aromatic compound derived from G-type lignin such as vanillin, and an aromatic compound derived from H-type lignin such as p -hydroxybenzoic acid or p -hydroxybenzaldehyde. It includes a step of obtaining vanillic acid by reacting a mixture with an aromatic compound and/or an aromatic compound derived from S-type lignin such as syringic acid and syringaldehyde on the transformed microorganism (1).
  • a production method of another aspect of the present invention includes a step of allowing acetovanillone to act on transformed microorganism (2) to obtain vanillic acid.
  • Acetovanilone is contained in decomposition products obtained by alkaline oxidative decomposition of lignin such as kraft cooking and soda cooking. A mixture may also be used.
  • the method of allowing the lignin-derived aromatic compound to act on the transformed microorganism is a method in which the lignin-derived aromatic compound and the transformed microorganism come into contact with each other, and vanillic acid is produced and/or accumulated by the enzymes of the transformed microorganism.
  • the transformed microorganism is cultured under various culture conditions suitable for the NGC7 strain.
  • a method for producing vanillic acid, etc. can be mentioned.
  • the culture method is not particularly limited, and examples thereof include a solid culture method and a liquid culture method performed under aerated conditions.
  • “Production of vanillic acid” means obtaining vanillic acid from a source of vanillic acid, for example, synthesizing and converting vanillic acid from compounds such as vanillic acid precursors; vanillic acid, syringic acid, In the mixture of aromatic compounds derived from S-type lignin such as p -hydroxybenzoic acid and / or aromatic compounds derived from H-type lignin, while decomposing aromatic compounds derived from S-type lignin and aromatic compounds derived from H-type lignin, vanillin Accumulation (concentration) of vanillic acid by not decomposing acid can be mentioned.
  • the medium may be a synthetic medium or a natural medium as long as it contains a normal medium for culturing Pseudomonas microorganisms such as the NGC7 strain, that is, a carbon source, a nitrogen source, inorganic substances, and other nutrients in appropriate proportions.
  • a carbon source a nitrogen source, inorganic substances, and other nutrients in appropriate proportions.
  • MMx-3 medium, Wx minimal medium, etc. as described in Examples below, can be used, but are not particularly limited.
  • Carbon sources that can be used include aromatic compounds derived from S-type lignin, aromatic compounds derived from H-type lignin, other carbon sources such as sugars and organic acids, and combinations thereof.
  • the medium components preferably contain components necessary for cell growth and enzyme activation, such as Mg 2+ and Fe 2+ . Iron ions, magnesium ions, etc. can be added to the medium as compounds, but they may also be added as mineral inclusions.
  • the aromatic compound derived from H-type lignin and the aromatic compound derived from S-type lignin are not particularly limited as long as they are either lignin of H-type lignin and S-type lignin and aromatic compounds that can be derived from these lignins.
  • Examples thereof include compounds corresponding to decomposition products of S-type lignin and H-type phenyllignin. Specific examples include acetosyringone, p -hydroxyacetophenone, syringic acid, syringaldehyde, p -coumaric acid, p- Hydroxybenzoic acid, protocatechuic acid and the like.
  • the medium contains vanillic acid or an aromatic compound derived from G-type lignin, which is a precursor of vanillic acid.
  • the aromatic compound derived from G-type lignin is not particularly limited as long as it is a G-type lignin and an aromatic compound that can be derived from G-type lignin. Specific examples include ferulic acid, vanillin, acetovanillone, and the like.
  • the lignin-derived aromatic compound is preferably a biomass containing lignin or a product obtained by subjecting the biomass to pretreatment and extracting it, but it may be chemically synthesized and purified regardless of the biomass. .
  • the aromatic compounds derived from lignin can be used alone or in combination of two or more.
  • Biomass containing lignin (hereinafter sometimes referred to as lignocellulose) is not particularly limited, but examples include natural products such as grasses and trees, products obtained by processing these natural products, and agricultural wastes. Specific examples include woody biomass such as broad-leaved trees and conifers. For example, broad-leaved trees are known to contain a large amount of S-type lignin, and coniferous trees are known to contain a large amount of G-type lignin.
  • Lignocellulose can be, for example, solid, suspended, liquid, etc., depending on the presence or absence of pretreatment.
  • a suspension obtained by adding pulverized lignocellulose to a liquid can be used.
  • the lignocellulose may be a lignin extract.
  • the lignin extract for example, powdered lignocellulose is used at 0.1% (w/v) to 50% (w/v), preferably 1% (w/v) to 20% (w/v).
  • v such as a suspension suspended in a solvent suitable for lignin extraction.
  • the lignin extract is prepared by heating the suspension at 10° C. to 150° C., preferably 20° C. to 130° C., more preferably 20° C. to 80° C. for several hours to several days, preferably 1 hour to 6 days.
  • a cedar lignin extract can be obtained by using cedar powder as the lignocellulose, and a birch lignin extract can be obtained by using white birch powder.
  • a method for preparing an aromatic compound from a lignin extract is not particularly limited, and includes, for example, the following methods. That is, put 50 mL of 2M NaOH, 0.5 g of lignin extract, and 3 mL of nitrobenzene in a stainless steel vessel of a small autoclave device (Portable Reactor TVS-1, Pressure Glass Industry Co., Ltd.), and stir at 500 rpm at 170 ° C. for 2.5 hours. process. Allow to cool to 60° C. or below, and collect the supernatant by centrifugation (6,000 ⁇ g, 10 min). The resulting supernatant is subjected to diethyl ether extraction three times (recovering the aqueous layer).
  • the diethyl ether extraction is repeated three times (collecting the ether layer). Add sodium sulfate to the ether layer and dehydrate overnight in the refrigerator. Collect the ether layer and dry the extract under reduced pressure. The ether extract is dissolved in ion-exchanged water (pH ⁇ 9) while sodium hydroxide is added to obtain an aromatic compound solution derived from the lignin extract.
  • Solvents suitable for extracting lignin and preparing aromatic compounds are not particularly limited, and examples include water, low-molecular-weight alcohols such as dioxane, methanol, and isopropanol, diethyl ether, and dimethylformamide.
  • a metal foam such as copper can be used as a lignin decomposition catalyst to obtain a lignin decomposition product.
  • a catalyst layer formed by loading a catalyst for lignin decomposition products [Cu(OH) 2 /CF] into a stainless steel tube is fed with an alkali-treated lignocellulose and oxygen gas, and then the solution that has passed through the catalyst layer is , pH is adjusted to pH 9 to 11 with an acid, then passed through a filtration membrane such as an MF membrane or a UF membrane to obtain a filtrate, and then the obtained filtrate is acidified with an acid to pH 2 to 4, for example.
  • a lignin decomposition product can be obtained.
  • the culture conditions for Pseudomonas microorganisms commonly known to those skilled in the art may be adopted.
  • the initial pH of the medium is adjusted to 5 to 10
  • the culture temperature is 20° C. to 40° C.
  • the culture time is several hours. It can be appropriately set to several days, preferably 1 to 7 days, more preferably 2 to 5 days.
  • the culture method is not particularly limited, and aeration and agitation deep culture, shaking culture, static culture, etc. can be employed, but it is preferable to culture under conditions such as aeration so that the dissolved oxygen concentration is sufficient.
  • fed-batch culture may be employed in which carbon sources, lignin extracts, G-type lignin-derived aromatic compounds, etc. are added according to the decrease in carbon sources and acetovanillone and the increase in vanillic acid.
  • MMx-3 medium containing syringic acid and/or p -hydroxybenzoic acid as a carbon source and ferulic acid and vanillin as substrates for vanillic acid was used at 30°C and 180 rpm. 1 hour to 5 days of shaking culture, stirring culture, and the like.
  • LB medium containing acetovanillone as a vanillic acid substrate was used at 30° C., 180 rpm, and a dissolved oxygen concentration of 5% to 20%, as described in the Examples below. 1 hour to 5 days of shaking culture, stirring culture, and the like.
  • the carbon source and other components can be added as appropriate after the culture is started.
  • the method of obtaining vanillic acid from the culture after the end of the culture is not particularly limited. Since vanillic acid accumulates in the culture medium, the cells and the culture supernatant are separated from the culture by normal solid-liquid separation operations such as filtration and centrifugation, and the collected culture supernatant is subjected to solidification using a column. Vanillic acid is extracted by phase extraction or solvent extraction using a solvent in which vanillic acid is soluble.
  • the extraction solvent is not particularly limited as long as it dissolves vanillic acid, and examples include ethyl acetate and diethyl ether.
  • Vanillic acid can be separated and purified from the culture medium by known methods such as precipitation, extraction, distillation, recrystallization, and adsorbents, or by partially modifying the methods.
  • hydrochloric acid is added to the culture supernatant to adjust the pH to 2 to 5, and then diethyl ether is added and mixed.
  • the diethyl ether layer is dried under reduced pressure using an evaporator, and the resulting precipitate is washed with deionized water, collected by suction filtration, and dried under reduced pressure.
  • the dried solid is dissolved in glacial acetic acid and recrystallized to obtain purified vanillic acid.
  • a method using a synthetic adsorbent such as the method described in the literature by Gomes et al. However, it is possible to obtain purified vanillic acid.
  • the qualitative or quantitative analysis of vanillic acid may be performed by HPLC described in the examples below.
  • vanillic acid can be selectively obtained without degrading it.
  • the transformed microorganism (1) is used and 5 mM vanillic acid and 5 mM syringic acid are used as carbon sources, a culture solution containing 4.4 mM vanillic acid and no syringic acid is obtained after culturing for 48 hours.
  • a culture solution containing 4.9 mM vanillic acid and no p -hydroxybenzoic acid can be obtained after culturing for 48 hours.
  • acetovanillone when using transformed microorganism (2), when 0.2 mM acetovanillone is used as a substrate, acetovanillone is converted to vanillic acid and vanilloyl acetic acid within 24 hours after culturing, and vanilloyl acetic acid is converted to vanillin by further culturing. After 30 hours of culturing, a culture medium containing vanillic acid and free of acetovanillone and vanilloyl acetic acid can be obtained.
  • Vanillic acid obtained using the transformed microorganism and production method of one embodiment of the present invention can be converted into various industrially useful compounds.
  • Vanillic acid has an aromatic ring structure that imparts heat resistance and rigidity to polymers, and is expected to be used in synthesizing functional polymers such as liquid crystal polyesters.
  • Vanillic acid and vanillic acid derivatives obtained by modifying vanillic acid can be used, for example, by themselves or together with other components as heat-resistant plastics, anti-tracking plastics, liquid-crystalline copolymerized polyester resins, and the like. Be expected.
  • NGC7 strain (hereinafter also simply referred to as NGC7 strain) is an independent administrative agency product with the identification of microorganisms as "NGC7" and the accession number as "NITE BP-03043". It has been deposited with the Patent Microorganisms Depositary Center of the Evaluation Technology Platform (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture 292-0818) with a deposit date of October 4, 2019. For NGC7, a copy of the receipt is shown in FIG. 1A and a certificate of viability is shown in FIG. 1B.
  • Tables 1 to 3 show the test results of investigating the properties of NGC7 strains.
  • the NGC7 strains were Pseudomonas putida strain NBRC14164, P. plecoglossicida strain FPC951 , P. taiwanensis BCRC17751 strain and Montemonas pseudomonas. monteilii ) showed 99.2% to 99.5% sequence identity with the 16S rRNA sequence of strain CIP104883 (Shinoda, E. et al., J. Ind. Microbiol. Biotechnol. 46(8), 1071-1080 .).
  • the NGC7 strain was a motile Gram-negative bacillus, was positive in catalase reaction and oxidase reaction, and oxidized glucose. From these results, the NGC7 strain was consistent with the properties of the genus Pseudomonas (see, for example, Palleroni NJ. Pseudomonads. In: Bergey's Manual of Systematics of Archeaea and Bacteria).
  • NGC7 strain When tested using an API20NE kit (bioMérieux Japan), NGC7 strain does not reduce nitrate, exhibits arginine dihydrolase activity, does not hydrolyze gelatin, and does not hydrolyze glucose, potassium gluconate and n-capric acid. but did not assimilate L-arabinose, adipic acid, etc. In addition, it must produce a fluorescent pigment on King's agar medium, exhibit no reticinase and lipase (Tween80) activities, grow at 4°C in the presence of 6% NaCl, and not grow at 41°C in the presence of 7% NaCl. was confirmed. The properties possessed by these NGC7 strains are similar to those of P. putida well matched. However, from the results of 16S rRNA gene analysis, the NGC7 strain is a P. P. putida may be classified as a species different from P. putida. Pseudomonas sp . estimated.
  • NGC7 ⁇ vanAB strain which is a mutant strain in which the vanillate O -demethylase gene ( vanAB ) was disrupted from the NGC7 strain, was prepared by the following procedure.
  • Genomic DNA of NGC7 strain was prepared by a conventional method, and the entire nucleotide sequence was analyzed.
  • a gene encoding a protein having an amino acid sequence highly identical to the deduced amino acid sequences of vanillate O -demethylase oxygenase component (VanA) and vanillate O -demethylase oxidoreductase component (VanB) derived from S. putida strain KT2440 was isolated from strain NGC7. Searched from the genome sequence. In the genomic DNA of the NGC7 strain, there are 4 genes encoding amino acid sequences that show a sequence identity of 25% or more with VanA (Table 4), and there are amino acids that show a sequence identity of 30% or more with VanB. It was confirmed that there were 6 genes encoding sequences (Table 5).
  • FIG. 1 shows a gene cluster derived from strain NGC7 that encodes amino acid sequences that have a sequence identity of 25% or more with the deduced amino acid sequence of VanA derived from S. putida strain KT2440.
  • Table 5 shows FIG. 1 shows a gene cluster derived from strain NGC7 that encodes amino acid sequences that have a sequence identity of 30% or more with the deduced amino acid sequence of VanB derived from S. putida strain KT2440.
  • vanillate O 2 -demethylase is composed of an oxygenase component (VanA) and an oxidoreductase component (VanB).
  • vanA1 and vanB1 , vanA2 and vanB2 , vanA3 and vanB3 , and vanA4 and vanB4 were considered as paired gene sets.
  • NGC7 ⁇ vanA1B1 strain, NGC7 ⁇ vanA2B2 strain, NGC7 ⁇ vanA3B3 strain and NGC7 ⁇ vanA4B4 strain lacking each gene set were prepared by the following procedure. This was done by deleting the entire region or almost the entire region of each gene.
  • Pseudomonas sp Using the genomic DNA of the NGC7 strain as a template, a primer set consisting of primers 1 and 2 of SEQ ID NOS: 19 and 20, and a primer set consisting of primers 3 and 4 of SEQ ID NOS: 21 and 22, respectively, were used to perform PCR to obtain the vanA1 gene.
  • a DNA fragment of about 1.0 kbp upstream of the 5' end of the vanB1 gene and downstream of the 3' end of the vanB1 gene was amplified, respectively. Each fragment was ligated with pK18 mobsacB (see Gene, Vol.
  • a DNA fragment of about 1.0 kbp upstream of the 5' end of the vanB2 gene and downstream of the 3' end of the vanA2 gene were amplified, respectively.
  • Plasmid pvanA2B2del for constructing vanA2 gene and vanB2 gene region deleted strains was prepared by ligating each fragment to pK18 mobsacB previously digested with BamHI by seamless cloning method using NEBuilder HiFi DNA assembly.
  • approximately 1.0 kbp DNA fragments upstream of the 5' end of the vanB3 gene and downstream of the 3' end of the vanA3 gene were amplified, respectively.
  • Plasmid pvanA3B3del for constructing vanA3 gene and vanB3 gene region deleted strains was prepared by ligating each fragment to pK18 mobsacB previously digested with BamHI and seamless cloning method using NEBuilder HiFi DNA assembly.
  • a DNA fragment of about 1.0 kbp upstream of the 5' end of the vanA4 gene and downstream of the 3' end of the vanB4 gene were amplified, respectively.
  • Plasmid pvanA4B4del for constructing vanA4 gene and vanB4 gene region deleted strains was prepared by ligating each fragment to pK18 mobsacB previously digested with BamHI by seamless cloning method using NEBuilder HiFi DNA assembly.
  • Plasmids pvanA1B1del, pvanA2B2del, pvanA3B3del and pvanA4B4del prepared as follows were electroporated into Pseudomonas sp. It was introduced into the NGC7 strain.
  • Pseudomonas sp. obtained by shaking culture in 10 mL of LB liquid medium. NGC7 strain cells were washed with 3 mL of 0.5 M sucrose aqueous solution, and then suspended in 1 mL of 0.5 M sucrose aqueous solution. The cell suspension and the plasmid were mixed and applied under the conditions of 200 ⁇ , 25 ⁇ F and 2.5 kV using Gene Pulser Xcell TM (Bio-Rad Laboratories).
  • the obtained Km-resistant strain was smeared on a YT agar medium (10 g/L yeast extract, 20 g/L tryptone, 18 g/L agar) containing 25% sucrose and statically cultured at 30°C.
  • Transformants in which it was confirmed that the internal region of each vanAB gene on the genomic DNA had been deleted by colony direct PCR were designated as NGC7 ⁇ vanA1B1 strain, NGC7 ⁇ vanA2B2 strain, NGC7 ⁇ vanA3B3 strain, and NGC7 ⁇ vanA4B4 strain, respectively.
  • NGC7 ⁇ vanAB strain using vanillic acid (VA) and syringic acid (SA) as substrates
  • VA vanillic acid
  • SA syringic acid
  • the resulting culture solution was subjected to centrifugation to collect the cells, and the obtained cells were added to a carbon source-free Wx-solution [9.8 g/L Na 2 HPO 4 ⁇ 12H 2 O, 1.7 g/ L KH 2 PO 4 , 1.0 g/L (NH 4 ) 2 SO 4 ], followed by washing with Wx liquid medium [9.
  • FIG. 2A shows the results of OD 600 measurements with VA as the carbon source
  • FIG. 2B shows the results of OD 600 measurements with SA as the carbon source.
  • strains NGC7, NGC7 ⁇ vanA1B1 , NGC7 ⁇ vanA2B2 and NGC7 ⁇ vanA3B3 grew , but NGC7 ⁇ vanA4B4 did not grow.
  • the NGC7 ⁇ vanA4B4 strain showed a growth profile similar to that of the NGC7 strain under the medium condition with SA as the sole carbon source. That is, the vanillate O -demethylase encoded by the vanA4 gene and the vanB4 gene is an oxygenation-type vanillate O -demethylase involved in the decomposition of VA in the NGC7 strain, and even if the vanA4 gene and the vanB4 gene are deleted, SA is degraded. I have found that I can maintain my sexuality.
  • NGC7 strain, NGC7 ⁇ vanA1B1 strain, NGC7 ⁇ vanA2B2 strain, NGC7 ⁇ vanA3B3 strain, and NGC7 ⁇ vanA4B4 strain were each inoculated into 10 mL of LB liquid medium and subjected to shaking culture overnight at 30°C. 0.1 mL of the obtained culture solution was inoculated into 10 mL of fresh LB liquid medium and subjected to shaking culture at 30° C. for 18 hours.
  • the obtained culture solution is subjected to centrifugation to collect the cells, and the obtained cells are suspended in a 50 mM Tris-HCl buffer (pH 7.5) medium to obtain a cell suspension having an OD 600 of 2. 1 mL of liquid was prepared. VA or SA was added to the resulting cell suspension to a final concentration of 0.1 mM, followed by shaking culture at 30°C.
  • the concentrations of SA and VA were measured using a high-performance liquid chromatograph (“Acquity ultraperformance liquid chromatography system”, Nippon Waters Co., Ltd.).
  • a TSKgel ODS-140HTP column (diameter: 2.1 mm, length: 100 mm, particle size: 2.3 ⁇ m; Tosoh Corporation) was used and kept at 30°C.
  • Solvents used were 90% (v/v) H2O , 0.1% (v/v) HCOOH, 10% (v/v) CH3CN in isocratic mode.
  • the mobile phase flow rate was 0.5 mL/min, and the measurement wavelength was 270 nm for SA and 260 nm for VA.
  • Figures 3A and 3B show the reduction rates of the SA and VA concentrations at each measurement time, based on the SA and VA concentrations at the start of the culture.
  • NGC7 strain degraded all the added VA, but NGC7 ⁇ vanA4B4 strain hardly degraded VA.
  • FIG. 3B the NGC7 ⁇ vanA4B4 strain degraded SA in the same manner as the NGC7 strain under SA-supplemented conditions.
  • vanillate O -demethylase encoded by the vanA4 gene and the vanB4 gene is an oxygenated vanillate O -demethylase involved in VA decomposition of the NGC7 strain, and the NGC7 strain lacking the vanA4 gene and the vanB4 gene. can maintain the degradability of SA.
  • NGC7 ⁇ vanA4B4 strain [5. Evaluation of VA accumulation from lignin-derived aromatic compounds using NGC7 ⁇ vanA4B4 strain]
  • the NGC7 ⁇ vanA4B4 strain was inoculated into 10 mL of LB liquid medium and cultured with shaking at 30°C overnight. 0.1 mL of the resulting culture solution was inoculated into 10 mL of new LB liquid medium and subjected to shaking culture at 30° C. for 16 hours.
  • the resulting culture solution is subjected to centrifugation to collect the cells, the cells are washed with physiological saline, and then suspended again in physiological saline to obtain a cell suspension having an OD 600 of 5. 1 mL of liquid was prepared.
  • 0.2 mL of the resulting cell suspension was added as a carbon source to SA, VA, p -hydroxybenzoic acid (HBA), a mixture of SA and VA (SA-VA), a mixture of SA and HBA (SA-HBA), MMx-3 liquid medium containing a mixture of VA and HBA (VA-HBA) or a mixture of SA, VA and HBA (SA-VA-HBA) [34.2 g/L Na 2 HPO 4.12H 2 O, 6.0 g /L KH2PO4 , 1.0 g/L NaCl, 2.5 g/L ( NH4) 2SO4 , 49.3 mg/L MgSO4.7H2O , 15 mg/ L CaCl2.2H2O , 5 mg /L FeSO 4 .7H 2 O] (final concentration of each compound is 5 mM) and subjected to shaking culture at 30°C.
  • HBA p -hydroxybenzoic acid
  • SA-VA a mixture of SA and VA
  • OD-Monitor C&T (Taitec Co., Ltd.) was used for OD 600 measurement.
  • the concentrations of SA, VA and HBA were measured using a high-performance liquid chromatograph (“Agilent 1200 series”, Agilent Technologies).
  • a ZORBAX Eclipse Plus C18 column (diameter: 4.6 mm, length: 150 mm, particle size: 0.5 ⁇ m) was used and kept at 40°C.
  • Gradient elution mode (solvent A: 5% (v/v) CH3OH , 1% (v/v) CH3COOH, solvent B: 50% (v/v) CH3OH , 1% (v /v) CH 3 COOH), equilibrated with solvent A, then increasing the percentage of solvent B to 20% over 8 minutes from the start of the analysis, then increasing the percentage of solvent B to 100% over 5 minutes. raised.
  • the flow rate of the mobile phase was 1.0 mL/min, and the measurement wavelengths were 254 nm and 280 nm.
  • FIGS. 4A to 4G The results of measurements using SA, VA, HBA, SA-VA, SA-HBA, VA-HBA and SA-VA-HBA as carbon sources are shown in FIGS. 4A to 4G.
  • the NGC7 ⁇ vanA4B4 strain grew when at least one of SA and HBA was used as the carbon source, but did not grow when VA was used as the sole carbon source.
  • the SYK-6 strain (hereinafter also referred to as SYK-6 strain) has the ability to decompose acetovanilone (AV) and acetosyringone, which are aromatic compounds derived from lignin.
  • the SYK-6 strain has the following AV degrading enzyme gene clusters involved in AV degradation: acvA gene, acvB gene, acvC gene, acvD gene, acvE gene, acvF gene, vceA gene and vceB gene.
  • AV degrading enzyme gene clusters involved in AV degradation: acvA gene, acvB gene, acvC gene, acvD gene, acvE gene, acvF gene, vceA gene and vceB gene.
  • a nucleic acid fragment containing the acvA gene, the acvB gene, the acvC gene, the acvD gene , the acvE gene and the acvF gene was obtained from the genomic DNA of the SYK-6 strain among the AV degrading enzyme gene group derived from the SYK-6 strain according to a conventional method.
  • the nucleic acid fragment was ligated under the control of E. coli-derived lactose promoter inserted into pSEVA241 plasmid DNA to obtain plasmid pSEVA241_P lac -acv.
  • nucleic acid fragment containing the vceA gene and the vceB gene was obtained from the genomic DNA of the SYK-6 strain and ligated under the control of the E. coli-derived lactose promoter inserted into the pJB866 plasmid DNA to obtain the plasmid pTS093_vceA-B. rice field.
  • the plasmid DNAs of pSEVA241_P lac -acv and pTS093_vceA-B were used to transform the NGC7 strain and the NGC7 ⁇ vanA4B4 strain to obtain the NGC7 [pSEVA241_P lac -acv, pTS093_vceA-B] strain and the NGC7 ⁇ vanA4B4, pSEVA241_vceA241_P lac -acv, pTS093_vceA-B4 B] strains were obtained respectively.
  • strains were inoculated into 10 mL of LB liquid medium containing 25 mg/L kanamycin (Km) and 15 mg/L tetracycline (Tc) and subjected to overnight shaking culture (180 rpm) at 30°C.
  • Km kanamycin
  • Tc tetracycline
  • 0.1 mL of the resulting culture solution was inoculated into 10 mL of new LB liquid medium (25 mg/L Km, 15 mg/L Tc included) and subjected to shaking culture at 30°C for 16 hours.
  • the resulting culture solution is subjected to centrifugation to collect the cells, and the cells obtained are washed with MMx-3 medium and then resuspended in MMx-3 medium until OD 600 reaches 10.
  • 1 mL of cell suspension was prepared so that AV was added to the resulting cell suspension to a final concentration of 0.2 mM, followed by shaking culture (1500 rpm) at 30°C.
  • the NGC7 strain does not have the ability to degrade AV, but by introducing the AV-degrading enzyme gene group, it acquired the ability to decompose AV. However, due to the degradability of VA, VA did not accumulate.
  • the NGC7 ⁇ vanA4B4 strain lacking the ability to degrade VA degrades AV and accumulates VA by introducing an AV degrading enzyme gene group. was found to be able to produce
  • the resulting lignin decomposition catalyst was cut into discs with a diameter of 10 mm, and 10 discs were placed in a stainless steel tube with an inner diameter of 10 mm to form a catalyst layer.
  • a lignin solution obtained by dissolving 10 g of sulfite lignin (Tokyo Kasei Kogyo Co., Ltd.) in 900 mL of a 2 M sodium hydroxide aqueous solution was added to the catalyst layer heated to 180°C with a heater, using a liquid feed pump at 0.225 mL/ The liquid was sent in mL.
  • oxygen gas was introduced into the catalyst layer at a rate of 2 mL/min in terms of standard conditions.
  • a back pressure valve was provided at the outlet of the catalyst layer, and the pressure in the catalyst layer was adjusted to be maintained at 8 atmospheres by gauge.
  • the pH of the solution obtained from the catalyst layer was adjusted to 10 using hydrochloric acid.
  • the prepared solution was tested using a flat membrane tester "HP4750" (Steritech) to test an MF membrane, a UF membrane ("MWCO 5,000 Da”; MT, Synder) and a UF membrane ("MWCO 1,000 Da”; GE, Suez) to collect the filtrate.
  • the resulting filtrate was adjusted to pH 3 using hydrochloric acid, and then extracted three times using ethyl acetate.
  • the resulting ethyl acetate layer was evaporated to dryness to obtain an extract, and then a solution of the obtained extract dissolved in a small amount of 2M NaOH was adjusted to pH 8 with hydrochloric acid to give lignin.
  • a decomposition product was obtained.
  • the concentration of major aromatic monomers in the lignin degradation product was measured using a high-performance liquid chromatograph using the apparatus and conditions described in Example 5 above.
  • the major aromatic monomers in the lignin degradation product were acetovanilone (AV), vanillin (VN) and vanillic acid (VA) at concentrations of 51.1 mM, 296.4 mM and 113.8 mM, respectively.
  • the NGC7 ⁇ vanA4B4 [pSEVA241_P lac -acv, pTS093_vceA-B] strain described in Example 6 above was inoculated into 10 mL of LB liquid medium containing 25 mg/L Km and 15 mg/L Tc, and cultured overnight at 30°C with shaking (180 rpm ).
  • 0.1 mL of the obtained culture solution was inoculated into 10 mL of fresh LB liquid medium (containing 25 mg/L Km and 15 mg/L Tc) and subjected to shaking culture at 30° C. for 16 hours.
  • the resulting culture solution is subjected to centrifugation to collect the cells, and the obtained cells are washed with MMx-3 medium and then suspended again in MMx-3 medium until the OD 600 reaches 10.
  • 1 mL of cell suspension was prepared so that
  • MMx-3 medium (containing 25 mg / L Km and 15 mg / L Tc) containing 0.75 mL of 200 g / L glucose solution and 0.1 mL of lignin degradation product model, cells so that the initial OD 600 is 0.1 Suspension was added.
  • the medium containing the obtained cells was subjected to shaking culture at 30°C. After the start of the culture, samples were taken at regular time intervals, the OD 600 of the culture solution was measured, and the culture supernatant obtained by centrifuging the culture solution was measured for AV, VN, VA and glucose concentrations.
  • OD 600 was measured using a spectrophotometer (“BioSpec-mini”, Shimadzu Corporation).
  • the concentrations of AV, VN and VA were measured using a high performance liquid chromatograph using the apparatus and conditions described in Example 5 above. Glucose concentration was measured using "Biosensor BF-5" (Oji Keisokuki Co., Ltd.).
  • the measurement results are shown in FIG. As shown in FIG. 6, the NGC7 ⁇ vanA4B4 [pSEVA241_P lac -acv, pTS093_vceA-B] strain degraded acetovanillone and vanillin in the lignin degradation product model to produce VA with a yield of 97.3%.
  • the VA yield was calculated from [VA amount (mol) after culturing for 78 h]/[AV amount (mol) + VN amount (mol) + VA amount (mol) at the start of culturing] x 100 (%).
  • NGC7 ⁇ vanA4B4 [pSEVA241_P lac -acv, pTS093_vceA-B] strain described in Example 6 above was inoculated into 10 mL of LB liquid medium containing 25 mg/L Km and 15 mg/L Tc, and cultured overnight at 30°C with shaking (180 rpm). ).
  • 0.1 mL of the resulting culture solution was inoculated into 10 mL of a new LB liquid medium (containing 25 mg/L Km and 15 mg/L Tc) and subjected to shaking culture at 30° C. for 16 hours.
  • the resulting culture solution is subjected to centrifugation to collect the cells, and the obtained cells are washed with MMx-3 medium and then suspended again in MMx-3 medium until the OD 600 reaches 10.
  • 1 mL of cell suspension was prepared so that
  • the VA yield was calculated from [VA amount (mol) after culturing for 68 h]/[AV amount (mol) + VN amount (mol) + VA amount (mol)] x 100 (%) at the start of culturing.
  • Each fragment was ligated with pK18 mobsacB previously digested with HindIII and BamHI by a seamless cloning method using NEBuilder HiFi DNA assembly to construct a new plasmid pvanA2B2del2 for constructing a vanB2 gene region deletion strain of the vanA2 gene.
  • Each fragment was ligated with pK18 mobsacB previously digested with HindIII and BamHI by a seamless cloning method using NEBuilder HiFi DNA assembly to construct a new plasmid pvanA4B4del2 for constructing a vanB4 gene region deletion strain of the vanA4 gene.
  • the genome of the NGC7 strain contains PSN — 1511 ( aph gene; SEQ ID NO: 37), a gene encoding an amino acid sequence showing 50.9% sequence identity with aminoglycoside-3′-phosphotransferase (P00552) derived from Klebsiella pneumoniae . Confirmed it exists. Since it was presumed that the NGC7 strain would have Km resistance due to the function of the aph gene, a mutant strain was prepared by disrupting this gene.
  • PCR using the genomic DNA of strain NGC7 as a template and a primer set consisting of primers 25 and 26 of SEQ ID NOs: 46 and 47 and a primer set consisting of primers 27 and 28 of SEQ ID NOs: 48 and 49, respectively.
  • a DNA fragment of about 1.2 kbp upstream of the 5'-end and downstream of the 3'-end of the aph gene was amplified by the method.
  • Each fragment was ligated with pK18 mobsacB previously digested with HindIII and BamHI by a seamless cloning method using NEBuilder HiFi DNA assembly to prepare a plasmid paphdel for constructing an aph gene region deletion strain.
  • the plasmid pvanA4B4del2 was introduced into the NGC7 ⁇ vanA1B1 strain to prepare the NGC7 ⁇ vanA1B1 ⁇ vanA4B4 strain lacking the internal regions of the vanA4 and vanB4 genes on the genomic DNA.
  • a plasmid paphdel was introduced into the obtained NGC7 ⁇ vanA1B1 ⁇ vanA4B4 strain to prepare an NGC7 ⁇ vanA1B1 ⁇ vanA4B4 ⁇ aph strain lacking the internal region of the aph gene on the genomic DNA.
  • the obtained NGC7 ⁇ vanA1B1 ⁇ vanA4B4 ⁇ aph strain was transfected with the plasmid pvanA2B2del2 to prepare the NGC7 ⁇ vanA1B1 ⁇ vanA4B4 ⁇ aph ⁇ vanA2B2 strain in which the internal regions of the vanA2 and vanB2 genes on the genomic DNA were deleted.
  • a plasmid pvanA3B3del was introduced into the obtained NGC7 ⁇ vanA1B1 ⁇ vanA4B4 ⁇ aph ⁇ vanA2B2 strain to prepare an NGC7 ⁇ vanA1B1 ⁇ vanA4B4 ⁇ aph ⁇ vanA2B2 ⁇ vanA3B3 strain in which the vanA3 gene on the genomic DNA and the internal region of the vanB3 gene were deleted. did.
