Method for producing vinyl catechol compound

Abstract

To provide a method for producing vinyl catechol compounds that enables the production of the vinyl catechol compounds without heavy metals.SOLUTION: A method for producing a vinyl catechol compound disclosed herein includes the step for reacting a vinylphenol compound represented by formula (I) with at least one selected from the group consisting of a first microbial cell containing 4-hydroxyphenylacetic acid-3-monooxygenase, a processed product of the first microbial cell, a second microbial cell containing phenol-2-monooxygenase, and a processed product of the second microbial cell. In formula (I), R represents hydrogen or a methyl group, and the position of the hydroxyl group is in the meta or para position relative to the -C(R)=CH2 group.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present disclosure relates to a method for producing a vinyl catechol compound. [Background technology]

[0002] Paraisopropenylcatechol is known as a raw material for photoresist (see Patent Document 1). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Application Laid-Open No. 62-173458 Summary of the Invention [Problem to be solved by the invention]

[0004] However, heavy metals are usually used in the chemical synthesis of para-isopropenyl catechol. Heavy metals may have an adverse effect on the environment. Therefore, there is a demand for a method for producing a vinyl catechol compound without using heavy metals.

[0005] In view of the above circumstances, the present disclosure relates to providing a method for producing a vinylcatechol compound, which can produce a vinylcatechol compound without using heavy metals. [Means for solving the problem]

[0006] Means for solving the above problems include the following aspects. <1> A vinylphenol compound represented by the following formula (I), At least one selected from the group consisting of a first bacterial body containing 4-hydroxyphenylacetate-3-monooxygenase, a processed product of the first bacterial body, a second bacterial body containing phenol-2-monooxygenase, and a processed product of the second bacterial body; A method for producing a vinylcatechol compound, comprising the step of reacting

[0007] [ka]

[0008] [In the formula (I), R represents hydrogen or a methyl group, The position of the hydroxyl group is -C(R)=CH 2 The position is meta or para to the group. <2> The 4-hydroxyphenylacetate-3-monooxygenase has an amino acid sequence having 60% or more identity to the amino acid sequence of SEQ ID NO:1, The phenol-2-monooxygenase has an amino acid sequence having 60% or more identity with the amino acid sequence of SEQ ID NO:2. <1> A method for producing the vinyl catechol compound according to claim 1. <3> R represents a methyl group, The position of the hydroxyl group is a para position. <1> or <2> A method for producing the vinyl catechol compound according to claim 1. <4> the step includes adding the vinylphenol compound all at once to a medium containing at least one selected from the group consisting of the first bacterial body, a treated product of the first bacterial body, the second bacterial body, and a treated product of the second bacterial body, and a liquid medium; The concentration of the vinylphenol compound in the liquid medium is 1.5 mmol / L or less. <1> ~ <3> 13. A method for producing a vinyl catechol compound according to claim 12, <5> the step includes adding the vinylphenol compound in two or more divided portions to a culture medium containing at least one selected from the group consisting of the first bacterial body, a treated product of the first bacterial body, the second bacterial body, and a treated product of the second bacterial body, and a liquid medium, and a final concentration of the vinylphenol compound is more than 1.5 mmol / L with respect to the liquid medium. <1> ~ <3> 13. A method for producing a vinyl catechol compound according to claim 12, <6> The final concentration of the vinylphenol compound is 6.0 mmol / L or more in the liquid medium. <5> A method for producing the vinyl catechol compound according to claim 1. <7> In the step, the vinylphenol compound is reacted with at least one selected from the group consisting of the second bacterial body and a treated product of the second bacterial body. <1> ~ <6> 13. A method for producing a vinyl catechol compound according to claim 12, Effect of the Invention

[0009] According to the present disclosure, there is provided a method for producing a vinylcatechol compound, which can produce a vinylcatechol compound without using heavy metals. [Brief description of the drawings]

[0010] [Figure 1] FIG. 1 is a chromatogram of the culture medium after carrying out the biocatalysis step of Experiment 1. [Diagram 2] FIG. 2 shows extracted ion chromatograms of the culture supernatant after the biocatalysis step using strain A in experiment 1 and Mirex® PM. [Diagram 3] FIG. 3 shows mass spectra detected for the culture supernatant after the biocatalytic reaction step using strain A in Experiment 1 and Mirex (registered trademark) PM. [Figure 4] FIG. 4 shows chromatograms of the culture medium after the biocatalytic reaction step using strain A in experiment 2 and the aqueous solution after ethyl acetate extraction. [Diagram 5] FIG. 5 is a graph showing the measurement results of Experiment 3. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The contents of the present disclosure will be described in detail below. The following description of the components may be based on a representative embodiment of the present disclosure, but the present disclosure is not limited to such an embodiment. In this specification, a numerical range expressed using "to" means a range that includes the numerical values ​​before and after "to" as the lower and upper limits. In this specification, the amount of each component in a composition means, when a plurality of substances corresponding to each component are present in the composition, the total amount of the plurality of substances present in the composition, unless otherwise specified.

[0012] (1) Method for producing vinylcatechol compound The method for producing a vinyl catechol compound according to the present disclosure (hereinafter also referred to as the "production method according to the present disclosure") includes a step of reacting a vinyl phenol compound represented by the following formula (I) (hereinafter also referred to as "vinyl phenol compound (I)") with at least one selected from the group consisting of a first bacterial cell containing 4-hydroxyphenylacetate-3-monooxygenase (hereinafter also referred to simply as "first bacterial cell"), a processed product of the first bacterial cell, a second bacterial cell containing phenol-2-monooxygenase (hereinafter also referred to simply as "second bacterial cell"), and a processed product of the second bacterial cell (hereinafter also referred to as "biocatalyst") (hereinafter also referred to as a "biocatalyst reaction step").

[0013] [ka]

[0014] In formula (I), R represents a hydrogen atom or a methyl group, and the position of the hydroxyl group is -C(R)=CH 2 The position is meta or para to the group.

[0015] In the present disclosure, "4-hydroxyphenylacetate-3-monooxygenase" refers to an enzyme (protein) having the ability (hereinafter also referred to as "hydroxylation activity") to form a catechol group by introducing one oxygen atom of an oxygen molecule in the form of a hydroxyl group into the phenol group of a vinylphenol compound (I) to produce a vinylcatechol compound corresponding to the substrate, vinylphenol compound (I). In the present disclosure, "phenol-2-monooxygenase" refers to an enzyme (protein) having hydroxylation activity that introduces one oxygen atom of an oxygen molecule in the form of a hydroxyl group to the phenol group of a vinylphenol compound (I) to form a catechol group. In the present disclosure, the term "treated bacterial cells" refers to a general term for bacterial cells that have been subjected to some kind of treatment. Details of the treated bacterial cells will be described later.

[0016] Since the manufacturing method of the present disclosure has the above-mentioned configuration, it can manufacture a vinylcatechol compound without using heavy metals. Furthermore, the manufacturing method of the present disclosure can increase the conversion efficiency (hereinafter, also simply referred to as "conversion efficiency") from a vinylphenol compound (I) to a vinylcatechol compound, compared to conventional chemical synthesis.

[0017] (1.1) Vinylcatechol compounds The vinyl catechol compound produced by the production method of the present disclosure (hereinafter also referred to as "vinyl catechol compound (II)") is represented by the following formula (II).

[0018] [ka]

[0019] In formula (II), R represents a hydrogen atom or a methyl group. R in formula (II) is the same as R in formula (I) of the vinylphenol compound (I) which is the raw material.

[0020] From the viewpoint of suitability as a raw material for photoresists (e.g., i-line resists, KrF (krypton fluoride) resists, and EUV (extreme ultraviolet) resists, etc.), the vinyl catechol compound (II) is preferably a compound represented by the following formula (II-1) (hereinafter also referred to as "paraisopropenyl catechol (II-1)").

[0021] [ka]

[0022] (1.2) Biocatalytic reaction process In the biocatalytic reaction step, the vinylphenol compound (I) is reacted with a biocatalyst. The biocatalyst functions as a catalyst in the reaction from the vinylphenol compound (I) to the vinylcatechol compound (II).

[0023] Hereinafter, the reaction of the vinylphenol compound (I) with a biocatalyst is also referred to as a "biocatalytic reaction".

[0024] (1.2.1) Vinylphenol compounds (I) The vinylphenol compound (I) is represented by the above formula (I). The vinylphenol compound (I) may be used alone or in combination.

[0025] It is preferable that the R represents a methyl group and the position of the hydroxyl group is a para position. Specifically, it is preferable that the vinylphenol compound (I) is a compound represented by the following formula (I-1) (hereinafter, also referred to as "paraisopropenylphenol (I-1)"). This gives paraisopropenylcatechol (II-1).

[0026] [ka]

[0027] The vinylphenol compound (I) may be a commercially available product or may be a synthesized product. The method for synthesizing the vinylphenol compound (I) is not particularly limited, and may be a known method. Examples of paraisopropenylphenol (I-1) products include "Mirex (registered trademark) PM" manufactured by Mitsui Chemicals, Inc.

[0028] (1.2.2) Biocatalysis The biocatalyst includes at least one member selected from the group consisting of a first bacterial body, a treated product of the first bacterial body, a second bacterial body, and a treated product of the second bacterial body.

[0029] In one embodiment, it is preferable to allow the vinylphenol compound (I) to react with at least one selected from the group consisting of the second bacterial body and a processed product of the second bacterial body in the biocatalytic reaction step, thereby achieving a higher conversion efficiency.