  • the NGC7 ⁇ vanA1B1 ⁇ vanA4B4 ⁇ aph ⁇ vanA2B2 ⁇ vanA3B3 strain was transformed using the plasmid pSEVA241_P lac -acv and the plasmid pTS093_vceA-B described in 6 above to obtain NGC7 ⁇ vanA1B1 ⁇ vanA4 ⁇ vanA4 ⁇ B3 ⁇ B3 ⁇ BAph .
  • pSEVA241_P lac -acv, pTS093_vceA-B] strains were obtained.
  • NGC7 ⁇ vanA4B4 [pSEVA241_P lac -acv, pTS093_vceA-B] strain
  • NGC7 ⁇ vanA1B1 ⁇ vanA4B4 ⁇ aph ⁇ vanA2B2 ⁇ vanA3B3 [pSEVA241_P lac -acv, pTS093_vceA-B] was used in the same manner as in the above example except for the strain.
  • VA production ability from the lignin degradation product was evaluated by the method described in 9.
  • Vanillic acid can be obtained from biomass containing lignin-derived aromatic compounds and vanillic acid by the transformed microorganism and production method of one embodiment of the present invention. Vanillic acid can be converted into various industrially useful compounds, and can be used, for example, as a raw material for vanillic acid derivatives that are used in heat-resistant and rigid plastics, coating agents, and the like.

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Abstract

The purpose of the present invention is to provide a microorganism which can selectively produce and/or accumulate vanillic acid while growing with use of not only H-type lignin-derived aromatic compounds but also S-type lignin-derived aromatic compounds as carbon sources, and a method for using the microorganism to produce vanillic acid. The purpose is achieved by, for example: a transformed microorganism, the host microorganism of which is Pseudomonas species (Pseudomonas sp.) NGC7 strain (accession number: NITE BP-03043), and in which a vanA4 gene (SEQ ID NO: 1) or the vanA4 gene and a vanB4 gene (SEQ ID NO: 2) on the chromosome are deleted; and a method for using the transformed microorganism to produce vanillic acid.

Description

バニリン酸産生形質転換微生物及びその利用Transformed microorganism producing vanillic acid and its use
　本発明は、バニリン酸が生産可能な形質転換微生物及び該形質転換微生物を利用したバニリン酸の製造方法に関する。特に、本発明は、ｐ－ヒドロキシフェニルリグニン、グアイアシルリグニン及びシリンギルリグニン由来の芳香族化合物の混合物からバニリン酸を選択的に生産することが可能な形質転換微生物に関する。 The present invention relates to a transformed microorganism capable of producing vanillic acid and a method for producing vanillic acid using the transformed microorganism. In particular, the present invention relates to transformed microorganisms capable of selectively producing vanillic acid from a mixture of aromatic compounds derived from p -hydroxyphenyllignin, guaiacyllignin and syringyllignin.
　国連主導の持続可能な開発目標（Ｓｕｓｔａｉｎａｂｌｅ　Ｄｅｖｅｌｏｐｍｅｎｔ　Ｇｏａｌｓ；ＳＤＧｓ）の達成に向けた国際協調及び我が国での２０５０年達成目標のカーボンニュートラル社会の実現から、石油原料に代えて非可食バイオマスから機能性材料を製造することが求められている。その中で、非可食バイオマスを原料とした、セルロース誘導体及び脂肪族ポリマー原料の開発が進められている。しかし、これまでに、剛性、耐熱性などが満足できるポリマーはほとんど知られていない。 From the international cooperation to achieve the Sustainable Development Goals (SDGs) led by the United Nations and the realization of a carbon-neutral society, which is the target to be achieved in Japan in 2050, non-edible biomass will replace petroleum raw materials with functionality. There is a need to manufacture materials. Among them, the development of raw materials for cellulose derivatives and aliphatic polymers using non-edible biomass as raw materials is underway. However, until now, almost no polymer has been known that satisfies rigidity, heat resistance, and the like.
　非可食バイオマスの一種にリグニンがある。リグニンは植物の維管束細胞壁成分として存在する無定形高分子物質であって、フェニルプロパン系の構成単位が複雑に縮合したものであり、メトキシ基を含有することが化学構造上の大きな特徴になっている。リグニンは木質化した植物細胞を相互に膠着し、組織を強化する働きをしており、木材中に約１８％～３６％、草本中には約１５％～２５％存在する。そこで、木材を有効利用するために、リグニンを分解し、有用化合物を得ようとする試みがなされている。木材などのバイオマスに由来するリグニンには、ｐ－ヒドロキシフェニルリグニン（Ｈ型リグニン）、グアイアシルリグニン（Ｇ型リグニン）及びシリンギルリグニン（Ｓ型リグニン）の３種があることが知られている。 One type of non-edible biomass is lignin. Lignin is an amorphous macromolecular substance that exists as a component of the vascular cell wall of plants. It is a complex condensation of phenylpropane-based structural units, and its chemical structure is characterized by the presence of methoxy groups. ing. Lignin has the function of binding lignified plant cells to each other and strengthening the tissue, and is present in wood at approximately 18% to 36% and in herbs at approximately 15% to 25%. Therefore, attempts have been made to decompose lignin to obtain useful compounds in order to effectively utilize wood. It is known that there are three types of lignin derived from biomass such as wood: p -hydroxyphenyl lignin (H-type lignin), guaiacyl lignin (G-type lignin) and syringyl lignin (S-type lignin). .
　リグニンを分解して得られる化合物の中には、分子内に芳香環を有するモノマーとして使用できるものがある。このような芳香族モノマーを原料とするポリマーは、剛性、耐熱性などの機能性が優れたものになり得る。 Among the compounds obtained by decomposing lignin, there are some that can be used as monomers with aromatic rings in the molecule. Polymers made from such aromatic monomers can be excellent in functionality such as rigidity and heat resistance.
　リグニン由来の芳香族モノマーの候補物質の一つとして、バニリン酸がある。バニリン酸を重合してなるポリマーは融点が非常に高いという特徴がある。例えば、バニリン酸を修飾等して得たモノマーを原料として得られる高機能性ポリマーは、融点が高く、機能性が高いという報告がある（例えば、下記特許文献１及び２を参照）。 Vanillic acid is one of the candidates for lignin-derived aromatic monomers. A polymer obtained by polymerizing vanillic acid is characterized by a very high melting point. For example, it has been reported that a highly functional polymer obtained using a monomer obtained by modifying vanillic acid as a raw material has a high melting point and high functionality (see, for example, Patent Documents 1 and 2 below).
　これまでに、リグニン又はリグニン由来芳香族化合物を分解する微生物として、スフィンゴビウム・スピーシーズ（Ｓｐｈｉｎｇｏｂｉｕｍ　ｓｐ．）ＳＹＫ－６株（以下、ＳＹＫ－６株ともよぶ。）が知られている。ＳＹＫ－６株は、Ｇ型リグニン由来のバニリン酸及びＳ型リグニン由来のシリンガ酸を分解することができ、またメチオニン存在下ではＨ型リグニン由来のｐ－ヒドロキシ安息香酸も分解することができる。ＳＹＫ－６株からｌｉｇＭ遺伝子を欠失するように形質転換した形質転換微生物（ＳＹＫ－６ΔｌｉｇＭ株）又はＳＹＫ－６株からｌｉｇＭ遺伝子及びｄｅｓＡ遺伝子を欠失するように形質転換した形質転換微生物（ＳＹＫ－６ΔｌｉｇＭΔｄｅｓＡ株）は、バニリン酸の分解性の一部又は全部を欠如するという報告がある（例えば、下記非特許文献１を参照）。 Sphingobium sp. SYK-6 strain (hereinafter also referred to as SYK-6 strain) is known as a microorganism that decomposes lignin or lignin-derived aromatic compounds. The SYK-6 strain can degrade vanillic acid derived from G-type lignin and syringic acid derived from S-type lignin, and can also degrade p -hydroxybenzoic acid derived from H-type lignin in the presence of methionine. A transformed microorganism transformed from the SYK-6 strain to delete the ligM gene (SYK-6Δ ligM strain) or a transformed microorganism transformed from the SYK-6 strain to delete the ligM gene and desA gene ( SYK-6Δ ligM Δ desA strain) is reported to lack some or all of the vanillic acid degradability (see, for example, Non-Patent Document 1 below).
　また、本発明者らは、リグニン由来芳香族化合物であるｐ－ヒドロキシ安息香酸、バニリン酸及びシリンガ酸を分解することができるシュードモナス・スピーシーズ（Ｐｓｅｕｄｏｍｏｎａｓ　ｓｐ．）　ＮＧＣ７株（寄託番号：ＮＩＴＥ　ＢＰ－０３０４３；以下、ＮＧＣ７株ともよぶ。）を単離し、ＮＧＣ７株を形質転換して上記リグニン由来芳香族化合物からムコン酸を得ることに成功している（例えば、下記特許文献３を参照）。 In addition, the present inventors have found that Pseudomonas sp. NGC7 strain (accession number: NITE BP-03043) is capable of degrading p -hydroxybenzoic acid, vanillic acid and syringic acid, which are lignin-derived aromatic compounds. ; hereinafter also referred to as NGC7 strain), and transformed the NGC7 strain to successfully obtain muconic acid from the lignin-derived aromatic compound (see, for example, Patent Document 3 below).
特許第５４２１０６０号Patent No. 5421060 特開２０１２－１１６９１３号公報JP 2012-116913 A ＷＯ２０２０／０８０４６７WO2020/080467
　非特許文献１に記載のＳＹＫ－６ΔｌｉｇＭΔｄｅｓＡ株は、バニリン酸を分解しないものの、バニリン酸のアナログであるシリンガ酸の分解能も失っており、メチオニン非存在下ではＨ型リグニン由来の芳香族化合物を炭素源として増殖できない。したがって、ＳＹＫ－６ΔｌｉｇＭΔｄｅｓＡ株を用いてバニリン酸を製造しようとする場合、Ｓ型リグニンを多く含む広葉樹などを利用できず、使用できるバイオマスの種類が限られるという問題がある。 Although the SYK-6Δ ligM Δ desA strain described in Non-Patent Document 1 does not degrade vanillic acid, it also loses the ability to decompose syringic acid, an analog of vanillic acid, and in the absence of methionine, aromatics derived from H-type lignin The compound cannot be grown as a carbon source. Therefore, when attempting to produce vanillic acid using the SYK-6Δ ligM Δ desA strain, there is a problem that the types of biomass that can be used are limited because broad-leaved trees containing a large amount of S-type lignin cannot be used.
　非特許文献１に記載のＳＹＫ－６ΔｌｉｇＭ株はシリンガ酸を分解するが、バニリン酸も分解することから、バニリン酸を蓄積することができない。したがって、ＳＹＫ－６ΔｌｉｇＭ株を用いても、選択的にバニリン酸を製造することができないという問題がある。 The SYK-6Δ ligM strain described in Non-Patent Document 1 degrades syringic acid, but also degrades vanillic acid, and therefore cannot accumulate vanillic acid. Therefore, even if the SYK-6Δ ligM strain is used, there is a problem that vanillic acid cannot be selectively produced.
　また、上記のとおり、非特許文献１に記載のいずれの微生物も、バニリン酸及びそのアナログであるシリンガ酸の一方だけを特異的に分解することはできない。 In addition, as described above, none of the microorganisms described in Non-Patent Document 1 can specifically decompose either vanillic acid or its analog syringic acid.
　一方、特許文献３に記載のＮＧＣ７株及びＮＧＣ７形質転換株は、バニリン酸を分解することから、バニリン酸を蓄積することはできない。また、本発明者らが調べたところによれば、ＮＧＣ７株において、ＳＹＫ－６株のｌｉｇＭ遺伝子及びｄｅｓＡ遺伝子に相当する遺伝子はこれまでのゲノム解析からは見出されていない。 On the other hand, the NGC7 strain and the NGC7 transformant described in Patent Document 3 cannot accumulate vanillic acid because they degrade vanillic acid. Further, according to investigations by the present inventors, no genes corresponding to the ligM gene and desA gene of the SYK-6 strain have been found in the NGC7 strain by genome analysis so far.
　仮にＮＧＣ７株がＳＹＫ－６株のｌｉｇＭ遺伝子及びｄｅｓＡ遺伝子に相当する遺伝子を有しており、それらを破壊したとしても、非特許文献１に記載のＳＹＫ－６ΔｌｉｇＭΔｄｅｓＡ株のようにシリンガ酸を分解できず、炭素源として増殖できない可能性があり、又は非特許文献１に記載のＳＹＫ－６ΔｌｉｇＭ株のようにシリンガ酸を炭素源として増殖しつつも、バニリン酸を分解する可能性がある。 Even if the NGC7 strain has genes corresponding to the ligM gene and desA gene of the SYK-6 strain and disrupts them, syringic acid like the SYK-6Δ ligM Δ desA strain described in Non-Patent Document 1 and may not grow as a carbon source, or, like the SYK-6Δ ligM strain described in Non-Patent Document 1, while growing using syringic acid as a carbon source, vanillic acid may be degraded. be.
　以上のとおり、バニリン酸及びシリンガ酸のいずれか一方のみを分解して炭素源としても利用しつつ、他方を分解せずに蓄積するという微生物株はこれまでにほとんど知られていない。すなわち、Ｈ型リグニン由来芳香族化合物だけでなく、Ｓ型リグニン由来芳香族化合物を分解して炭素源としても利用しつつ、バニリン酸を分解せずに蓄積する微生物についてはこれまでほとんど知られていない。その結果として、Ｓ型リグニン由来の芳香族化合物を含む広葉樹に由来するバイオマスを原料とした場合、従来の微生物を利用しては、培養液中にバニリン酸以外の芳香族化合物が蓄積し、バニリン酸の精製工程が複雑になるという問題が生じている。したがって、これまでに、微生物を利用して、幅広いバイオマスのリグニン由来芳香族化合物からバニリン酸を選択的に生産することができていない。 As described above, there are almost no known microbial strains that decompose only one of vanillic acid and syringic acid and use it as a carbon source while accumulating the other without decomposing it. That is, microorganisms that decompose not only H-type lignin-derived aromatic compounds but also S-type lignin-derived aromatic compounds and use them as carbon sources while accumulating vanillic acid without decomposing have been known so far. do not have. As a result, when biomass derived from broadleaf trees containing aromatic compounds derived from S-type lignin is used as a raw material, aromatic compounds other than vanillic acid accumulate in the culture medium using conventional microorganisms, and vanillin A problem arises that the acid purification process is complicated. Therefore, to date, it has not been possible to selectively produce vanillic acid from lignin-derived aromatic compounds in a wide range of biomass using microorganisms.
　そこで、本発明は、Ｈ型リグニン由来芳香族化合物だけでなく、Ｓ型リグニン由来芳香族化合物を分解し、炭素源としても利用しつつ、バニリン酸を選択的に生産及び／又は選択的に蓄積することが可能である微生物及び該微生物を利用したバニリン酸の製造方法を提供することを、発明が解決しようとする課題とする。 Therefore, the present invention decomposes not only H-type lignin-derived aromatic compounds but also S-type lignin-derived aromatic compounds, and selectively produces and / or selectively accumulates vanillic acid while using it as a carbon source. It is an object of the present invention to provide a microorganism that can be used to produce vanillic acid and a method for producing vanillic acid using the microorganism.
　本発明者らは、上記課題を解決するために、Ｓ型リグニン由来芳香族化合物を炭素源として増殖しつつ、バニリン酸の分解性が低減又は欠失した微生物について鋭意検討した。その中で、本発明者らは、バニリン酸の分解に関与する数多くの遺伝子群のうち、酸素添加型（オキシゲナーゼタイプ）のバニレート　Ｏ－デメチラーゼに着眼するに至った。バニレート　Ｏ－デメチラーゼは、バニレート　Ｏ－デメチラーゼ　オキシゲナーゼコンポーネント（ＶａｎＡ）及びバニレート　Ｏ－デメチラーゼ　オキシドレダクターゼコンポーネント（ＶａｎＢ）からなる。 In order to solve the above problems, the present inventors diligently studied microorganisms in which the degradability of vanillic acid was reduced or lost while growing using an S-type lignin-derived aromatic compound as a carbon source. Among many gene groups involved in the degradation of vanillic acid, the present inventors have focused on oxygenation type (oxygenase type) vanillate O 2 -demethylase. Vanillate O 2 -demethylase consists of a vanillate O 2 -demethylase oxygenase component (VanA) and a vanillate O 2 -demethylase oxidoreductase component (VanB).
　そこで、本発明者らは、ＮＧＣ７株はシュードモナス・プチダ（Ｐ．ｐｕｔｉｄａ）と近縁種であることから、Ｐ．ｐｕｔｉｄａ　ＫＴ２４４０株由来のＶａｎＡ及びＶａｎＢをコードするｖａｎＡ遺伝子及びｖａｎＢ遺伝子に相当する遺伝子をＮＧＣ７株のゲノム配列から探索することを試みた。 Therefore, the present inventors found that the NGC7 strain is closely related to Pseudomonas putida . An attempt was made to search the genome sequence of the NGC7 strain for genes corresponding to the vanA and vanB genes encoding VanA and VanB derived from the S. putida KT2440 strain.
　その結果、予想外に、ＮＧＣ７株は、ｖａｎＡ遺伝子に相当する４種類の遺伝子を有し、さらにｖａｎＢ遺伝子に相当する６種類の遺伝子を有することを見出した。このようにｖａｎＡ遺伝子及びｖａｎＢ遺伝子のそれぞれを複数種類で有する微生物はこれまでにほとんど知られていない。 As a result, it was unexpectedly found that the NGC7 strain has 4 genes corresponding to the vanA gene and 6 genes corresponding to the vanB gene. Microorganisms having multiple types of vanA genes and vanB genes have thus far been scarcely known.
　本発明者らは、複数種類のｖａｎＡ遺伝子及び複数種類のｖａｎＢ遺伝子について、ペアになる遺伝子のセットを試行錯誤し、４対のｖａｎＡ遺伝子及びｖａｎＢ遺伝子のセットを選ぶに至った。そして、この４対の遺伝子セットをそれぞれ欠損したＮＧＣ７形質転換株を製造したところ、シリンガ酸を分解して増殖できる形質転換株もあれば、シリンガ酸の資化性が劣る形質転換株もあること、バニリン酸を分解する形質転換株もあれば、バニリン酸をほとんど分解しない形質転換株もあることを見出した。 The inventors of the present invention conducted trial and error to set pairs of genes for multiple types of vanA genes and multiple types of vanB genes, and came to select sets of four pairs of vanA genes and vanB genes. Then, when NGC7 transformant strains lacking each of these four pairs of gene sets were produced, some transformant strains were able to degrade syringic acid and proliferate, while others were inferior in assimilation of syringic acid. , some transformants degrade vanillic acid, and some transformants hardly degrade vanillic acid.
　驚くべきことに、ある特定のｖａｎＡ遺伝子及びｖａｎＢ遺伝子のセットを欠損したＮＧＣ７形質転換株は、野生型のＮＧＣ７株と比較して同等以上にシリンガ酸を分解して増殖しつつも、バニリン酸をほとんど分解しなかった。 Surprisingly, an NGC7 transformant strain lacking a specific set of vanA and vanB genes degrades syringic acid to a level equal to or greater than that of the wild-type NGC7 strain and proliferates, while producing vanillic acid. almost did not decompose.
　したがって、該ＮＧＣ７形質転換株を用いれば、Ｓ型リグニン由来の芳香族化合物を利用して増殖しつつ、Ｇ型リグニン由来の芳香族化合物からバニリン酸を製造し得ることを見出した。また、例えば、現行工業レベルで行われているリグニン分解方法であるクラフト蒸解液及びソーダ蒸解液に含まれる主要な芳香族化合物であるアセトバニロンからも、該ＮＧＣ７形質転換株にアセトバニロンからバニリン酸への変換を可能とする遺伝子群を組み込むことで、バニリン酸を製造することできた。 Therefore, it was found that by using the NGC7 transformant strain, vanillic acid can be produced from aromatic compounds derived from G-type lignin while growing using aromatic compounds derived from S-type lignin. In addition, for example, acetovanillone, which is a major aromatic compound contained in kraft cooking liquor and soda cooking liquor, which are lignin decomposition methods currently used at the industrial level, is also used in the NGC7 transformant to convert acetovanillone to vanillic acid. By incorporating a gene group that enables conversion, vanillic acid could be produced.
　上記した知見を基にして、本発明者らは、遂に、本発明の課題を解決するものとして、Ｈ型リグニン由来芳香族化合物やＳ型リグニン由来芳香族化合物などのＧ型リグニン由来芳香族化合物以外の化合物を分解し炭素源としても増殖しつつ、バニリン酸を選択的に生産及び／又は蓄積することが可能である微生物及び該微生物を利用したバニリン酸の製造方法を創作することに成功した。本発明は、これらの本発明者らによって初めて得られた知見及び成功例に基づき完成された発明である。 Based on the above findings, the present inventors have finally found that the problems of the present invention are solved by finding G-type lignin-derived aromatic compounds such as H-type lignin-derived aromatic compounds and S-type lignin-derived aromatic compounds. We succeeded in creating a microorganism capable of selectively producing and/or accumulating vanillic acid while decomposing compounds other than . The present invention is an invention completed based on the findings and successful examples obtained for the first time by these inventors.
　したがって、本発明によれば、以下の各一態様が提供される。
［１］宿主微生物がシュードモナス・スピーシーズ（Ｐｓｅｕｄｏｍｏｎａｓ　ｓｐ．）　ＮＧＣ７株（受託番号：ＮＩＴＥ　ＢＰ－０３０４３）であり、
染色体上にある、ｖａｎＡ４遺伝子（配列番号１）又は該ｖａｎＡ４遺伝子及びｖａｎＢ４遺伝子（配列番号２）が欠失している
形質転換微生物。
［２］宿主微生物がシュードモナス・スピーシーズ（Ｐｓｅｕｄｏｍｏｎａｓ　ｓｐ．）　ＮＧＣ７株（受託番号：ＮＩＴＥ　ＢＰ－０３０４３）であり、
染色体上にあるｖａｎＡ４遺伝子（配列番号１）又は該ｖａｎＡ４遺伝子及びｖａｎＢ４遺伝子（配列番号２）が欠失しており、かつ、
挿入されたａｃｖＡ遺伝子、ａｃｖＢ遺伝子、ａｃｖＣ遺伝子、ａｃｖＤ遺伝子、ａｃｖＥ遺伝子、ａｃｖＦ遺伝子、ｖｃｅＡ遺伝子及びｖｃｅＢ遺伝子を発現する
形質転換微生物。
［３］グアイアシルリグニン由来の芳香族化合物を、［１］又は［２］に記載の形質転換微生物に作用させることにより、バニリン酸を得る工程
を含む、バニリン酸の製造方法。
［４］グアイアシルリグニン由来の芳香族化合物と、ｐ－ヒドロキシフェニルリグニン由来の芳香族化合物及び／又はシリンギルリグニン由来の芳香族化合物との混合物を、［１］又は［２］に記載の形質転換微生物に作用させることにより、バニリン酸を得る工程
を含む、バニリン酸の製造方法。
［５］アセトバニロンを含むグアイアシルリグニン由来の芳香族化合物の混合物を、［２］に記載の形質転換微生物に作用させることにより、バニリン酸を得る工程
を含む、バニリン酸の製造方法。 Therefore, according to the present invention, the following aspects are provided.
[1] The host microorganism is Pseudomonas sp. NGC7 strain (accession number: NITE BP-03043),
A transformed microorganism lacking the vanA4 gene (SEQ ID NO: 1) or the vanA4 gene and the vanB4 gene (SEQ ID NO: 2) on the chromosome.
[2] the host microorganism is Pseudomonas sp. NGC7 strain (accession number: NITE BP-03043);
The vanA4 gene (SEQ ID NO: 1) or the vanA4 gene and the vanB4 gene (SEQ ID NO: 2) on the chromosome are deleted, and
A transformed microorganism expressing the inserted acvA gene, acvB gene, acvC gene, acvD gene, acvE gene, acvF gene, vceA gene and vceB gene.
[3] A method for producing vanillic acid, comprising a step of obtaining vanillic acid by allowing an aromatic compound derived from guaiacyllignin to act on the transformed microorganism of [1] or [2].
[4] A mixture of an aromatic compound derived from guaiacyl lignin and an aromatic compound derived from p -hydroxyphenyl lignin and/or an aromatic compound derived from syringyl lignin is added to the trait of [1] or [2]. A method for producing vanillic acid, comprising a step of obtaining vanillic acid by allowing the transformed microorganism to act.
[5] A method for producing vanillic acid, comprising a step of obtaining vanillic acid by allowing a mixture of aromatic compounds derived from guaiacyl lignin containing acetovanillone to act on the transformed microorganism of [2].
　本発明によれば、Ｈ型リグニン由来芳香族化合物及び／又はＳ型リグニン由来芳香族化合物を分解し、炭素源としても利用しつつ、微生物学的にＧ型リグニン由来芳香族化合物からバニリン酸を選択的に生産及び／又は蓄積することが可能である。本発明によれば、炭素源となるリグニン分解物としてはＨ型リグニン及びＳ型リグニンのいずれの由来のものも利用することができることから、グルコースなどのリグニン以外の炭素源を用いずに、リグニン分解物を炭素源として用いることもでき、安価かつリグニンの種類に依拠せずに、Ｇ型リグニン由来の芳香族化合物からバニリン酸を製造することができる。したがって、本発明によれば、リグニンを含むバイオマスの有効利用の一環として、工業的規模でのバニリン酸の製造が期待される。 According to the present invention, H-type lignin-derived aromatic compounds and / or S-type lignin-derived aromatic compounds are decomposed and used as a carbon source, while microbiologically vanillic acid is produced from G-type lignin-derived aromatic compounds. It can be selectively produced and/or accumulated. According to the present invention, since it is possible to use either of H-type lignin and S-type lignin as a lignin decomposition product that serves as a carbon source, lignin can be obtained without using a carbon source other than lignin such as glucose. A decomposition product can also be used as a carbon source, and vanillic acid can be produced from an aromatic compound derived from G-type lignin inexpensively and independently of the type of lignin. Therefore, according to the present invention, production of vanillic acid on an industrial scale is expected as part of effective utilization of biomass containing lignin.
図１Ａは、受託番号ＮＩＴＥ　ＢＰ－０３０４３（シュードモナス・スピーシーズ　ＮＧＣ７株）の受託証の写しである。FIG. 1A is a copy of the receipt of accession number NITE BP-03043 (Pseudomonas sp. NGC7 strain). 図１Ｂは、受託番号ＮＩＴＥ　ＢＰ－０３０４３（シュードモナス・スピーシーズ　ＮＧＣ７株）の生存に関する証明書の写しである。FIG. 1B is a copy of the certificate of survival of accession number NITE BP-03043 (Pseudomonas sp. NGC7 strain). 図２Ａは、後述する実施例に記載するとおり、バニリン酸（ＶＡ）を炭素源としてＮＧＣ７株、ＮＧＣ７ΔｖａｎＡ１Ｂ１株、ＮＧＣ７ΔｖａｎＡ２Ｂ２株、ＮＧＣ７ΔｖａｎＡ３Ｂ３株及びＮＧＣ７ΔｖａｎＡ４Ｂ４株を培養した場合において、経時的にＯＤ６００を測定した結果を示した図である。FIG. 2A shows the OD over time when the NGC7 strain, NGC7Δ vanA1B1 strain, NGC7Δ vanA2B2 strain, NGC7Δ vanA3B3 strain, and NGC7Δ vanA4B4 strain were cultured using vanillic acid (VA) as a carbon source, as described in Examples below. 6 is a diagram showing the results of measuring 600 ; FIG. 図２Ｂは、後述する実施例に記載するとおり、シリンガ酸（ＳＡ）を炭素源としてＮＧＣ７株、ＮＧＣ７ΔｖａｎＡ１Ｂ１株、ＮＧＣ７ΔｖａｎＡ２Ｂ２株、ＮＧＣ７ΔｖａｎＡ３Ｂ３株及びＮＧＣ７ΔｖａｎＡ４Ｂ４株を培養した場合において、経時的にＯＤ６００を測定した結果を示した図である。FIG. 2B shows the OD over time when the NGC7 strain, NGC7Δ vanA1B1 strain, NGC7Δ vanA2B2 strain, NGC7Δ vanA3B3 strain, and NGC7Δ vanA4B4 strain were cultured using syringic acid (SA) as a carbon source, as described in Examples below. 6 is a diagram showing the results of measuring 600 ; FIG. 図３Ａは、後述する実施例に記載するとおり、ＮＧＣ７株、ＮＧＣ７ΔｖａｎＡ１Ｂ１株、ＮＧＣ７ΔｖａｎＡ２Ｂ２株、ＮＧＣ７ΔｖａｎＡ３Ｂ３株及びＮＧＣ７ΔｖａｎＡ４Ｂ４株によるＶＡの分解性を評価した結果を示した図である。FIG. 3A is a diagram showing the results of evaluating the degradability of VA by the NGC7 strain, the NGC7Δ vanA1B1 strain, the NGC7Δ vanA2B2 strain, the NGC7Δ vanA3B3 strain, and the NGC7Δ vanA4B4 strain, as described in Examples below. 図３Ｂは、後述する実施例に記載するとおり、ＮＧＣ７株、ＮＧＣ７ΔｖａｎＡ１Ｂ１株、ＮＧＣ７ΔｖａｎＡ２Ｂ２株、ＮＧＣ７ΔｖａｎＡ３Ｂ３株及びＮＧＣ７ΔｖａｎＡ４Ｂ４株によるＳＡの分解性を評価した結果を示した図である。FIG. 3B is a diagram showing the results of evaluating the degradability of SA by the NGC7 strain, the NGC7Δ vanA1B1 strain, the NGC7Δ vanA2B2 strain, the NGC7Δ vanA3B3 strain, and the NGC7Δ vanA4B4 strain, as described in Examples below. 図４Ａは、後述する実施例に記載するとおり、炭素源としてＳＡを用いてＮＧＣ７ΔｖａｎＡ４Ｂ４株を培養した場合のＳＡ濃度及びＯＤ６００を経時的に測定した結果を示した図である。FIG. 4A is a diagram showing the results of measuring the SA concentration and OD 600 over time when the NGC7Δ vanA4B4 strain was cultured using SA as a carbon source, as described in Examples below. 図４Ｂは、後述する実施例に記載するとおり、炭素源としてＶＡを用いてＮＧＣ７ΔｖａｎＡ４Ｂ４株を培養した場合のＶＡ濃度及びＯＤ６００を経時的に測定した結果を示した図である。FIG. 4B shows the results of measuring the VA concentration and OD 600 over time when the NGC7Δ vanA4B4 strain was cultured using VA as a carbon source, as described in Examples below. 図４Ｃは、後述する実施例に記載するとおり、炭素源としてｐ－ヒドロキシ安息香酸（ＨＢＡ）を用いてＮＧＣ７ΔｖａｎＡ４Ｂ４株を培養した場合のＨＢＡ濃度及びＯＤ６００を経時的に測定した結果を示した図である。FIG. 4C shows the results of measuring the HBA concentration and OD 600 over time when the NGC7Δ vanA4B4 strain was cultured using p -hydroxybenzoic acid (HBA) as a carbon source, as described in Examples below. It is a diagram. 図４Ｄは、後述する実施例に記載するとおり、炭素源としてＳＡ及びＶＡの混合物を用いてＮＧＣ７ΔｖａｎＡ４Ｂ４株を培養した場合のＳＡ濃度、ＶＡ濃度及びＯＤ６００を経時的に測定した結果を示した図である。FIG. 4D shows the results of measuring the SA concentration, VA concentration, and OD 600 over time when the NGC7Δ vanA4B4 strain was cultured using a mixture of SA and VA as the carbon source, as described in Examples below. It is a diagram. 図４Ｅは、後述する実施例に記載するとおり、炭素源としてＳＡ及びＨＢＡの混合物を用いてＮＧＣ７ΔｖａｎＡ４Ｂ４株を培養した場合のＳＡ濃度、ＨＢＡ濃度及びＯＤ６００を経時的に測定した結果を示した図である。FIG. 4E shows the results of measuring the SA concentration, HBA concentration, and OD 600 over time when the NGC7Δ vanA4B4 strain was cultured using a mixture of SA and HBA as the carbon source, as described in Examples below. It is a diagram. 図４Ｆは、後述する実施例に記載するとおり、炭素源としてＶＡ及びＨＢＡの混合物を用いてＮＧＣ７ΔｖａｎＡ４Ｂ４株を培養した場合のＶＡ濃度、ＨＢＡ濃度及びＯＤ６００を経時的に測定した結果を示した図である。FIG. 4F shows the results of measuring the VA concentration, HBA concentration, and OD 600 over time when the NGC7Δ vanA4B4 strain was cultured using a mixture of VA and HBA as the carbon source, as described in Examples below. It is a diagram. 図４Ｇは、後述する実施例に記載するとおり、炭素源としてＳＡ、ＶＡ及びＨＢＡの混合物を用いてＮＧＣ７ΔｖａｎＡ４Ｂ４株を培養した場合のＳＡ濃度、ＶＡ濃度、ＨＢＡ濃度及びＯＤ６００を経時的に測定した結果を示した図である。FIG. 4G shows the SA concentration, VA concentration, HBA concentration, and OD 600 measured over time when the NGC7Δ vanA4B4 strain was cultured using a mixture of SA, VA, and HBA as the carbon source, as described in Examples below. FIG. 10 is a diagram showing the results of the experiment. 図５Ａは、後述する実施例に記載するとおり、ＮＧＣ７［ｐＳＥＶＡ２４１＿Ｐ ｌａｃ －ａｃｖ，ｐＴＳ０９３＿ｖｃｅＡ－Ｂ］株によるアセトバニロン（ＡＶ）の分解性を評価し、ＡＶ濃度、バニロイル酢酸（ＶＡＡ）濃度及びＶＡ濃度を経時的に測定した結果を示した図である。FIG. 5A shows the degradability of acetovanilone (AV) by the NGC7 [pSEVA241_P lac -acv, pTS093_vceA-B] strain as described in the Examples below, and the AV concentration, vanilloyl acetic acid (VAA) concentration, and VA concentration were evaluated. It is the figure which showed the result of having measured with time. 図５Ｂは、後述する実施例に記載するとおり、ＮＧＣ７ΔｖａｎＡ４Ｂ４［ｐＳＥＶＡ２４１＿Ｐ ｌａｃ －ａｃｖ，ｐＴＳ０９３＿ｖｃｅＡ－Ｂ］株によるＡＶの分解性を評価し、ＡＶ濃度、ＶＡＡ濃度及びＶＡ濃度を経時的に測定した結果を示した図である。FIG. 5B shows the results of evaluating the degradability of AV by the NGC7Δ vanA4B4 [pSEVA241_P lac -acv, pTS093_vceA-B] strain and measuring the AV concentration, VAA concentration, and VA concentration over time, as described in Examples below. It is a figure showing. 図６は、後述する実施例に記載するとおり、ＮＧＣ７ΔｖａｎＡ４Ｂ４［ｐＳＥＶＡ２４１＿Ｐ ｌａｃ －ａｃｖ，ｐＴＳ０９３＿ｖｃｅＡ－Ｂ］株によるリグニン分解物モデルの分解性を評価し、ＡＶ濃度、ＶＮ濃度、ＶＡ濃度及びグルコース濃度並びにＯＤ６００を経時的に測定した結果を示した図である。FIG. 6 shows the degradability of the lignin degradation product model by the NGC7Δ vanA4B4 [pSEVA241_P lac -acv, pTS093_vceA-B] strain, as described in the Examples described later. FIG. 4 is a diagram showing the results of measuring OD 600 over time; 図７は、後述する実施例に記載するとおり、ＮＧＣ７ΔｖａｎＡ４Ｂ４［ｐＳＥＶＡ２４１＿Ｐ ｌａｃ －ａｃｖ，ｐＴＳ０９３＿ｖｃｅＡ－Ｂ］株によるリグニン分解物の分解性を評価し、ＡＶ濃度、ＶＮ濃度、ＶＡ濃度及びグルコース濃度並びにＯＤ６００を経時的に測定した結果を示した図である。FIG. 7 shows the evaluation of the degradability of lignin degradation products by the NGC7Δ vanA4B4 [pSEVA241_P lac -acv, pTS093_vceA-B] strain, as described in the Examples below, and the AV concentration, VN concentration, VA concentration, glucose concentration, and OD 600 is a diagram showing the results of measuring 600 over time. FIG. 図８は、後述する実施例に記載するとおり、ＮＧＣ７ΔｖａｎＡ１Ｂ１ΔｖａｎＡ４Ｂ４ΔａｐｈΔｖａｎＡ２Ｂ２ΔｖａｎＡ３Ｂ３［ｐＳＥＶＡ２４１＿Ｐ ｌａｃ －ａｃｖ，ｐＴＳ０９３＿ｖｃｅＡ－Ｂ］株によるリグニン分解物の分解性を評価し、ＡＶ濃度、ＶＮ濃度、ＶＡ濃度及びグルコース濃度並びにＯＤ６００を経時的に測定した結果を示した図である。Figure 8, as described in the Examples below, NGC7 Δ vanA1B1 Δ vanA4B4 Δ aph Δ vanA2B2 Δ vanA3B3 [pSEVA241_P lac -acv, pTS093_vceA-B] strain to evaluate the degradability of lignin degradation products, AV concentration, VN concentration , VA concentration, glucose concentration, and OD 600 over time.