[0030] (1.2.2.1) First bacterial body The first bacterial body contains 4-hydroxyphenylacetate-3-monooxygenase.

[0031] The first bacterial cell is not particularly limited as long as it produces 4-hydroxyphenylacetate-3-monooxygenase and retains the hydroxylation activity of the vinylphenol compound (I) in a medium (e.g., a liquid medium such as an aqueous medium or an oil-based medium). These first bacterial cells may be used alone or in combination of two or more kinds.

[0032] The first bacterial cell may be a bacterial cell into which a gene for 4-hydroxyphenylacetate-3-monooxygenase has been introduced (hereinafter, also referred to as a "first gene-introduced cell"). The method for introducing the gene for 4-hydroxyphenylacetate-3-monooxygenase into the bacterial cell is not particularly limited. The gene may be introduced using a vector. Examples of the vector include a viral vector and a non-viral vector (e.g., a plasmid). The gene may be introduced transiently or may be integrated into a chromosome. Examples of a method for incorporating a gene into a chromosome include a method of transforming a host cell using a gene insertion cassette into a chromosome.

[0033] The first gene introducer may be a transformant in which a cloned 4-hydroxyphenylacetate-3-monooxygenase gene is highly expressed in an arbitrary host, or a transformant in which an artificially synthesized 4-hydroxyphenylacetate-3-monooxygenase gene is highly expressed in an arbitrary host. The first gene introducer may also be a transformant in which a mutant 4-hydroxyphenylacetate-3-monooxygenase gene with further improved temperature resistance or the like is expressed in an arbitrary host, or a transformant in which a 4-hydroxyphenylacetate-3-monooxygenase gene modified with a histidine tag or the like is highly expressed in an arbitrary host.

[0034] Any host cell may include general bacteria (eg, Escherichia coli, Bacillus sp. (eg, Bacillus subtilis), etc.), yeast, actinomycetes, and the like. The term "mutant 4-hydroxyphenylacetate-3-monooxygenase" refers to a 4-hydroxyphenylacetate-3-monooxygenase that contains one or more amino acid substitutions, deletions, or insertions that constitute the wild-type 4-hydroxyphenylacetate-3-monooxygenase. Mutant 4-hydroxyphenylacetate-3-monooxygenase can be produced using recombinant DNA technology.

[0035] The vector used to prepare the first gene introducer contains a gene encoding 4-hydroxyphenylacetate-3-monooxygenase, and can be obtained by linking the gene encoding 4-hydroxyphenylacetate-3-monooxygenase to the vector. The type of vector is not particularly limited, and may be a commercially available expression plasmid, such as pET-21a(+), pKK223-3, pUC19, pBluescriptKS(+), pBR322, pAUR101, pAUR112, pAUR123, pAUR224 DNA, pPICZ, pPICZα, pPTR II DNA, pBE-S, and pBIC. The expression plasmid for 4-hydroxyphenylacetate-3-monooxygenase can be constructed by incorporating a gene encoding 4-hydroxyphenylacetate-3-monooxygenase into the expression plasmid. The host used for transformation is not particularly limited as long as the vector is stable, self-replicating, and capable of expressing the traits of the foreign DNA. The host is not particularly limited, and examples thereof include the above-mentioned hosts.

[0036] The form of the first bacterial body is not particularly limited as long as it contains the first bacterial body, and examples thereof include a culture solution containing the first bacterial body, a bacterial harvest obtained by separating and recovering the bacterial body by centrifuging the culture solution, and further a product obtained by washing the bacterial harvest with physiological saline or the like.

[0037] (1.2.2.1.1) 4-Hydroxyphenylacetate-3-monooxygenase The 4-hydroxyphenylacetate-3-monooxygenase is not particularly limited as long as it has hydroxylation activity, and examples thereof include 4-hydroxyphenylacetate-3-monooxygenase derived from Escherichia coli, preferably Escherichia coli BL21, etc.

[0038] The 4-hydroxyphenylacetate-3-monooxygenase may be any known one. From the viewpoint of efficiently producing the vinylphenol compound (I), the 4-hydroxyphenylacetate-3-monooxygenase preferably includes 4-hydroxyphenylacetate-3-monooxygenase derived from Escherichia coli, more preferably includes 4-hydroxyphenylacetate-3-monooxygenase derived from Escherichia coli BL21, and further preferably is 4-hydroxyphenylacetate-3-monooxygenase derived from Escherichia coli BL21. The 4-hydroxyphenylacetate-3-monooxygenase may be used alone or in combination of two or more kinds.

[0039] The 4-hydroxyphenylacetate-3-monooxygenase preferably has an amino acid sequence having 60% or more identity with the amino acid sequence of SEQ ID NO: 1. SEQ ID NO: 1 is the amino acid sequence of 4-hydroxyphenylacetate-3-monooxygenase derived from Escherichia coli.

[0040] SEQ ID NO:1: MKPEDFRASTQRPFTGEEYLKSLQDGREIYIYGERVKDVTTHPAFRNAAASVAQLYDALHKPEMQDSLCWNTDTGSGGYTHKFFRVAKSADDLRQQRDAIAEWSRLSYGWMGRTPDYKAAFGCALGANPG FYGQFEQNARNWYTRIQETGLYFNHAIVNPPIDRHLPTDKVKDVYIKLEKETDAGIIVSGAKVVATNSALTHYNMIGFGSAQVMGENPDFALMFVAPMDADGVKLISRASYEMVAGATGSPYDYPLSSRF DENDAILVMDNVLIPWENVLIYRDFDRCRRWTMEGGFARMYPLQACVRLAVKLDFITALLKKSLECTGTLEFRGVQADLGEVVAWRNTFWALSDSMCSEATPWVNGAYLPDHAALQTYRVLAPMAYAKIK NIIERNVTSGLIYLPSSARDLNNPQIDQYLAKYVRGSNGMDHVQRIKILKLMWDAIGSEFGGRHELYEINYSGSQDEIRLQCLRQAQNSGNMDKMMAMVDRCLSEYDQDGWTVPHLHNNDDINMLDKLLK

[0041] In the present disclosure, "amino acid sequence identity" refers to the percentage (%) of identical amino acids in one polypeptide relative to the total number of amino acids in the other polypeptide when the two polypeptides being compared are aligned and, if necessary, gaps are introduced into either polypeptide to maximize the homology between the amino acid sequences of the two polypeptides. Amino acid sequence homology can be easily determined using known programs such as the homology search program BLAST (Basic local alignment search tool; Altschul, SF et al., J. Mol. Biol., 215, 403-410, 1990) search.

[0042] The identity to SEQ ID NO: 1 is more preferably 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more. The identity to SEQ ID NO: 1 may be 100%. That is, the 4-hydroxyphenylacetate-3-monooxygenase may be derived from Escherichia coli (NCBI-Protein ID: ACT46003).

[0043] Examples of 4-hydroxyphenylacetate-3-monooxygenase having an amino acid sequence having 60% or more identity with the amino acid sequence of SEQ ID NO:1 include those in the table below.

[0044] [Table 1]

[0045] (1.2.2.2) Treated product of first bacterial body Examples of the treated product of the first bacterial body include extracts or ground products from the first bacterial body, crude enzyme preparations obtained by separating the hydroxylating active fractions of these extracts and ground products, purified enzyme products obtained by purifying the crude enzyme preparations, immobilized products obtained by immobilizing the extracts, ground products, crude enzyme preparations, or purified enzyme products using an appropriate means, heat-treated products of the first bacterial body, organic solvent-treated products of the first bacterial body (e.g., acetone-treated products, ethanol-treated products, etc.), and surfactant-treated products of the first bacterial body. In the production method of the present disclosure, the above-mentioned 4-hydroxyphenylacetate-3-monooxygenase-producing bacterial cells may be subjected to various treatments, such as collection by centrifugation or disruption, in order to increase the efficiency of the reaction with the vinylphenol compound (I).

[0046] (1.2.2.3) Second bacterial body The second bacterial body contains phenol-2-monooxygenase.

[0047] The second bacterial cell is not particularly limited as long as it produces phenol-2-monooxygenase and retains the hydroxylation activity of the vinylphenol compound (I) in a medium (e.g., a liquid medium such as an aqueous medium or an oil-based medium). These bacterial cells may be used alone or in combination of two or more kinds.

[0048] The second bacterial cell may be a bacterial cell into which a gene for phenol-2-monooxygenase has been introduced (hereinafter also referred to as a "second gene-introduced body"). The method for introducing the gene for phenol-2-monooxygenase into the bacterial cell is not particularly limited. The gene may be introduced using a vector. Examples of the vector include a viral vector and a non-viral vector (e.g., a plasmid). The gene may be introduced transiently or may be integrated into a chromosome. Examples of a method for incorporating a gene into a chromosome include a method of transforming a host cell using a gene insertion cassette into a chromosome.

[0049] The second gene introducer may be a transformant in which a cloned phenol-2-monooxygenase gene is highly expressed in an arbitrary host, or a transformant in which an artificially synthesized phenol-2-monooxygenase gene is highly expressed in an arbitrary host. The second gene introducer may also be a transformant in which a mutant phenol-2-monooxygenase gene with further improved temperature resistance or the like is expressed in an arbitrary host, or a transformant in which a phenol-2-monooxygenase gene modified with a histidine tag or the like is highly expressed in an arbitrary host.