　以下、本発明の各態様の詳細について説明するが、本発明は、本項目の事項によってのみに限定されず、本発明の目的を達成する限りにおいて種々の態様をとり得る。 The details of each aspect of the present invention will be described below, but the present invention is not limited only by the matters of this item, and can take various aspects as long as the object of the present invention is achieved.
　本明細書における各用語は、別段の定めがない限り、微生物学分野などの技術分野の当業者により通常用いられている意味で使用され、不当に限定的な意味を有するものとして解釈されるべきではない。また、本明細書においてなされている推測及び理論は、本発明者らのこれまでの知見及び経験によってなされたものであることから、本発明はこのような推測及び理論のみによって拘泥されるものではない。 Unless otherwise specified, each term used herein has the meaning commonly used by those skilled in the art of microbiology and other technical fields, and should be interpreted as having an unduly restrictive meaning. is not. In addition, the speculations and theories made in the present specification are based on the knowledge and experience of the present inventors, so the present invention is not bound solely by such speculations and theories. do not have.
　「含む」は、含まれるものとして明示されている要素以外の要素を付加できることを意味する（「少なくとも含む」と同義である）が、「からなる」及び「から本質的になる」を包含する。すなわち、「含む」は、明示されている要素及び任意の１種若しくは２種以上の要素を含み、明示されている要素からなり、又は明示されている要素から本質的になることを意味し得る。要素としては、成分、工程、条件、パラメーターなどの制限事項などが挙げられる。
　「及び／又は」との用語は、列記した複数の関連項目のいずれか１つ、又は２つ以上の任意の組み合わせ若しくは全ての組み合わせを意味する。 "Contains" means that it can add elements other than those explicitly included (which is synonymous with "including at least"), but includes "consisting of" and "consisting essentially of" . That is, "comprising" can mean including the specified element and any one or more elements, consisting of, or consisting essentially of the specified element. . Factors include limitations such as components, steps, conditions, parameters, and the like.
The term "and/or" means any one, or any or all combinations of two or more of the associated listed items.
　「遺伝子の欠失」は、遺伝子が正常に転写されないこと、遺伝子の発現によって産生されるべきタンパク質が正常に翻訳されないことなどのように、遺伝子が正常に機能せずに遺伝子の発現が妨げられていることを意味する。遺伝子の欠失は、例えば、遺伝子の全部又は一部が破壊、欠損、置換、挿入などにより遺伝子の構造が変化することによって生じ得る。ただし、遺伝子の欠失は、遺伝子の構造に変化が生じずに、例えば、遺伝子の制御領域をブロックするなどの手段によって遺伝子の発現が抑えられることによっても生じ得る。
　「遺伝子の発現」は、転写や翻訳などを介して、遺伝子によってコードされるタンパク質が本来の構造や活性を有するように生産されることを意味する。 "Gene deletion" means that the gene does not function normally and the expression of the gene is prevented, such as the gene not being transcribed normally or the protein that should be produced by the expression of the gene not being translated normally. means that Gene deletion can occur, for example, by disruption, deletion, substitution, insertion, or the like of all or part of a gene, resulting in a change in the structure of the gene. However, gene deletion can also occur by suppressing gene expression by, for example, blocking the regulatory region of the gene without altering the structure of the gene.
"Expression of a gene" means that a protein encoded by a gene is produced through transcription, translation, etc. so as to have the original structure and activity.
（形質転換微生物の概要）
　本発明の一態様の形質転換微生物は、宿主微生物の染色体上にある特定の遺伝子が欠失するように、宿主微生物を形質転換した微生物である。また、本発明の一態様である形質転換微生物は、さらに外来遺伝子として挿入したアセトバニロン分解酵素遺伝子群を発現するように、宿主微生物を形質転換していることが好ましい。 (Overview of transformed microorganisms)
A transformed microorganism of one aspect of the present invention is a microorganism transformed from a host microorganism such that a specific gene on the chromosome of the host microorganism is deleted. In addition, the transformed microorganism that is one aspect of the present invention preferably transforms the host microorganism so as to express the acetovanilone-degrading enzyme gene group inserted as a foreign gene.
（欠失又は挿入する遺伝子）
　宿主微生物であるシュードモナス・スピーシーズ（Ｐｓｅｕｄｏｍｏｎａｓ　ｓｐ．）　ＮＧＣ７株（受託番号：ＮＩＴＥ　ＢＰ－０３０４３）は、染色体上にｖａｎＡ４遺伝子（配列番号１）及びｖａｎＢ４遺伝子（配列番号２）を有する。このうち、バニリン酸に直接作用するのはオキシゲナーゼコンポーネントあるｖａｎＡ４遺伝子である。そこで、本発明の一態様の形質転換微生物は、染色体上にあるこれらの遺伝子のうち、ｖａｎＡ４遺伝子が欠失している。本発明の一態様の形質転換微生物は、ＮＧＣ７株が染色体上に本来有するｖａｎＡ４遺伝子を欠失するものであれば特に限定されないが、バニリン酸への作用を高度に低減するために、ｖａｎＡ４遺伝子及びｖａｎＢ４遺伝子の両方が欠失していることが好ましい。 (Gene to be deleted or inserted)
The host microorganism, Pseudomonas sp. NGC7 strain (accession number: NITE BP-03043), has vanA4 gene (SEQ ID NO: 1) and vanB4 gene (SEQ ID NO: 2) on the chromosome. Among them, vanA4 gene, which is an oxygenase component, directly acts on vanillic acid. Therefore, the transformed microorganism of one aspect of the present invention lacks the vanA4 gene among these genes on the chromosome. The transformed microorganism of one aspect of the present invention is not particularly limited as long as it lacks the vanA4 gene that the NGC7 strain originally has on the chromosome. Preferably both vanB4 genes are deleted.
　本発明者らが調べたところによれば、ＮＧＣ７株は、ｖａｎＡ遺伝子と推定される遺伝子を４種類有し、及びｖａｎＢ遺伝子として推定される遺伝子を６種類有する。ＮＧＣ７株のように、複数種類のｖａｎＡ遺伝子及びｖａｎＢ遺伝子と推定される遺伝子を有する微生物は非常に稀である。 According to investigations conducted by the present inventors, the NGC7 strain has 4 types of presumed vanA genes and 6 types of presumed vanB genes. Microorganisms that have multiple types of presumed vanA and vanB genes , such as the NGC7 strain, are extremely rare.
　ｖａｎＡ遺伝子は、バニレート　Ｏ－デメチラーゼ　オキシゲナーゼコンポーネント（ｖａｎｉｌｌａｔｅ　Ｏ－ｄｅｍｅｔｈｙｌａｓｅ　ｏｘｙｇｅｎａｓｅ　ｃｏｍｐｏｎｅｎｔ）を発現する遺伝子である。バニレート　Ｏ－デメチラーゼ　オキシゲナーゼコンポーネント（ＥＣ　１．１４．１３．８２）は、Ｏｘｉｄｏｒｅｄｕｃｔａｓｅ　ｃｏｍｐｏｎｅｎｔを介して供給されるＮＡＤＨ又はＮＡＤＰＨ由来の電子と、分子状酸素から供給される酸素原子とを利用して、バニリン酸のメチルエーテル結合を開裂し、プロトカテク酸、ホルムアルデヒド及び水を生成する。 The vanA gene is the gene that expresses the vanillate O - demethylase oxygenase component. The vanillate O -demethylase oxygenase component (EC 1.14.13.82) utilizes electrons from NADH or NADPH supplied via the oxygenductase component and oxygen atoms supplied from molecular oxygen to convert vanillin Cleavage the methyl ether bond of the acid to produce protocatechuic acid, formaldehyde and water.
　バニレート　Ｏ－デメチラーゼ　オキシゲナーゼコンポーネントは、そのアミノ酸配列中にＲｉｅｓｋｅ［２Ｆｅ－２Ｓ］ｉｒｏｎ－ｓｕｌｆｕｒ　ｄｏｍａｉｎ（Ｗ７－Ｖ１０７，ＰＲＯＳＩＴＥ　ｅｎｔｒｙ　ｎｏ．ＰＳ５１２９６）を有し、該ドメイン中のＣ及びＨ（Ｃ４７，Ｈ４９，Ｃ６６，Ｈ６９）がＦｅ－Ｓの結合に関わる。 The vanillate O -demethylase oxygenase component has a Rieske [2Fe-2S] iron-sulfur domain (W7-V107, PROSITE entry no. PS51296) in its amino acid sequence, and C and H (C47, H49, C66, H69) are involved in Fe—S bonding.
　ｖａｎＢ遺伝子は、バニレート　Ｏ－デメチラーゼ　オキシドレダクターゼコンポーネント（ｖａｎｉｌｌａｔｅ　Ｏ－ｄｅｍｅｔｈｙｌａｓｅ　ｏｘｉｄｏｒｅｄｕｃｔａｓｅ　ｃｏｍｐｏｎｅｎｔ）を発現する遺伝子である。バニレート　Ｏ－デメチラーゼ　オキシドレダクターゼコンポーネント（ＥＣ　１．１４．１３．８２）は、ＮＡＤＨ又はＮＡＤＰＨから電子を抜き取り、酸素添加酵素（オキシゲナーゼ）へと伝達する酸化還元酵素の一つとして知られている。バニレート　Ｏ－デメチラーゼ　オキシドレダクターゼコンポーネントは、バニレート　Ｏ－デメチラーゼ　オキシゲナーゼコンポーネントであるＶａｎＡにＮＡＤＨ又はＮＡＤＰＨ由来の電子を伝達する。 The vanB gene is the gene that expresses the vanillate O - demethylase oxidoreductase component. Vanillate O -demethylase oxidoreductase component (EC 1.14.13.82) is known as one of the oxidoreductases that extract electrons from NADH or NADPH and transfer them to oxygenases. The vanillate O 2 -demethylase oxidoreductase component transfers electrons from NADH or NADPH to the vanillate O 2 -demethylase oxygenase component, VanA.
　バニレート　Ｏ－デメチラーゼ　オキシドレダクターゼコンポーネントは、そのアミノ酸配列中に、２Ｆｅ－２Ｓ　Ｆｅｒｒｅｄｏｘｉｎ　ｔｙｐｅ　ｉｒｏｎ－ｓｕｌｆｅｒ　ｂｉｎｄｉｎｇ　ｄｏｍａｉｎ（Ｇ２２９－Ｉ３１６、ＰＲＯＳＩＴＥ　ｅｎｔｒｙ　ｎｏ．ＰＳ５１０８５）を有し、該アミノ酸配列中のＣ（Ｃ２６５、Ｃ２７０、Ｃ２７３、Ｃ３０３）がＦｅ－Ｓの結合に関わる。また、バニレート　Ｏ－デメチラーゼ　オキシドレダクターゼコンポーネントは、そのアミノ酸配列中に、ＮＡＤ－ｂｉｎｄｉｎｇ　ｄｏｍａｉｎ（Ｌ１０９－Ｄ２０１，Ｐｆａｍ　ｅｎｔｒｙ　ｎｏ．ＰＦ００１７５）及びＦｅｒｒｅｄｏｘｉｎ　ｒｅｄｕｃｔａｓｅ　ｔｙｐｅ　ＦＡＤ－ｂｉｎｄｉｎｇ　ｄｏｍａｉｎ（Ｍ１－Ａ１０１、ＰＲＯＳＩＴＥ　ｅｎｔｏｒｙ ｎｏ．ＰＳ５１３８４）を有する。 The vanillate O -demethylase oxidoreductase component has a 2Fe-2S Ferredoxin type iron-sulfer binding domain (G229-I316, PROSITE entry no. PS51085) in its amino acid sequence, and C (C265, C270 , C273, C303) are involved in the bonding of Fe—S. In addition, the vanillate O -demethylase oxidoreductase component has in its amino acid sequence the NAD-binding domain (L109-D201, Pfam entry no. PF00175) and the Ferredoxin reductase type FAD-binding domain (M1-A101, PROSITE entry no. PS514). ).
　ｖａｎＡ遺伝子の発現産物（ＶａｎＡ）及びｖａｎＢ遺伝子の発現産物（ＶａｎＢ）が共同して機能することでバニレート　Ｏ－デメチラーゼ（ＶａｎＡＢ）を構成する。ＶａｎＡＢは、バニリン酸の脱メチル化を担うが、シリンガ酸を３－Ｏ－メチルガリック酸へ、３－Ｏ－メチルガリック酸をガリック酸へそれぞれ変換することができるので、シリンガ酸　Ｏ－デメチラーゼ、３－Ｏ－メチルガリック酸　デメチラーゼとしても機能することができる。なお、Ｓｐｈｉｎｇｏｂｉｕｍ　ｓｐ．ＳＹＫ－６株はシリンガ酸　Ｏ－デメチラーゼをコードする遺伝子としてｄｅｓＡ遺伝子を有し、バニリン酸／３－Ｏ－メチルガリック酸　Ｏ－デメチラーゼをコードする遺伝子としてｌｉｇＭ遺伝子を有する。 The expression product of the vanA gene (VanA) and the expression product of the vanB gene (VanB) function together to constitute vanylate O -demethylase (VanAB). VanAB, which is responsible for the demethylation of vanillate, can convert syringate to 3- O-methylgallic acid and 3-O-methylgallic acid to gallic acid, respectively, thus syringate O - demethylase , It can also function as a 3- O -methylgallate demethylase. In addition, Sphingobium sp. The SYK-6 strain has the desA gene as a gene encoding syringate O -demethylase and the ligM gene as a gene encoding vanillate/3-O-methylgallate O -demethylase.
　ＳＹＫ－６株が有するｄｅｓＡ遺伝子及びｌｉｇＭ遺伝子がコードする酵素は、テトラヒドロ葉酸へのメチル転移反応を触媒し、その反応の進行にはテトラヒドロ葉酸を要求する。テトラヒドロ葉酸の再生にはメチオニン、チミジン、プリン生合成などに要求されるＣ１化合物を供給するＣ１代謝が必要であることから、これらの酵素はＣ１代謝と共役して機能させなければならない。これに対し、ＮＧＣ７株が発現するバニレート　Ｏ－デメチラーゼは、酸素添加型であり、オキシゲナーゼコンポーネントとオキシドレダクターゼコンポーネントとからなることにより、Ｃ１代謝との共役などを要せず、複雑さが低減されている。 Enzymes encoded by the desA gene and the ligM gene of the SYK-6 strain catalyze a methyl transfer reaction to tetrahydrofolic acid, and require tetrahydrofolic acid for the progress of the reaction. Since regeneration of tetrahydrofolate requires C1 metabolism to supply C1 compounds required for methionine, thymidine, purine biosynthesis, etc., these enzymes must function in conjunction with C1 metabolism. In contrast, the vanillate O -demethylase expressed by the NGC7 strain is oxygenated and consists of an oxygenase component and an oxidoreductase component. there is
　以上のとおり、ｖａｎＡ遺伝子及びｖａｎＢ遺伝子は１組の遺伝子セットとして機能すると考えられる。そこで、本発明者らは、ＮＧＣ７株が有するｖａｎＡ遺伝子及びｖａｎＢ遺伝子についてＮＧＣ７株のゲノムＤＮＡを用いて探索したところ、予想外に、ｖａｎＡ１遺伝子（配列番号１１）、ｖａｎＡ２遺伝子（配列番号１２）、ｖａｎＡ３遺伝子（配列番号１３）及びｖａｎＡ４遺伝子（配列番号１）という４種類のｖａｎＡ遺伝子を有すること、並びにｖａｎＢ１遺伝子（配列番号１４）、ｖａｎＢ２遺伝子（配列番号１５）、ｖａｎＢ３遺伝子（配列番号１６）、ｖａｎＢ４遺伝子（配列番号２）、ｖａｎＢ５遺伝子（配列番号１７）及びｖａｎＢ６遺伝子（配列番号１８）という６種類のｖａｎＢ遺伝子を有することを見出した。本発明者らは、さらに検討を進めて、これらの遺伝子情報を基に、それぞれ対になる遺伝子を選択して、合計４組のｖａｎＡ遺伝子及びｖａｎＢ遺伝子の遺伝子セットを想定した。そして、これらの４対の遺伝子セットを、ｖａｎＡ１遺伝子及びｖａｎＢ１遺伝子の遺伝子セット、ｖａｎＡ２遺伝子及びｖａｎＢ２遺伝子の遺伝子セット、ｖａｎＡ３遺伝子及びｖａｎＢ３遺伝子の遺伝子セット、並びにｖａｎＡ４遺伝子及びｖａｎＢ４遺伝子セットとした。 As described above, the vanA gene and vanB gene are considered to function as a set of genes. Therefore, when the present inventors searched for the vanA gene and vanB gene possessed by the NGC7 strain using the genomic DNA of the NGC7 strain, they unexpectedly found vanA1 gene (SEQ ID NO: 11), vanA2 gene (SEQ ID NO: 12), Having four types of vanA genes, vanA3 gene (SEQ ID NO: 13) and vanA4 gene (SEQ ID NO: 1), and vanB1 gene (SEQ ID NO: 14), vanB2 gene (SEQ ID NO: 15), vanB3 gene (SEQ ID NO: 16), It was found to have six types of vanB genes, vanB4 gene (SEQ ID NO: 2), vanB5 gene (SEQ ID NO: 17) and vanB6 gene (SEQ ID NO: 18). The present inventors proceeded with further studies, and based on these gene information, selected genes to be paired with each other, and assumed a total of four gene sets of vanA genes and vanB genes. These four pairs of gene sets were defined as a gene set of vanA1 gene and vanB1 gene, a gene set of vanA2 gene and vanB2 gene, a gene set of vanA3 gene and vanB3 gene, and a gene set of vanA4 gene and vanB4 gene.
　本発明者らは、上記４対の遺伝子セットの欠損株を作製して、バニリン酸の分解性について試験したところ、ｖａｎＡ４遺伝子及びｖａｎＢ４遺伝子のセットがバニリン酸の分解に深く関与していることを見出し、本発明を完成するに至った。 The present inventors produced strains lacking the above-mentioned four pairs of gene sets and tested the vanillic acid degradability. The discovery led to the completion of the present invention.
　上記事情を鑑みれば、本発明の一態様の形質転換微生物は、複数種類あると想定されるバニレート　Ｏ－デメチラーゼのうち、バニリン酸の分解に深く関与しているバニレート　Ｏ－デメチラーゼの発現を阻止するために、ｖａｎＡ４遺伝子及びｖａｎＢ４遺伝子の両方が欠失したＮＧＣ７形質転換株（以下、ＮＧＣ７ΔｖａｎＡ４Ｂ４株ともよぶ。）であることが好ましい。 In view of the above circumstances, the transformed microorganism of one embodiment of the present invention inhibits the expression of vanillate O -demethylase, which is deeply involved in the decomposition of vanillic acid, among a plurality of vanillate O -demethylases that are assumed to exist. Therefore, it is preferably an NGC7 transformant strain lacking both the vanA4 gene and the vanB4 gene (hereinafter also referred to as NGC7ΔvanA4B4 strain).
　本発明の一態様の形質転換微生物は、ｖａｎＡ４遺伝子及びｖａｎＢ４遺伝子に加えて、その他のｖａｎＡ遺伝子及び／又はｖａｎＢ遺伝子が欠失していてもよい。ただし、ｖａｎＡ１遺伝子及びｖａｎＢ１遺伝子を欠失したＮＧＣ７形質転換株、並びにｖａｎＡ２遺伝子及びｖａｎＢ２遺伝子を欠失したＮＧＣ７形質転換株は、ＮＧＣ７株及びＮＧＣ７ΔｖａｎＡ４Ｂ４株に比べて、シリンガ酸の資化性が劣るという結果が得られている。そこで、シリンガ酸などのＳ型リグニン由来の芳香族化合物を炭素源として用いる場合は、本発明の一態様の形質転換微生物は、ｖａｎＡ１遺伝子、ｖａｎＢ１遺伝子、ｖａｎＡ２遺伝子及びｖａｎＢ２遺伝子からなる群から選択される１種、２種、３種又は４種の遺伝子が欠失していないことが好ましい。一方、Ｈ型リグニン由来及び／又はＧ型リグニン由来の芳香族化合物やグルコースなどの炭素源を用いる場合は、バニリン酸の収率高めること、バニリン酸の分解をより低減することなどが期待できることから、本発明の一態様の形質転換微生物は、ｖａｎＡ４遺伝子及びｖａｎＢ４遺伝子に加えて、他のｖａｎＡ遺伝子及び／又は他のｖａｎＢ遺伝子が欠失していることが好ましい。 The transformed microorganism of one aspect of the present invention may lack other vanA and/or vanB genes in addition to the vanA4 and vanB4 genes. However, the NGC7 transformant lacking the vanA1 and vanB1 genes and the NGC7 transformant lacking the vanA2 and vanB2 genes are inferior in syringic acid assimilation compared to the NGC7 and NGC7Δ vanA4B4 strains. The result is obtained. Therefore, when an aromatic compound derived from S-type lignin such as syringic acid is used as a carbon source, the transformed microorganism of one aspect of the present invention is selected from the group consisting of vanA1 gene, vanB1 gene, vanA2 gene and vanB2 gene. Preferably, 1, 2, 3 or 4 of the genes are not deleted. On the other hand, when using a carbon source such as an aromatic compound derived from H-type lignin and / or G-type lignin and glucose, it is possible to increase the yield of vanillic acid and further reduce the decomposition of vanillic acid. In addition to the vanA4 gene and the vanB4 gene, the transformed microorganism of one aspect of the present invention preferably lacks another vanA gene and/or another vanB gene.
　具体的には、Ｈ型リグニン由来芳香族化合物だけでなく、Ｓ型リグニン由来芳香族化合物を分解し、炭素源としても利用しつつ、バニリン酸を選択的に生産及び／又は選択的に蓄積することを目的とする場合は、Ｓ型リグニン由来の芳香族化合物の資化性を高めるために、本発明の一態様の形質転換微生物は、ｖａｎＡ４遺伝子及びｖａｎＢ４遺伝子を欠失しており、かつｖａｎＡ１遺伝子及びｖａｎＢ１遺伝子を保持することが好ましく；ｖａｎＡ４遺伝子及びｖａｎＢ４遺伝子を欠失しており、かつｖａｎＡ２遺伝子及びｖａｎＢ２遺伝子を保持することが好ましく；又は、ｖａｎＡ４遺伝子及びｖａｎＢ４遺伝子を欠失しており、かつｖａｎＡ１遺伝子及びｖａｎＢ１遺伝子並びにｖａｎＡ２遺伝子及びｖａｎＢ２遺伝子を保持することが好ましい。 Specifically, not only H-type lignin-derived aromatic compounds but also S-type lignin-derived aromatic compounds are decomposed, and vanillic acid is selectively produced and / or selectively accumulated while also being used as a carbon source. For this purpose, in order to enhance the assimilation of aromatic compounds derived from S-type lignin, the transformed microorganism of one aspect of the present invention lacks the vanA4 gene and the vanB4 gene, and vanA1 preferably retains the vanA4 and vanB4 genes; and preferably retains the vanA2 and vanB2 genes; or lacks the vanA4 and vanB4 genes ; Moreover, it is preferable to retain the vanA1 gene, the vanB1 gene, and the vanA2 gene and the vanB2 gene.
　また、Ｇ型リグニン由来芳香族化合物及び／又はＨ型リグニン由来芳香族化合物を分解し、炭素源としても利用しつつ、バニリン酸を選択的に生産及び／又は選択的に蓄積することを目的とする場合や、ｐ－ヒドロキシフェニルリグニン由来の芳香族化合物やグルコースなどの炭素源を用いる場合は、バニリン酸の回収率を高めるに、本発明の一態様の形質転換微生物は、ｖａｎＡ４遺伝子及びｖａｎＢ４遺伝子を欠失しており、かつｖａｎＡ１遺伝子及びｖａｎＢ１遺伝子を欠失することが好ましく；ｖａｎＡ４遺伝子及びｖａｎＢ４遺伝子を欠失しており、かつｖａｎＡ２遺伝子及びｖａｎＢ２遺伝子を欠失していることが好ましく；ｖａｎＡ４遺伝子及びｖａｎＢ４遺伝子を欠失しており、かつｖａｎＡ３遺伝子及びｖａｎＢ３遺伝子を欠失していることが好ましく；又は、ｖａｎＡ４遺伝子及びｖａｎＢ４遺伝子を欠失しており、かつｖａｎＡ１遺伝子及びｖａｎＢ１遺伝子、ｖａｎＡ２遺伝子及びｖａｎＢ２遺伝子並びにｖａｎＡ３遺伝子及びｖａｎＢ３遺伝子を欠失していることが好ましい。なお、ｖａｎＡ４遺伝子及びｖａｎＢ４遺伝子を欠失しており、かつｖａｎＡ１遺伝子及びｖａｎＢ１遺伝子、ｖａｎＡ２遺伝子及びｖａｎＢ２遺伝子並びにｖａｎＡ３遺伝子及びｖａｎＢ３遺伝子を欠失している本発明の一態様の形質転換微生物が備える高収率でバニリン酸を生産できるという特徴は、ｖａｎＡ１遺伝子及びｖａｎＢ１遺伝子を欠失している形質転換微生物、ｖａｎＡ２遺伝子及びｖａｎＢ２遺伝子を欠失している形質転換微生物、並びにｖａｎＡ３遺伝子及びｖａｎＢ３遺伝子を欠失している形質転換微生物からは予想することができない、驚くべき特徴である。 In addition, the purpose is to selectively produce and / or selectively accumulate vanillic acid while decomposing G-type lignin-derived aromatic compounds and / or H-type lignin-derived aromatic compounds and using them as carbon sources. or when using an aromatic compound derived from p -hydroxyphenyl lignin or a carbon source such as glucose, in order to increase the recovery rate of vanillic acid, the transformed microorganism of one embodiment of the present invention comprises vanA4 gene and vanB4 gene. and preferably deletes the vanA1 gene and the vanB1 gene; preferably deletes the vanA4 gene and the vanB4 gene, and preferably deletes the vanA2 gene and the vanB2 gene; preferably lacks the vanA3 gene and the vanB3 gene; or lacks the vanA4 gene and the vanB4 gene ; and the vanB2 gene and the vanA3 and vanB3 genes are preferably deleted. The transformed microorganism of one aspect of the present invention lacking the vanA4 gene and the vanB4 gene, and lacking the vanA1 gene, the vanB1 gene, the vanA2 gene, the vanB2 gene, and the vanA3 gene and the vanB3 gene. The feature of being able to produce vanillic acid at a high yield is the transformed microorganism lacking the vanA1 gene and the vanB1 gene, the transformed microorganism lacking the vanA2 gene and the vanB2 gene, and the vanillin gene lacking the vanA3 gene and the vanB3 gene. It is a surprising feature that cannot be expected from a transformed microorganism that is missing.
　ＳＹＫ－６株は、リグニン由来の芳香族化合物の一つであるアセトバニロンを分解することができる。ＳＹＫ－６株のアセトバニロンの分解性には、ａｃｖＡ遺伝子（配列番号３）、ａｃｖＢ遺伝子（配列番号４）、ａｃｖＣ遺伝子（配列番号５）、ａｃｖＤ遺伝子（配列番号６）、ａｃｖＥ遺伝子（配列番号７）、ａｃｖＦ遺伝子（配列番号８）、ｖｃｅＡ遺伝子（配列番号９）及びｖｃｅＢ遺伝子（配列番号１０）といったアセトバニロン分解酵素が関与している。 The SYK-6 strain is capable of degrading acetovanilone, one of the aromatic compounds derived from lignin. The acetovanilone degradability of the SYK-6 strain includes acvA gene (SEQ ID NO: 3), acvB gene (SEQ ID NO: 4), acvC gene (SEQ ID NO: 5), acvD gene (SEQ ID NO: 6), acvE gene (SEQ ID NO: 7 ), acvF gene (SEQ ID NO: 8), vceA gene (SEQ ID NO: 9) and vceB gene (SEQ ID NO: 10) are involved.
　そこで、本発明の一態様の形質転換微生物は、アセトバニロンの分解性の観点から、外来遺伝子として、ａｃｖＡ遺伝子（配列番号３）、ａｃｖＢ遺伝子（配列番号４）、ａｃｖＣ遺伝子（配列番号５）、ａｃｖＤ遺伝子（配列番号６）、ａｃｖＥ遺伝子（配列番号７）、ａｃｖＦ遺伝子（配列番号８）、ｖｃｅＡ遺伝子（配列番号９）及びｖｃｅＢ遺伝子（配列番号１０）が挿入され、かつ発現することが好ましい。本発明の一態様の形質転換微生物は、外来遺伝子であるアセトバニロン分解酵素遺伝子群が挿入され、かつ発現することにより、アセトバニロンを分解してバニリン酸を生産及び蓄積することができる。 Therefore, from the viewpoint of acetovanilone degradability, the transformed microorganism of one embodiment of the present invention has the following foreign genes: acvA gene (SEQ ID NO: 3), acvB gene (SEQ ID NO: 4), acvC gene (SEQ ID NO: 5), acvD The gene (SEQ ID NO: 6), acvE gene (SEQ ID NO: 7), acvF gene (SEQ ID NO: 8), vceA gene (SEQ ID NO: 9) and vceB gene (SEQ ID NO: 10) are preferably inserted and expressed. The transformed microorganism of one aspect of the present invention is capable of degrading acetovanillone and producing and accumulating vanillic acid by inserting and expressing the acetovanillone-degrading enzyme gene group as a foreign gene.
　なお、挿入する遺伝子は、ＳＹＫ－６株が本来保有する遺伝子（すなわち、野生型遺伝子）と完全に同一でなくともよく、少なくとも野生型遺伝子が発現するタンパク質（すなわち、野生型タンパク質）と同一の、又は近似する酵素学的性質を有するタンパク質を発現する遺伝子である限り、野生型遺伝子のヌクレオチド配列に相補的なヌクレオチド配列とストリンジェントな条件下でハイブリダイズするヌクレオチド配列を有するヌクレオチド配列などであってもよい。 The inserted gene may not be completely identical to the gene originally possessed by the SYK-6 strain (that is, the wild-type gene), but at least is identical to the protein that the wild-type gene expresses (that is, the wild-type protein). or a nucleotide sequence that hybridizes under stringent conditions with a nucleotide sequence complementary to that of the wild-type gene, as long as the gene expresses a protein with similar enzymatic properties. may
　「ストリンジェントな条件下でハイブリダイズするヌクレオチド配列」は、野生型遺伝子のヌクレオチド配列を有するＤＮＡをプローブとして使用し、コロニーハイブリダイゼーション法、プラークハイブリダイゼーション法、サザンブロットハイブリダイゼーション法などを用いることにより得られるＤＮＡのヌクレオチド配列を意味する。 A "nucleotide sequence that hybridizes under stringent conditions" is obtained by colony hybridization, plaque hybridization, Southern blot hybridization, etc. using DNA having the nucleotide sequence of a wild-type gene as a probe. It refers to the nucleotide sequence of the resulting DNA.
　「ストリンジェントな条件」は、特異的なハイブリッドのシグナルが非特異的なハイブリッドのシグナルと明確に識別される条件であり、使用するハイブリダイゼーションの系と、プローブの種類、配列及び長さによって異なる。そのような条件は、ハイブリダイゼーションの温度を変えること、洗浄の温度及び塩濃度を変えることにより決定可能である。例えば、非特異的なハイブリッドのシグナルまで強く検出されてしまう場合には、ハイブリダイゼーション及び洗浄の温度を上げるとともに、必要により洗浄の塩濃度を下げることにより特異性を上げることができる。また、特異的なハイブリッドのシグナルも検出されない場合には、ハイブリダイゼーション及び洗浄の温度を下げるとともに、必要により洗浄の塩濃度を上げることにより、ハイブリッドを安定化させることができる。 "Stringent conditions" are conditions under which a specific hybrid signal is clearly distinguished from a non-specific hybrid signal, and vary depending on the hybridization system used and the type, sequence and length of the probe. . Such conditions can be determined by varying the temperature of hybridization, varying the temperature and salt concentration of washing. For example, when even non-specific hybrid signals are strongly detected, the specificity can be increased by raising the temperature of hybridization and washing and, if necessary, lowering the salt concentration of washing. If no specific hybrid signal is detected, hybrids can be stabilized by lowering the hybridization and washing temperatures and, if necessary, increasing the washing salt concentration.
　ストリンジェントな条件の具体例としては、例えば、プローブとしてＤＮＡプローブを用い、ハイブリダイゼーションは、５×ＳＳＣ、１．０％（ｗ／ｖ）　核酸ハイブリダイゼーション用ブロッキング試薬（ロシュ・ダイアグノスティクス社）、０．１％（ｗ／ｖ）　Ｎ－ラウロイルサルコシン、０．０２％（ｗ／ｖ）　ＳＤＳを用い、一晩（８時間～１６時間程度）で行う。洗浄は、０．１～０．５×ＳＳＣ、０．１％（ｗ／ｖ）　ＳＤＳ、好ましくは０．１×ＳＳＣ、０．１％（ｗ／ｖ）　ＳＤＳを用い、１５分間、２回行う。ハイブリダイゼーション及び洗浄を行う温度は６５℃以上、好ましくは６８℃以上である。 As a specific example of stringent conditions, for example, a DNA probe is used as a probe, and hybridization is performed using 5×SSC, 1.0% (w/v) blocking reagent for nucleic acid hybridization (Roche Diagnostics). , 0.1% (w/v) N-lauroyl sarcosine, 0.02% (w/v) SDS, overnight (8 to 16 hours). Wash twice for 15 minutes using 0.1-0.5×SSC, 0.1% (w/v) SDS, preferably 0.1×SSC, 0.1% (w/v) SDS conduct. The temperature for hybridization and washing is 65°C or higher, preferably 68°C or higher.
　また、ストリンジェントな条件下でハイブリダイズするヌクレオチド配列を有するＤＮＡとしては、例えば、コロニー若しくはプラーク由来の野生型遺伝子のヌクレオチド配列を有するＤＮＡ又は該ＤＮＡの断片を固定化したフィルターを用いて、上記したストリンジェントな条件下でハイブリダイゼーションすることによって得られるＤＮＡや０．５Ｍ～２．０ＭのＮａＣｌ存在下にて、４０℃～７５℃でハイブリダイゼーションを実施した後、好ましくは０．７Ｍ～１．０ＭのＮａＣｌ存在下にて、６５℃でハイブリダイゼーションを実施した後、０．１～１×ＳＳＣ溶液（１×ＳＳＣ溶液は、１５０ｍＭ　塩化ナトリウム、１５ｍＭ　クエン酸ナトリウム）を用い、６５℃条件下でフィルターを洗浄することにより同定できるＤＮＡなどを挙げることができる。プローブの調製やハイブリダイゼーションの方法は、Ｍｏｌｅｃｕｌａｒ　Ｃｌｏｎｉｎｇ：Ａ　ｌａｂｏｒａｔｏｒｙ　Ｍａｎｕａｌ，２ｎｄ－Ｅｄ．，Ｃｏｌｄ　Ｓｐｒｉｎｇ　Ｈａｒｂｏｒ　Ｌａｂｏｒａｔｏｒｙ，Ｃｏｌｄ　Ｓｐｒｉｎｇ　Ｈａｒｂｏｒ，ＮＹ．，１９８９、Ｃｕｒｒｅｎｔ　Ｐｒｏｔｏｃｏｌｓ　ｉｎ　Ｍｏｌｅｃｕｌａｒ　Ｂｉｏｌｏｇｙ，Ｓｕｐｐｌｅｍｅｎｔ　１－３８，Ｊｏｈｎ　Ｗｉｌｅｙ＆Ｓｏｎｓ，１９８７－１９９７（以下、これらの文献を参考技術文献とよぶ場合がある。）などに記載されている方法に準じて実施することができる。なお、当業者であれば、このようなバッファーの塩濃度や温度などの条件に加えて、その他のプローブ濃度、プローブ長さ、反応時間などの諸条件を加味して、野生型遺伝子のヌクレオチド配列に相補的なヌクレオチド配列とストリンジェントな条件下でハイブリダイズするヌクレオチド配列を有するＤＮＡを得るための条件を適宜設定することができる。 As the DNA having a nucleotide sequence that hybridizes under stringent conditions, for example, DNA having the nucleotide sequence of a wild-type gene derived from colonies or plaques, or a filter immobilized with a fragment of the DNA, DNA obtained by hybridization under stringent conditions, or in the presence of 0.5 M to 2.0 M NaCl, after hybridization at 40 ° C. to 75 ° C., preferably 0.7 M to 1 After performing hybridization at 65°C in the presence of 0 M NaCl, 0.1 to 1 x SSC solution (1 x SSC solution is 150 mM sodium chloride and 15 mM sodium citrate) was used to perform hybridization at 65°C. Examples include DNA that can be identified by washing the filter with . Probe preparation and hybridization methods are described in Molecular Cloning: A Laboratory Manual, 2nd-Ed. , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. , 1989, Current Protocols in Molecular Biology, Supplement 1-38, John Wiley & Sons, 1987-1997 (hereinafter, these documents may be referred to as reference technical documents), etc. can be done. It should be noted that those skilled in the art would be able to determine the nucleotide sequence of the wild-type gene by taking into account various conditions such as probe concentration, probe length, reaction time, etc., in addition to conditions such as buffer salt concentration and temperature. Conditions for obtaining a DNA having a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence complementary to , can be appropriately set.
　ストリンジェントな条件下でハイブリダイズするヌクレオチド配列を含むＤＮＡとしては、プローブとして使用する野生型遺伝子のヌクレオチド配列を有するＤＮＡのヌクレオチド配列と一定以上の配列同一性を有するＤＮＡが挙げられ、例えば、野生型遺伝子のヌクレオチド配列と８０％以上、好ましくは８５％以上、より好ましくは９０％以上、９１％以上、９２％以上、９３％以上、９４％以上、９５％以上、９６％以上、９７％以上、９８％以上又は９９％以上、さらに好ましくは９９．５％以上の配列同一性を有するＤＮＡが挙げられる。該配列同一性の上限は特に限定されず、典型的には１００％である。 Examples of DNAs containing nucleotide sequences that hybridize under stringent conditions include DNAs having a certain or more sequence identity with the nucleotide sequence of the DNA having the nucleotide sequence of the wild-type gene used as a probe. 80% or more, preferably 85% or more, more preferably 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more of the nucleotide sequence of the type gene , 98% or more, or 99% or more, more preferably 99.5% or more of sequence identity. The upper limit of the sequence identity is not particularly limited and is typically 100%.
　野生型遺伝子のヌクレオチド配列に相補的なヌクレオチド配列とストリンジェントな条件下でハイブリダイズするヌクレオチド配列としては、例えば、ヌクレオチド配列におけるヌクレオチド数１００個を一単位とすれば、野生型遺伝子のヌクレオチド配列において、該一単位あたり、１から数個、好ましくは１から２０個、より好ましくは１から１５個、さらに好ましくは１、２、３、４、５、６、７、８、９又は１０個のヌクレオチドの欠失、置換、付加などを有するヌクレオチド配列を含む。ここで、「ヌクレオチドの欠失」とは配列中のヌクレオチドに欠落又は消失があることを意味し、「ヌクレオチドの置換」は配列中のヌクレオチドが別のヌクレオチドに置き換えられていることを意味し、「ヌクレオチドの付加」とは新たなヌクレオチドが挿入するように付け加えられていることを意味する。 As a nucleotide sequence that hybridizes under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence of the wild-type gene, for example, if 100 nucleotides in the nucleotide sequence are one unit, the nucleotide sequence of the wild-type gene , 1 to several, preferably 1 to 20, more preferably 1 to 15, still more preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 per unit It includes nucleotide sequences with nucleotide deletions, substitutions, additions, and the like. Here, "nucleotide deletion" means that there is a missing or missing nucleotide in the sequence, and "nucleotide substitution" means that a nucleotide in the sequence is replaced with another nucleotide, "Addition of a nucleotide" means that a new nucleotide is added to insert.
　野生型遺伝子のヌクレオチド配列に相補的なヌクレオチド配列とストリンジェントな条件下でハイブリダイズするヌクレオチド配列によってコードされるタンパク質は、野生型遺伝子のヌクレオチド配列によってコードされるタンパク質が有するアミノ酸配列において１から数個のアミノ酸の欠失、置換、付加などを有するアミノ酸配列を有するタンパク質である蓋然性があるが、野生型遺伝子のヌクレオチド配列によってコードされるタンパク質と同じ酵素活性を有するものである。 A protein encoded by a nucleotide sequence that hybridizes under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence of the wild-type gene has one to several amino acids in the protein encoded by the nucleotide sequence of the wild-type gene. It is likely that the protein has an amino acid sequence with single amino acid deletions, substitutions, additions, etc., but has the same enzymatic activity as the protein encoded by the nucleotide sequence of the wild-type gene.
　野生型タンパク質と同一の、又は近似する酵素学的性質を有するタンパク質は、そのアミノ酸配列が、野生型タンパク質が有するアミノ酸配列において１から数個のアミノ酸の欠失、置換、付加などを有するアミノ酸配列からなるものであってもよい。ここで、アミノ酸配列の「１から数個のアミノ酸の欠失、置換、付加」における「１から数個」の範囲は特に限定されないが、例えば、アミノ酸配列におけるアミノ酸数１００個を一単位とすれば、該一単位あたり、１、２、３、４、５、６、７、８、９、１０、１１、１２、１３、１４、１５、１６、１７、１８、１９又は２０個程度、好ましくは１、２、３、４、５、６、７、８、９又は１０個程度、より好ましくは１、２、３、４又は５個程度を意味する。また、「アミノ酸の欠失」とは配列中のアミノ酸残基の欠落又は消失を意味し、「アミノ酸の置換」は配列中のアミノ酸残基が別のアミノ酸残基に置き換えられていることを意味し、「アミノ酸の付加」とは配列中に新たなアミノ酸残基が挿入するように付け加えられていることを意味する。 A protein having the same or similar enzymatic properties as a wild-type protein is an amino acid sequence in which one to several amino acids are deleted, substituted, added, etc. in the amino acid sequence of the wild-type protein. It may consist of Here, the range of "1 to several" in the "deletion, substitution or addition of one to several amino acids" of the amino acid sequence is not particularly limited, but for example, 100 amino acids in the amino acid sequence may be regarded as one unit. For example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 per unit, preferably means about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, more preferably about 1, 2, 3, 4 or 5. In addition, "amino acid deletion" means deletion or disappearance of an amino acid residue in a sequence, and "amino acid substitution" means that an amino acid residue in a sequence is replaced with another amino acid residue. and "addition of an amino acid" means that a new amino acid residue is added to insert into the sequence.
　「１から数個のアミノ酸の欠失、置換、付加」の具体的な態様としては、１から数個のアミノ酸が別の化学的に類似したアミノ酸で置き換えられた態様がある。例えば、ある疎水性アミノ酸を別の疎水性アミノ酸に置換する場合、ある極性アミノ酸を同じ電荷を有する別の極性アミノ酸に置換する場合などを挙げることができる。このような化学的に類似したアミノ酸は、アミノ酸毎に当該技術分野において知られている。具体例を挙げると、非極性（疎水性）アミノ酸としては、アラニン、バリン、イソロイシン、ロイシン、プロリン、トリプトファン、フェニルアラニン、メチオニンなどが挙げられる。極性（中性）アミノ酸としては、グリシン、セリン、スレオニン、チロシン、グルタミン、アスパラギン、システインなどが挙げられる。陽電荷をもつ塩基性アミノ酸としては、アルギニン、ヒスチジン、リジンなどが挙げられる。また、負電荷をもつ酸性アミノ酸としては、アスパラギン酸、グルタミン酸などが挙げられる。 Specific embodiments of "deletion, substitution, addition of one to several amino acids" include embodiments in which one to several amino acids are replaced with other chemically similar amino acids. For example, replacement of a hydrophobic amino acid with another hydrophobic amino acid, replacement of a polar amino acid with another polar amino acid having the same charge, and the like can be mentioned. Such chemically similar amino acids are known in the art for each amino acid. Specific examples of nonpolar (hydrophobic) amino acids include alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, methionine, and the like. Polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, cysteine, and the like. Examples of positively charged basic amino acids include arginine, histidine, and lysine. Moreover, aspartic acid, glutamic acid, etc. are mentioned as an acidic amino acid with a negative charge.
　野生型タンパク質が有するアミノ酸配列において１から数個のアミノ酸の欠失、置換、付加などを有するアミノ酸配列としては、野生型タンパク質が有するアミノ酸配列と一定以上の配列同一性を有するアミノ酸配列が挙げられ、例えば、野生型タンパク質が有するアミノ酸配列と８０％以上、好ましくは８５％以上、より好ましくは９０％以上、９１％以上、９２％以上、９３％以上、９４％以上、９５％以上、９６％以上、９７％以上、９８％以上又は９９％以上、さらに好ましくは９９．５％以上の配列同一性を有するアミノ酸配列などが挙げられる。該配列同一性の上限は特に限定されず、典型的には１００％である。 Amino acid sequences having one to several amino acid deletions, substitutions, additions, etc. in the amino acid sequence of the wild-type protein include amino acid sequences having a certain degree or more of sequence identity with the amino acid sequence of the wild-type protein. , for example, 80% or more, preferably 85% or more, more preferably 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% of the amino acid sequence of the wild-type protein As mentioned above, amino acid sequences having sequence identity of 97% or more, 98% or more, or 99% or more, more preferably 99.5% or more can be mentioned. The upper limit of the sequence identity is not particularly limited and is typically 100%.
（配列同一性を算出するための手段）
　ヌクレオチド配列及びアミノ酸配列の配列同一性を求める方法は特に限定されないが、例えば、通常知られる方法を利用して、野生型遺伝子のヌクレオチド配列及び野生型遺伝子が発現する野生型タンパク質のアミノ酸配列と対象となるヌクレオチド配列及びアミノ酸配列とをアラインメントし、両者の配列の一致率を算出するためのプログラムを用いることにより求められる。 (Means for calculating sequence identity)
The method for determining the sequence identity of nucleotide sequences and amino acid sequences is not particularly limited. It is determined by using a program for aligning the nucleotide sequence and amino acid sequence and calculating the rate of identity between the two sequences.
　２つのヌクレオチド配列及びアミノ酸配列における一致率を算出するためのプログラムとしては、例えば、Ｋａｒｌｉｎ及びＡｌｔｓｃｈｕｌのアルゴリズム（Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ　８７：２２６４－２２６８、１９９０；Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ　９０：５８７３－５８７７、１９９３）が知られており、このアルゴリズムを用いたＢＬＡＳＴプログラムがＡｌｔｓｃｈｕｌなどによって開発されている（Ｊ．Ｍｏｌ．Ｂｉｏｌ．２１５：４０３－４１０、１９９０）。さらに、ＢＬＡＳＴより感度よく配列同一性を決定するプログラムであるＧａｐｐｅｄ　ＢＬＡＳＴも知られている（Ｎｕｃｌｅｉｃ　Ａｃｉｄｓ　Ｒｅｓ．２５：３３８９－３４０２、１９９７）。したがって、当業者は例えば上記のプログラムを利用して、与えられた配列に対し、高い配列同一性を示す配列をデータベース中から検索することができる。これらは、例えば、米国Ｎａｔｉｏｎａｌ　Ｃｅｎｔｅｒ　ｆｏｒ　Ｂｉｏｔｅｃｈｎｏｌｏｇｙ　Ｉｎｆｏｒｍａｔｉｏｎのインターネット上のウェブサイト（ｈｔｔｐ：／／ｂｌａｓｔ．ｎｃｂｉ．ｎｌｍ．ｎｉｈ．ｇｏｖ／Ｂｌａｓｔ．ｃｇｉ）において利用可能である。 Examples of programs for calculating the rate of identity between two nucleotide sequences and amino acid sequences include the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87:2264-2268, 1990; Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993), and a BLAST program using this algorithm has been developed by Altschul et al. (J. Mol. Biol. 215:403-410, 1990). Furthermore, Gapped BLAST, which is a program for determining sequence identity with higher sensitivity than BLAST, is also known (Nucleic Acids Res. 25:3389-3402, 1997). Therefore, a person skilled in the art can, for example, use the programs described above to search the database for sequences showing high sequence identity to a given sequence. These are available, for example, on the Internet website of the US National Center for Biotechnology Information (http://blast.ncbi.nlm.nih.gov/Blast.cgi).
　上記の各方法は、データベース中から配列同一性を示す配列を検索するために通常的に用いられ得るが、個別の配列の配列同一性を決定する手段としては、Ｇｅｎｅｔｙｘネットワーク版　ｖｅｒｓｉｏｎ　１２．０．１（ジェネティックス社）のホモロジー解析を用いることもできる。この方法は、Ｌｉｐｍａｎ－Ｐｅａｒｓｏｎ法（Ｓｃｉｅｎｃｅ　２２７：１４３５－１４４１、１９８５）に基づくものである。ヌクレオチド配列の配列同一性を解析する際は、可能であればタンパク質をコードしている領域（ＣＤＳ又はＯＲＦ）を用いる。 Each of the above methods can be commonly used to retrieve sequences exhibiting sequence identity from databases, but Genetyx network version version 12.0. 1 (Genetics Inc.) can also be used. This method is based on the Lipman-Pearson method (Science 227:1435-1441, 1985). When analyzing nucleotide sequences for sequence identity, the protein coding region (CDS or ORF) is used whenever possible.
（挿入する遺伝子の由来）
　挿入するアセトバニロン分解酵素遺伝子群は、Ｓｐｈｉｎｇｏｂｉｕｍ　ｓｐ．ＳＹＫ－６株及びその近縁のアセトバニロンを分解することが可能な微生物、例えば、ａｌｐｈａ　ｐｒｏｔｅｏｂａｃｔｅｒｉａ網、Ｓｐｈｉｎｇｏｍｏｎａｄａｌｅｓ目、Ｓｐｈｉｎｇｏｍｏｎａｄａｃｅａｅ科Ｓｐｈｉｎｇｏｂｉｕｍ属に属し、リグニン由来の芳香族化合物を分解する能力を有する微生物株などに由来することが好ましい。ＳＹＫ－６株の「近縁種」は、ＳＹＫ－６株と、１６Ｓ　ｒＲＮＡ遺伝子のヌクレオチド配列の同一性が９９．０％～９９．９％であるスフィンゴビウム属微生物を指す。また、生物分類学上Ｓｐｈｉｎｇｏｂｉｕｍ属ではないが、アセトバニロンの分解能力を保持している微生物株由来であれば、挿入するアセトバニロン分解酵素遺伝子群としては好ましく、例えば、ａｌｐｈａ　ｐｒｏｔｅｏｂａｃｔｅｒｉａ網、Ｓｐｈｉｎｇｏｍｏｎａｄａｌｅｓ目、Ｓｐｈｉｎｇｏｍｏｎａｄａｃｅａｅ科に属し、アセトバニロン及びそのアナログ化合物を分解する能力を有する微生物株など、ｇａｍｍａ　ｐｒｏｔｅｏｂａｃｔｅｒｉａ網、Ｐｓｅｕｄｏｍｏｄａｌｅｓ目、Ｐｓｅｕｄｏｍｏｎａｄａｃｅａｅ科、Ｐｓｅｕｄｏｍｏｎａｓ属に属し、アセトバニロン及びそのアナログ化合物を分解する能力を有する微生物株に由来することも除外されない。 (Origin of inserted gene)
The acetovanilone degrading enzyme gene group to be inserted is Sphingobium sp. SYK-6 strain and its related microorganisms capable of degrading acetovanilone, such as alpha proteobacteria, belonging to the order Sphingomonadales, Sphingomonadaceae family Sphingobium genus, and having the ability to degrade lignin-derived aromatic compounds, etc. It is preferably derived from A "closely related species" of strain SYK-6 refers to a microorganism of the genus Sphingobium having 99.0% to 99.9% nucleotide sequence identity in the 16S rRNA gene with strain SYK-6. In addition, if it is derived from a microorganism strain that is not taxonomically belonging to the genus Sphingobium but retains the ability to decompose acetovanilone, it is preferable for the acetovanilone-degrading enzyme gene group to be inserted. and belonging to the gamma proteobacteria network, the order Pseudomodales, the family Pseudomonadaceae, the genus Pseudomonas , and having the ability to degrade acetovanilone and analogues thereof. are also not excluded.
（宿主微生物）
　宿主微生物は、シュードモナス・スピーシーズ（Ｐｓｅｕｄｏｍｏｎａｓ　ｓｐ．）　ＮＧＣ７株（受託番号：ＮＩＴＥ　ＢＰ－０３０４３）である。 (host microorganism)
The host microorganism is Pseudomonas sp. strain NGC7 (accession number: NITE BP-03043).
　本発明者らが調べたところによれば、ＮＧＣ７株のゲノムには、ＮＧＣ７株がカナマイシン耐性を示す遺伝的要因であると考えられるａｐｈ遺伝子（配列番号３７）が存在する。そこで、形質転換株の選抜のために、カナマイシンを用いた薬剤選択を利用する場合は、ＮＧＣ７株のゲノム上にあるａｐｈ遺伝子を欠失させることが好ましい。このように、形質転換株の選抜効率を高めるために、選択条件に応じてＮＧＣ７株のゲノム上にある遺伝子を欠失させてもよい。 According to investigations by the present inventors, the genome of the NGC7 strain contains the aph gene (SEQ ID NO: 37), which is thought to be a genetic factor that causes the NGC7 strain to exhibit kanamycin resistance. Therefore, when drug selection using kanamycin is used for selection of transformants, it is preferable to delete the aph gene on the genome of the NGC7 strain. Thus, in order to increase the selection efficiency of transformants, genes on the genome of the NGC7 strain may be deleted depending on the selection conditions.
（遺伝子工学的手法による遺伝子のクローニング）
　欠失及び挿入する遺伝子は、適当な公知の各種ベクター中に挿入することができる。さらに、このベクターを宿主微生物であるＮＧＣ７株に導入して、遺伝子を欠失した、又は遺伝子を挿入した形質転換体（形質転換微生物）を作製できる。欠失する遺伝子は野生型遺伝子の全部又は一部が破壊、欠損、置換、挿入などにより遺伝子の構造が変化したものであることが好ましい。挿入する遺伝子は、野生型遺伝子と同一又は近似するタンパク質を発現する遺伝子であることが好ましい。 (Gene cloning by genetic engineering technique)
Genes to be deleted and inserted can be inserted into various known appropriate vectors. Furthermore, this vector can be introduced into the NGC7 strain, which is a host microorganism, to produce a transformant (transformed microorganism) in which the gene has been deleted or the gene has been inserted. The gene to be deleted is preferably a wild-type gene whose structure has been altered by disruption, deletion, substitution, insertion, or the like, in whole or in part. The gene to be inserted is preferably a gene that expresses the same or similar protein as the wild-type gene.
　欠失及び挿入する遺伝子の取得方法、これらの遺伝子のヌクレオチド配列やこれらの遺伝子が発現するタンパク質のアミノ酸配列に関する情報の取得方法、各種ベクターの製造方法や形質転換微生物の作製方法などは、当業者にとって適宜選択することができる。また、本明細書では、形質転換や形質転換体にはそれぞれ形質導入や形質導入体を包含する。欠失及び挿入する遺伝子のクローニングの一例を非限定的に後述する。 Methods for obtaining genes to be deleted and inserted, methods for obtaining information on the nucleotide sequences of these genes and amino acid sequences of proteins expressed by these genes, methods for producing various vectors, methods for producing transformed microorganisms, etc., are well known to those skilled in the art. can be selected as appropriate for In addition, in this specification, transformation and transformants include transduction and transductants, respectively. A non-limiting example of cloning genes to be deleted and inserted is described below.
　例えば、欠失又は挿入する遺伝子に関連する野生型遺伝子を有する由来生物や種々の微生物から、常法、例えば、参考技術文献に記載の方法により、染色体ＤＮＡ及びｍＲＮＡを抽出することができる。抽出したｍＲＮＡを鋳型としてｃＤＮＡを合成することができる。このようにして得られた染色体ＤＮＡ及びｃＤＮＡを用いて、染色体ＤＮＡ及びｃＤＮＡのライブラリーを作製することができる。 For example, chromosomal DNA and mRNA can be extracted from derived organisms and various microorganisms that have wild-type genes related to the gene to be deleted or inserted by a conventional method, for example, the method described in the reference technical document. cDNA can be synthesized using the extracted mRNA as a template. Using the chromosomal DNA and cDNA thus obtained, a library of chromosomal DNA and cDNA can be constructed.
　例えば、挿入する遺伝子は、該遺伝子に関連する野生型遺伝子を有する由来生物の染色体ＤＮＡ及びｃＤＮＡを鋳型としたクローニングにより得ることができる。野生型遺伝子の由来生物は上記したとおりのＳＹＫ－６株及びその近縁種である。例えば、ＳＹＫ－６株を培養し、得られた菌体から水分を取り除き、液体窒素中で冷却しながら乳鉢などを用いて物理的に磨砕することにより細かい粉末状の菌体片とし、該菌体片から通常の方法により染色体ＤＮＡ画分を抽出する。染色体ＤＮＡ抽出操作には、ＤＮｅａｓｙ　Ｂｌｏｏｄ　＆　ＴｉｓｓｕｅＫｉｔ（キアゲン社）などの市販の染色体ＤＮＡ抽出キットなどが利用できる。本明細書において、染色体ＤＮＡとゲノムＤＮＡとは同義である。 For example, the gene to be inserted can be obtained by cloning using, as templates, the chromosomal DNA and cDNA of the derived organism containing the wild-type gene related to the gene. Wild-type genes are derived from the SYK-6 strain and its relatives as described above. For example, the SYK-6 strain is cultured, water is removed from the obtained cells, and the cells are ground physically using a mortar or the like while cooling in liquid nitrogen to obtain fine powdery cells. A chromosomal DNA fraction is extracted from the cell piece by a conventional method. A commercially available chromosomal DNA extraction kit such as DNeasy Blood & Tissue Kit (Qiagen) can be used for the chromosomal DNA extraction procedure. As used herein, chromosomal DNA and genomic DNA are synonymous.
　次いで、染色体ＤＮＡを鋳型として、５’末端配列及び３’末端配列に相補的な合成プライマーを用いてポリメラーゼ連鎖反応（ｐｏｌｙｍｅｒａｓｅ　ｃｈａｉｎ　ｒｅａｃｔｉｏｎ；ＰＣＲ）を行うことにより、ＤＮＡを増幅する。プライマーとしては、挿入する遺伝子を含むＤＮＡ断片の増幅が可能であれば特に限定されない。その例としては、ｖａｎＡ４遺伝子を増幅するものとして、ＮＧＣ７株のゲノム配列を参考として設計した配列番号３５及び３６で表されるプライマーなどが挙げられる。なお、このようなプライマーを用いると、目的遺伝子全長が増幅できる。別の方法として、ショットガンライブラリーからの目的遺伝子クローンのスクリーニングや、Ｉｎｖｅｒｓｅ　ＰＣＲ、Ｎｅｓｔｅｄ　ＰＣＲ、５’ＲＡＣＥ法、３’ＲＡＣＥ法などの適当なＰＣＲにより、目的の遺伝子断片を含むＤＮＡを増幅させ、これらを連結させて全長の目的遺伝子を含むＤＮＡを得ることなどができる。 DNA is then amplified by polymerase chain reaction (PCR) using the chromosomal DNA as a template and synthetic primers complementary to the 5' and 3' terminal sequences. The primer is not particularly limited as long as it can amplify a DNA fragment containing the gene to be inserted. Examples thereof include primers represented by SEQ ID NOs: 35 and 36 designed with reference to the genome sequence of the NGC7 strain for amplifying the vanA4 gene. The full-length target gene can be amplified using such primers. As another method, screening of target gene clones from a shotgun library, or appropriate PCR such as Inverse PCR, Nested PCR, 5'RACE method, 3'RACE method, etc., is used to amplify DNA containing the target gene fragment. , and ligating them to obtain a DNA containing the full-length target gene.
　また、欠失又は挿入する遺伝子を取得する方法は、上記したとおりに特に限定されず、遺伝子工学的手法によらなくとも、例えば、化学合成法を用いて遺伝子を構築することが可能である。 In addition, the method of obtaining the gene to be deleted or inserted is not particularly limited as described above, and it is possible to construct the gene using, for example, a chemical synthesis method without using genetic engineering techniques.
　ＰＣＲにより増幅された増幅産物及び化学合成した遺伝子におけるヌクレオチド配列の確認は、例えば、次のように行うことができる。
　まず、配列を確認したいＤＮＡを通常の方法に準じて適当なベクターに挿入して組換え体ＤＮＡを作製する。ベクターへのクローニングには、Ｉｎ－Ｆｕｓｉｏｎ　ＨＤ　Ｃｌｏｎｉｎｇ　Ｋｉｔ（タカラバイオ社）、ＴＡ　Ｃｌｏｎｉｎｇ　Ｋｉｔ（インビトロジェン社）などの公知又は市販のキット；ｐＵＣ４Ｋ（Ｇｅｎｅ，ｖｏｌ．１９、ｐ２５９－２６８、１９８２を参照）、ｐＥＸ１８Ａｍｐ（Ｇｅｎｅ，ｖｏｌ．２１２，ｐ７７－８６，１９９８を参照）、ｐＰＳ８５８（Ｇｅｎｅ，ｖｏｌ．２１２，ｐ７７－８６，１９９８を参照）、ｐＵＣ１１８（タカラバイオ社）、ｐＪＢ８６６（Ｐｌａｓｍｉｄ，ｖｏｌ．３８，ｐ３５－５１，１９９７を参照）、ｐＭＣＬ２００（Ｇｅｎｅ，ｖｏｌ．１６２，ｐ１５７－１５８，１９９５を参照）、ｐＱＥ３０（キアゲン社）、ｐＵＣ１１９（タカラバイオ社）、ｐＵＣ１８（タカラバイオ社）、ｐＢＲ３２２（タカラバイオ社）、ｐＡＫ４０５（Ａｎｄｒｅａｓ　Ｋａｃｚｍａｒｃｚｙｋら、Ａｐｐｌｉｅｄ　ａｎｄ　Ｅｎｖｉｒｏｎｍｅｎｔａｌ　Ｍｉｃｒｏｂｉｏｌｏｇｙ，２０１２，ｖｏｌ．７８（１０），ｐ３７７４－３７７７を参照）、ｐＫ１８ｍｏｂｓａｃＢ（Ａｎｄｒｅａｓ　Ｓｃｈａｆｅｒら、Ｇｅｎｅ，１９９４，ｖｏｌ．１４５，ｐ６９－７３を参照）などの公知又は市販のプラスミドベクター；λＥＭＢＬ３（ストラタジーン社）などの公知又は市販のバクテリオファージベクターなどが使用できる。 Nucleotide sequences of amplification products amplified by PCR and chemically synthesized genes can be confirmed, for example, as follows.
First, a recombinant DNA is prepared by inserting a DNA whose sequence is to be confirmed into an appropriate vector according to a conventional method. For cloning into a vector, known or commercially available kits such as In-Fusion HD Cloning Kit (Takara Bio) and TA Cloning Kit (Invitrogen); pUC4K (see Gene, vol.19, p259-268, 1982). , pEX18Amp (see Gene, vol.212, p77-86, 1998), pPS858 (see Gene, vol.212, p77-86, 1998), pUC118 (Takara Bio), pJB866 (Plasmid, vol.38, p35-51, 1997), pMCL200 (Gene, vol.162, p157-158, 1995), pQE30 (Qiagen), pUC119 (Takara Bio), pUC18 (Takara Bio), pBR322 (Takara Bio ), pAK405 (see Andreas Kaczmarczyk et al., Applied and Environmental Microbiology, 2012, vol.78(10), p3774-3777), pK18 mobsacB (see Andreas Schafer et al., Gene, 1994, vol.6-9-3777) ) or other known or commercially available plasmid vectors; λEMBL3 (Stratagene) or other known or commercially available bacteriophage vectors.