[0050] Any host cell may include general bacteria (eg, Escherichia coli, Bacillus sp. (eg, Bacillus subtilis), etc.), yeast, actinomycetes, and the like. The term "mutant phenol-2-monooxygenase" refers to a phenol-2-monooxygenase that contains one or more amino acid substitutions, deletions, or insertions that constitute the wild-type phenol-2-monooxygenase. Mutant phenol-2-monooxygenases can be produced using recombinant DNA technology.

[0051] The vector used to prepare the second gene introducer contains a gene encoding phenol-2-monooxygenase, and can be obtained by ligating the gene encoding phenol-2-monooxygenase to the vector. The type of vector is not particularly limited, and may be a commercially available expression plasmid, such as pET-21a(+), pKK223-3, pUC19, pBluescriptKS(+), pBR322, pAUR101, pAUR112, pAUR123, pAUR224 DNA, pPICZ, pPICZα, pPTR II DNA, pBE-S, and pBIC. The expression plasmid for phenol-2-monooxygenase can be constructed by incorporating a gene encoding phenol-2-monooxygenase into the expression plasmid. The host used for transformation is not particularly limited as long as the vector is stable, self-replicating, and capable of expressing the traits of the foreign DNA. The host is not particularly limited, and examples thereof include the above-mentioned hosts.

[0052] The form of the second bacterial cells is not particularly limited as long as it contains bacterial cells that produce phenol-2-monooxygenase, and examples thereof include a culture medium containing the second bacterial cells, bacterial harvests separated and recovered by centrifuging the culture medium, and the bacterial harvests washed with physiological saline or the like.

[0053] (1.2.2.3.1) Phenol-2-monooxygenase The phenol-2-monooxygenase is not particularly limited as long as it has hydroxylation activity, and examples thereof include phenol-2-monooxygenase derived from Parageobacillus thermoglucosidasius, preferably Parageobacillus thermoglucosidasius NBRC107763, etc.

[0054] The phenol-2-monooxygenase may be any known one. From the viewpoint of efficiently producing the vinylphenol compound (I), the phenol-2-monooxygenase preferably contains phenol-2-monooxygenase derived from Parageobacillus thermoglucosidasius, more preferably contains phenol-2-monooxygenase derived from Parageobacillus thermoglucosidasius NBRC107763, and further preferably is phenol-2-monooxygenase derived from Parageobacillus thermoglucosidasius NBRC107763. The phenol-2-monooxygenase may be used alone or in combination of two or more kinds.

[0055] The phenol-2-monooxygenase preferably has an amino acid sequence having 60% or more identity with the amino acid sequence of SEQ ID NO: 2. SEQ ID NO: 2 is the amino acid sequence of phenol-2-monooxygenase derived from Parageobacillus thermoglucosidasius.

[0056] SEQ ID NO:2: MKDMMNGKEYLESLRDNRVVYLNGEKIDDVTTHPAYENAARSIARMYDALHDEQMGKILTTTTEEGYRTHKFFKEPKNAQDLLEARDAIAQWAKLSYGFMGRTPDYKASFTAHLKAFADYYEGFED NARNWYKKTTKEIPFINHTIINPQVDRSKPLHENKDVFVRAVKERDDGIIVSGAKMVGTAAALTHYNFVSNYGAQDLGDGDQSHALIFFVPPMNAPGVKMISRQSYEQIAKTLGSPFDYPLSSRFDE NDAVIVLDNVFIPWENVFAYKNVKVVNGFFVETGFVNRFTFQGCTRFAVKLDFMVGLLMKATEAAGTKQFRGVQANIGEVVAFRNMFWALSTAMAANCEMRNGVALPNLQYGAAYRVLAPMVWPRV KQIFEQVVAGGLIQLPSSANDFLNPELRPYLDRYYRGSGIGAEERVKLMKMIWDAIGTEFGGRHELYEINYAGNHENIRLETLKIADVTGDSERFKAFVDSALDDYDLHGWVNDTWVSPKKEIVQK

[0057] The homology to SEQ ID NO: 2 is more preferably 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more. The homology to SEQ ID NO: 2 may be 100%. That is, the phenol-2-monooxygenase may be derived from Parageobacillus thermoglucosidasius (UNIPROT ID: Q9LAG3).

[0058] Examples of phenol-2-monooxygenases having an amino acid sequence that has 60% or more identity with the amino acid sequence of SEQ ID NO:2 include those in the table below.

[0059] [Table 2]

[0060] (1.2.2.4) Treatment product of the second bacterial cell Examples of treated products of the second bacterial cells include extracts or ground products from the second bacterial cells, crude enzyme preparations obtained by separating the hydroxylating active fractions of these extracts and ground products, purified enzyme products obtained by purifying the crude enzyme preparations, immobilized products obtained by immobilizing the extracts, ground products, crude enzyme preparations, or purified enzyme products using an appropriate means, heat-treated products of the second bacterial cells, organic solvent-treated products of the second bacterial cells (e.g., acetone-treated products, ethanol-treated products, etc.), surfactant-treated products of the second bacterial cells, etc. In the production method of the present disclosure, the above-mentioned phenol-2-monooxygenase-producing bacterial cells may be subjected to various treatments, such as collection by centrifugation or disruption, in order to increase the efficiency of the reaction with the vinylphenol compound (I).

[0061] It is preferred that the 4-hydroxyphenylacetate-3-monooxygenase has an amino acid sequence having 60% or more identity with SEQ ID NO:1, and the phenol-2-monooxygenase has an amino acid sequence having 60% or more identity with the amino acid sequence of SEQ ID NO:2.

[0062] (1.2.3) Biocatalysis In the biocatalytic reaction, the vinylphenol compound (I) is contacted with the biocatalyst and reacted to produce the vinylcatechol compound (II).

[0063] The method of the biocatalytic reaction is not particularly limited, and examples thereof include a batch system, a fed-batch system, and a continuous system. In the batch system, the biocatalyst, the liquid medium, and the vinylphenol compound (I) are all charged into a reaction tank at once, and then the biocatalytic reaction is allowed to proceed. In the fed-batch system, the biocatalyst, a part of the vinylphenol compound (I), and the liquid medium are charged into a reaction tank, and then the remaining vinylphenol compound (I) is continuously or intermittently supplied to allow the biocatalytic reaction to proceed. In the continuous system, the biocatalyst, the vinylphenol compound (I), and the liquid medium are continuously or intermittently supplied, and the reaction liquid is continuously or intermittently removed, while the reaction is continuously carried out without removing the entire amount of the reaction liquid in the reaction tank. The reaction liquid includes the biocatalyst, the unreacted vinylphenol compound (I), the liquid medium, and the produced vinylcatechol compound (II), etc. For the biocatalytic reaction, a device capable of mass culture (e.g., a jar fermenter, a bioreactor, etc.) may be used.

[0064] Alternatively, the biocatalytic reaction may be carried out by inducing expression of the 4-hydroxyphenylacetate-3-monooxygenase or the phenol-2-monooxygenase in cells containing the biocatalyst that have been cultured to a predetermined amount, and then adding the vinylphenol compound (I).

[0065] The culture method and biocatalytic reaction include ordinary culture methods (eg, batch culture, fed-batch culture, continuous culture, etc.).

[0066] In batch culture, for example, the bacteria may be inoculated into a medium, cultured, induced to express, and then cultured, after which a solution of the vinylphenol compound (I) as the raw material may be added all at once. For example, in batch culture, a preculture is performed, the bacteria are inoculated into a minimal medium at a concentration of 1%, and cultured until the OD 660When the β-amino acid sequence reaches 0.5 to 20, expression is induced (for example, 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) is added in the case of T7 promoter) and the mixture is cultured for 2 to 5 hours, after which a solution of the raw material vinylphenol compound (I) is added all at once and reacted for 20 to 35 hours to obtain a vinylcatechol compound (II). Alternatively, a solution of the vinylphenol compound (I) may be added to the medium before or during inoculation. The species of bacteria to be inoculated is, for example, Escherichia coli.

[0067] In fed-batch culture, for example, the bacteria are inoculated into a medium, cultured, induced for expression, and then cultured, and the raw material, a vinylphenol compound (I) solution, is fed at a predetermined supply rate. For example, in fed-batch culture, preculture is performed, the bacteria are inoculated into a minimal medium at a concentration of 1%, and cultured at OD 660 When the β-amino acid ratio reaches 0.5 to 20, expression is induced (for example, 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) is added in the case of a T7 promoter) and the mixture is cultured for 2 to 5 hours, after which a solution of the raw material vinylphenol compound (I) is fed at a predetermined feed rate and reacted for 20 to 35 hours to obtain a vinylcatechol compound (II). The species of bacteria to be inoculated is, for example, Escherichia coli.

[0068] The culture conditions are not particularly limited and may be appropriately selected depending on the type of culture and the culture method, as long as the bacterial cells can grow and produce the 4-hydroxyphenylacetate-3-monooxygenase or phenol-2-monooxygenase. The culture temperature may be any temperature at which the cells containing the biocatalyst can grow, and may be 25°C to 40°C, or 30°C to 37°C. The culture pH may be 6.0 to 7.5, or may be around 7.0, or 6.2 to 7.2.

[0069] The conditions of the biocatalytic reaction are not particularly limited, and may be conventional reaction conditions. The reaction temperature may be the same as the culture temperature. The reaction pH may be the same as the culture pH. The total amount of the vinylphenol compound (I) to be added is appropriately selected depending on the type of culture method, etc., and is preferably an amount in which almost all of the vinylphenol compound (I) to be added is converted to the vinylcatechol compound (II). The reaction time is not particularly limited. The biocatalytic reaction is preferably carried out until the added vinylphenol compound (I) is used up.

[0070] As the medium used for the culture and biocatalytic reaction, either a synthetic medium or a natural medium can be used so long as it contains an appropriate amount of a carbon source, a nitrogen source, inorganic salts and other nutrients.

[0071] (1.2.3.1) Batch culture The biocatalytic reaction step includes adding the vinylphenol compound (I) all at once to a culture medium containing at least one selected from the group consisting of the first bacterial body, a treated product of the first bacterial body, the second bacterial body, and a treated product of the second bacterial body, and a liquid medium (i.e., the biocatalytic reaction is a batch culture), and the concentration of the vinylphenol compound (I) is preferably 1.5 mmol / L or less relative to the liquid medium. This leads to a higher conversion efficiency. This effect is presumably due to, but not limited to, the following reasons. When the biocatalyst acts on the vinylphenol compound (I), the biocatalyst needs to metabolize glucose, etc. to obtain reducing power (NADH, etc.). When the biocatalytic reaction is a batch culture, if the concentration of the vinylphenol compound (I) is 1.5 mmol / L or less, the biocatalyst can easily metabolize glucose, etc. and obtain reducing power. As a result, it is presumed that the conversion efficiency will be higher.

[0072] When the biocatalytic reaction is a batch culture, the concentration of the vinylphenol compound (I) is more preferably 0.01 mmol / L to 1.5 mmol / L, and even more preferably 0.1 mmol / L to 1.0 mmol / L, relative to the liquid medium, from the viewpoint of increasing the concentration of the product.

[0073] (1.2.3.2) Fed-batch culture The biocatalytic reaction step includes adding the vinylphenol compound (I) in two or more divided portions to a culture medium containing at least one selected from the group consisting of the first bacterial body, a treated product of the first bacterial body, the second bacterial body, and a treated product of the second bacterial body, and a liquid medium (i.e., the biocatalytic reaction is a fed-batch culture), and it is preferable that the final concentration of the vinylphenol compound (I) is more than 1.5 mmol / L with respect to the liquid medium. This leads to a higher conversion efficiency. This effect is presumably due to, but not limited to, the following reasons. When the biocatalyst acts on the vinylphenol compound (I), the biocatalyst needs to metabolize glucose, etc. to obtain reducing power (NADH, etc.). When the biocatalytic reaction is a fed-batch culture, even if the final concentration of the vinylphenol compound (I) exceeds 1.5 mmol / L, adding the vinylphenol compound (I) in two or more portions makes it easier for the biocatalyst to efficiently metabolize glucose, etc., and to efficiently obtain reducing power. As a result, it is presumed that the conversion efficiency will be higher.

[0074] When the biocatalytic reaction is a fed-batch culture, the final concentration of the vinylphenol compound (I) is more preferably 6.0 mmol / L or more relative to the liquid medium, thereby improving the concentration of the vinylcatechol compound (II) obtained. From the viewpoint of further improving the conversion efficiency, the final concentration of the vinylphenol compound (I) is more preferably 1 mmol / L to 10 mmol / L, and particularly preferably 1.5 mmol / L to 6 mmol / L, relative to the liquid medium.

[0075] When the biocatalytic reaction is a fed-batch culture, the number of times and the amount of the vinylphenol compound (I) are appropriately selected depending on the type and amount of the biocatalyst. The addition rate of the vinylphenol compound (I) is preferably a rate at which the added vinylphenol compound (I) is rapidly converted to the vinylcatechol compound (II). The addition rate of the vinylphenol compound (I) is preferably 0.1 mg / L / min to 10 mg / L / min, more preferably 1 mg / L / min to 5 mg / L / min.

[0076] (1.2.3.3)Liquid medium Examples of the liquid medium include an aqueous medium, an oil-based medium, etc. Among these, the liquid medium is preferably an aqueous medium.

[0077] The aqueous medium is not particularly limited, and usually water is used, but a mixture of water and a water-soluble organic solvent can also be used. The water used as the aqueous medium is not particularly limited, and examples thereof include pure water, purified water (e.g., distilled water, ion-exchanged water, etc.), etc. Examples of the water-soluble organic solvent to be mixed with water include alcohol solvents, ether solvents, ketone solvents, and aprotic polar solvents. Examples of the alcohol solvents include methanol, ethanol, 1-propanol, 2-propanol, 2-methoxyethanol, 2-ethoxyethanol, ethylene glycol, and propylene glycol. Examples of the ether solvents include dimethyl ether, diethyl ether, diisopropyl ether, dioxane, and ethylene glycol dimethyl ether. Examples of the ketone solvents include acetone, methyl ethyl ketone, and methyl isobutyl ketone. Examples of the aprotic polar solvents include dimethylformamide, dimethylacetamide, dimethylsulfoxide, 1-methyl-2-pyrrolidinone, and 1,3-dimethyl-2-imidazolidinone. When water and a water-soluble organic solvent are used as a mixture, the mixing ratio of water to the water-soluble organic solvent (water / water-soluble organic solvent) is not particularly limited as long as it is within a range in which the hydroxylation activity of the biocatalyst can be well maintained, and is preferably 100 / 100 to 100 / 1, more preferably 100 / 50 to 100 / 1, and even more preferably 100 / 30 to 100 / 1, in terms of volume ratio.

[0078] (1.2.3.4) pH adjusters In the biocatalytic reaction step, a pH adjusting agent may be used, if necessary. The pH regulator can adjust the pH of the mixture of the biocatalyst with the vinylphenol compound (I) and the vinylcatechol compound (II) to a suitable range for maintaining good activity of the biocatalyst. When the pH suitable for the reaction is lower than 7, an acid can be used as the pH regulator, and when the pH suitable for the reaction is higher than 7, a base can be used as the pH regulator.

[0079] Acids used as pH adjusters include inorganic acids (e.g., hydrochloric acid, hypochlorous acid, sulfuric acid, nitric acid, phosphoric acid, boric acid, etc.), and organic acids (e.g., citric acid, formic acid, acetic acid, methanesulfonic acid, ethanesulfonic acid, etc.). Examples of the base used as a pH adjuster include inorganic bases (e.g., potassium hydroxide, aqueous ammonia, sodium hydroxide, magnesium hydroxide, lithium carbonate, lithium hydrogen carbonate, etc.), and organic bases (e.g., trimethylamine, triethylamine, aniline, etc.).

[0080] (1.3) Other processes The production method of the present disclosure may further include a step other than the biocatalytic reaction step (hereinafter also referred to as "other step"), if necessary.

[0081] Other steps include a step of purifying the vinylcatechol compound (II). EXAMPLES

[0082] Hereinafter, the embodiments of the present disclosure will be described in detail with reference to examples. Note that the present disclosure is not limited to the description of these examples. Examples of the present disclosure will be described below, but the present disclosure is not limited to the following examples. In the following, "%" means "% by mass" unless otherwise specified.

[0083] [1] Preparation [1.1] Culture medium [1.1.1] LB medium 10 g of hypopeptone (Nihon Seiyaku), 5 g of dried yeast extract D-3H (Nihon Seiyaku), and 10 g of sodium chloride were added to 1 L of distilled water, and the pH was adjusted to 7.0 with NaOH, followed by sterilization in an autoclave. This produced the LB medium.

[0084] [1.1.2]LB agar medium 10 g of hypopeptone (Nihon Seiyaku), 5 g of dried yeast extract D-3H (Nihon Seiyaku), and 10 g of sodium chloride were added to 1 L of distilled water, and the pH was adjusted to 7.0 with NaOH. Then, 20 g of agar was added and sterilized in an autoclave. This produced the LB agar medium.

[0085] [1.1.3]HCF medium To 873 mL of distilled water, 100 mL of phosphate / citrate buffer (133 g of potassium dihydrogen phosphate, 40 g of ammonium dihydrogen phosphate, 17 g of citric acid, and 22 g of sodium hydroxide in 1 L of distilled water), 14.3 mL of 70% glucose solution, 2.4 mL of 50% magnesium sulfate heptahydrate solution, 10 mL of trace metal solution (10 g of iron(III) citrate n-hydrate, 250 mg of cobalt chloride hexahydrate, 150 mg of manganese chloride tetrahydrate, 150 mg of copper chloride dihydrate, 300 mg of boric acid, 250 mg of sodium molybdate dihydrate, 1.3 g of zinc acetate dihydrate, and 840 mg of ethylenediaminetetraacetate dihydrate (EDTA) in 1 L of distilled water), and 0.23 mL of 2% thiamine solution were added to obtain HCF medium. This HCF medium was sterilized by a filter and used as the HCF medium (HCF medium containing 1% glucose) in Test 1 (test tube culture) described below and Test 2 (flask culture) described below.

[0086] 844mL of distilled water and 100mL of phosphate / citrate buffer were added to the culture tank of the jar fermenter and autoclaved. After the solution had cooled sufficiently, 42.9mL and 2.4mL of 70% glucose solution and 50% magnesium sulfate heptahydrate solution, which had been separately autoclaved, respectively, and 10mL and 0.23mL of trace metal solution and 2% thiamine solution, which had been filter-sterilized, respectively, were aseptically added (HCF medium containing 3% glucose).