　構築した組換え体ＤＮＡを大量に得たい場合は、組換え体ＤＮＡを、例えば、大腸菌（Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ）、好ましくは大腸菌　ＪＭ１０９株（タカラバイオ社）及び大腸菌　ＤＨ５α株（タカラバイオ社）などに導入して形質転換し、次いで得られた形質転換体に含まれる組換え体ＤＮＡを、ＱＩＡＧＥＮ　Ｐｌａｓｍｉｄ　Ｍｉｎｉ　Ｋｉｔ（キアゲン社）などを用いて精製することができる。 When a large amount of the constructed recombinant DNA is desired, the recombinant DNA is introduced into, for example, Escherichia coli , preferably Escherichia coli JM109 strain (Takara Bio Inc.) and Escherichia coli DH5α strain (Takara Bio Inc.). Then, the recombinant DNA contained in the resulting transformant can be purified using QIAGEN Plasmid Mini Kit (Qiagen) or the like.
　組換え体ＤＮＡに挿入されている各遺伝子のヌクレオチド配列の決定は、ジデオキシ法（Ｍｅｔｈｏｄｓ　ｉｎ　Ｅｎｚｙｍｏｌｏｇｙ，１０１，ｐ２０－７８，１９８３などを参照）などにより行う。ヌクレオチド配列の決定の際に使用する配列解析装置は特に限定されないが、例えば、Ｌｉ－ＣＯＲ　ＭＯＤＥＬ　４２００Ｌシークエンサー（アロカ社）、３７０ＤＮＡシークエンスシステム（パーキンエルマー社）、ＣＥＱ２０００ＸＬ　ＤＮＡアナリシスシステム（ベックマン社）などが挙げられる。そして、決定されたヌクレオチド配列を元に、翻訳されるタンパク質のアミノ酸配列を知り得る。 The nucleotide sequence of each gene inserted into the recombinant DNA is determined by the dideoxy method (see Methods in Enzymology, 101, p20-78, 1983, etc.). The sequence analyzer used for nucleotide sequence determination is not particularly limited, but examples include Li-COR MODEL 4200L Sequencer (Aloka), 370 DNA Sequence System (PerkinElmer), CEQ2000XL DNA Analysis System (Beckman), etc. mentioned. Then, based on the determined nucleotide sequence, the amino acid sequence of the protein to be translated can be known.
（遺伝子を含む組換えベクターの構築）
　欠失又は挿入する遺伝子を含む組換えベクター（組換え体ＤＮＡ）は、欠失又は挿入する遺伝子を含むＰＣＲ増幅産物と各種ベクターとを、遺伝子の欠失又は発現が可能な形で結合することにより構築することができる。なお、欠失する遺伝子の場合は、組換えベクターを宿主微生物に導入して、相同組換えによって、組換えベクター中の遺伝子が宿主微生物中の遺伝子に置き換わるために、組換えベクターは欠失する遺伝子の上流及び下流の領域を含むことが好ましい。 (Construction of Recombinant Vector Containing Gene)
Recombinant vectors (recombinant DNA) containing genes to be deleted or inserted should combine PCR amplification products containing genes to be deleted or inserted with various vectors in a form that allows gene deletion or expression. can be constructed by In the case of the deleted gene, the recombinant vector is introduced into the host microorganism, and the gene in the recombinant vector is replaced with the gene in the host microorganism by homologous recombination, so the recombinant vector is deleted. It preferably includes upstream and downstream regions of the gene.
　非限定的な例として挿入する遺伝子を含む組換えベクターを作製する方法は、例えば、適当な制限酵素で挿入する遺伝子のいずれかを含むＤＮＡ断片を切り出し、該ＤＮＡ断片を適当な制限酵素で切断したプラスミドベクターとを、Ｉｎ－Ｆｕｓｉｏｎ　ＨＤ　Ｃｌｏｎｉｎｇ　Ｋｉｔ（タカラバイオ社）などの市販の組換えベクター作製キットなどを用いて連結することにより構築することができる。または、プラスミドベクターと相同的な配列を両末端に付加した遺伝子を含むＤＮＡ断片と、インバースＰＣＲにより増幅したプラスミド由来のＤＮＡ断片とを、Ｉｎ－Ｆｕｓｉｏｎ　ＨＤ　Ｃｌｏｎｉｎｇ　Ｋｉｔ（タカラバイオ社）などの市販の組換えベクター作製キットを用いて連結させることにより得ることができる。 As a non-limiting example, a method for producing a recombinant vector containing a gene to be inserted is, for example, excising a DNA fragment containing any of the genes to be inserted with an appropriate restriction enzyme, and cleaving the DNA fragment with an appropriate restriction enzyme. It can be constructed by ligating the resulting plasmid vector using a commercially available recombinant vector preparation kit such as In-Fusion HD Cloning Kit (Takara Bio). Alternatively, a DNA fragment containing a gene with sequences homologous to the plasmid vector added to both ends and a plasmid-derived DNA fragment amplified by inverse PCR are combined with a commercially available product such as the In-Fusion HD Cloning Kit (Takara Bio Inc.). It can be obtained by ligation using a recombinant vector preparation kit.
　欠失又は挿入する遺伝子を含む組換えベクターは、欠失又は挿入する遺伝子とプラスミドベクター由来の遺伝子（ヌクレオチド配列）とを少なくとも含む。組換えベクターの一例として、ｖａｎＡ４遺伝子及び／又はｖａｎＢ４遺伝子を含む組換えベクター；アセトバニロン分解酵素遺伝子群を含む組換えベクターなどが挙げられる。また、組換えベクターは、上記した遺伝子以外の遺伝子を、本発明の課題解決を妨げない限りに含むものであってもよい。 A recombinant vector containing a gene to be deleted or inserted contains at least the gene to be deleted or inserted and a gene (nucleotide sequence) derived from a plasmid vector. Examples of recombinant vectors include recombinant vectors containing vanA4 gene and/or vanB4 gene; recombinant vectors containing acetovanilone degrading enzyme gene group; In addition, the recombinant vector may contain genes other than the genes described above as long as they do not interfere with the solution of the problems of the present invention.
　組換えベクターは、異種遺伝子又は異種ヌクレトチド配列を含んでもよい。異種遺伝子はＮＧＣ７株に本来的に存在しない（ｎｏｔ　ｎａｔｕｒａｌｌｙ　ｏｃｃｕｒｉｎｇ）遺伝子であれば特に限定されず、例えば、ＮＧＣ７株由来のヌクレオチド配列に依拠しない合成遺伝子、挿入する遺伝子と由来生物が相違する生物に由来する遺伝子、ＮＧＣ７株と相違する他の微生物、植物、動物、ウイルスなどの生物に由来する遺伝子などが挙げられる。異種遺伝子の具体例としては、ｐＵＣ１１８由来のＤＮＡ断片、例えば、ラクトースプロモーター領域（Ｐ ｌａｃ ）などが挙げられるが、これに限定されない。 A recombinant vector may contain a heterologous gene or heterologous nucleotide sequence. The heterologous gene is not particularly limited as long as it is a gene not naturally occurring in the NGC7 strain. Examples include genes derived from NGC7 strain, genes derived from organisms such as other microorganisms different from the NGC7 strain, plants, animals, and viruses. Specific examples of heterologous genes include, but are not limited to, DNA fragments derived from pUC118, such as the lactose promoter region (P lac ).
（形質転換微生物の作製方法）
　形質転換微生物の作製方法は特に限定されず、例えば、常法に従って、遺伝子の欠失又は挿入が実現する態様でＮＧＣ７株に挿入する方法などが挙げられる。具体的には、挿入する遺伝子のいずれかを発現誘導プロモーター及びターミネーターの間に挿入したＤＮＡコンストラクトを作製し、次いで該ＤＮＡコンストラクトによってＮＧＣ７株を形質転換することなどにより、挿入する遺伝子を発現する形質転換微生物が得られる。又は、欠失する遺伝子並びに該遺伝子の上流及び下流の領域を含むＤＮＡコンストラクトを作製し、次いで該ＤＮＡコンストラクトによって宿主微生物を形質転換することなどにより、遺伝子を欠失する形質転換微生物が得られる。本明細書では、ＮＧＣ７株を形質転換するために作製された組換えベクターをＤＮＡコンストラクトと総称してよぶ。 (Method for producing transformed microorganism)
The method for producing the transformed microorganism is not particularly limited, and includes, for example, a method of inserting into the NGC7 strain in such a manner that gene deletion or insertion is achieved according to a conventional method. Specifically, a DNA construct is prepared by inserting any of the genes to be inserted between an expression-inducing promoter and a terminator, and then the NGC7 strain is transformed with the DNA construct to express the gene to be inserted. A transformed microorganism is obtained. Alternatively, a transformed microorganism lacking the gene can be obtained by preparing a DNA construct containing the gene to be deleted and the upstream and downstream regions of the gene, and then transforming the host microorganism with the DNA construct. Recombinant vectors constructed to transform strain NGC7 are collectively referred to herein as DNA constructs.
　ＤＮＡコンストラクトをＮＧＣ７株に導入する方法は特に限定されないが、例えば、当業者に知られているとおりである、導入したＤＮＡコンストラクトが自律的に増殖して遺伝子を発現するようにＮＧＣ７株に導入する方法；相同組換えを利用することによりＤＮＡコンストラクトをＮＧＣ７株の染色体上に直接的に挿入する方法などが挙げられる。 The method of introducing the DNA construct into the NGC7 strain is not particularly limited. For example, as known to those skilled in the art, the introduced DNA construct is introduced into the NGC7 strain so as to autonomously proliferate and express the gene. Method: a method of directly inserting a DNA construct into the chromosome of the NGC7 strain by utilizing homologous recombination.
　挿入する遺伝子を含むＤＮＡコンストラクトをＮＧＣ７株に導入する方法として、相同組換えを利用する方法では、染色体上の組換え部位の上流領域及び下流領域と相同な配列の間に、ＤＮＡコンストラクトを連結し、ＮＧＣ７株のゲノム中に挿入することができる。 As a method for introducing a DNA construct containing a gene to be inserted into the NGC7 strain, in a method using homologous recombination, the DNA construct is ligated between sequences homologous to the upstream and downstream regions of the recombination site on the chromosome. , can be inserted into the genome of strain NGC7.
ＮＧＣ７株の作製に用いられるベクター－宿主系は、ＮＧＣ７株中において、挿入する遺伝子が発現し得る系又は染色体上の遺伝子が欠失し得る系であれば特に限定されないが、例えば、ｐＪＢ８６６（Ｐｌａｓｍｉｄ，ｖｏｌ．３８（１），ｐ３５－５１，１９９７）－シュードモナス属微生物の系、ｐＫＴ２３０（Ｇｅｎｅ，ｖｏｌ．１６，ｐ２３７－２４７，１９８１）－シュードモナス属微生物の系、ｐＳＥＶＡ（Ｎｕｃｌｅｉｃ　Ａｃｉｄｓ　Ｒｅｓｅａｒｃｈ，ｖｏｌ．４８（Ｄ１），ｐＤ１１６４－Ｄ１１７０，２０２０）－シュードモナス属微生物の系などが挙げられる。 The vector-host system used for the preparation of the NGC7 strain is not particularly limited as long as it is a system in which the inserted gene can be expressed in the NGC7 strain or a system in which the gene on the chromosome can be deleted. , vol.38(1), p35-51, 1997)-Pseudomonas microorganism system, pKT230 (Gene, vol.16, p237-247, 1981)-Pseudomonas microorganism system, pSEVA (Nucleic Acids Research, vol. 48(D1), pD1164-D1170, 2020)-Pseudomonas microorganism system.
　挿入する遺伝子を含むＤＮＡコンストラクトは、ＮＧＣ７株の染色体に導入しない形で、自律的に増幅して、挿入する遺伝子を発現しても、ＮＧＣ７株の染色体に導入した形で挿入する遺伝子を発現しても、どちらでもよい。 The DNA construct containing the gene to be inserted is amplified autonomously without being introduced into the chromosome of the NGC7 strain, and even if the gene to be inserted is expressed, the gene to be inserted into the chromosome of the NGC7 strain is expressed. or either.
　ＤＮＡコンストラクトには、形質転換された細胞を選択することを可能にするためのマーカー遺伝子が含まれていてもよい。マーカー遺伝子は特に限定されず、例えば、ゲンタマイシン、カナマイシン、テトラサイクリン、アンピシリン、カルベニシリンなどの薬剤に対する薬剤耐性遺伝子などが挙げられる。マーカー遺伝子は、欠失する遺伝子の途中に、又は欠失する遺伝子に置換するように含まれていてもよい。 The DNA construct may contain a marker gene to enable selection of transformed cells. The marker gene is not particularly limited, and examples thereof include drug resistance genes for drugs such as gentamicin, kanamycin, tetracycline, ampicillin and carbenicillin. A marker gene may be included in the middle of the deleted gene or to replace the deleted gene.
　挿入する遺伝子を含むＤＮＡコンストラクトは、遺伝子の種類によっては、ＮＧＣ７株中で遺伝子を発現することを可能にするプロモーター及びターミネーターに加えて、その他の制御配列（例えば、オペレーターなどの転写制御に関わるシス配列など）を含むことができる。 Depending on the type of gene, the DNA construct containing the gene to be inserted may contain promoters and terminators that enable expression of the gene in the NGC7 strain, as well as other regulatory sequences (e.g., transcription control sequences such as operators). arrays, etc.).
　ＤＮＡコンストラクトの一実施態様は、例えば、後述する実施例に記載があるｐｖａｎＡ１Ｂ１ｄｅｌプラスミドＤＮＡ、ｐｖａｎＡ２Ｂ２ｄｅｌプラスミドＤＮＡ、ｐｖａｎＡ３Ｂ３ｄｅｌプラスミドＤＮＡ、ｐｖａｎＡ４Ｂ４ｄｅｌプラスミドＤＮＡ、ｐＳＥＶＡ２４１＿Ｐ ｌａｃ －ａｃｖプラスミドＤＮＡ、ｐＴＳ０９３＿ｖｃｅＡ－ＢプラスミドＤＮＡなどが挙げられるが、これらに限定されない。 One embodiment of the DNA construct includes, for example, pvanA1B1del plasmid DNA, pvanA2B2del plasmid DNA, pvanA3B3del plasmid DNA, pvanA4B4del plasmid DNA, pSEVA241_P lac -acv plasmid DNA, pTS093_vceA-B plasmid DNA, etc., which are described in Examples below. but not limited to these.
　ＮＧＣ７株への形質転換方法としては、当業者に知られる方法を適宜選択することができ、例えば、エレクトロポレーション（電気穿孔）法、接合伝達法などによって実施できる。 As a method for transforming the NGC7 strain, methods known to those skilled in the art can be appropriately selected, for example, an electroporation method, a conjugative transfer method, and the like.
　形質転換微生物を選択及び増殖させるための培地は、ＮＧＣ７株の生育に適した培地を用いればよい。例えば、マーカー遺伝子としてカナマイシン、ゲンタマイシン及びテトラサイクリンの耐性遺伝子を用いた場合は、形質転換微生物の選択及び増殖は、例えば、形質転換微生物をこれらの薬剤を含むＬＢ培地で培養することなどによって実施できる。 A medium suitable for the growth of the NGC7 strain should be used as the medium for selecting and growing transformed microorganisms. For example, when kanamycin, gentamicin and tetracycline resistance genes are used as marker genes, selection and growth of transformed microorganisms can be carried out by, for example, culturing the transformed microorganisms in LB medium containing these drugs.
　形質転換微生物が作製されたことの確認は、例えば、遺伝子の欠失した形質転換微生物のみが生存し得る条件や挿入する遺伝子を発現する形質転換微生物のみが生存し得る条件下で形質転換微生物を培養することなどにより達成し得る。また、形質転換微生物を培養し、次いで培養後に得られた培養物におけるバニリン酸の量が、同じ条件下で培養したＮＧＣ７株の培養物におけるバニリン酸の量よりも大きいことを確認することなどにより、形質転換微生物が作製されたことを確認することができる。 To confirm that a transformed microorganism has been produced, for example, the transformed microorganism is placed under conditions in which only the transformed microorganism lacking the gene can survive or under conditions in which only the transformed microorganism expressing the gene to be inserted can survive. It can be achieved by culturing and the like. Alternatively, by culturing the transformed microorganism and then confirming that the amount of vanillic acid in the culture obtained after culturing is greater than the amount of vanillic acid in the culture of the NGC7 strain cultured under the same conditions. , it is possible to confirm that a transformed microorganism has been produced.
　形質転換微生物が作製されたことの確認は、形質転換微生物から染色体ＤＮＡを抽出し、これを鋳型としてＰＣＲを行い、形質転換が起きた場合に増幅が可能なＰＣＲ産物が生じることやＰＣＲ産物の特性やヌクレオチド配列を確認することなどにより行ってもよい。 To confirm that a transformed microorganism has been produced, chromosomal DNA is extracted from the transformed microorganism, PCR is performed using this as a template, and if transformation occurs, a PCR product that can be amplified is generated or the PCR product This may be done, for example, by confirming the properties or the nucleotide sequence.
　例えば、欠失又は挿入する遺伝子のプロモーターのヌクレオチド配列に対するフォワードプライマーと、マーカー遺伝子のヌクレオチド配列に対するリバースプライマーとの組み合わせでＰＣＲを行い、想定の長さの産物が生じることを確認する。 For example, perform PCR using a combination of a forward primer for the nucleotide sequence of the promoter of the gene to be deleted or inserted and a reverse primer for the nucleotide sequence of the marker gene, and confirm that a product of the expected length is generated.
　相同組換えにより形質転換を行う場合には、用いた上流側の相同領域より上流に位置するフォワードプライマーと、用いた下流側の相同領域より下流に位置するリバースプライマーとの組み合わせでＰＣＲを行い、相同組換えが起きた場合に想定される長さの産物が生じることを確認することが好ましい。  When transformation is performed by homologous recombination, PCR is performed with a combination of a forward primer located upstream from the used upstream homologous region and a reverse primer located downstream from the used downstream homologous region, It is preferable to confirm that a product of the expected length is produced when homologous recombination occurs. 
　ＳＹＫ－６株の近縁種等のアセトバニロン分解酵素遺伝子群を用いる場合、該アセトバニロン分解酵素遺伝子群は、ＮＧＣ７株に発現させるために、コドン、二次構造、ＧＣ含量などを最適化した遺伝子であってもよい。 When using the acetovanilone degrading enzyme gene cluster of closely related species of the SYK-6 strain, the acetovanillone degrading enzyme gene cluster is a gene optimized for codons, secondary structure, GC content, etc. for expression in the NGC7 strain. There may be.
（形質転換微生物の具体的な一実施態様）
　形質転換微生物の具体的一態様は、宿主微生物がＮＧＣ７株であって、染色体上にあるｖａｎＡ４遺伝子（配列番号１）が欠失している、形質転換微生物である。形質転換微生物の具体的一態様は、宿主微生物がＮＧＣ７株であって、染色体上にあるｖａｎＡ４遺伝子（配列番号１）に加えて、ｖａｎＢ４遺伝子（配列番号２）が欠失している形質転換微生物である。形質転換微生物の具体的一態様は、宿主微生物がＮＧＣ７株であって、染色体上にあるｖａｎＡ４遺伝子（配列番号１）及びｖａｎＢ４遺伝子に加えて、ｖａｎＡ１遺伝子（配列番号１１）、ｖａｎＡ２遺伝子（配列番号１２）、ｖａｎＡ３遺伝子（配列番号１３）、ｖａｎＢ１遺伝子（配列番号１４）、ｖａｎＢ２遺伝子（配列番号１５）、ｖａｎＢ３遺伝子（配列番号１６）、ｖａｎＢ５遺伝子（配列番号１７）及びｖａｎＢ６遺伝子（配列番号１８）からなる群から選ばれる１種以上の遺伝子が欠失している、形質転換微生物である。これらの形質転換微生物を総称して形質転換微生物（１）ともよぶ。 (One specific embodiment of the transformed microorganism)
A specific embodiment of the transformed microorganism is a transformed microorganism in which the host microorganism is strain NGC7 and the vanA4 gene (SEQ ID NO: 1) on the chromosome is deleted. A specific embodiment of the transformed microorganism is a transformed microorganism in which the host microorganism is the NGC7 strain and lacks the vanB4 gene (SEQ ID NO: 2) in addition to the vanA4 gene (SEQ ID NO: 1) on the chromosome. is. In one specific embodiment of the transformed microorganism, the host microorganism is the NGC7 strain, and in addition to the vanA4 gene (SEQ ID NO: 1) and vanB4 gene on the chromosome, vanA1 gene (SEQ ID NO: 11), vanA2 gene (SEQ ID NO: 12), vanA3 gene (SEQ ID NO: 13), vanB1 gene (SEQ ID NO: 14), vanB2 gene (SEQ ID NO: 15), vanB3 gene (SEQ ID NO: 16), vanB5 gene (SEQ ID NO: 17) and vanB6 gene (SEQ ID NO: 18) A transformed microorganism lacking one or more genes selected from the group consisting of These transformed microorganisms are also collectively referred to as transformed microorganisms (1).
　形質転換微生物の具体的一態様（以下、形質転換微生物（２）ともよぶ。）は、上記遺伝子の欠失に加えて、挿入されたａｃｖＡ遺伝子、ａｃｖＢ遺伝子、ａｃｖＣ遺伝子、ａｃｖＤ遺伝子、ａｃｖＥ遺伝子、ａｃｖＦ遺伝子、ｖｃｅＡ遺伝子及びｖｃｅＢ遺伝子からなるアセトバニロン分解酵素遺伝子群を発現する、形質転換微生物である。 A specific embodiment of the transformed microorganism (hereinafter, also referred to as transformed microorganism (2)) has, in addition to deletion of the above genes, the inserted acvA gene, acvB gene, acvC gene, acvD gene, acvE gene, It is a transformed microorganism expressing an acetovanilone-degrading enzyme gene group consisting of the acvF gene, the vceA gene and the vceB gene.
　形質転換微生物（１）は、染色体上にあるｖａｎＡ４遺伝子（配列番号１）又は該ｖａｎＡ４遺伝子及びｖａｎＢ４遺伝子（配列番号２）が欠失しているという態様を少なくともとることにより、バニリン酸の分解作用が欠失しつつも、シリンガ酸の資化作用を維持することにより、特許文献３に記載の形質転換微生物では不可能である、ｐ－ヒドロキシ安息香酸などのＨ型リグニン由来の芳香族化合物及び／又はシリンガ酸などのＳ型リグニン由来の芳香族化合物を炭素源として増殖しつつ、フェルラ酸やバニリンなどのＧ型リグニン由来の芳香族化合物からバニリン酸の製造が可能である。 The transformed microorganism (1) lacks at least the vanA4 gene (SEQ ID NO: 1) or the vanA4 gene and the vanB4 gene (SEQ ID NO: 2) on the chromosome, thereby degrading vanillic acid. is deleted, but by maintaining the assimilation of syringic acid, aromatic compounds derived from H-type lignin such as p -hydroxybenzoic acid, which is impossible with the transformed microorganism described in Patent Document 3, and / Or, it is possible to produce vanillic acid from aromatic compounds derived from G-type lignin such as ferulic acid and vanillin while growing using aromatic compounds derived from S-type lignin such as syringic acid as a carbon source.
　形質転換微生物（２）は、上記遺伝子の欠失に加えて、挿入されたアセトバニロン分解酵素遺伝子群が発現するという態様を少なくともとることにより、ｐ－ヒドロキシ安息香酸などのＨ型リグニン由来の芳香族化合物及び／又はシリンガ酸などのＳ型リグニン由来の芳香族化合物を炭素源として増殖しつつ、フェルラ酸やバニリンなどのＧ型リグニン由来の芳香族化合物及びアセトバニロン又はアセトバニロンを含むＧ型リグニン由来の芳香族化合物の混合物からバニリン酸の製造が可能である。 In the transformed microorganism (2), in addition to the deletion of the above genes, the inserted acetovanilone degrading enzyme gene group is expressed at least, so that aromatic aromatics derived from H-type lignin such as p -hydroxybenzoic acid G-type lignin-derived aromatic compounds such as ferulic acid and vanillin and G-type lignin-derived aromatic compounds such as ferulic acid and vanillin and acetovanilone or G-type lignin-derived aromatics containing acetovanillone It is possible to produce vanillic acid from mixtures of family compounds.
　形質転換微生物（１）及び（２）の具体的態様は、後述する実施例に記載があるＮＧＣ７ΔｖａｎＡ４Ｂ４株、ＮＧＣ７ΔｖａｎＡ４Ｂ４［ｐＳＥＶＡ２４１＿Ｐ ｌａｃ －ａｃｖ，ｐＴＳ０９３＿ｖｃｅＡ－Ｂ］株などであるが、これらに限定されない。 Specific embodiments of transformed microorganisms (1) and (2) include, but are not limited to, NGC7Δ vanA4B4 strain and NGC7Δ vanA4B4 [pSEVA241_P lac -acv, pTS093_vceA-B] strains, which are described in Examples below. .
（製造方法）
　本発明の一態様の製造方法（以下、製造方法（１）とよぶ。）は、バニリンといったＧ型リグニン由来の芳香族化合物と、ｐ－ヒドロキシ安息香酸やｐ－ヒドロキシベンズアルデヒドといったＨ型リグニン由来の芳香族化合物及び／又はシリンガ酸やシリンガアルデヒドといったＳ型リグニン由来の芳香族化合物との混合物を、形質転換微生物（１）に作用させることにより、バニリン酸を得る工程を含む。 (Production method)
A production method of one embodiment of the present invention (hereinafter referred to as production method (1)) comprises an aromatic compound derived from G-type lignin such as vanillin, and an aromatic compound derived from H-type lignin such as p -hydroxybenzoic acid or p -hydroxybenzaldehyde. It includes a step of obtaining vanillic acid by reacting a mixture with an aromatic compound and/or an aromatic compound derived from S-type lignin such as syringic acid and syringaldehyde on the transformed microorganism (1).
　本発明の別の一態様の製造方法（以下、製造方法（２）とよぶ。）は、アセトバニロンを、形質転換微生物（２）に作用させることにより、バニリン酸を得る工程を含む。アセトバニロンは、クラフト蒸解、ソーダ蒸解といったリグニンのアルカリ酸化分解によって得られる分解物に含まれることから、アセトバニロンの供給源を主にＧ型リグニンから成る針葉樹リグニンのアルカリ酸化分解物などの芳香族化合物の混合物としてもよい。 A production method of another aspect of the present invention (hereinafter referred to as production method (2)) includes a step of allowing acetovanillone to act on transformed microorganism (2) to obtain vanillic acid. Acetovanilone is contained in decomposition products obtained by alkaline oxidative decomposition of lignin such as kraft cooking and soda cooking. A mixture may also be used.
　本明細書では、製造方法（１）及び（２）をまとめて指すときには、単に「製造方法」とよぶ。 In this specification, when manufacturing methods (1) and (2) are collectively referred to, they are simply referred to as "manufacturing method".
　リグニン由来の芳香族化合物を形質転換微生物に作用させる方法は、リグニン由来の芳香族化合物と形質転換微生物とが接触して、形質転換微生物が有する酵素によってバニリン酸が生産及び／又は蓄積できる方法であれば特に限定されないが、例えば、リグニン由来の芳香族化合物を含有し、かつ、ＮＧＣ７株の生育に適した培地を用いて、ＮＧＣ７株に適した各種培養条件下で形質転換微生物を培養することによって、バニリン酸を製造する方法などが挙げられる。培養方法は特に限定されず、例えば、通気条件下で行う固体培養法や液体培養法などが挙げられる。 The method of allowing the lignin-derived aromatic compound to act on the transformed microorganism is a method in which the lignin-derived aromatic compound and the transformed microorganism come into contact with each other, and vanillic acid is produced and/or accumulated by the enzymes of the transformed microorganism. However, for example, using a medium containing a lignin-derived aromatic compound and suitable for the growth of the NGC7 strain, the transformed microorganism is cultured under various culture conditions suitable for the NGC7 strain. A method for producing vanillic acid, etc., can be mentioned. The culture method is not particularly limited, and examples thereof include a solid culture method and a liquid culture method performed under aerated conditions.
　「バニリン酸の製造」は、バニリン酸の供給源からバニリン酸を得ることを意味し、例えば、バニリン酸の前駆体などの化合物からバニリン酸を合成及び変換すること；バニリン酸と、シリンガ酸、ｐ－ヒドロキシ安息香酸といったＳ型リグニン由来芳香族化合物及び／又はＨ型リグニン由来芳香族化合物が混在する中で、Ｓ型リグニン由来芳香族化合物及びＨ型リグニン由来芳香族化合物を分解しつつ、バニリン酸を分解しないことにより、バニリン酸を蓄積（濃縮）することなどが挙げられる。 "Production of vanillic acid" means obtaining vanillic acid from a source of vanillic acid, for example, synthesizing and converting vanillic acid from compounds such as vanillic acid precursors; vanillic acid, syringic acid, In the mixture of aromatic compounds derived from S-type lignin such as p -hydroxybenzoic acid and / or aromatic compounds derived from H-type lignin, while decomposing aromatic compounds derived from S-type lignin and aromatic compounds derived from H-type lignin, vanillin Accumulation (concentration) of vanillic acid by not decomposing acid can be mentioned.
　培地は、ＮＧＣ７株などのシュードモナス属微生物を培養する通常の培地、すなわち、炭素源、窒素源、無機物、その他の栄養素を適切な割合で含有するものであれば、合成培地及び天然培地のいずれでも使用でき、例えば、後述する実施例に記載があるようなＭＭｘ－３培地、Ｗｘ最少培地などを利用することができるが、特に限定されない。炭素源は、Ｓ型リグニン由来芳香族化合物、Ｈ型リグニン由来芳香族化合物、糖や有機酸などのその他の炭素源又はこれらの組み合わせを用いることができる。ただし、培地成分には、細胞の増殖や酵素の活性化に必要な成分、例えば、Ｍｇ２＋、Ｆｅ２＋など含まれることが好ましい。鉄イオン、マグネシウムイオンなどを化合物として培地に添加することができるが、ミネラル含有物として添加してもよい。 The medium may be a synthetic medium or a natural medium as long as it contains a normal medium for culturing Pseudomonas microorganisms such as the NGC7 strain, that is, a carbon source, a nitrogen source, inorganic substances, and other nutrients in appropriate proportions. For example, MMx-3 medium, Wx minimal medium, etc., as described in Examples below, can be used, but are not particularly limited. Carbon sources that can be used include aromatic compounds derived from S-type lignin, aromatic compounds derived from H-type lignin, other carbon sources such as sugars and organic acids, and combinations thereof. However, the medium components preferably contain components necessary for cell growth and enzyme activation, such as Mg 2+ and Fe 2+ . Iron ions, magnesium ions, etc. can be added to the medium as compounds, but they may also be added as mineral inclusions.
　Ｈ型リグニン由来の芳香族化合物及びＳ型リグニン由来の芳香族化合物は、Ｈ型リグニン及びＳ型リグニンのいずれかのリグニン並びにこれらのリグニンから誘導し得る芳香族化合物であれば特に限定されないが、例えば、Ｓ型リグニン、Ｈ型フェニルリグニンの分解物に相当する化合物などが挙げられ、具体的には、アセトシリンゴン、ｐ－ヒドロキシアセトフェノン、シリンガ酸、シリンガアルデヒド、ｐ－クマル酸、ｐ－ヒドロキシ安息香酸、プロトカテク酸などが挙げられる。 The aromatic compound derived from H-type lignin and the aromatic compound derived from S-type lignin are not particularly limited as long as they are either lignin of H-type lignin and S-type lignin and aromatic compounds that can be derived from these lignins. Examples thereof include compounds corresponding to decomposition products of S-type lignin and H-type phenyllignin. Specific examples include acetosyringone, p -hydroxyacetophenone, syringic acid, syringaldehyde, p -coumaric acid, p- Hydroxybenzoic acid, protocatechuic acid and the like.
　培地には、バニリン酸又はバニリン酸の前駆体となるＧ型リグニン由来の芳香族化合物が含まれる。Ｇ型リグニン由来の芳香族化合物は、Ｇ型リグニン及びＧ型リグニンから誘導し得る芳香族化合物であれば特に限定されないが、例えば、Ｇ型リグニンの分解物に相当する化合物などが挙げられ、具体的には、フェルラ酸、バニリン、アセトバニロンなどが挙げられる。 The medium contains vanillic acid or an aromatic compound derived from G-type lignin, which is a precursor of vanillic acid. The aromatic compound derived from G-type lignin is not particularly limited as long as it is a G-type lignin and an aromatic compound that can be derived from G-type lignin. Specific examples include ferulic acid, vanillin, acetovanillone, and the like.
　リグニン由来の芳香族化合物は、リグニンを含むバイオマスや該バイオマスを前処理に供して抽出したものであることが好ましいが、該バイオマスとは関係なく化学的に合成及び精製したものであってもよい。リグニン由来の芳香族化合物は、上記したものを単独で、又は２種以上を組み合わせて使用できる。 The lignin-derived aromatic compound is preferably a biomass containing lignin or a product obtained by subjecting the biomass to pretreatment and extracting it, but it may be chemically synthesized and purified regardless of the biomass. . The aromatic compounds derived from lignin can be used alone or in combination of two or more.
　リグニンを含むバイオマス（以下、リグノセルロースとよぶ場合がある。）は特に限定されないが、例えば、草や木などの天然物、これら天然物に処理を加えて得られるもの、農業廃棄物などが挙げられるが、具体的には広葉樹や針葉樹などの木質系のバイオマスなどが挙げられる。例えば、広葉樹はＳ型リグニンを多く含むことが知られており、針葉樹はＧ型リグニンを多く含むことが知られている。 Biomass containing lignin (hereinafter sometimes referred to as lignocellulose) is not particularly limited, but examples include natural products such as grasses and trees, products obtained by processing these natural products, and agricultural wastes. Specific examples include woody biomass such as broad-leaved trees and conifers. For example, broad-leaved trees are known to contain a large amount of S-type lignin, and coniferous trees are known to contain a large amount of G-type lignin.