[0087] [1.2] Preparation of plasmids [1.2.1] Construction of pET21a-FRT-hpaBC The hpaBC gene was amplified by PCR using the chromosomal DNA of Escherichia coli BL21 strain as a template and the primer set hpaBC-F (5'-GTTTAACTTTAAGAAGGAGATATACATATGAAACCAGAAGATTTCCGC-3') (SEQ ID NO: 3) and hpaBC-R (5'-GTGGTGGTGGTGCTCGAGTTAAATCGCAGCTTCCATTTC-3') (SEQ ID NO: 4). Phusion HS2 DNA polymerase (New England BioLabs) was used as the PCR enzyme, and the reaction solution composition and reaction conditions were in accordance with the attached instructions. The PCR amplified product of about 2100 bp was purified using QIAquick PCR Purification Kit (Qiagen) and digested overnight at 37°C with the restriction enzyme DpnI (New England Biolabs). The PCR product was purified again using the QIAquick PCR Purification Kit, and assembly reactions were performed using pET21a-FRT digested with restriction enzymes NdeI-XhoI and Gibson assembly reagent (New England BioLabs). Escherichia coli DH5α was transformed using the resulting reaction solution, which was then spread onto LB agar medium containing 20 μg / mL kanamycin and cultured overnight at 37°C. From the colonies obtained after culture, those carrying a plasmid containing an insert of the desired size were selected by colony direct PCR. OneTaq DNA polymerase (New England Biolabs) was used for colony direct PCR, and the reaction solution composition and reaction conditions were in accordance with the attached instructions. The resulting colonies were inoculated into LB liquid medium containing kanamycin and cultured overnight at 37°C. Plasmid pET21a-FRT-hpaBC was obtained from the culture solution using NucleoSpin Plasmid QuickPure (Macherey-Nagel).

[0088] [1.2.2] Preparation of pET21a-FRT-tmo The tmo operon gene was amplified by PCR using the chromosomal DNA of Cupriavidus metallidurans NBRC 102507 strain as a template and the primer set cmtmo-F (5'-GTTTAACTTTAAGAAGGAGATATACATATGGCTTTGTTAAATCGGATGG-3') (SEQ ID NO: 5) and cmtmo-R (5'-GTGGTGGTGGTGCTCGAGTTAAAAGAAACGATCGAAATGAAGC-3') (SEQ ID NO: 6). Phusion HS2 DNA polymerase (New England BioLabs) was used as the PCR enzyme, and the reaction solution composition and reaction conditions were in accordance with the attached instructions. The PCR amplified product of about 5000 bp was purified using QIAquick PCR Purification Kit (Qiagen) and digested with the restriction enzyme DpnI (New England Biolabs) at 37°C overnight. The PCR product was purified again using the QIAquick PCR Purification Kit, and assembly reactions were performed using pET21a-FRT digested with restriction enzymes NdeI-XhoI and Gibson assembly reagent (New England BioLabs). Escherichia coli DH5α was transformed using the resulting reaction solution, which was then spread onto LB agar medium containing 20 μg / mL kanamycin and cultured overnight at 37°C. From the colonies obtained after culture, those carrying a plasmid containing an insert of the desired size were selected by colony direct PCR. OneTaq DNA polymerase (New England Biolabs) was used for colony direct PCR, and the reaction solution composition and reaction conditions were as per the attached instructions. The resulting colonies were inoculated into LB liquid medium containing kanamycin and cultured overnight at 37°C. Plasmid pET21a-FRT-tmo was obtained from the culture solution using NucleoSpin Plasmid QuickPure (Macherey-Nagel).

[0089] [1.2.3] Construction of pET21a-FRT-pheA1A2 The pheA1A2 gene was amplified by PCR using the chromosomal DNA of Parageobacillus thermoglucosidasius NBRC107763 strain as a template and the primer set ptpheAB-F (5'-GTTTAACTTTAAGAAGGAGATATACATATGAAAGATATGATGAATGGCAAG-3') (SEQ ID NO: 7) and ptpheAB-R (5'-GTGGTGGTGGTGCTCGAGTTAGCTCGACGTCTCCACTTTTTC-3') (SEQ ID NO: 8). Phusion HS2 DNA polymerase (New England BioLabs) was used as the PCR enzyme, and the reaction solution composition and reaction conditions were as per the attached instructions. The PCR amplified product of about 2000 bp was purified using QIAquick PCR Purification Kit (Qiagen) and digested with the restriction enzyme DpnI (New England Biolabs) at 37°C overnight. The PCR product was purified again using the QIAquick PCR Purification Kit, and assembly reactions were performed using pET21a-FRT digested with restriction enzymes NdeI-XhoI and Gibson assembly reagent (New England BioLabs). Escherichia coli DH5α was transformed using the resulting reaction solution, which was then spread onto LB agar medium containing 20 μg / mL kanamycin and cultured overnight at 37°C. From the colonies obtained after culture, those carrying a plasmid containing an insert of the desired size were selected by colony direct PCR. OneTaq DNA polymerase (New England Biolabs) was used for colony direct PCR, and the reaction solution composition and reaction conditions were as per the attached instructions. The resulting colonies were inoculated into LB liquid medium containing kanamycin and cultured overnight at 37°C. Plasmid pET21a-FRT-pheA1A2 was obtained from the culture solution using NucleoSpin Plasmid QuickPure (Macherey-Nagel).

[0090] [1.2.4] Construction of pET21a-FRT-pobA The pobA gene was amplified by PCR using the chromosomal DNA of Pseudomonas putida KT2440 strain as a template and the primer set pppobA-F (5'-GTTTAACTTTAAGAAGGAGATATACATATGAAAACTCAGGTTGCAATTATTG-3') (SEQ ID NO: 9) and pppobA-R (5'-GTGGTGGTGGTGCTCGAGTCAGGCAACTTCCTCGAACG-3') (SEQ ID NO: 10). Phusion HS2 DNA polymerase (New England BioLabs) was used as the PCR enzyme, and the reaction solution composition and reaction conditions were in accordance with the attached instructions. The PCR amplified product of about 1200 bp was purified using QIAquick PCR Purification Kit (Qiagen) and digested overnight at 37°C with the restriction enzyme DpnI (New England Biolabs). The PCR product was purified again using the QIAquick PCR Purification Kit, and assembly reactions were performed using pET21a-FRT digested with restriction enzymes NdeI-XhoI and Gibson assembly reagent (New England BioLabs). Escherichia coli DH5α was transformed using the resulting reaction solution, which was then spread onto LB agar medium containing 20 μg / mL kanamycin and cultured overnight at 37°C. From the colonies obtained after culture, those carrying a plasmid containing an insert of the desired size were selected by colony direct PCR. OneTaq DNA polymerase (New England Biolabs) was used for colony direct PCR, and the reaction solution composition and reaction conditions were in accordance with the attached instructions. The resulting colonies were inoculated into LB liquid medium containing kanamycin and cultured overnight at 37°C. Plasmid pET21a-FRT-pobA was obtained from the culture solution using NucleoSpin Plasmid QuickPure (Macherey-Nagel).

[0091] [1.2.5] Construction of pET21a-FRT-phhAB The phhAB gene was amplified by PCR using the chromosomal DNA of Pseudomonas putida KT2440 strain as a template and the primer set ppphhAB-F (5'-GTTTAACTTTAAGAAGGAGATATACATATGAAACAGACGCAATACGTGG-3') (SEQ ID NO: 11) and ppphhAB-R (5'-GTGGTGGTGGTGCTCGAGTTACTTCCGGCCTTCAGCCG-3') (SEQ ID NO: 12). Phusion HS2 DNA polymerase (New England BioLabs) was used as the PCR enzyme, and the reaction solution composition and reaction conditions were as described in the attached instructions. The PCR amplified product of about 1200 bp was purified using QIAquick PCR Purification Kit (Qiagen) and digested overnight at 37°C with the restriction enzyme DpnI (New England Biolabs). The PCR product was purified again using the QIAquick PCR Purification Kit, and assembly reactions were performed using pET21a-FRT digested with restriction enzymes NdeI-XhoI and Gibson assembly reagent (New England BioLabs). Escherichia coli DH5α was transformed using the resulting reaction solution, which was then spread onto LB agar medium containing 20 μg / mL kanamycin and cultured overnight at 37°C. From the colonies obtained after culture, those carrying a plasmid containing an insert of the desired size were selected by colony direct PCR. OneTaq DNA polymerase (New England Biolabs) was used for colony direct PCR, and the reaction solution composition and reaction conditions were in accordance with the attached instructions. The resulting colonies were inoculated into LB liquid medium containing kanamycin and cultured overnight at 37°C. The plasmid pET21a-FRT-phhAB was obtained from the culture solution using NucleoSpin Plasmid QuickPure (Macherey-Nagel).

[0092] [1.3] Preparation of strains [1.3.1] Preparation of strain A Using pET21a-FRT-hpaBC as a template, a primer set of delta-pflBA-F (5'-CGAAGTACGCAGTAAATAAAAAATCCACTTAAGAAGGTAGGTGTTACATGATTCCGGGGATCCGTCGACC-3') (SEQ ID NO: 13) and delta-pflBA-FRT-R (5'-CTCAATAAAGTTGCCGCTTTACGGGGAAATTAGAACATTACCTTATGACCATTCGCCAATCCGGATATAG-3') (SEQ ID NO: 14) was used to insert the gene cassette FRT-Km-FRT-P into the chromosome. T7 -hpaBC was amplified by PCR. PrimeStar GXL (Takara Bio) was used as the PCR enzyme, and PCR reaction was performed according to the attached instructions. The PCR amplified product was purified using a QIAquick PCR Purification Kit (Qiagen) and digested overnight at 37°C with the restriction enzyme DpnI (New England Biolabs). The digested product was purified again using a QIAquick PCR Purification Kit. When eluting DNA with the purification kit, sterile distilled water was used instead of the elution buffer included with the kit. The obtained DNA solution was purified using FRT-Km-FRT-P T7 This was used as a -hpaBC cassette solution in the insertion experiment into the Escherichia coli chromosome described below.

[0093] Escherichia coli MG1655(DE3) / pKD46 strain was cultured in 5 mL of LB medium containing 10 mM L-arabinose and 50 μg / mL carbenicillin to reach an OD 660 The culture was incubated with shaking at 30°C until the value of was about 0.5. 2 mL of the culture solution was centrifuged at 5,000 x g (4°C) for 5 minutes to collect the bacterial cells, which were then washed with 1 mL of sterile distilled water at 4°C. The culture solution was centrifuged again at 5,000 x g (4°C) for 5 minutes to collect the bacterial cells, which were then washed with 1 mL of sterile distilled water at 4°C. The culture solution was centrifuged again at 5,000 x g (4°C) for 5 minutes to collect the bacterial cells, which were then suspended in 50 μL of sterile distilled water. FRT-Km-FRT-P at a concentration of 100 to 200 ng / μL was added to the culture solution. T71 μL of the -hpaBC cassette solution was added to the bacterial cell suspension, and the entire volume was transferred to a 2 mm-wide electroporation cuvette. Electroporation was performed using a MicroPulser electroporator (Bio-Rad) with the EC2 setting (output voltage 2.5 kV). 1 mL of LB medium was added to the cuvette to suspend the bacterial cells, and the entire volume was transferred to a 1.5 mL tube and cultured (rehabilitated) with shaking at 37°C for 3 hours. The entire volume was spread onto LB agar medium containing 20 μg / mL kanamycin, and cultured overnight at 37°C.

[0094] The colonies obtained after cultivation were examined to see whether the target gene had been inserted at the target site on the chromosomal DNA. Using a primer set of K2 (5'-CGGTGCCCTGAATGAACTGC-3') (sequence number 15) and D-pflBA (5'-CGTTATTCTCCAGAGGTTCATCAGC-3') (sequence number 16), colony direct PCR was performed using OneTaq DNA polymerase (New England Biolabs) and the colonies as template DNA. The PCR reaction solution composition and reaction conditions were as per the attached instructions. For colonies in which amplification was observed around 3100 bp, single colony isolation was performed using LB agar medium containing kanamycin, and then the strain A (MG1655(DE3) pflBA::P T7 The strain was suspended in LB medium containing 15% glycerol as 100% glycerol (-hpaBC) and stored at -80°C. When using the strain, the stored strain was spread on LB agar medium containing kanamycin and cultured overnight at 37°C, and the resulting colonies were used.

[0095] [1.3.2] Preparation of strain B Using pET21a-FRT-tmo as a template, the gene insertion cassette FRT-Km-FRT-P was inserted into the chromosome using a primer set of delta-mngAB-F (5'-TACAAATACAATACAAATAATTTCAATCAAGTGAAATTGATCACATAATGATTCCGGGGATCCGTCGACC-3') (SEQ ID NO: 17) and delta-mngAB-FRT-R (5'-CGCATATGTGGCTTCGGTTTGATTGCTATTCAGGCAAGCCGGTAACTGAAATTCGCCAATCCGGATATAG-3') (SEQ ID NO: 18). T7 -tmo was amplified by PCR. PrimeStar GXL (Takara Bio) was used as the PCR enzyme, and PCR reaction was performed according to the attached instructions. The PCR amplified product was purified using a QIAquick PCR Purification Kit (Qiagen) and digested overnight at 37°C with the restriction enzyme DpnI (New England Biolabs). The digested product was purified again using a QIAquick PCR Purification Kit. When eluting DNA with the purification kit, sterile distilled water was used instead of the elution buffer included with the kit. The obtained DNA solution was purified using FRT-Km-FRT-P T7 This was used as a -tmo cassette solution in the insertion experiment into the Escherichia coli chromosome described below.

[0096] Escherichia coli MG1655(DE3) / pKD46 strain was cultured in 5 mL of LB medium containing 10 mM L-arabinose and 50 μg / mL carbenicillin to reach an OD 660 The culture was incubated with shaking at 30°C until the value of was about 0.5. 2 mL of the culture solution was centrifuged at 5,000 x g (4°C) for 5 minutes to collect the bacterial cells, which were then washed with 1 mL of sterile distilled water at 4°C. The culture solution was centrifuged again at 5,000 x g (4°C) for 5 minutes to collect the bacterial cells, which were then washed with 1 mL of sterile distilled water at 4°C. The culture solution was centrifuged again at 5,000 x g (4°C) for 5 minutes to collect the bacterial cells, which were then suspended in 50 μL of sterile distilled water. FRT-Km-FRT-P at a concentration of 100 to 200 ng / μL was added to the culture solution. T71 μL of the -tmo cassette solution was added to the bacterial cell suspension, and the entire volume was transferred to a 2 mm-wide electroporation cuvette. Electroporation was performed using a MicroPulser electroporator (Bio-Rad) with the EC2 setting (output voltage 2.5 kV). 1 mL of LB medium was added to the cuvette to suspend the bacterial cells, and the entire volume was transferred to a 1.5 mL tube and cultured (rehabilitated) with shaking at 37°C for 3 hours. The entire volume was spread on LB agar medium containing 20 μg / mL kanamycin, and cultured overnight at 37°C.

[0097] The colonies obtained after cultivation were examined to see whether the target gene had been inserted at the target site on the chromosomal DNA. Using a primer set of K2 (5'-CGGTGCCCTGAATGAACTGC-3') (sequence number 15) and D-mngAB (5'-GGCTGAAATTAGTTTCGGTTGG-3') (sequence number 19), colony direct PCR was performed using OneTaq DNA polymerase (New England Biolabs) with the colonies as template DNA. The PCR reaction solution composition and reaction conditions were as per the attached instructions. For colonies in which amplification was observed around 6000 bp, single colony isolation was performed using LB agar medium containing kanamycin, and then the strain B (MG1655(DE3)mngAB::P T7 The strains were suspended in LB medium containing 15% glycerol as sucrose (-tmo) and stored at -80°C. When using the strains, the stored strains were spread on LB agar medium containing kanamycin and cultured overnight at 37°C, and the resulting colonies were used.

[0098] [1.3.3] Preparation of strain C Using pET21a-FRT-pheA1A2 as a template, the gene insertion cassette FRT-Km-FRT-P was inserted into the chromosome using a primer set of delta-mngAB-F (5'-TACAAATACAATACAAATAATTTCAATCAAGTGAAATTGATCACATAATGATTCCGGGGATCCGTCGACC-3') (SEQ ID NO: 17) and delta-mngAB-FRT-R (5'-CGCATATGTGGCTTCGGTTTGATTGCTATTCAGGCAAGCCGGTAACTGAAATTCGCCAATCCGGATATAG-3') (SEQ ID NO: 18). T7 -pheA1A2 was amplified by PCR. PrimeStar GXL (Takara Bio) was used as the PCR enzyme, and PCR reaction was performed according to the attached instructions. The PCR amplified product was purified using a QIAquick PCR Purification Kit (Qiagen) and digested overnight at 37°C with the restriction enzyme DpnI (New England Biolabs). The digested product was purified again using a QIAquick PCR Purification Kit. When eluting DNA with the purification kit, sterile distilled water was used instead of the elution buffer included with the kit. The obtained DNA solution was diluted with FRT-Km-FRT-P T7 This was used as a -pheA1A2 cassette solution in the insertion experiment into the Escherichia coli chromosome described below.

[0099] Escherichia coli MG1655(DE3) / pKD46 strain was cultured in 5 mL of LB medium containing 10 mM L-arabinose and 50 μg / mL carbenicillin to reach an OD 660 The culture was incubated with shaking at 30°C until the value of was about 0.5. 2 mL of the culture solution was centrifuged at 5,000 x g (4°C) for 5 minutes to collect the bacterial cells, which were then washed with 1 mL of sterile distilled water at 4°C. The culture solution was centrifuged again at 5,000 x g (4°C) for 5 minutes to collect the bacterial cells, which were then washed with 1 mL of sterile distilled water at 4°C. The culture solution was centrifuged again at 5,000 x g (4°C) for 5 minutes to collect the bacterial cells, which were then suspended in 50 μL of sterile distilled water. FRT-Km-FRT-P at a concentration of 100 to 200 ng / μL was added to the culture solution. T71 μL of the -pheA1A2 cassette solution was added to the bacterial cell suspension, and the entire volume was transferred to a 2 mm-wide electroporation cuvette. Electroporation was performed using a MicroPulser electroporator (Bio-Rad) at EC2 settings (output voltage 2.5 kV). 1 mL of LB medium was added to the cuvette to suspend the bacterial cells, and the entire volume was transferred to a 1.5 mL tube and cultured (rehabilitated) with shaking at 37°C for 3 hours. The entire volume was spread onto LB agar medium containing 20 μg / mL kanamycin, and cultured overnight at 37°C.

[0100] The colonies obtained after cultivation were examined to see whether the target gene had been inserted at the target site on the chromosomal DNA. Using a primer set of K2 (5'-CGGTGCCCTGAATGAACTGC-3') (sequence number 15) and D-mngAB (5'-GGCTGAAATTAGTTTCGGTTGG-3') (sequence number 19), colony direct PCR was performed using OneTaq DNA polymerase (New England Biolabs) with the colonies as template DNA. The PCR reaction solution composition and reaction conditions were as per the attached instructions. For colonies in which amplification was observed around 3000 bp, single colony isolation was performed using LB agar medium containing kanamycin, and then the strain C (MG1655(DE3)mngAB::P T7 The strains were suspended in LB medium containing 15% glycerol as pheA1A2 and stored at -80°C. When using the strains, the stored strains were spread on LB agar medium containing kanamycin and cultured overnight at 37°C, and the resulting colonies were used.