　リグノセルロースは、前処理の有無などによって、例えば、固体状、懸濁状、液体状などであり得る。例えば、リグノセルロースを粉砕したものを液体に加えて得られる懸濁液とすることもできる。 Lignocellulose can be, for example, solid, suspended, liquid, etc., depending on the presence or absence of pretreatment. For example, a suspension obtained by adding pulverized lignocellulose to a liquid can be used.
　リグノセルロースは、リグニン抽出物であってもよい。リグニン抽出物としては、例えば、リグノセルロースの粉末化したものを、０．１％（ｗ／ｖ）～５０％（ｗ／ｖ）、好ましくは１％（ｗ／ｖ）～２０％（ｗ／ｖ）となるように、リグニンの抽出に適した溶媒中に懸濁した懸濁液などが挙げられる。また、リグニン抽出物は、該懸濁液を１０℃～１５０℃、好ましくは２０℃～１３０℃、より好ましくは２０℃～８０℃で、数時間～数日間、好ましくは１時間～６日間の抽出処理に供し、次いで抽出処理液から固形分を除いた液体状のリグニン抽出物、又は液体状のリグニン抽出物から溶媒を留去し、乾固することにより得られる、固体状のリグニン抽出物などであってもよい。リグノセルロースとしてスギ粉末を用いることによりスギリグニン抽出物、シラカバ粉末を用いることでシラカバリグニン抽出物が得られる。 The lignocellulose may be a lignin extract. As the lignin extract, for example, powdered lignocellulose is used at 0.1% (w/v) to 50% (w/v), preferably 1% (w/v) to 20% (w/v). v), such as a suspension suspended in a solvent suitable for lignin extraction. In addition, the lignin extract is prepared by heating the suspension at 10° C. to 150° C., preferably 20° C. to 130° C., more preferably 20° C. to 80° C. for several hours to several days, preferably 1 hour to 6 days. A liquid lignin extract obtained by subjecting it to an extraction treatment and then removing solids from the extraction treatment liquid, or a solid lignin extract obtained by distilling off the solvent from the liquid lignin extract and drying it. and so on. A cedar lignin extract can be obtained by using cedar powder as the lignocellulose, and a birch lignin extract can be obtained by using white birch powder.
　リグニン抽出物からの芳香族化合物の調製方法は特に限定されないが、例えば、以下の方法などが挙げられる。
　すなわち、小型オートクレーブ装置（耐圧硝子工業株式会社、ポータブルリアクター　ＴＶＳ－１）のステンレスベッセルに２Ｍ　ＮａＯＨ　５０ｍＬ、リグニン抽出物　０．５ｇ、ニトロベンゼン　３ｍＬを入れ、５００ｒｐｍで撹拌しながら１７０℃で２．５時間処理する。６０℃以下まで放冷し、遠心分離（６，０００ｘｇ、１０ｍｉｎ）により上清を回収する。得られた上清を、ジエチルエーテル抽出を３回繰り返す（水層を回収）。水層を塩酸で酸性化した後、ジエチルエーテル抽出を３回繰り返す（エーテル層を回収）。エーテル層に硫酸ナトリウムを加え、冷蔵庫内で一晩脱水処理する。エーテル層を回収し、抽出物を減圧乾固する。エーテル抽出物を、水酸化ナトリウムを加えながらイオン交換水に溶解し（ｐＨ≒９）、リグニン抽出物由来の芳香族化合物溶液とする。 A method for preparing an aromatic compound from a lignin extract is not particularly limited, and includes, for example, the following methods.
That is, put 50 mL of 2M NaOH, 0.5 g of lignin extract, and 3 mL of nitrobenzene in a stainless steel vessel of a small autoclave device (Portable Reactor TVS-1, Pressure Glass Industry Co., Ltd.), and stir at 500 rpm at 170 ° C. for 2.5 hours. process. Allow to cool to 60° C. or below, and collect the supernatant by centrifugation (6,000×g, 10 min). The resulting supernatant is subjected to diethyl ether extraction three times (recovering the aqueous layer). After acidifying the aqueous layer with hydrochloric acid, the diethyl ether extraction is repeated three times (collecting the ether layer). Add sodium sulfate to the ether layer and dehydrate overnight in the refrigerator. Collect the ether layer and dry the extract under reduced pressure. The ether extract is dissolved in ion-exchanged water (pH≈9) while sodium hydroxide is added to obtain an aromatic compound solution derived from the lignin extract.
　リグニンの抽出や芳香族化合物調製に適した溶媒は特に限定されず、例えば、水、ジオキサン、メタノール、イソプロパノールなどの低分子アルコール、ジエチルエーテル、ジメチルホルムアミドなどが挙げられる。 Solvents suitable for extracting lignin and preparing aromatic compounds are not particularly limited, and examples include water, low-molecular-weight alcohols such as dioxane, methanol, and isopropanol, diethyl ether, and dimethylformamide.
　また、例えば、銅などの金属フォームをリグニン分解用触媒として用いて、リグニン分解物を得ることもできる。例えば、リグニン分解物用触媒［Ｃｕ（ＯＨ）２／ＣＦ］をステンレス管中に装填して形成した触媒層に、リグノセルロースのアルカリ処理物及び酸素ガスを流し、次いで触媒層を通過した溶液を、酸によりアルカリ性、例えば、ｐＨ９～１１に調整し、次いでＭＦ膜、ＵＦ膜といったろ過膜に通過させてろ液を得て、次いで得られたろ液を、酸により酸性、例えば、ｐＨ２～４になるように調整し、次いで酢酸エチルを用いた抽出処理に供し、次いで得られた酢酸エチル層を濃縮、蒸発乾固することなどにより抽出物を得て、次いで得られた抽出物をアルカリ水溶液に溶解し、さらに酸により弱アルカリ性～中性に中和することにより、リグニン分解物を得ることができる。 Further, for example, a metal foam such as copper can be used as a lignin decomposition catalyst to obtain a lignin decomposition product. For example, a catalyst layer formed by loading a catalyst for lignin decomposition products [Cu(OH) 2 /CF] into a stainless steel tube is fed with an alkali-treated lignocellulose and oxygen gas, and then the solution that has passed through the catalyst layer is , pH is adjusted to pH 9 to 11 with an acid, then passed through a filtration membrane such as an MF membrane or a UF membrane to obtain a filtrate, and then the obtained filtrate is acidified with an acid to pH 2 to 4, for example. and then subjected to an extraction treatment using ethyl acetate, then the ethyl acetate layer obtained is concentrated and evaporated to dryness to obtain an extract, and then the obtained extract is dissolved in an aqueous alkaline solution. Then, by neutralizing to weakly alkaline to neutral with an acid, a lignin decomposition product can be obtained.
　培養条件は、当業者により通常知られるシュードモナス属微生物の培養条件を採用すればよく、例えば、培地の初発ｐＨは５～１０に調整し、培養温度は２０℃～４０℃、培養時間は数時間～数日間、好ましくは１日間～７日間、より好ましくは２日間～５日間など、適宜設定することができる。培養手段は特に限定されず、通気撹拌深部培養、振盪培養、静置培養などを採用することができるが、通気をするなどして溶存酸素濃度が十分になるような条件で培養することが好ましい。また、炭素源及びアセトバニロンの減少、バニリン酸の増加などに応じて、炭素源、リグニン抽出物、Ｇ型リグニン由来芳香族化合物などを追加する流加培養を採用してもよい。 As the culture conditions, the culture conditions for Pseudomonas microorganisms commonly known to those skilled in the art may be adopted. For example, the initial pH of the medium is adjusted to 5 to 10, the culture temperature is 20° C. to 40° C., and the culture time is several hours. It can be appropriately set to several days, preferably 1 to 7 days, more preferably 2 to 5 days. The culture method is not particularly limited, and aeration and agitation deep culture, shaking culture, static culture, etc. can be employed, but it is preferable to culture under conditions such as aeration so that the dissolved oxygen concentration is sufficient. . In addition, fed-batch culture may be employed in which carbon sources, lignin extracts, G-type lignin-derived aromatic compounds, etc. are added according to the decrease in carbon sources and acetovanillone and the increase in vanillic acid.
　例えば、培地及び培養条件の一例として、炭素源としてシリンガ酸及び／又はｐ－ヒドロキシ安息香酸を含み、バニリン酸の基質としてフェルラ酸やバニリンを含むＭＭｘ－３培地を用いた、３０℃、１８０ｒｐｍでの１時間～５日間の振盪培養や撹拌培養などが挙げられる。培地及び培養条件の別の一例として、後述する実施例に記載があるとおりの、バニリン酸の基質としてアセトバニロンを含むＬＢ培地を用いた、３０℃、１８０ｒｐｍ、溶存酸素濃度が５％～２０％での１時間～５日間の振盪培養や撹拌培養などが挙げられる。なお、炭素源その他の成分は、培養開始後に適宜追加することができる。 For example, as an example of the medium and culture conditions, MMx-3 medium containing syringic acid and/or p -hydroxybenzoic acid as a carbon source and ferulic acid and vanillin as substrates for vanillic acid was used at 30°C and 180 rpm. 1 hour to 5 days of shaking culture, stirring culture, and the like. As another example of the medium and culture conditions, LB medium containing acetovanillone as a vanillic acid substrate was used at 30° C., 180 rpm, and a dissolved oxygen concentration of 5% to 20%, as described in the Examples below. 1 hour to 5 days of shaking culture, stirring culture, and the like. Incidentally, the carbon source and other components can be added as appropriate after the culture is started.
　培養終了後に培養物からバニリン酸を取得する方法は特に限定されない。バニリン酸は培養液中に蓄積することから、培養物からろ過、遠心分離などの通常の固液分離操作により菌体と培養上清とを分離し、回収した培養上清からカラムを用いた固相抽出やバニリン酸が可溶性のある溶媒を用いた溶媒抽出などによりバニリン酸を抽出する。 The method of obtaining vanillic acid from the culture after the end of the culture is not particularly limited. Since vanillic acid accumulates in the culture medium, the cells and the culture supernatant are separated from the culture by normal solid-liquid separation operations such as filtration and centrifugation, and the collected culture supernatant is subjected to solidification using a column. Vanillic acid is extracted by phase extraction or solvent extraction using a solvent in which vanillic acid is soluble.
　抽出溶媒はバニリン酸が溶解するものであれば特に限定されず、例えば、酢酸エチル、ジエチルエーテルなどが挙げられる。 The extraction solvent is not particularly limited as long as it dissolves vanillic acid, and examples include ethyl acetate and diethyl ether.
　バニリン酸は沈殿、抽出、蒸留、再結晶、吸着剤など公知の方法や該方法を一部変更した方法などで培養液から分離精製することができる。精製方法の具体的一態様としては、例えば、培養上清に塩酸を加え、ｐＨを２～５に調整した後、ジエチルエーテルを添加して混合する。ジエチルエーテル層をエバポレーターで減圧乾固して、得られた析出物はイオン交換水で洗浄した後、吸引濾過により回収し、減圧乾燥する。乾燥した固体を氷酢酸に溶解して再結晶して、精製バニリン酸を得る。また、Ｇｏｍｅｓらの文献に記載の方法（Ｓｅｐａｒａｔｉｏｎ　ａｎｄ　Ｐｕｒｉｆｉｃａｔｉｏｎ　Ｔｅｃｈｎｏｌｏｇｙ、ｖｏｌ．２１６、ｐ９２－１０１、２０１９）など合成吸着剤を用いた方法、特開２０１７―１７１５９１号公報に記載の減圧蒸留による方法などでも精製バニリン酸を得ることはできる。 Vanillic acid can be separated and purified from the culture medium by known methods such as precipitation, extraction, distillation, recrystallization, and adsorbents, or by partially modifying the methods. As a specific embodiment of the purification method, for example, hydrochloric acid is added to the culture supernatant to adjust the pH to 2 to 5, and then diethyl ether is added and mixed. The diethyl ether layer is dried under reduced pressure using an evaporator, and the resulting precipitate is washed with deionized water, collected by suction filtration, and dried under reduced pressure. The dried solid is dissolved in glacial acetic acid and recrystallized to obtain purified vanillic acid. In addition, a method using a synthetic adsorbent such as the method described in the literature by Gomes et al. However, it is possible to obtain purified vanillic acid.
　バニリン酸の定性的又は定量的分析は、後述する実施例に記載されているＨＰＬＣにより実施すればよい。 The qualitative or quantitative analysis of vanillic acid may be performed by HPLC described in the examples below.
　本発明の一態様の形質転換微生物を用いれば、バニリン酸を分解することなく、選択的に得ることができる。
　例えば、形質転換微生物（１）を用いれば、５ｍＭ　バニリン酸及び５ｍＭ　シリンガ酸を炭素源とした場合は４８時間の培養で４．４ｍＭのバニリン酸を含み、かつシリンガ酸を含まない培養液を得ることができ；５ｍＭ　ｐ－ヒドロキシ安息香酸及び５ｍＭ　バニリン酸を炭素源とした場合は４８時間の培養で４．９ｍＭのバニリン酸を含み、かつｐ－ヒドロキシ安息香酸を含まない培養液を得ることができ；５ｍＭ　バニリン酸、５ｍＭ　ｐ－ヒドロキシ安息香酸及び５ｍＭ　シリンガ酸を炭素源とした場合は４８時間の培養で４．０ｍＭのバニリン酸を含み、かつｐ－ヒドロキシ安息香酸とシリンガ酸を含まない培養液を得ることができる。収率の上限は特に限定されず、典型的には消費されたＧ型リグニン由来の芳香族化合物（例えば、フェルラ酸、バニリン、バニリン酸）と培養液中に蓄積したバニリン酸から算出される量である。 By using the transformed microorganism of one embodiment of the present invention, vanillic acid can be selectively obtained without degrading it.
For example, if the transformed microorganism (1) is used and 5 mM vanillic acid and 5 mM syringic acid are used as carbon sources, a culture solution containing 4.4 mM vanillic acid and no syringic acid is obtained after culturing for 48 hours. When 5 mM p -hydroxybenzoic acid and 5 mM vanillic acid are used as carbon sources, a culture solution containing 4.9 mM vanillic acid and no p -hydroxybenzoic acid can be obtained after culturing for 48 hours. When 5 mM vanillic acid, 5 mM p -hydroxybenzoic acid and 5 mM syringic acid are used as a carbon source, culture containing 4.0 mM vanillic acid and containing no p -hydroxybenzoic acid and syringic acid after 48 hours of culture. You can get the liquid. The upper limit of the yield is not particularly limited, typically consumed aromatic compounds derived from G-type lignin (e.g., ferulic acid, vanillin, vanillic acid) and the amount calculated from vanillic acid accumulated in the culture medium is.
　例えば、形質転換微生物（２）を用いれば、０．２ｍＭ　アセトバニロンを基質とした場合は、培養後２４時間までにアセトバニロンをバニリン酸及びバニロイル酢酸に転換して、さらに培養することによりバニロイル酢酸はバニリン酸へ転換され、培養後３０時間でバニリン酸を含み、かつアセトバニロン及びバニロイル酢酸を含まない培養液を得ることができる。 For example, when using transformed microorganism (2), when 0.2 mM acetovanillone is used as a substrate, acetovanillone is converted to vanillic acid and vanilloyl acetic acid within 24 hours after culturing, and vanilloyl acetic acid is converted to vanillin by further culturing. After 30 hours of culturing, a culture medium containing vanillic acid and free of acetovanillone and vanilloyl acetic acid can be obtained.
　本発明の製造方法では、本発明の目的を達成し得る限り、上記した工程の前段若しくは後段又は工程中に、種々の工程や操作を加入することができる。 In the production method of the present invention, various steps and operations can be added before, after, or during the above steps as long as the object of the present invention can be achieved.
（バニリン酸の用途）
　本発明の一態様の形質転換微生物及び製造方法を利用して得られたバニリン酸は、種々の産業上有用な化合物に変換することができる。バニリン酸は、重合体に耐熱性及び剛性を付与する芳香環構造を有し、液晶ポリエステルなどの機能性ポリマー合成への利用が期待される。バニリン酸及びバニリン酸を修飾して得られるバニリン酸誘導体は、例えば、それ自体で、又は他の成分と共に使用して、耐熱性プラスチック、耐トラッキングプラスチック、液晶性共重合ポリエステル樹脂などとしての利用が期待される。 (Use of vanillic acid)
Vanillic acid obtained using the transformed microorganism and production method of one embodiment of the present invention can be converted into various industrially useful compounds. Vanillic acid has an aromatic ring structure that imparts heat resistance and rigidity to polymers, and is expected to be used in synthesizing functional polymers such as liquid crystal polyesters. Vanillic acid and vanillic acid derivatives obtained by modifying vanillic acid can be used, for example, by themselves or together with other components as heat-resistant plastics, anti-tracking plastics, liquid-crystalline copolymerized polyester resins, and the like. Be expected.
　以下、本発明を実施例によってさらに具体的に説明するが、本発明はこれら実施例に限定されるものではなく、本発明の課題を解決し得る限り、本発明は種々の態様をとることができる。 The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples, and the present invention can take various forms as long as the problems of the present invention can be solved. can.
［１．Ｐｓｅｕｄｏｍｏｎａｓ　ｓｐ．　ＮＧＣ７株の生物学的分類］
　シュードモナス・スピーシーズ（Ｐｓｅｕｄｏｍｏｎａｓ　ｓｐ．）　ＮＧＣ７株（以下、単にＮＧＣ７株ともよぶ。）は、微生物の識別の表示を「ＮＧＣ７」とし、かつ、受託番号を「ＮＩＴＥ　ＢＰ－０３０４３」として、独立行政法人製品評価技術基盤機構の特許微生物寄託センター（〒２９２－０８１８　千葉県木更津市かずさ鎌足２－５－８）に２０１９年１０月４日付けの受託日により寄託されている。ＮＧＣ７について、受託証の写しを図１Ａに示し、生存に関する証明書を図１Ｂに示す。 [1. Pseudomonas sp. Biological Classification of NGC7 Strain]
Pseudomonas sp. NGC7 strain (hereinafter also simply referred to as NGC7 strain) is an independent administrative agency product with the identification of microorganisms as "NGC7" and the accession number as "NITE BP-03043". It has been deposited with the Patent Microorganisms Depositary Center of the Evaluation Technology Platform (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture 292-0818) with a deposit date of October 4, 2019. For NGC7, a copy of the receipt is shown in FIG. 1A and a certificate of viability is shown in FIG. 1B.
　ＮＧＣ７株の性状を調べた試験結果を表１～表３に示す。 Tables 1 to 3 show the test results of investigating the properties of NGC7 strains.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
　１６Ｓ ｒＲＮＡ配列の解析から、ＮＧＣ７株はシュードモナス・プチダ（Ｐｓｅｕｄｏｍｏｎａｓ　ｐｕｔｉｄａ）ＮＢＲＣ１４１６４株、シュードモナス・プレコグロッシシダ（Ｐ．ｐｌｅｃｏｇｌｏｓｓｉｃｉｄａ）ＦＰＣ９５１株、シュードモナス・タイワネンシス（Ｐ．ｔａｉｗａｎｅｎｓｉｓ）ＢＣＲＣ１７７５１株及びシュードモナス・モンテイリー（Ｐ．ｍｏｎｔｅｉｌｉｉ）ＣＩＰ１０４８８３株の１６Ｓ　ｒＲＮＡ配列と９９．２％～９９．５％の配列同一性を示した（Ｓｈｉｎｏｄａ，Ｅ．ｅｔ　ａｌ．，Ｊ．Ｉｎｄ．Ｍｉｃｒｏｂｉｏｌ．Ｂｉｏｔｅｃｈｎｏｌ．４６（８），１０７１－１０８０．を参照）。 From the analysis of 16S rRNA sequences, the NGC7 strains were Pseudomonas putida strain NBRC14164, P. plecoglossicida strain FPC951 , P. taiwanensis BCRC17751 strain and Montemonas pseudomonas. monteilii ) showed 99.2% to 99.5% sequence identity with the 16S rRNA sequence of strain CIP104883 (Shinoda, E. et al., J. Ind. Microbiol. Biotechnol. 46(8), 1071-1080 .).
　表１～表３の結果から、ＮＧＣ７株は、運動性を有するグラム陰性桿菌であり、カタラーゼ反応及びオキシダーゼ反応は陽性を示し、グルコースを酸化した。これらの結果からＮＧＣ７株は、Ｐｓｅｕｄｏｍｏｎａｓ属の性状と一致した（例えば、Ｐａｌｌｅｒｏｎｉ　ＮＪ．Ｐｓｅｕｄｏｍｏｎａｄｓ．Ｉｎ：　Ｂｅｒｇｅｙ’ｓ　Ｍａｎｕａｌ　ｏｆ　Ｓｙｓｔｅｍａｔｉｃｓ　ｏｆ　Ａｒｃｈｅａｅａ　ａｎｄ　Ｂａｃｔｅｒｉａを参照）。 From the results in Tables 1 to 3, the NGC7 strain was a motile Gram-negative bacillus, was positive in catalase reaction and oxidase reaction, and oxidized glucose. From these results, the NGC7 strain was consistent with the properties of the genus Pseudomonas (see, for example, Palleroni NJ. Pseudomonads. In: Bergey's Manual of Systematics of Archeaea and Bacteria).
　ＡＰＩ２０ＮＥキット（ビオメリュー・ジャパン社）を用いて試験したところ、ＮＧＣ７株は、硝酸塩を還元せず、アルギニンジヒドロラーゼ活性を示し、ゼラチンを加水分解せず、グルコース、グルコン酸カリウム及びｎ－カプリン酸などを資化し、Ｌ－アラビノース、アジピン酸などを資化しなかった。また、Ｋｉｎｇ’ｓ寒天培地上で蛍光色素を産生し、レチシナーゼ及びリパーゼ（Ｔｗｅｅｎ８０）活性を示さず、４℃及び６％ＮａＣｌ存在下で生育し、４１℃及び７％ＮａＣｌ存在下では生育しないことが確認できた。これらのＮＧＣ７株が有する性状はＰ．ｐｕｔｉｄａの性状とよく一致した。しかし、１６Ｓ　ｒＲＮＡ遺伝子解析の結果から、ＮＧＣ７株がＰ．ｐｕｔｉｄａと異なる種に分類される可能性が示唆されることから、Ｐ．ｐｕｔｉｄａと似た性質を有するＰｓｅｕｄｏｍｏｎａｓ　ｓｐ．と推定した。 When tested using an API20NE kit (bioMérieux Japan), NGC7 strain does not reduce nitrate, exhibits arginine dihydrolase activity, does not hydrolyze gelatin, and does not hydrolyze glucose, potassium gluconate and n-capric acid. but did not assimilate L-arabinose, adipic acid, etc. In addition, it must produce a fluorescent pigment on King's agar medium, exhibit no reticinase and lipase (Tween80) activities, grow at 4°C in the presence of 6% NaCl, and not grow at 41°C in the presence of 7% NaCl. was confirmed. The properties possessed by these NGC7 strains are similar to those of P. putida well matched. However, from the results of 16S rRNA gene analysis, the NGC7 strain is a P. P. putida may be classified as a species different from P. putida. Pseudomonas sp . estimated.
［２．ＮＧＣ７ΔｖａｎＡＢ株の作製］
　以下の手順により、ＮＧＣ７株からバニレート　Ｏ－デメチラーゼ遺伝子（ｖａｎＡＢ）を破壊した変異株である、ＮＧＣ７ΔｖａｎＡＢ株を作製した。 [2. Preparation of NGC7Δ vanAB strain]
The NGC7Δ vanAB strain, which is a mutant strain in which the vanillate O -demethylase gene ( vanAB ) was disrupted from the NGC7 strain, was prepared by the following procedure.
　常法によりＮＧＣ７株のゲノムＤＮＡを調製し、全ヌクレオチド配列を解析した。Ｐ．ｐｕｔｉｄａ　ＫＴ２４４０株由来のバニレート　Ｏ－デメチラーゼ　オキシゲナーゼコンポーネント（ＶａｎＡ）及びバニレート　Ｏ－デメチラーゼ　オキシドレダクターゼコンポーネント（ＶａｎＢ）の推定アミノ酸配列と配列同一性が高いアミノ酸配列を有するタンパク質をコードしている遺伝子をＮＧＣ７株のゲノム配列から検索した。ＮＧＣ７株のゲノムＤＮＡには、ＶａｎＡと２５％以上の配列同一性を示したアミノ酸配列をコードしている遺伝子が４個あり（表４）、ＶａｎＢと３０％以上の配列同一性を示したアミノ酸配列をコードしている遺伝子が６個あることを確認した（表５）。 Genomic DNA of NGC7 strain was prepared by a conventional method, and the entire nucleotide sequence was analyzed. P. A gene encoding a protein having an amino acid sequence highly identical to the deduced amino acid sequences of vanillate O -demethylase oxygenase component (VanA) and vanillate O -demethylase oxidoreductase component (VanB) derived from S. putida strain KT2440 was isolated from strain NGC7. Searched from the genome sequence. In the genomic DNA of the NGC7 strain, there are 4 genes encoding amino acid sequences that show a sequence identity of 25% or more with VanA (Table 4), and there are amino acids that show a sequence identity of 30% or more with VanB. It was confirmed that there were 6 genes encoding sequences (Table 5).
　表４は、Ｐ．ｐｕｔｉｄａ　ＫＴ２４４０株由来ＶａｎＡの推定アミノ酸配列と２５％以上の配列同一性を示したアミノ酸配列をコードしているＮＧＣ７株由来の遺伝子群を示す。また、表５は、Ｐ．ｐｕｔｉｄａ　ＫＴ２４４０株由来ＶａｎＢの推定アミノ酸配列と３０％以上の配列同一性を示したアミノ酸配列をコードしているＮＧＣ７株由来の遺伝子群を示す。 Table 4 shows the results of P.S. FIG. 1 shows a gene cluster derived from strain NGC7 that encodes amino acid sequences that have a sequence identity of 25% or more with the deduced amino acid sequence of VanA derived from S. putida strain KT2440. Also, Table 5 shows FIG. 1 shows a gene cluster derived from strain NGC7 that encodes amino acid sequences that have a sequence identity of 30% or more with the deduced amino acid sequence of VanB derived from S. putida strain KT2440.
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
　酸素添加型のバニレート　Ｏ－デメチラーゼは、オキシゲナーゼコンポーネント（ＶａｎＡ）とオキシドレダクターゼコンポーネント（ＶａｎＢ）とから構成される。ｖａｎＢ５及びｖａｎＢ６の両遺伝子の周辺領域にはオキシゲナーゼコンポーネントであるＶａｎＡと同一性を示すアミノ酸配列をコードする領域は存在しなかった。そこで、ｖａｎＡ１及びｖａｎＢ１、ｖａｎＡ２及びｖａｎＢ２、ｖａｎＡ３及びｖａｎＢ３、並びにｖａｎＡ４及びｖａｎＢ４をペアの遺伝子セットと考えた。そして、それぞれの遺伝子セットを欠失させたＮＧＣ７ΔｖａｎＡ１Ｂ１株、ＮＧＣ７ΔｖａｎＡ２Ｂ２株、ＮＧＣ７ΔｖａｎＡ３Ｂ３株及びＮＧＣ７ΔｖａｎＡ４Ｂ４株を以下の手順により作製した。各遺伝子の全領域又はほぼ全領域を欠失させることにより行った。 The oxygenated form of vanillate O 2 -demethylase is composed of an oxygenase component (VanA) and an oxidoreductase component (VanB). In the peripheral regions of both vanB5 and vanB6 genes, there was no region encoding an amino acid sequence identical to the oxygenase component VanA. Therefore, vanA1 and vanB1 , vanA2 and vanB2 , vanA3 and vanB3 , and vanA4 and vanB4 were considered as paired gene sets. Then, NGC7Δ vanA1B1 strain, NGC7Δ vanA2B2 strain, NGC7Δ vanA3B3 strain and NGC7Δ vanA4B4 strain lacking each gene set were prepared by the following procedure. This was done by deleting the entire region or almost the entire region of each gene.
　Ｐｓｅｕｄｏｍｏｎａｓ　ｓｐ．　ＮＧＣ７株のゲノムＤＮＡを鋳型として、配列番号１９及び２０のプライマー１及び２からなるプライマーセットと、配列番号２１及び２２のプライマー３及び４からなるプライマーセットとをそれぞれ用いたＰＣＲ法によって、ｖａｎＡ１遺伝子の５’末端上流及びｖａｎＢ１遺伝子の３’末端下流の約１．０ｋｂｐのＤＮＡ断片をそれぞれ増幅した。それぞれの断片をあらかじめＢａｍＨＩで消化したｐＫ１８ｍｏｂｓａｃＢ（Ｇｅｎｅ、Ｖｏｌ．１４５、ｐ６９－７３、１９９４を参照）とＮＥＢｕｉｌｄｅｒ　ＨｉＦｉ　ＤＮＡアッセンブリー（Ｎｅｗ　Ｅｎｇｌａｎｄ　Ｂｉｏｌａｂｓ）を用いたシームレスクローニング法により連結させることにより、ｖａｎＡ１遺伝子、ｖａｎＢ１遺伝子領域欠失株作製用プラスミドｐｖａｎＡ１Ｂ１ｄｅｌを作製した。 Pseudomonas sp. Using the genomic DNA of the NGC7 strain as a template, a primer set consisting of primers 1 and 2 of SEQ ID NOS: 19 and 20, and a primer set consisting of primers 3 and 4 of SEQ ID NOS: 21 and 22, respectively, were used to perform PCR to obtain the vanA1 gene. A DNA fragment of about 1.0 kbp upstream of the 5' end of the vanB1 gene and downstream of the 3' end of the vanB1 gene was amplified, respectively. Each fragment was ligated with pK18 mobsacB (see Gene, Vol. 145, p69-73, 1994) previously digested with BamHI by a seamless cloning method using NEBuilder HiFi DNA assembly (New England Biolabs) to create the vanA1 gene. , a plasmid pvanA1B1del for producing a vanB1 gene region deletion strain was produced.
　同様に、ＮＧＣ７株のゲノムＤＮＡを鋳型として、配列番号２３及び２４のプライマー５及び６からなるプライマーセットと、配列番号２５及び２６のプライマー７及び８からなるプライマーセットとをそれぞれ用いたＰＣＲ法によって、ｖａｎＢ２遺伝子の５’末端上流とｖａｎＡ２遺伝子の３’末端下流の約１．０ｋｂｐのＤＮＡ断片をそれぞれ増幅した。それぞれの断片をあらかじめＢａｍＨＩで消化したｐＫ１８ｍｏｂｓａｃＢとＮＥＢｕｉｌｄｅｒ　ＨｉＦｉ　ＤＮＡアッセンブリーを用いたシームレスクローニング法により連結させることにより、ｖａｎＡ２遺伝子、ｖａｎＢ２遺伝子領域欠失株作製用プラスミドｐｖａｎＡ２Ｂ２ｄｅｌを作製した。 Similarly, by PCR using the genomic DNA of strain NGC7 as a template, a primer set consisting of primers 5 and 6 of SEQ ID NOs: 23 and 24, and a primer set consisting of primers 7 and 8 of SEQ ID NOs: 25 and 26, respectively. , a DNA fragment of about 1.0 kbp upstream of the 5' end of the vanB2 gene and downstream of the 3' end of the vanA2 gene were amplified, respectively. Plasmid pvanA2B2del for constructing vanA2 gene and vanB2 gene region deleted strains was prepared by ligating each fragment to pK18 mobsacB previously digested with BamHI by seamless cloning method using NEBuilder HiFi DNA assembly.
　同様に、ＮＧＣ７株のゲノムＤＮＡを鋳型として、配列番号２７及び２８のプライマー９及び１０からなるプライマーセットと、配列番号２９及び３０のプライマー１１及び１２からなるプライマーセットとをそれぞれ用いたＰＣＲ法によって、ｖａｎＢ３遺伝子の５’末端上流とｖａｎＡ３遺伝子の３’末端下流の約１．０ｋｂｐのＤＮＡ断片をそれぞれ増幅した。それぞれの断片をあらかじめＢａｍＨＩで消化したｐＫ１８ｍｏｂｓａｃＢとＮＥＢｕｉｌｄｅｒ　ＨｉＦｉ　ＤＮＡアッセンブリーを用いたシームレスクローニング法により連結させることにより、ｖａｎＡ３遺伝子、ｖａｎＢ３遺伝子領域欠失株作製用プラスミドｐｖａｎＡ３Ｂ３ｄｅｌを作製した。 Similarly, by PCR using the genomic DNA of the NGC7 strain as a template, a primer set consisting of primers 9 and 10 of SEQ ID NOs: 27 and 28 and a primer set consisting of primers 11 and 12 of SEQ ID NOs: 29 and 30, respectively. , approximately 1.0 kbp DNA fragments upstream of the 5' end of the vanB3 gene and downstream of the 3' end of the vanA3 gene were amplified, respectively. Plasmid pvanA3B3del for constructing vanA3 gene and vanB3 gene region deleted strains was prepared by ligating each fragment to pK18 mobsacB previously digested with BamHI and seamless cloning method using NEBuilder HiFi DNA assembly.
　同様に、ＮＧＣ７株のゲノムＤＮＡを鋳型として、配列番号３１及び３２のプライマー１３及び１４からなるプライマーセットと、配列番号３３及び３４のプライマー１５及び１６からなるプライマーセットとをそれぞれ用いたＰＣＲ法によって、ｖａｎＡ４遺伝子の５’末端上流とｖａｎＢ４遺伝子の３’末端下流の約１．０ｋｂｐのＤＮＡ断片をそれぞれ増幅した。それぞれの断片をあらかじめＢａｍＨＩで消化したｐＫ１８ｍｏｂｓａｃＢとＮＥＢｕｉｌｄｅｒ　ＨｉＦｉ　ＤＮＡアッセンブリーを用いたシームレスクローニング法により連結させることにより、ｖａｎＡ４遺伝子、ｖａｎＢ４遺伝子領域欠失株作製用プラスミドｐｖａｎＡ４Ｂ４ｄｅｌを作製した。 Similarly, by PCR using the genomic DNA of the NGC7 strain as a template, a primer set consisting of primers 13 and 14 of SEQ ID NOS: 31 and 32, and a primer set consisting of primers 15 and 16 of SEQ ID NOS: 33 and 34, respectively. , a DNA fragment of about 1.0 kbp upstream of the 5' end of the vanA4 gene and downstream of the 3' end of the vanB4 gene were amplified, respectively. Plasmid pvanA4B4del for constructing vanA4 gene and vanB4 gene region deleted strains was prepared by ligating each fragment to pK18 mobsacB previously digested with BamHI by seamless cloning method using NEBuilder HiFi DNA assembly.