[0101] [1.3.4] Preparation of strain D Using pET21a-FRT-pobA as a template and a primer set of delta-mngAB-F (5'-TACAAATACAATACAAATAATTTCAATCAAGTGAAATTGATCACATAATGATTCCGGGGATCCGTCGACC-3') (SEQ ID NO: 17) and delta-mngAB-FRT-R (5'-CGCATATGTGGCTTCGGTTTGATTGCTATTCAGGCAAGCCGGTAACTGAAATTCGCCAATCCGGATATAG-3') (SEQ ID NO: 18), the gene insertion cassette FRT-Km-FRT-P was inserted into the chromosome. T7 -pobA was amplified by PCR. PrimeStar GXL (Takara Bio) was used as the PCR enzyme, and PCR reaction was performed according to the attached instructions. The PCR amplified product was purified using a QIAquick PCR Purification Kit (Qiagen) and digested overnight at 37°C with the restriction enzyme DpnI (New England Biolabs). The digested product was purified again using a QIAquick PCR Purification Kit. When eluting DNA with the purification kit, sterile distilled water was used instead of the elution buffer included with the kit. The obtained DNA solution was diluted with FRT-Km-FRT-P T7 This was used as a -pobA cassette solution in the insertion experiment into the Escherichia coli chromosome described below.

[0102] Escherichia coli MG1655(DE3) / pKD46 strain was cultured in 5 mL of LB medium containing 10 mM L-arabinose and 50 μg / mL carbenicillin to reach an OD 660 The culture was incubated with shaking at 30°C until the value of was about 0.5. 2 mL of the culture solution was centrifuged at 5,000 x g (4°C) for 5 minutes to collect the bacterial cells, which were then washed with 1 mL of sterile distilled water at 4°C. The culture solution was centrifuged again at 5,000 x g (4°C) for 5 minutes to collect the bacterial cells, which were then washed with 1 mL of sterile distilled water at 4°C. The culture solution was centrifuged again at 5,000 x g (4°C) for 5 minutes to collect the bacterial cells, which were then suspended in 50 μL of sterile distilled water. FRT-Km-FRT-P at a concentration of 100 to 200 ng / μL was added to the culture solution. T71 μL of the -pobA cassette solution was added to the bacterial cell suspension, and the entire volume was transferred to a 2 mm-wide electroporation cuvette. Electroporation was performed using a MicroPulser electroporator (Bio-Rad) at EC2 settings (output voltage 2.5 kV). 1 mL of LB medium was added to the cuvette to suspend the bacterial cells, and the entire volume was transferred to a 1.5 mL tube and cultured (rehabilitated) with shaking at 37°C for 3 hours. The entire volume was spread on LB agar medium containing 20 μg / mL kanamycin, and cultured overnight at 37°C.

[0103] The colonies obtained after cultivation were examined to see whether the target gene had been inserted at the target site on the chromosomal DNA. Using a primer set of K2 (5'-CGGTGCCCTGAATGAACTGC-3') (sequence number 15) and D-mngAB (5'-GGCTGAAATTAGTTTCGGTTGG-3') (sequence number 19), colony direct PCR was performed using OneTaq DNA polymerase (New England Biolabs) with the colonies as template DNA. The PCR reaction solution composition and reaction conditions were as per the attached instructions. For colonies in which amplification was observed around 2200 bp, single colony isolation was performed using LB agar medium containing kanamycin, and then the strain D (MG1655(DE3)mngAB::P T7 The strain was suspended in LB medium containing 15% glycerol as β-pobA and stored at -80°C. When using the strain, the stored strain was spread on LB agar medium containing kanamycin and cultured overnight at 37°C, and the resulting colonies were used.

[0104] [1.3.5] Preparation of strain E Using pET21a-FRT-phhAB as a template, a primer set of delta-mngAB-F (5'-TACAAATACAATACAAATAATTTCAATCAAGTGAAATTGATCACATAATGATTCCGGGGATCCGTCGACC-3') (SEQ ID NO: 17) and delta-mngAB-FRT-R (5'-CGCATATGTGGCTTCGGTTTGATTGCTATTCAGGCAAGCCGGTAACTGAAATTCGCCAATCCGGATATAG-3') (SEQ ID NO: 18) was used to insert the gene cassette FRT-Km-FRT-P into the chromosome. T7 -phhAB was amplified by PCR. PrimeStar GXL (Takara Bio) was used as the PCR enzyme, and PCR reaction was performed according to the attached instructions. The PCR amplified product was purified using a QIAquick PCR Purification Kit (Qiagen) and digested overnight at 37°C with the restriction enzyme DpnI (New England Biolabs). The digested product was purified again using a QIAquick PCR Purification Kit. When eluting DNA with the purification kit, sterile distilled water was used instead of the elution buffer included with the kit. The obtained DNA solution was diluted with FRT-Km-FRT-P T7 This was used as a -phhAB cassette solution in the insertion experiment into the Escherichia coli chromosome described below.

[0105] Escherichia coli MG1655(DE3) / pKD46 strain was cultured in 5 mL of LB medium containing 10 mM L-arabinose and 50 μg / mL carbenicillin to reach an OD 660 The culture was incubated with shaking at 30°C until the value of was about 0.5. 2 mL of the culture solution was centrifuged at 5,000 x g (4°C) for 5 minutes to collect the bacterial cells, which were then washed with 1 mL of sterile distilled water at 4°C. The culture solution was centrifuged again at 5,000 x g (4°C) for 5 minutes to collect the bacterial cells, which were then washed with 1 mL of sterile distilled water at 4°C. The culture solution was centrifuged again at 5,000 x g (4°C) for 5 minutes to collect the bacterial cells, which were then suspended in 50 μL of sterile distilled water. FRT-Km-FRT-P at a concentration of 100 to 200 ng / μL was added to the culture solution. T71 μL of the -phhAB cassette solution was added to the bacterial cell suspension, and the entire volume was transferred to a 2 mm-wide electroporation cuvette. Electroporation was performed using a MicroPulser electroporator (Bio-Rad) with the EC2 setting (output voltage 2.5 kV). 1 mL of LB medium was added to the cuvette to suspend the bacterial cells, and the entire volume was transferred to a 1.5 mL tube and cultured (rehabilitated) with shaking at 37°C for 3 hours. The entire volume was spread on agar LB medium containing 20 μg / mL kanamycin, and cultured overnight at 37°C.

[0106] The colonies obtained after cultivation were examined to see whether the target gene had been inserted at the target site on the chromosomal DNA. Using a primer set of K2 (5'-CGGTGCCCTGAATGAACTGC-3') (sequence number 15) and D-mngAB (5'-GGCTGAAATTAGTTTCGGTTGG-3') (sequence number 19), colony direct PCR was performed using OneTaq DNA polymerase (New England Biolabs) with the colonies as template DNA. The PCR reaction solution composition and reaction conditions were as per the attached instructions. For colonies in which amplification was observed around 2200 bp, single colony isolation was performed using LB agar medium containing kanamycin, and then the strain E (MG1655(DE3)mngAB::P T7 The strain was suspended in LB medium containing 15% glycerol as phhAB (-phhAB) and stored at -80°C. When using the strain, the stored strain was spread on LB agar medium containing kanamycin and cultured overnight at 37°C, and the resulting colonies were used.

[0107] The list of the strains prepared is shown in Table 3. The strains prepared and the classification of the genes and enzymes they possess are shown in Table 4.

[0108] [Table 3]

[0109] [Table 4]

[0110] [2] Experiment 1 (test tube culture) Using strain A, strain B, strain C, strain D, or strain E, an oxidation reaction experiment of p-isopropenylphenol in test tube culture was carried out as follows.

[0111] [2.1] Biocatalytic reaction process A colony of each of strains A, B, C, D, or E on the LB agar medium was inoculated into 5 mL of LB medium and pre-cultured at 33°C for 8 hours. 50 μL of the pre-culture was inoculated into 5 mL of HCF medium and pre-cultured at 33°C for 16 hours. 50 μL of the pre-culture was inoculated into 5 mL of new HCF medium and the OD 660 The culture was shaken at 33°C until the value of was about 0.5. 50 μL of 100 mM isopropyl β-D-thiogalactopyranoside (IPTG) was added, and the culture was further cultured at 33°C for 2 hours. 500 μL of 5 mM paraisopropenylphenol (I-1) (trade name: Mirex (registered trademark) PM) solution sterilized with a 0.22 μm sterilized filter was added, and the culture was cultured at 33°C for 20 hours. The concentration of paraisopropenylphenol (I-1) was 0.45 mmol / L in water. Paraisopropenylphenol was added to HCF medium without inoculation of the strain, and the medium was shaken at 33°C in the same manner as above to serve as a control.