　以下のようにして、作製したプラスミドｐｖａｎＡ１Ｂ１ｄｅｌ、ｐｖａｎＡ２Ｂ２ｄｅｌ、ｐｖａｎＡ３Ｂ３ｄｅｌ及びｐｖａｎＡ４Ｂ４ｄｅｌを、それぞれエレクトロポレーション法によりＰｓｅｕｄｏｍｏｎａｓ　ｓｐ．ＮＧＣ７株に導入した。 Plasmids pvanA1B1del, pvanA2B2del, pvanA3B3del and pvanA4B4del prepared as follows were electroporated into Pseudomonas sp. It was introduced into the NGC7 strain.
　ＬＢ液体培地１０ｍＬで振盪培養して得たＰｓｅｕｄｏｍｏｎａｓ　ｓｐ．ＮＧＣ７株細胞を、３ｍＬの０．５Ｍシュークロース水溶液で洗浄した後、１ｍＬの０．５Ｍシュークロース水溶液に懸濁した。菌体懸濁液と前記プラスミドとを混合し、Ｇｅｎｅ　Ｐｕｌｓｅｒ　ＸｃｅｌｌＴＭ（Ｂｉｏ－Ｒａｄ　Ｌａｂｏｒａｔｏｒｉｅｓ）を用いて、２００Ω、２５μＦ、２．５ｋＶの条件で印加した。印加後ただちに１ｍＬのＳＯＣ（２０ｇ／Ｌ　トリプトン、０．５ｇ／Ｌ　酵母エキス、１０ｍＭ　ＮａＣｌ、２．５ｍＭ　ＫＣｌ、１０ｍＭ　ＭｇＣｌ２・６Ｈ２Ｏ、１０ｍＭ　ＭｇＳＯ４・７Ｈ２Ｏ）液体培地を加えて、３０℃で１時間振盪培養し、カナマイシン（Ｋｍ）５０ｍｇ／Ｌを含むＬＢ寒天培地上で生育可能であるＫｍ耐性株として選抜した。 Pseudomonas sp. obtained by shaking culture in 10 mL of LB liquid medium. NGC7 strain cells were washed with 3 mL of 0.5 M sucrose aqueous solution, and then suspended in 1 mL of 0.5 M sucrose aqueous solution. The cell suspension and the plasmid were mixed and applied under the conditions of 200Ω, 25 μF and 2.5 kV using Gene Pulser Xcell ™ (Bio-Rad Laboratories). Immediately after application, 1 mL of SOC (20 g/L tryptone, 0.5 g/L yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl 2.6H 2 O, 10 mM MgSO 4.7H 2 O ) liquid medium was added, Shaking culture was carried out at 30° C. for 1 hour, and a Km-resistant strain that was able to grow on LB agar medium containing 50 mg/L of kanamycin (Km) was selected.
　得られたＫｍ耐性株を、２５％のスクロースを含むＹＴ寒天培地（１０ｇ／Ｌ　酵母エキス、２０ｇ／Ｌ　トリプトン、１８ｇ／Ｌ　寒天）上に塗抹し、３０℃で静置培養した。コロニーダイレクトＰＣＲによりゲノムＤＮＡ上の各ｖａｎＡＢ遺伝子の内部領域が欠失したことが確認できた形質転換体を、それぞれＮＧＣ７ΔｖａｎＡ１Ｂ１株、ＮＧＣ７ΔｖａｎＡ２Ｂ２株、ＮＧＣ７ΔｖａｎＡ３Ｂ３株、及びＮＧＣ７ΔｖａｎＡ４Ｂ４株とした。 The obtained Km-resistant strain was smeared on a YT agar medium (10 g/L yeast extract, 20 g/L tryptone, 18 g/L agar) containing 25% sucrose and statically cultured at 30°C. Transformants in which it was confirmed that the internal region of each vanAB gene on the genomic DNA had been deleted by colony direct PCR were designated as NGC7Δ vanA1B1 strain, NGC7Δ vanA2B2 strain, NGC7Δ vanA3B3 strain, and NGC7Δ vanA4B4 strain, respectively.
［３．バニリン酸（ＶＡ）及びシリンガ酸（ＳＡ）を基質とした、ＮＧＣ７ΔｖａｎＡＢ株の増殖性の評価］
　ＮＧＣ７株、ＮＧＣ７ΔｖａｎＡ１Ｂ１株、ＮＧＣ７ΔｖａｎＡ２Ｂ２株、ＮＧＣ７ΔｖａｎＡ３Ｂ３株及びＮＧＣ７ΔｖａｎＡ４Ｂ４株をそれぞれＬＢ液体培地１０ｍＬに接種し、３０℃、１８時間の振盪培養に供した。得られた培養液を遠心分離に供して菌体を回収し、得られた菌体を炭素源を含まないＷｘ－ｓｏｌｕｔｉｏｎ［９．８ｇ／Ｌ　Ｎａ２ＨＰＯ４・１２Ｈ２Ｏ、１．７ｇ／Ｌ　ＫＨ２ＰＯ４、１．０ｇ／Ｌ（ＮＨ４）２ＳＯ４］で洗浄した後、唯一の炭素源として５ｍＭ　バニリン酸（ＶＡ）又は５ｍＭ　シリンガ酸（ＳＡ）を含むＷｘ液体培地［９．８ｇ／Ｌ　Ｎａ２ＨＰＯ４・１２Ｈ２Ｏ、１．７ｇ／Ｌ　ＫＨ２ＰＯ４、１．０ｇ／Ｌ　（ＮＨ４）２ＳＯ４、１００ｍｇ／Ｌ　ＭｇＳＯ４・７Ｈ２Ｏ、９．５ｍｇ／Ｌ　ＦｅＳＯ４・７Ｈ２Ｏ、１０．７５ｍｇ／Ｌ　ＭｇＯ、２ｍｇ／Ｌ　ＣａＣＯ３、１．４４ｍｇ／Ｌ　ＺｎＳＯ４・７Ｈ２Ｏ、１．１２ｍｇ／Ｌ　ＭｎＳＯ４・４Ｈ２Ｏ、０．２５ｍｇ／Ｌ　ＣｕＳＯ４・５Ｈ２Ｏ、０．２８ｍｇ／Ｌ　ＣｏＳＯ４・７Ｈ２Ｏ、０．０６ｍｇ／Ｌ　Ｈ３ＢＯ３、５１．３μＬ　１２Ｎ　ＨＣｌ］０．２ｍＬに、６００ｎｍの波長を用いたときの光学密度（ｏｐｔｉｃａｌ　ｄｅｎｓｉｔｙ；ＯＤ６００）が０．１になるように接種し、３０℃の振盪培養（５６７ｃｐｍ）に供した。 [3. Evaluation of proliferation of NGC7ΔvanAB strain using vanillic acid (VA) and syringic acid (SA) as substrates]
NGC7 strain, NGC7ΔvanA1B1 strain, NGC7ΔvanA2B2 strain, NGC7ΔvanA3B3 strain and NGC7ΔvanA4B4 strain were each inoculated into 10 mL of LB liquid medium and subjected to shaking culture at 30° C. for 18 hours. The resulting culture solution was subjected to centrifugation to collect the cells, and the obtained cells were added to a carbon source-free Wx-solution [9.8 g/L Na 2 HPO 4 ·12H 2 O, 1.7 g/ L KH 2 PO 4 , 1.0 g/L (NH 4 ) 2 SO 4 ], followed by washing with Wx liquid medium [9. 8g/L Na2HPO4.12H2O , 1.7g /L KH2PO4 , 1.0g /L ( NH4 ) 2SO4 , 100mg /L MgSO4.7H2O , 9.5mg /L FeSO4.7H2O , 10.75 mg/L MgO, 2 mg/L CaCO3 , 1.44 mg/L ZnSO4.7H2O , 1.12 mg/L MnSO4.4H2O , 0.25 mg/L CuSO 4.5H2O , 0.28 mg/L CoSO4.7H2O , 0.06 mg/L H3BO3 , 51.3 μL 12N HCl] in 0.2 mL optical density using a wavelength of 600 nm ( The seedlings were inoculated so that the optical density (OD 600 ) was 0.1, and subjected to shaking culture (567 cpm) at 30°C.
　培養開始後、一定時間毎に培養液のＯＤ６００を測定した。ＯＤ測定には分光光度計「Ｅｐｏｃｈ　２」（ＢｉｏＴｅｋ　Ｉｎｓｔｒｕｍｅｎｔｓ社）で測定した。ＶＡを炭素源とした場合のＯＤ６００の測定結果を図２Ａに示し、ＳＡを炭素源とした場合のＯＤ６００の測定結果を図２Ｂに示す。 After starting the culture, the OD 600 of the culture solution was measured at regular intervals. The OD was measured with a spectrophotometer "Epoch 2" (BioTek Instruments). FIG. 2A shows the results of OD 600 measurements with VA as the carbon source, and FIG. 2B shows the results of OD 600 measurements with SA as the carbon source.
　図２Ａに示すとおり、ＶＡを唯一の炭素源とした培地条件では、ＮＧＣ７株、ＮＧＣ７ΔｖａｎＡ１Ｂ１株、ＮＧＣ７ΔｖａｎＡ２Ｂ２株及びＮＧＣ７ΔｖａｎＡ３Ｂ３株は増殖したが、ＮＧＣ７ΔｖａｎＡ４Ｂ４株は増殖しなかった。 As shown in FIG. 2A, in the medium condition with VA as the only carbon source, strains NGC7, NGC7Δ vanA1B1 , NGC7Δ vanA2B2 and NGC7Δ vanA3B3 grew , but NGC7Δ vanA4B4 did not grow.
　しかし、図２Ｂに示すとおり、ＳＡを唯一の炭素源とした培地条件では、ＮＧＣ７ΔｖａｎＡ４Ｂ４株は、ＮＧＣ７株と同様の増殖プロファイルを示した。すなわち、ｖａｎＡ４遺伝子及びｖａｎＢ４遺伝子にコードされるバニレート　Ｏ－デメチラーゼが、ＮＧＣ７株のＶＡ分解に関わる酸素添加型のバニレート　Ｏ－デメチラーゼであること、及びｖａｎＡ４遺伝子及びｖａｎＢ４遺伝子を欠損してもＳＡの分解性を維持できることがわかった。 However, as shown in FIG. 2B, the NGC7Δ vanA4B4 strain showed a growth profile similar to that of the NGC7 strain under the medium condition with SA as the sole carbon source. That is, the vanillate O -demethylase encoded by the vanA4 gene and the vanB4 gene is an oxygenation-type vanillate O -demethylase involved in the decomposition of VA in the NGC7 strain, and even if the vanA4 gene and the vanB4 gene are deleted, SA is degraded. I have found that I can maintain my sexuality.
［４．ＮＧＣ７ΔｖａｎＡＢ株が有するＶＡ及びＳＡの分解性の評価］
　ＮＧＣ７株、ＮＧＣ７ΔｖａｎＡ１Ｂ１株、ＮＧＣ７ΔｖａｎＡ２Ｂ２株、ＮＧＣ７ΔｖａｎＡ３Ｂ３株及びＮＧＣ７ΔｖａｎＡ４Ｂ４株を、それぞれＬＢ液体培地１０ｍＬに接種し、３０℃、一晩の振盪培養に供した。得られた培養液０．１ｍＬを新しいＬＢ液体培地１０ｍＬに接種し、３０℃、１８時間の振盪培養に供した。 [4. Evaluation of degradability of VA and SA possessed by NGC7Δ vanAB strain]
NGC7 strain, NGC7Δ vanA1B1 strain, NGC7Δ vanA2B2 strain, NGC7Δ vanA3B3 strain, and NGC7Δ vanA4B4 strain were each inoculated into 10 mL of LB liquid medium and subjected to shaking culture overnight at 30°C. 0.1 mL of the obtained culture solution was inoculated into 10 mL of fresh LB liquid medium and subjected to shaking culture at 30° C. for 18 hours.
　得られた培養液を遠心分離に供して菌体を回収し、得られた菌体を５０ｍＭ　Ｔｒｉｓ－ＨＣｌ緩衝液（ｐＨ７．５）培地に懸濁し、ＯＤ６００が２になるような細胞懸濁液１ｍＬを調製した。得られた細胞懸濁液に、終濃度が０．１ｍＭになるようにＶＡ又はＳＡを添加し、次いで３０℃の振盪培養に供した。 The obtained culture solution is subjected to centrifugation to collect the cells, and the obtained cells are suspended in a 50 mM Tris-HCl buffer (pH 7.5) medium to obtain a cell suspension having an OD 600 of 2. 1 mL of liquid was prepared. VA or SA was added to the resulting cell suspension to a final concentration of 0.1 mM, followed by shaking culture at 30°C.
　培養開始後、一定時間毎にサンプリングし、培養液を遠心分離して得た培養上清について、ＳＡ及びＶＡの濃度を測定した。 After the start of the culture, samples were taken at regular intervals, and the concentrations of SA and VA were measured for the culture supernatant obtained by centrifuging the culture solution.
　ＯＤ６００測定には「ＧｅｎｅＱｕａｎｔ　１００」（ＧＥヘルスケア・ジャパン社）を用いた。 "GeneQuant 100" (GE Healthcare Japan) was used for OD600 measurement.
　ＳＡ及びＶＡの濃度は、高速液体クロマトグラフ（「Ａｃｑｕｉｔｙ　ｕｌｔｒａｐｅｒｆｏｒｍａｎｃｅ　ｌｉｑｕｉｄ　ｃｈｒｏｍａｔｏｇｒａｐｈｙ　ｓｙｓｔｅｍ」、日本ウォーターズ社）を用いて測定した。カラムはＴＳＫｇｅｌ　ＯＤＳ－１４０ＨＴＰ　ｃｏｌｕｍｎ（径　２．１ｍｍ、長さ　１００ｍｍ、粒径　２．３μｍ；東ソー社）を使用し、３０℃で保温した。アイソクラチックモードで溶媒を９０％（ｖ／ｖ）Ｈ２Ｏ、０．１％（ｖ／ｖ）ＨＣＯＯＨ、１０％（ｖ／ｖ）ＣＨ３ＣＮを使用した。移動相の流速は０．５ｍＬ／ｍｉｎとし、測定波長はＳＡについて２７０ｎｍとし、ＶＡについて２６０ｎｍとした。 The concentrations of SA and VA were measured using a high-performance liquid chromatograph (“Acquity ultraperformance liquid chromatography system”, Nippon Waters Co., Ltd.). A TSKgel ODS-140HTP column (diameter: 2.1 mm, length: 100 mm, particle size: 2.3 µm; Tosoh Corporation) was used and kept at 30°C. Solvents used were 90% (v/v) H2O , 0.1% (v/v) HCOOH, 10% (v/v) CH3CN in isocratic mode. The mobile phase flow rate was 0.5 mL/min, and the measurement wavelength was 270 nm for SA and 260 nm for VA.
　培養開始時のＳＡ濃度及びＶＡ濃度を基準にした、各測定時間でのＳＡ濃度及びＶＡ濃度の減少率を図３Ａ及び図３Ｂにそれぞれ示す。 　Figures 3A and 3B show the reduction rates of the SA and VA concentrations at each measurement time, based on the SA and VA concentrations at the start of the culture.
　図３Ａに示すとおり、ＶＡを添加した条件では、ＮＧＣ７株は添加したＶＡを全て分解したが、ＮＧＣ７ΔｖａｎＡ４Ｂ４株はＶＡをほとんど分解しなかった。一方、図３Ｂに示すとおり、ＳＡを添加した条件では、ＮＧＣ７株と同様にＮＧＣ７ΔｖａｎＡ４Ｂ４株はＳＡを分解した。 As shown in FIG. 3A, under the condition of adding VA, NGC7 strain degraded all the added VA, but NGC7ΔvanA4B4 strain hardly degraded VA. On the other hand, as shown in FIG. 3B, the NGC7ΔvanA4B4 strain degraded SA in the same manner as the NGC7 strain under SA-supplemented conditions.
　以上の結果より、ｖａｎＡ４遺伝子及びｖａｎＢ４遺伝子にコードされるバニレート　Ｏ－デメチラーゼは、ＮＧＣ７株のＶＡ分解に関わる酸素添加型のバニレート　Ｏ－デメチラーゼであること、及びｖａｎＡ４遺伝子及びｖａｎＢ４遺伝子を欠損したＮＧＣ７株はＳＡの分解性を維持できることがわかった。 From the above results, the vanillate O -demethylase encoded by the vanA4 gene and the vanB4 gene is an oxygenated vanillate O -demethylase involved in VA decomposition of the NGC7 strain, and the NGC7 strain lacking the vanA4 gene and the vanB4 gene. can maintain the degradability of SA.
［５．ＮＧＣ７ΔｖａｎＡ４Ｂ４株を用いたリグニン由来芳香族化合物からのＶＡ蓄積性の評価］
　ＮＧＣ７ΔｖａｎＡ４Ｂ４株を、ＬＢ液体培地　１０ｍＬに接種し、３０℃、一晩の振盪培養に供した。得られた培養液　０．１ｍＬを新しいＬＢ液体培地　１０ｍＬに接種し、３０℃、１６時間の振盪培養に供した。 [5. Evaluation of VA accumulation from lignin-derived aromatic compounds using NGC7Δ vanA4B4 strain]
The NGC7Δ vanA4B4 strain was inoculated into 10 mL of LB liquid medium and cultured with shaking at 30°C overnight. 0.1 mL of the resulting culture solution was inoculated into 10 mL of new LB liquid medium and subjected to shaking culture at 30° C. for 16 hours.
　得られた培養液を遠心分離に供して菌体を回収し、得られた菌体を生理食塩水で洗浄した後、再度生理食塩水に懸濁し、ＯＤ６００が５になるように細胞懸濁液　１ｍＬを調製した。得られた細胞懸濁液　０．２ｍＬを、炭素源としてＳＡ、ＶＡ、ｐ－ヒドロキシ安息香酸（ＨＢＡ）、ＳＡ及びＶＡの混合物（ＳＡ－ＶＡ）、ＳＡ及びＨＢＡの混合物（ＳＡ－ＨＢＡ）、ＶＡ及びＨＢＡの混合物（ＶＡ－ＨＢＡ）又はＳＡ、ＶＡ及びＨＢＡの混合物（ＳＡ－ＶＡ－ＨＢＡ）を含むＭＭｘ－３液体培地［３４．２ｇ／Ｌ　Ｎａ２ＨＰＯ４・１２Ｈ２Ｏ、６．０ｇ／Ｌ　ＫＨ２ＰＯ４、１．０ｇ／Ｌ　ＮａＣｌ、２．５ｇ／Ｌ（ＮＨ４）２ＳＯ４、４９．３ｍｇ／Ｌ　ＭｇＳＯ４・７Ｈ２Ｏ、１５ｍｇ／Ｌ　ＣａＣｌ２・２Ｈ２Ｏ、５ｍｇ／Ｌ　ＦｅＳＯ４・７Ｈ２Ｏ］（それぞれの化合物の終濃度はそれぞれ５ｍＭ）１０ｍＬに添加し、３０℃の振盪培養に供した。 The resulting culture solution is subjected to centrifugation to collect the cells, the cells are washed with physiological saline, and then suspended again in physiological saline to obtain a cell suspension having an OD 600 of 5. 1 mL of liquid was prepared. 0.2 mL of the resulting cell suspension was added as a carbon source to SA, VA, p -hydroxybenzoic acid (HBA), a mixture of SA and VA (SA-VA), a mixture of SA and HBA (SA-HBA), MMx-3 liquid medium containing a mixture of VA and HBA (VA-HBA) or a mixture of SA, VA and HBA (SA-VA-HBA) [34.2 g/L Na 2 HPO 4.12H 2 O, 6.0 g /L KH2PO4 , 1.0 g/L NaCl, 2.5 g/L ( NH4) 2SO4 , 49.3 mg/L MgSO4.7H2O , 15 mg/ L CaCl2.2H2O , 5 mg /L FeSO 4 .7H 2 O] (final concentration of each compound is 5 mM) and subjected to shaking culture at 30°C.
　培養開始後、一定時間毎にサンプリングし、培養液のＯＤ６００を測定し、さらに培養液を遠心分離して得た培養上清について、ＳＡ、ＶＡ及びＨＢＡの濃度を測定した。 After the start of the culture, samples were taken at regular time intervals, the OD 600 of the culture solution was measured, and the culture supernatant obtained by centrifuging the culture solution was measured for the concentrations of SA, VA and HBA.
　ＯＤ６００測定には「ＯＤ－ＭｏｎｉｔｏｒＣ＆Ｔ」（タイテック社）を用いた。 “OD-Monitor C&T” (Taitec Co., Ltd.) was used for OD 600 measurement.
　ＳＡ、ＶＡ及びＨＢＡの濃度は、高速液体クロマトグラフ（「Ａｇｉｌｅｎｔ１２００シリーズ」、アジレントテクノロジー社）を用いて測定した。カラムはＺＯＲＢＡＸ　Ｅｃｌｉｐｓｅ　Ｐｌｕｓ　Ｃ１８　ｃｏｌｕｍｎ（径　４．６ｍｍ、長さ　１５０ｍｍ、粒径　０．５μｍ）を使用し、４０℃で保温した。勾配（グラジエント）溶離モード（溶媒Ａ：５％（ｖ／ｖ）ＣＨ３ＯＨ、１％（ｖ／ｖ）ＣＨ３ＣＯＯＨ、溶媒Ｂ：５０％（ｖ／ｖ）ＣＨ３ＯＨ、１％（ｖ／ｖ）ＣＨ３ＣＯＯＨ）を使用し、溶媒Ａで平衡化した後、分析開始から８分かけて溶媒Ｂの割合を２０％まで上昇させ、その後５分かけて溶媒Ｂの割合を１００％まで上昇させた。移動相の流速は１．０ｍＬ／ｍｉｎとし、測定波長は２５４ｎｍ及び２８０ｎｍを用いた。 The concentrations of SA, VA and HBA were measured using a high-performance liquid chromatograph (“Agilent 1200 series”, Agilent Technologies). A ZORBAX Eclipse Plus C18 column (diameter: 4.6 mm, length: 150 mm, particle size: 0.5 µm) was used and kept at 40°C. Gradient elution mode (solvent A: 5% (v/v) CH3OH , 1% (v/v) CH3COOH, solvent B: 50% (v/v) CH3OH , 1% (v /v) CH 3 COOH), equilibrated with solvent A, then increasing the percentage of solvent B to 20% over 8 minutes from the start of the analysis, then increasing the percentage of solvent B to 100% over 5 minutes. raised. The flow rate of the mobile phase was 1.0 mL/min, and the measurement wavelengths were 254 nm and 280 nm.
　炭素源としてＳＡ、ＶＡ、ＨＢＡ、ＳＡ－ＶＡ、ＳＡ－ＨＢＡ、ＶＡ－ＨＢＡ及びＳＡ－ＶＡ－ＨＢＡを用いて測定した結果を図４Ａ～図４Ｇに示す。 The results of measurements using SA, VA, HBA, SA-VA, SA-HBA, VA-HBA and SA-VA-HBA as carbon sources are shown in FIGS. 4A to 4G.
　図４Ａ～図４Ｇが示すように、ＮＧＣ７ΔｖａｎＡ４Ｂ４株は、ＳＡ及びＨＢＡの少なくともいずれか一方を炭素源とした場合は増殖し、ＶＡを唯一の炭素源とした場合は増殖しなかった。 As shown in FIGS. 4A to 4G, the NGC7Δ vanA4B4 strain grew when at least one of SA and HBA was used as the carbon source, but did not grow when VA was used as the sole carbon source.
　特に、図４Ｆが示すとおり、ＶＡ－ＨＢＡを炭素源とした場合は、ＮＧＣ７ΔｖａｎＡ４Ｂ４株は、ＨＢＡを分解したものの、９８％以上のＶＡが分解されずに蓄積した。図４Ｄが示すとおり、ＳＡ－ＶＡを炭素源とした場合も同様に、ＳＡが優先して分解され、９０％以上のＶＡは分解されずに蓄積した。そして、図４Ｇが示すとおり、ＳＡ－ＶＡ－ＨＢＡを炭素源とした場合でも、８０％以上のＶＡは分解されずに蓄積した。 In particular, as shown in FIG. 4F, when VA-HBA was used as the carbon source, NGC7Δ vanA4B4 strain degraded HBA, but accumulated 98% or more of VA without degradation. As shown in FIG. 4D, when SA-VA was used as the carbon source, SA was preferentially decomposed, and 90% or more of VA accumulated without being decomposed. Then, as shown in FIG. 4G, even when SA-VA-HBA was used as the carbon source, 80% or more of VA accumulated without being decomposed.
［６．ＮＧＣ７ΔｖａｎＡ４Ｂ４株を用いたリグニン由来芳香族化合物からのＶＡ生産性の評価］
　Ｓｐｈｉｎｇｏｂｉｕｍ　ｓｐ．ＳＹＫ－６株（以下、ＳＹＫ－６株ともよぶ。）はリグニン由来の芳香族化合物の一つであるアセトバニロン（ＡＶ）及びアセトシリンゴンの分解能を有する。そして、ＳＹＫ－６株は、ＡＶの分解に関わるＡＶ分解酵素遺伝子群として、ａｃｖＡ遺伝子、ａｃｖＢ遺伝子、ａｃｖＣ遺伝子、ａｃｖＤ遺伝子、ａｃｖＥ遺伝子、ａｃｖＦ遺伝子、ｖｃｅＡ遺伝子及びｖｃｅＢ遺伝子を有する。しかし、これらの酵素遺伝子群を利用してＡＶからＶＡを生産したという報告は無い。そこで、以下のとおりに、ＮＧＣ７ΔｖａｎＡ４Ｂ４株にＡＶ分解酵素遺伝子群を導入することにより、ＡＶからＶＡを生産することを試みた。 [6. Evaluation of VA productivity from lignin-derived aromatic compounds using NGC7Δ vanA4B4 strain]
Sphingobium sp. The SYK-6 strain (hereinafter also referred to as SYK-6 strain) has the ability to decompose acetovanilone (AV) and acetosyringone, which are aromatic compounds derived from lignin. The SYK-6 strain has the following AV degrading enzyme gene clusters involved in AV degradation: acvA gene, acvB gene, acvC gene, acvD gene, acvE gene, acvF gene, vceA gene and vceB gene. However, there is no report that VA was produced from AV using these enzyme gene clusters. Therefore, an attempt was made to produce VA from AV by introducing an AV degrading enzyme gene group into the NGC7Δ vanA4B4 strain as follows.
　常法に従って、ＳＹＫ－６株由来のＡＶ分解酵素遺伝子群のうち、ＳＹＫ－６株のゲノムＤＮＡからａｃｖＡ遺伝子、ａｃｖＢ遺伝子、ａｃｖＣ遺伝子、ａｃｖＤ遺伝子、ａｃｖＥ遺伝子及びａｃｖＦ遺伝子を含む核酸断片を得て、該核酸断片を、ｐＳＥＶＡ２４１プラスミドＤＮＡに挿入した大腸菌由来ラクトースプロモーター制御下に連結し、プラスミドｐＳＥＶＡ２４１＿Ｐ ｌａｃ －ａｃｖを得た。 A nucleic acid fragment containing the acvA gene, the acvB gene, the acvC gene, the acvD gene , the acvE gene and the acvF gene was obtained from the genomic DNA of the SYK-6 strain among the AV degrading enzyme gene group derived from the SYK-6 strain according to a conventional method. , the nucleic acid fragment was ligated under the control of E. coli-derived lactose promoter inserted into pSEVA241 plasmid DNA to obtain plasmid pSEVA241_P lac -acv.
　また、同様に、ＳＹＫ－６株のゲノムＤＮＡからｖｃｅＡ遺伝子及びｖｃｅＢ遺伝子を含む核酸断片を得て、ｐＪＢ８６６プラスミドＤＮＡに挿入した大腸菌由来ラクトースプロモーターの制御下に連結して、プラスミドｐＴＳ０９３＿ｖｃｅＡ－Ｂを得た。 Similarly, a nucleic acid fragment containing the vceA gene and the vceB gene was obtained from the genomic DNA of the SYK-6 strain and ligated under the control of the E. coli-derived lactose promoter inserted into the pJB866 plasmid DNA to obtain the plasmid pTS093_vceA-B. rice field.
　ｐＳＥＶＡ２４１＿Ｐ ｌａｃ －ａｃｖ及びｐＴＳ０９３＿ｖｃｅＡ－ＢのプラスミドＤＮＡを用いて、ＮＧＣ７株及びＮＧＣ７ΔｖａｎＡ４Ｂ４株を形質転換して、ＮＧＣ７［ｐＳＥＶＡ２４１＿Ｐ ｌａｃ －ａｃｖ，ｐＴＳ０９３＿ｖｃｅＡ－Ｂ］株及びＮＧＣ７ΔｖａｎＡ４Ｂ４［ｐＳＥＶＡ２４１＿Ｐ ｌａｃ －ａｃｖ，ｐＴＳ０９３＿ｖｃｅＡ－Ｂ］株をそれぞれ得た。これらの菌株を、２５ｍｇ／Ｌ　カナマイシン（Ｋｍ）及び１５ｍｇ／Ｌ　テトラサイクリン（Ｔｃ）を含むＬＢ液体培地　１０ｍＬに接種し、３０℃、一晩の振盪培養（１８０ｒｐｍ）に供した。 The plasmid DNAs of pSEVA241_P lac -acv and pTS093_vceA-B were used to transform the NGC7 strain and the NGC7Δ vanA4B4 strain to obtain the NGC7 [pSEVA241_P lac -acv, pTS093_vceA-B] strain and the NGC7Δ vanA4B4, pSEVA241_vceA241_P lac -acv, pTS093_vceA-B4 B] strains were obtained respectively. These strains were inoculated into 10 mL of LB liquid medium containing 25 mg/L kanamycin (Km) and 15 mg/L tetracycline (Tc) and subjected to overnight shaking culture (180 rpm) at 30°C.
　得られた培養液　０．１ｍＬを新しいＬＢ液体培地（２５ｍｇ／Ｌ　Ｋｍ、１５ｍｇ／Ｌ　Ｔｃを含む）　１０ｍＬに接種し、３０℃、１６時間の振盪培養に供した。 0.1 mL of the resulting culture solution was inoculated into 10 mL of new LB liquid medium (25 mg/L Km, 15 mg/L Tc included) and subjected to shaking culture at 30°C for 16 hours.
　得られた培養液を遠心分離に供して菌体を回収し、得られた菌体を、ＭＭx－３培地を用いて洗浄し、次いで再度ＭＭｘ－３培地に懸濁して、ＯＤ６００が１０になるように細胞懸濁液　１ｍＬを調製した。得られた細胞懸濁液に、終濃度が０．２ｍＭになるようにＡＶを添加し、次いで３０℃の振盪培養（１５００ｒｐｍ）に供した。 The resulting culture solution is subjected to centrifugation to collect the cells, and the cells obtained are washed with MMx-3 medium and then resuspended in MMx-3 medium until OD 600 reaches 10. 1 mL of cell suspension was prepared so that AV was added to the resulting cell suspension to a final concentration of 0.2 mM, followed by shaking culture (1500 rpm) at 30°C.
　培養開始後、一定時間毎にサンプリングし、培養液の一部を遠心分離して得た培養上清について、ＡＶ、バニロイル酢酸（ＶＡＡ）及びＶＡの濃度を測定した。測定には、上記例５に記載した装置及び条件により高速液体クロマトグラフを用いて測定した。ＮＧＣ７［ｐＳＥＶＡ２４１＿Ｐ ｌａｃ －ａｃｖ，ｐＴＳ０９３＿ｖｃｅＡ－Ｂ］株を用いて測定した結果を図５Ａに示し、ＮＧＣ７ΔｖａｎＡ４Ｂ４［ｐＳＥＶＡ２４１＿Ｐ ｌａｃ －ａｃｖ，ｐＴＳ０９３＿ｖｃｅＡ－Ｂ］株を用いて結果を図５Ｂに示す。 After the start of the culture, samples were taken at regular time intervals, and the culture supernatant obtained by centrifuging a portion of the culture medium was measured for AV, vanilloyl acetic acid (VAA) and VA concentrations. The measurement was performed using a high-performance liquid chromatograph using the apparatus and conditions described in Example 5 above. The results measured using the NGC7 [pSEVA241_P lac -acv, pTS093_vceA-B] strain are shown in FIG. 5A, and the results using the NGC7Δ vanA4B4 [pSEVA241_P lac -acv, pTS093_vceA-B] strain are shown in FIG. 5B.
　図５Ａに示すとおり、ＮＧＣ７株はＡＶの分解性を有さないが、ＡＶ分解酵素遺伝子群を導入することにより、ＡＶの分解能を獲得した。しかし、ＶＡの分解性を有することから、ＶＡは蓄積しなかった。 As shown in Fig. 5A, the NGC7 strain does not have the ability to degrade AV, but by introducing the AV-degrading enzyme gene group, it acquired the ability to decompose AV. However, due to the degradability of VA, VA did not accumulate.
　それに対して、図５Ｂが示すとおり、ＶＡの分解性を欠失させたＮＧＣ７ΔｖａｎＡ４Ｂ４株は、ＡＶ分解酵素遺伝子群を導入することにより、ＡＶを分解してＶＡを蓄積し、結果としてＡＶからＶＡを生産できることがわかった。 On the other hand, as shown in FIG. 5B, the NGC7Δ vanA4B4 strain lacking the ability to degrade VA degrades AV and accumulates VA by introducing an AV degrading enzyme gene group. was found to be able to produce
［７．リグニン分解物の調製］
　アセトン及び蒸留水で洗浄した銅フォーム（１．７ｃｍ×５ｃｍ、０．５ｇ；Ｘｉａｍｅｎ　ＴＯＢ　Ｎｅｗ　Ｅｎｅｒｇｙ　Ｔｅｃｈｎｏｌｏｇｙ社）を、０．１２Ｍ　過硫酸アンモニウム水溶液　１００ｍＬ及び３Ｍ　水酸化ナトリウム水溶液　１００ｍＬの混合液に、３０℃で１時間浸漬した。上記処理後の銅フォームを蒸留水及びエタノールで洗浄し、リグニン分解用触媒として［Ｃｕ（ＯＨ）２／ＣＦ］を調製した。 [7. Preparation of lignin degradation product]
A copper foam (1.7 cm x 5 cm, 0.5 g; Xiamen TOB New Energy Technology) washed with acetone and distilled water was added to a mixture of 100 mL of 0.12 M ammonium persulfate aqueous solution and 100 mL of 3 M sodium hydroxide aqueous solution at 30°C. for 1 hour. The copper foam after the above treatment was washed with distilled water and ethanol to prepare [Cu(OH) 2 /CF] as a lignin decomposition catalyst.
　得られたリグニン分解用触媒を、直径１０ｍｍの円盤状に切り出し、これを内径１０ｍｍのステンレス管中に１０枚装填して、触媒層を形成した。ヒーターにて１８０℃に加熱した本触媒層に、サルファイトリグニン（東京化成工業社）　１０ｇを２Ｍ　水酸化ナトリウム水溶液　９００ｍＬに溶解して得たリグニン溶液を、送液ポンプを用いて０．２２５ｍＬ／ｍＬで送液した。触媒層にはリグニン溶液の他に、標準状態換算で２ｍＬ／ｍｉｎの酸素ガスを導入した。触媒層の出口には背圧弁を設け、触媒層内の圧力がゲージで８気圧に保たれるように調整した。 The resulting lignin decomposition catalyst was cut into discs with a diameter of 10 mm, and 10 discs were placed in a stainless steel tube with an inner diameter of 10 mm to form a catalyst layer. A lignin solution obtained by dissolving 10 g of sulfite lignin (Tokyo Kasei Kogyo Co., Ltd.) in 900 mL of a 2 M sodium hydroxide aqueous solution was added to the catalyst layer heated to 180°C with a heater, using a liquid feed pump at 0.225 mL/ The liquid was sent in mL. In addition to the lignin solution, oxygen gas was introduced into the catalyst layer at a rate of 2 mL/min in terms of standard conditions. A back pressure valve was provided at the outlet of the catalyst layer, and the pressure in the catalyst layer was adjusted to be maintained at 8 atmospheres by gauge.
　触媒層より得られた溶液のｐＨを、塩酸を用いて１０に調整した。調整後の溶液を、平膜試験機「ＨＰ４７５０」（Ｓｔｅｒｉｔｅｃｈ社）を用いて、ＭＦ膜、ＵＦ膜（「ＭＷＣＯ　５，０００Ｄａ」；ＭＴ、Ｓｙｎｄｅｒ社）及びＵＦ膜（「ＭＷＣＯ　１，０００Ｄａ」；ＧＥ、Ｓｕｅｚ社）に順次通過し、ろ液を回収した。 The pH of the solution obtained from the catalyst layer was adjusted to 10 using hydrochloric acid. The prepared solution was tested using a flat membrane tester "HP4750" (Steritech) to test an MF membrane, a UF membrane ("MWCO 5,000 Da"; MT, Synder) and a UF membrane ("MWCO 1,000 Da"; GE, Suez) to collect the filtrate.
　得られたろ液を、塩酸を用いてｐＨが３になるように調整し、次いで酢酸エチルを用いて３回抽出処理に供した。得られた酢酸エチル層を蒸発乾固して抽出物を得て、次いで得られた抽出物を少量の２Ｍ　ＮａＯＨに溶解した溶液を、塩酸でｐＨが８になるように調整することにより、リグニン分解物を得た。 The resulting filtrate was adjusted to pH 3 using hydrochloric acid, and then extracted three times using ethyl acetate. The resulting ethyl acetate layer was evaporated to dryness to obtain an extract, and then a solution of the obtained extract dissolved in a small amount of 2M NaOH was adjusted to pH 8 with hydrochloric acid to give lignin. A decomposition product was obtained.
　リグニン分解物中の主要な芳香族モノマーの濃度は、上記例５に記載した装置及び条件により高速液体クロマトグラフを用いて測定した。リグニン分解物中の主要な芳香族モノマーは、アセトバニロン（ＡＶ）、バニリン（ＶＮ）及びバニリン酸（ＶＡ）であり、濃度はそれぞれ５１．１ｍＭ、２９６．４ｍＭ及び１１３．８ｍＭであった。 The concentration of major aromatic monomers in the lignin degradation product was measured using a high-performance liquid chromatograph using the apparatus and conditions described in Example 5 above. The major aromatic monomers in the lignin degradation product were acetovanilone (AV), vanillin (VN) and vanillic acid (VA) at concentrations of 51.1 mM, 296.4 mM and 113.8 mM, respectively.
［８．ＮＧＣ７ΔｖａｎＡ４Ｂ４株を用いたリグニン分解物モデルからのＶＡ生産性の評価］
　リグニン分解物モデルとして、８１．５ｍＭ　ＡＶ、２９２．０ｍＭ　ＶＮ及び１２６．５ｍＭ　ＶＡを含む溶液（ｐＨ　８．０）を調製した。 [8. Evaluation of VA productivity from lignin degradation product model using NGC7Δ vanA4B4 strain]
A solution (pH 8.0) containing 81.5 mM AV, 292.0 mM VN and 126.5 mM VA was prepared as a lignin degradation product model.
　上記例６に記載のＮＧＣ７ΔｖａｎＡ４Ｂ４［ｐＳＥＶＡ２４１＿Ｐ ｌａｃ －ａｃｖ，ｐＴＳ０９３＿ｖｃｅＡ－Ｂ］株を、２５ｍｇ／Ｌ　Ｋｍ及び１５ｍｇ／Ｌ　Ｔｃを含むＬＢ液体培地　１０ｍＬに接種し、３０℃で一晩の振盪培養（１８０ｒｐｍ）に供した。得られた培養液　０．１ｍＬを新しいＬＢ液体培地（２５ｍｇ／Ｌ　Ｋｍ及び１５ｍｇ／Ｌ　Ｔｃを含む）１０ｍＬに接種し、３０℃で１６時間の振盪培養に供した。得られた培養液を遠心分離に供して菌体を回収し、得られた菌体を、ＭＭｘ－３培地を用いて洗浄し、次いで再度ＭＭｘ－３培地に懸濁して、ＯＤ６００が１０になるように細胞懸濁液　１ｍＬを調製した。 The NGC7Δ vanA4B4 [pSEVA241_P lac -acv, pTS093_vceA-B] strain described in Example 6 above was inoculated into 10 mL of LB liquid medium containing 25 mg/L Km and 15 mg/L Tc, and cultured overnight at 30°C with shaking (180 rpm ). 0.1 mL of the obtained culture solution was inoculated into 10 mL of fresh LB liquid medium (containing 25 mg/L Km and 15 mg/L Tc) and subjected to shaking culture at 30° C. for 16 hours. The resulting culture solution is subjected to centrifugation to collect the cells, and the obtained cells are washed with MMx-3 medium and then suspended again in MMx-3 medium until the OD 600 reaches 10. 1 mL of cell suspension was prepared so that
　２００ｇ／Ｌ　Ｇｌｕｃｏｓｅ溶液　０．７５ｍＬ及びリグニン分解物モデル　０．１ｍＬを含むＭＭｘ－３培地（２５ｍｇ／Ｌ　Ｋｍ及び１５ｍｇ／Ｌ　Ｔｃを含む）１０ｍＬに、初期ＯＤ６００が０．１になるように細胞懸濁液を添加した。得られた細胞を含む培地を、３０℃の振盪培養に供した。培養開始後、一定時間毎にサンプリングし、培養液のＯＤ６００を測定し、さらに培養液を遠心分離して得た培養上清について、ＡＶ、ＶＮ、ＶＡ及びグルコースの濃度を測定した。ＯＤ６００は分光光度計（「ＢｉｏＳｐｅｃ－ｍｉｎｉ」、島津製作所社）を用いて測定した。ＡＶ、ＶＮ及びＶＡの濃度は、上記例５に記載した装置及び条件により高速液体クロマトグラフを用いて測定した。グルコース濃度は、「バイオセンサー　ＢＦ－５」（王子計測機器社）を用いて測定した。 MMx-3 medium (containing 25 mg / L Km and 15 mg / L Tc) containing 0.75 mL of 200 g / L glucose solution and 0.1 mL of lignin degradation product model, cells so that the initial OD 600 is 0.1 Suspension was added. The medium containing the obtained cells was subjected to shaking culture at 30°C. After the start of the culture, samples were taken at regular time intervals, the OD 600 of the culture solution was measured, and the culture supernatant obtained by centrifuging the culture solution was measured for AV, VN, VA and glucose concentrations. OD 600 was measured using a spectrophotometer (“BioSpec-mini”, Shimadzu Corporation). The concentrations of AV, VN and VA were measured using a high performance liquid chromatograph using the apparatus and conditions described in Example 5 above. Glucose concentration was measured using "Biosensor BF-5" (Oji Keisokuki Co., Ltd.).
　測定結果を図６に示す。図６に示すとおり、ＮＧＣ７ΔｖａｎＡ４Ｂ４［ｐＳＥＶＡ２４１＿Ｐ ｌａｃ －ａｃｖ，ｐＴＳ０９３＿ｖｃｅＡ－Ｂ］株は、リグニン分解物モデル中のアセトバニロン及びバニリンを分解して、９７．３　％の収率でＶＡを生産した。なお、ＶＡ収率は、［７８ｈ培養後のＶＡ量（ｍｏｌ）］／［培養開始時点のＡＶ量（ｍｏｌ）＋ＶＮ量（ｍｏｌ）＋ＶＡ量（ｍｏｌ）］×１００（％）から算出した。 The measurement results are shown in FIG. As shown in FIG. 6, the NGC7Δ vanA4B4 [pSEVA241_P lac -acv, pTS093_vceA-B] strain degraded acetovanillone and vanillin in the lignin degradation product model to produce VA with a yield of 97.3%. The VA yield was calculated from [VA amount (mol) after culturing for 78 h]/[AV amount (mol) + VN amount (mol) + VA amount (mol) at the start of culturing] x 100 (%).
［９．ＮＧＣ７ΔｖａｎＡ４Ｂ４株を用いたリグニン分解物からのＶＡ生産性の評価］
　上記例６に記載のＮＧＣ７ΔｖａｎＡ４Ｂ４［ｐＳＥＶＡ２４１＿Ｐ ｌａｃ －ａｃｖ，ｐＴＳ０９３＿ｖｃｅＡ－Ｂ］株を、２５ｍｇ／Ｌ　Ｋｍ及び１５ｍｇ／Ｌ　Ｔｃを含むＬＢ液体培地　１０ｍＬに接種し、３０℃、一晩の振盪培養（１８０ｒｐｍ）に供した。得られた培養液　０．１ｍＬを新しいＬＢ液体培地（２５ｍｇ／Ｌ　Ｋｍ及び１５ｍｇ／Ｌ　Ｔｃを含む）１０ｍＬに接種し、３０℃、１６時間の振盪培養に供した。得られた培養液を遠心分離に供して菌体を回収し、得られた菌体を、ＭＭｘ－３培地を用いて洗浄し、次いで再度ＭＭｘ－３培地に懸濁して、ＯＤ６００が１０になるように細胞懸濁液　１ｍＬを調製した。 [9. Evaluation of VA productivity from lignin degradation product using NGC7Δ vanA4B4 strain]
The NGC7Δ vanA4B4 [pSEVA241_P lac -acv, pTS093_vceA-B] strain described in Example 6 above was inoculated into 10 mL of LB liquid medium containing 25 mg/L Km and 15 mg/L Tc, and cultured overnight at 30°C with shaking (180 rpm). ). 0.1 mL of the resulting culture solution was inoculated into 10 mL of a new LB liquid medium (containing 25 mg/L Km and 15 mg/L Tc) and subjected to shaking culture at 30° C. for 16 hours. The resulting culture solution is subjected to centrifugation to collect the cells, and the obtained cells are washed with MMx-3 medium and then suspended again in MMx-3 medium until the OD 600 reaches 10. 1 mL of cell suspension was prepared so that
　２００ｇ／Ｌ　Ｇｌｕｃｏｓｅ溶液　０．７５ｍＬ及び上記例７で調製したリグニン分解物　０．１０８ｍＬを含むＭＭｘ－３培地（２５ｍｇ／Ｌ　Ｋｍ及び１５ｍｇ／Ｌ　Ｔｃを含む）１０ｍＬに、初期ＯＤ６００が０．１になるように細胞懸濁液を添加した。得られた細胞を含む培地を、３０℃の振盪培養に供した。培養開始後、一定時間毎にサンプリングし、培養液のＯＤ６００を測定し、さらに培養液を遠心分離して得た培養上清について、ＡＶ、ＶＮ、ＶＡ及びグルコースの濃度を測定した。ＡＶ、ＶＮ、ＶＡ及びグルコースの濃度並びにＯＤ６００は、上記例８と同様に測定した。 10 mL of MMx-3 medium (containing 25 mg/L Km and 15 mg/L Tc) containing 0.75 mL of 200 g/L glucose solution and 0.108 mL of the lignin degradation product prepared in Example 7 above, and an initial OD 600 of 0.1 The cell suspension was added so that the The medium containing the obtained cells was subjected to shaking culture at 30°C. After the start of the culture, samples were taken at regular time intervals, the OD 600 of the culture solution was measured, and the culture supernatant obtained by centrifuging the culture solution was measured for AV, VN, VA and glucose concentrations. AV, VN, VA and glucose concentrations and OD 600 were measured as in Example 8 above.
結果を図７に示す。図７に示すとおり、ＮＧＣ７ΔｖａｎＡ４Ｂ４［ｐＳＥＶＡ２４１＿Ｐ ｌａｃ －ａｃｖ，ｐＴＳ０９３＿ｖｃｅＡ－Ｂ］株は、リグニン分解物中のアセトバニロン及びバニリンを分解して、９０．７％の収率でＶＡを生産した。ＶＡ収率は［６８ｈ培養後のＶＡ量（ｍｏｌ）］／［培養開始時点のＡＶ量（ｍｏｌ）＋ＶＮ量（ｍｏｌ）＋ＶＡ量（ｍｏｌ）］×１００（％）から算出した。 The results are shown in FIG. As shown in FIG. 7, the NGC7Δ vanA4B4 [pSEVA241_P lac -acv, pTS093_vceA-B] strain degraded acetovanillon and vanillin in the lignin degradation product to produce VA with a yield of 90.7%. The VA yield was calculated from [VA amount (mol) after culturing for 68 h]/[AV amount (mol) + VN amount (mol) + VA amount (mol)] x 100 (%) at the start of culturing.
［１０．ＮＧＣ７ΔｖａｎＡ１Ｂ１ΔｖａｎＡ４Ｂ４ΔａｐｈΔｖａｎＡ２Ｂ２ΔｖａｎＡ３Ｂ３株を用いたリグニン分解物からのＶＡ生産性の評価］
　ＮＧＣ７株のゲノムＤＮＡを鋳型として、配列番号３８及び３９のプライマー１７及び１８からなるプライマーセットと、配列番号４０及び４１のプライマー１９及び２０からなるプライマーセットとをそれぞれ用いたＰＣＲ法によって、ｖａｎＢ２遺伝子の５’末端上流及びｖａｎＡ２遺伝子の３’末端下流の約１．２ｋｂｐのＤＮＡ断片をそれぞれ増幅した。それぞれの断片をあらかじめＨｉｎｄＩＩＩ及びＢａｍＨＩで消化したｐＫ１８ｍｏｂｓａｃＢとＮＥＢｕｉｌｄｅｒ　ＨｉＦｉ　ＤＮＡアッセンブリーを用いたシームレスクローニング法により連結させることにより、新たなｖａｎＡ２遺伝子ｖａｎＢ２遺伝子領域欠失株作製用プラスミドｐｖａｎＡ２Ｂ２ｄｅｌ２を作製した。 [10. Evaluation of VA productivity from lignin degradation product using NGC7Δ vanA1B1 ΔvanA4B4 Δaph ΔvanA2B2 ΔvanA3B3 strain ]
Using the genomic DNA of the NGC7 strain as a template, a primer set consisting of primers 17 and 18 of SEQ ID NOs: 38 and 39 and a primer set consisting of primers 19 and 20 of SEQ ID NOs: 40 and 41, respectively, were used to obtain the vanB2 gene. A DNA fragment of about 1.2 kbp upstream of the 5' end of the vanA2 gene and downstream of the 3' end of the vanA2 gene was amplified, respectively. Each fragment was ligated with pK18 mobsacB previously digested with HindIII and BamHI by a seamless cloning method using NEBuilder HiFi DNA assembly to construct a new plasmid pvanA2B2del2 for constructing a vanB2 gene region deletion strain of the vanA2 gene.
　同様に、ＮＧＣ７株のゲノムＤＮＡを鋳型として、配列番号４２及び４３のプライマー２１及び２２からなるプライマーセットと、配列番号４４及び４５のプライマー２３及び２４からなるプライマーセットとをそれぞれ用いたＰＣＲ法によって、ｖａｎＡ４遺伝子の５’末端上流及びｖａｎＢ４遺伝子の３’末端下流の約１．２ｋｂｐのＤＮＡ断片をそれぞれ増幅した。それぞれの断片をあらかじめＨｉｎｄＩＩＩ及びＢａｍＨＩで消化したｐＫ１８ｍｏｂｓａｃＢとＮＥＢｕｉｌｄｅｒ　ＨｉＦｉ　ＤＮＡアッセンブリーを用いたシームレスクローニング法により連結させることにより、新たなｖａｎＡ４遺伝子ｖａｎＢ４遺伝子領域欠失株作製用プラスミドｐｖａｎＡ４Ｂ４ｄｅｌ２を作製した。 Similarly, by PCR using the genomic DNA of the NGC7 strain as a template, a primer set consisting of primers 21 and 22 of SEQ ID NOs: 42 and 43, and a primer set consisting of primers 23 and 24 of SEQ ID NOs: 44 and 45, respectively. , approximately 1.2 kbp DNA fragments upstream of the 5' end of the vanA4 gene and downstream of the 3' end of the vanB4 gene were amplified, respectively. Each fragment was ligated with pK18 mobsacB previously digested with HindIII and BamHI by a seamless cloning method using NEBuilder HiFi DNA assembly to construct a new plasmid pvanA4B4del2 for constructing a vanB4 gene region deletion strain of the vanA4 gene.
　ＮＧＣ７株のゲノムにはＫｌｅｂｓｉｅｌｌａ　ｐｎｅｕｍｏｎｉａｅ由来のアミノグリコシド－３’－ホスホトランスフェラーゼ（Ｐ００５５２）と５０．９％の配列同一性を示したアミノ酸配列をコードする遺伝子であるＰＳＮ＿１５１１（ａｐｈ遺伝子；配列番号３７）が存在することを確認した。ａｐｈ遺伝子の機能によりＮＧＣ７株がＫｍ耐性を有するようになることが推定されたため本遺伝子を破壊した変異株を作製した。 The genome of the NGC7 strain contains PSN — 1511 ( aph gene; SEQ ID NO: 37), a gene encoding an amino acid sequence showing 50.9% sequence identity with aminoglycoside-3′-phosphotransferase (P00552) derived from Klebsiella pneumoniae . Confirmed it exists. Since it was presumed that the NGC7 strain would have Km resistance due to the function of the aph gene, a mutant strain was prepared by disrupting this gene.
　上記と同様に、ＮＧＣ７株のゲノムＤＮＡを鋳型として、配列番号４６及び４７のプライマー２５及び２６からなるプライマーセットと、配列番号４８及び４９のプライマー２７及び２８からなるプライマーセットとをそれぞれ用いたＰＣＲ法によって、ａｐｈ遺伝子の５’末端上流及び３’末端下流の約１．２ｋｂｐのＤＮＡ断片をそれぞれ増幅した。それぞれの断片をあらかじめＨｉｎｄＩＩＩ及びＢａｍＨＩで消化したｐＫ１８ｍｏｂｓａｃＢとＮＥＢｕｉｌｄｅｒ　ＨｉＦｉ　ＤＮＡアッセンブリーを用いたシームレスクローニング法により連結させることにより、ａｐｈ遺伝子領域欠失株作製用プラスミドｐａｐｈｄｅｌを作製した。 In the same manner as described above, PCR using the genomic DNA of strain NGC7 as a template and a primer set consisting of primers 25 and 26 of SEQ ID NOs: 46 and 47 and a primer set consisting of primers 27 and 28 of SEQ ID NOs: 48 and 49, respectively. A DNA fragment of about 1.2 kbp upstream of the 5'-end and downstream of the 3'-end of the aph gene was amplified by the method. Each fragment was ligated with pK18 mobsacB previously digested with HindIII and BamHI by a seamless cloning method using NEBuilder HiFi DNA assembly to prepare a plasmid paphdel for constructing an aph gene region deletion strain.
　プラスミドｐｖａｎＡ４Ｂ４ｄｅｌ２をＮＧＣ７ΔｖａｎＡ１Ｂ１株に導入し、ゲノムＤＮＡ上のｖａｎＡ４遺伝子及びｖａｎＢ４遺伝子の内部領域を欠失したＮＧＣ７ΔｖａｎＡ１Ｂ１ΔｖａｎＡ４Ｂ４株を作製した。同様に、得られたＮＧＣ７ΔｖａｎＡ１Ｂ１ΔｖａｎＡ４Ｂ４株にプラスミドｐａｐｈｄｅｌを導入し、ゲノムＤＮＡ上のａｐｈ遺伝子の内部領域を欠失したＮＧＣ７ΔｖａｎＡ１Ｂ１ΔｖａｎＡ４Ｂ４Δａｐｈ株を作製した。さらに、得られたＮＧＣ７ΔｖａｎＡ１Ｂ１ΔｖａｎＡ４Ｂ４Δａｐｈ株にプラスミドｐｖａｎＡ２Ｂ２ｄｅｌ２を導入し、ゲノムＤＮＡ上のｖａｎＡ２遺伝子及びｖａｎＢ２遺伝子の内部領域を欠失したＮＧＣ７ΔｖａｎＡ１Ｂ１ΔｖａｎＡ４Ｂ４ΔａｐｈΔｖａｎＡ２Ｂ２株を作製した。そして、得られたＮＧＣ７ΔｖａｎＡ１Ｂ１ΔｖａｎＡ４Ｂ４ΔａｐｈΔｖａｎＡ２Ｂ２株にプラスミドｐｖａｎＡ３Ｂ３ｄｅｌを導入し、ゲノムＤＮＡ上のｖａｎＡ３遺伝子及びｖａｎＢ３遺伝子の内部領域を欠失したＮＧＣ７ΔｖａｎＡ１Ｂ１ΔｖａｎＡ４Ｂ４ΔａｐｈΔｖａｎＡ２Ｂ２ΔｖａｎＡ３Ｂ３株を作製した。 The plasmid pvanA4B4del2 was introduced into the NGC7ΔvanA1B1 strain to prepare the NGC7ΔvanA1B1ΔvanA4B4 strain lacking the internal regions of the vanA4 and vanB4 genes on the genomic DNA. Similarly, a plasmid paphdel was introduced into the obtained NGC7ΔvanA1B1ΔvanA4B4 strain to prepare an NGC7ΔvanA1B1ΔvanA4B4Δaph strain lacking the internal region of the aph gene on the genomic DNA. Further, the obtained NGC7Δ vanA1B1 ΔvanA4B4 Δaph strain was transfected with the plasmid pvanA2B2del2 to prepare the NGC7ΔvanA1B1 ΔvanA4B4 Δaph ΔvanA2B2 strain in which the internal regions of the vanA2 and vanB2 genes on the genomic DNA were deleted. Then, a plasmid pvanA3B3del was introduced into the obtained NGC7Δ vanA1B1 ΔvanA4B4 Δaph ΔvanA2B2 strain to prepare an NGC7ΔvanA1B1 ΔvanA4B4 Δaph ΔvanA2B2 ΔvanA3B3 strain in which the vanA3 gene on the genomic DNA and the internal region of the vanB3 gene were deleted. did.
　常法に従って、上記６に記載のプラスミドｐＳＥＶＡ２４１＿Ｐ ｌａｃ －ａｃｖ、プラスミドｐＴＳ０９３＿ｖｃｅＡ－Ｂを用いてＮＧＣ７ΔｖａｎＡ１Ｂ１ΔｖａｎＡ４Ｂ４ΔａｐｈΔｖａｎＡ２Ｂ２ΔｖａｎＡ３Ｂ３株を形質転換して、ＮＧＣ７ΔｖａｎＡ１Ｂ１ΔｖａｎＡ４Ｂ４ΔａｐｈΔｖａｎＡ２Ｂ２ΔｖａｎＡ３Ｂ３［ｐＳＥＶＡ２４１＿Ｐ ｌａｃ －ａｃｖ，ｐＴＳ０９３＿ｖｃｅＡ－Ｂ］株を得た。 According to a conventional method, the NGC7Δ vanA1B1 ΔvanA4B4 Δaph ΔvanA2B2 ΔvanA3B3 strain was transformed using the plasmid pSEVA241_P lac -acv and the plasmid pTS093_vceA-B described in 6 above to obtain NGC7Δ vanA1B1 ΔvanA4ΔvanA4ΔB3 ΔB3 ΔBAph . pSEVA241_P lac -acv, pTS093_vceA-B] strains were obtained.
　ＮＧＣ７ΔｖａｎＡ４Ｂ４［ｐＳＥＶＡ２４１＿Ｐ ｌａｃ －ａｃｖ，ｐＴＳ０９３＿ｖｃｅＡ－Ｂ］株に代えて、ＮＧＣ７ΔｖａｎＡ１Ｂ１ΔｖａｎＡ４Ｂ４ΔａｐｈΔｖａｎＡ２Ｂ２ΔｖａｎＡ３Ｂ３［ｐＳＥＶＡ２４１＿Ｐ ｌａｃ －ａｃｖ，ｐＴＳ０９３＿ｖｃｅＡ－Ｂ］株を用いたこと以外は同様にして、上記例９に記載の方法により、リグニン分解物からのＶＡ生産能を評価した。 Instead of the NGC7Δ vanA4B4 [pSEVA241_P lac -acv, pTS093_vceA-B] strain, NGC7Δ vanA1B1 Δ vanA4B4 Δ aph Δ vanA2B2 Δ vanA3B3 [pSEVA241_P lac -acv, pTS093_vceA-B] was used in the same manner as in the above example except for the strain. VA production ability from the lignin degradation product was evaluated by the method described in 9.
　結果を図８に示す。図８に示すとおり、ＮＧＣ７ΔｖａｎＡ１Ｂ１ΔｖａｎＡ４Ｂ４ΔａｐｈΔｖａｎＡ２Ｂ２ΔｖａｎＡ３Ｂ３［ｐＳＥＶＡ２４１＿Ｐ ｌａｃ －ａｃｖ，ｐＴＳ０９３＿ｖｃｅＡ－Ｂ］株は、リグニン分解物中のＡＶ及びＶＮを分解して、９２．８％の収率でＶＡを生産した。ＶＡ収率は［６８ｈ培養後のＶＡ量（ｍｏｌ）］／［培養開始時点のＡＶ量（ｍｏｌ）＋ＶＮ量（ｍｏｌ）＋ＶＡ量（ｍｏｌ）］×１００（％）から算出した。なお、図２ＡにおいてΔｖａｎＡ１Ｂ１株、ΔｖａｎＡ２Ｂ２株及びΔｖａｎＡ３Ｂ３株はＶＡを炭素源として増殖したにもかかわらず、図８においてΔｖａｎＡ１Ｂ１ΔｖａｎＡ４Ｂ４ΔａｐｈΔｖａｎＡ２Ｂ２ΔｖａｎＡ３Ｂ３株が高収率でＶＡを生産したことは、驚くべき結果である。 The results are shown in FIG. As shown in FIG. 8, the NGC7ΔvanA1B1ΔvanA4B4ΔaphΔvanA2B2ΔvanA3B3 [ pSEVA241_P lac -acv , pTS093_vceA -B] strain degraded AV and VN in the lignin hydrolyzate with a yield of 92.8%. VA was produced. The VA yield was calculated from [VA amount (mol) after culturing for 68 h]/[AV amount (mol) + VN amount (mol) + VA amount (mol)] x 100 (%) at the start of culturing. Although the ΔvanA1B1 , ΔvanA2B2 , and ΔvanA3B3 strains grew on VA as a carbon source in FIG . What you have produced is an astonishing result.
［１１．配列表］
　配列表に記載の配列は以下の表６Ａ及び表６Ｂに示すとおりである。 [11. sequence list]
The sequences listed in the Sequence Listing are shown in Tables 6A and 6B below.
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000007
　本発明の一態様の形質転換微生物及び製造方法によって、リグニン由来の芳香族化合物及びバニリン酸を含むバイオマスから、バニリン酸が得られる。バニリン酸は、種々の産業上有用な化合物に変換することができ、例えば、耐熱性及び剛性のあるプラスチック、コーティング剤などの用途があるバニリン酸誘導体の原料として利用することができる。 Vanillic acid can be obtained from biomass containing lignin-derived aromatic compounds and vanillic acid by the transformed microorganism and production method of one embodiment of the present invention. Vanillic acid can be converted into various industrially useful compounds, and can be used, for example, as a raw material for vanillic acid derivatives that are used in heat-resistant and rigid plastics, coating agents, and the like.
関連出願の相互参照Cross-reference to related applications
　本出願は、２０２１年３月３１日出願の日本特願２０２１－０５９６２１号の優先権を主張し、その全記載は、ここに開示として援用される。さらに、本願の発明の詳細な説明において挙げられている全文献の全記載はここに開示として援用される。 This application claims priority from Japanese Patent Application No. 2021-059621 filed on March 31, 2021, the entire description of which is incorporated herein by reference. Further, the entire disclosures of all documents cited in the Detailed Description of the Invention herein are hereby incorporated by reference.

Claims (5)

  1. 　宿主微生物がシュードモナス・スピーシーズ（Ｐｓｅｕｄｏｍｏｎａｓ　ｓｐ．）　ＮＧＣ７株（受託番号：ＮＩＴＥ　ＢＰ－０３０４３）であり、
    　染色体上にある、ｖａｎＡ４遺伝子（配列番号１）又は該ｖａｎＡ４遺伝子及びｖａｎＢ４遺伝子（配列番号２）が欠失している
    形質転換微生物。     The host microorganism is Pseudomonas sp. NGC7 strain (accession number: NITE BP-03043),
    A transformed microorganism lacking the vanA4 gene (SEQ ID NO: 1) or the vanA4 gene and the vanB4 gene (SEQ ID NO: 2) on the chromosome.
  2. 　宿主微生物がシュードモナス・スピーシーズ（Ｐｓｅｕｄｏｍｏｎａｓ　ｓｐ．）　ＮＧＣ７株（受託番号：ＮＩＴＥ　ＢＰ－０３０４３）であり、
    　染色体上にあるｖａｎＡ４遺伝子（配列番号１）又は該ｖａｎＡ４遺伝子及びｖａｎＢ４遺伝子（配列番号２）が欠失しており、かつ、
    　挿入されたａｃｖＡ遺伝子、ａｃｖＢ遺伝子、ａｃｖＣ遺伝子、ａｃｖＤ遺伝子、ａｃｖＥ遺伝子、ａｃｖＦ遺伝子、ｖｃｅＡ遺伝子及びｖｃｅＢ遺伝子を発現する
    形質転換微生物。     The host microorganism is Pseudomonas sp. NGC7 strain (accession number: NITE BP-03043),
    The vanA4 gene (SEQ ID NO: 1) or the vanA4 gene and the vanB4 gene (SEQ ID NO: 2) on the chromosome are deleted, and
    A transformed microorganism expressing the inserted acvA gene, acvB gene, acvC gene, acvD gene, acvE gene, acvF gene, vceA gene and vceB gene.
  3. 　グアイアシルリグニン由来の芳香族化合物を、請求項１又は２に記載の形質転換微生物に作用させることにより、バニリン酸を得る工程
    を含む、バニリン酸の製造方法。     A method for producing vanillic acid, comprising a step of obtaining vanillic acid by allowing an aromatic compound derived from guaiacyl lignin to act on the transformed microorganism according to claim 1 or 2.
  4. 　グアイアシルリグニン由来の芳香族化合物と、ｐ－ヒドロキシフェニルリグニン由来の芳香族化合物及び／又はシリンギルリグニン由来の芳香族化合物との混合物を、請求項１又は２に記載の形質転換微生物に作用させることにより、バニリン酸を得る工程
    を含む、バニリン酸の製造方法。     A mixture of an aromatic compound derived from guaiacyl lignin and an aromatic compound derived from p -hydroxyphenyl lignin and/or an aromatic compound derived from syringyl lignin is allowed to act on the transformed microorganism according to claim 1 or 2. Thus, a method for producing vanillic acid, comprising the step of obtaining vanillic acid.
  5. 　アセトバニロンを含むグアイアシルリグニン由来の芳香族化合物の混合物を、請求項２に記載の形質転換微生物に作用させることにより、バニリン酸を得る工程
    を含む、バニリン酸の製造方法。     A method for producing vanillic acid, comprising the step of allowing a mixture of aromatic compounds derived from guaiacyl lignin containing acetovanillone to act on the transformed microorganism according to claim 2 to obtain vanillic acid.
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