[0112] [2.2] Analysis The culture solution after the biocatalytic reaction process was analyzed using a liquid chromatograph (HPLC) (Shimadzu Corporation) equipped with a C18 column (COSMOSIL 5 C18-MS-II 3.0 × 150 mm, Nacalai Tesque) and a photodiode array. Eluent A was 0.1 v / v% trifluoroacetic acid, and eluent B was 80 v / v% methanol containing 0.1 v / v% trifluoroacetic acid. The column temperature was 40 °C, the flow rate was 0.4 mL / min, and a gradient was applied so that eluent B had an initial concentration of 20 v / v% to 100 v / v% in 5 minutes, and the column was operated in this state for another 5 minutes. Under these conditions, paraisopropenylphenol (I-1) eluted at around 9.2 minutes, and a compound expected to be paraisopropenylcatechol (II-1) eluted at around 8.3 minutes. A wavelength of 280 nm was used for detection. The HPLC analysis results are shown in Figure 1.

[0113] In FIG. 1, "Control" indicates a culture medium obtained by carrying out a biocatalytic reaction process without inoculating a strain. In FIG. 1, "Strain A" indicates a culture medium obtained by carrying out a biocatalytic reaction process using strain A. In FIG. 1, "Strain B" indicates a culture medium obtained by carrying out a biocatalytic reaction process using strain B. In FIG. 1, "Strain C" indicates a culture medium obtained by carrying out a biocatalytic reaction process using strain C. In FIG. 1, "Strain D" indicates a culture medium obtained by carrying out a biocatalytic reaction process using strain D. In FIG. 1, "Strain E" indicates a culture medium obtained by carrying out a biocatalytic reaction process using strain E.

[0114] As shown in FIG. 1, only in the culture solutions of strains A and C, the p-isopropenylphenol peak at around 9.2 minutes disappeared, and a new peak was detected at around 8.3 minutes.

[0115] [2.3] Peak Identification To identify the newly obtained peaks, the exact masses were measured using liquid chromatography-mass spectrometry (LC-MS). The LC-MS conditions were as follows:

[0116] [2.3.1] LC conditions LC: Agilent 1260 LC Column: COSMOSIL 5 C18-MRS-II (3.0 mm id × 150 mm, Nacalai Tesque) Column temperature: 40℃ Eluent A: 0.1v / v% formic acid (LC / MS grade, Thermo Fisher Scientific) Eluent B: Methanol (LC / MS grade, Merck) with 0.1v / v% formic acid ·Flow rate: 0.4 mL / min Gradient conditions: as shown in Table 5.

[0117] [Table 5]

[0118] [2.3.2] MS conditions ·MS instrument: Agilent 6530C Accurate-Mass Q-ToF MS Ion source: Dual Agilent Jet Stream ion source Ionization mode: negative ion mode Sheath gas temperature: 325℃ Sheath gas flow rate: 10 L / min Nebulizer gas pressure: 45 psi Dry gas temperature: 275℃ Dry gas flow rate: 8.0 L / min Ionization voltage: 4000 V

[0119] [2.3.3] Results The results of LC-MS are shown in Figures 2 and 3. In Figures 2 and 3, "culture supernatant of strain A" refers to the culture supernatant obtained by carrying out the biocatalytic reaction process using strain A. In Figures 2 and 3, "paraisopropenylphenol standard" refers to "Mirex (registered trademark) PM" manufactured by Mitsui Chemicals, Inc.

[0120] As shown in Figures 2 and 3, in the culture supernatant (post-reaction sample) of strain A, new peaks of m / z 149.0606 and m / z 195.0645 were detected at a retention time of approximately 8.8 minutes, which was shorter than the peak at approximately 9.9 minutes corresponding to the standard paraisopropenylphenol (1-1). Analysis of the mass spectrum of the new peaks revealed that m / z 149.0606 was [MH] - The ion m / z 195.0645 is a formic acid adduct ([M+HCOO] - ) ions. The molecular formula of the compound contained in the new peak was calculated from these m / z values, and it was found to be C 9 H 10 O 2 It was estimated to be para-isopropenyl catechol (II-1).

[0121] From these results, it was found that the method for producing a vinyl catechol compound, which includes a biocatalytic reaction step in which para-isopropenylphenol (I-1) is reacted with strain A (corresponding to the first bacterial body) or strain C (corresponding to the second bacterial body), can produce para-isopropenyl catechol (II-1) without using heavy metals.

[0122] [3] Experiment 2 (flask culture) Strain A was used in a flask culture to carry out an oxidation experiment of p-isopropenylphenol (I-1).

[0123] [3.1] Biocatalytic reaction process A colony of strain A on the LB agar medium was inoculated into 5 mL of LB medium and pre-cultured at 33°C for 8 hours. 50 μL of the pre-culture was inoculated into 5 mL of HCF medium and pre-cultured at 33°C for 16 hours. Then, 1 mL of the pre-culture was inoculated into 100 mL of the main culture medium. As the main culture medium, 100 mL of HCF medium not containing paraisopropenylphenol (I-1) or HCF medium containing paraisopropenylphenol (I-1) at a concentration of 1 mmol / L (hereinafter also referred to as "1 mM") in water was prepared, filtered, and then placed in a 500 mL baffled Erlenmeyer flask sterilized by autoclaving. In the HCF medium and the HCF medium containing 1 mM paraisopropenylphenol (I-1), the medium was cultured at 33°C for 6 hours, and 1 mL of 100 mM IPTG solution was added and cultured at 33°C for another 18 hours.

[0124] [3.2] Analysis The culture solution was diluted appropriately and analyzed by HPLC using the method described above. The HPLC analysis results are shown in Figure 4. In Figure 4, "1 mM" indicates the culture solution (hereinafter also referred to as "culture solution A") obtained by inoculating strain A into an HCF medium containing paraisopropenylphenol (I-1) at a concentration of 1 mM (1 mmol / L) in water and carrying out the biocatalytic reaction process. In Figure 1, "After ethyl acetate extraction (1 mM)" indicates the aqueous solution in the water layer after adding an equal amount of ethyl acetate to the supernatant of culture solution A, stirring, and centrifuging at 10,000 x g for 1 minute.

[0125] [3.3] Results The turbidity (OD) of each culture solution after incubation 660) was 5.57 in HCF medium containing 1 mM paraisopropenylphenol (I-1). Growth of strain A was confirmed in the presence of 1 mM paraisopropenylphenol (I-1). As shown in Figure 4, in the medium containing 1 mM paraisopropenylphenol (I-1), the 9.2 min peak of paraisopropenylphenol (I-1) disappeared in HPLC analysis, and instead, a peak of paraisopropenylcatechol (II-1) was observed at 8.3 min. In addition, when an equal amount of ethyl acetate was added to the supernatant of culture solution A, the mixture was stirred, and centrifuged at 10,000 × g for 1 minute, and the aqueous layer was analyzed by HPLC, the peak of paraisopropenylcatechol (II-1) disappeared as shown in Figure 4 (it is considered that it can be extracted from the culture supernatant with ethyl acetate).

[0126] [4] Experiment 3 (Jar Fermenter Cultivation) Using strain A, an experiment on the oxidation of p-isopropenylphenol (I-1) was carried out in a jar fermenter culture.

[0127] [4.1] Biocatalytic reaction process A colony of strain A on the LB agar medium was inoculated into two 5 mL tubes of LB medium and precultured at 37°C for 4 hours. 10 mL of the preculture was inoculated into a 3 L jar fermenter (Bioneer MODEL-8c, Marubishi Bio Engineering) containing 1 L of HCF medium, and incubated until the glucose in the medium was depleted (OD 660Culture was performed at 30°C (about 35°C). The feed solution was added to start glucose fed-batch culture, and 10 mL of 100 mM IPTG solution was added and culture was continued at 30°C for another 4 hours. The temperature was set at 33°C, and 100 mg of paraisopropenylphenol (I-1) powder (non-sterile) was added every 10 minutes for a total of 1.2 g. The culture solution was sampled at appropriate times, stored at -20°C, and appropriately diluted and analyzed by HPLC. The feed solution for fed-batch culture was prepared by dissolving 280 g of glucose in 186 mL of distilled water, which was sterilized by autoclaving, and additives were added aseptically. The additives were 16 mL of 50% magnesium sulfate heptahydrate solution, 4 mL of trace metal solution (10 g of iron (III) citrate n-hydrate, 250 mg of cobalt chloride hexahydrate, 150 mg of manganese chloride tetrahydrate, 150 mg of copper chloride dihydrate, 300 mg of boric acid, 250 mg of sodium molybdate dihydrate, 1.3 g of zinc acetate dihydrate, and 840 mg of ethylenediaminetetraacetic acid dihydrate (EDTA) in 1 L of distilled water), and 0.9 mL of 2% thiamine solution. The DO value of the jar fermenter was set to 2.26 ppm, and the DO value was controlled by controlling the stirring speed (100 rpm to 1000 rpm). The pH was set to 6.7 and controlled by adding 27% ammonia water. The addition of the feed solution was performed using the DO value as an index. That is, when the glucose in the medium was depleted and the DO value exceeded 4.5 ppm, 18 g of feed solution (equivalent to 10 g of glucose) was added as needed (the addition reduced the DO value to approximately the control value of 2.26).

[0128] [4.2] Analysis The results of the HPLC analysis are shown in Figure 5. The horizontal axis of the graph shown in Figure 5 indicates the time elapsed from the start of cultivation in the jar fermenter. The vertical axis of the graph shown in Figure 5 indicates the peak area of ​​para-isopropenylphenol (I-1) obtained from the HPLC chromatogram and the peak area of ​​para-isopropenylcatechol (II-1) obtained from the HPLC chromatogram.

[0129] [4.3] Results When paraisopropenylphenol (I-1) was added stepwise to a high-concentration culture medium, a peak of paraisopropenylcatechol (II-1) was detected, as shown in Figure 5. The peak of paraisopropenylcatechol (II-1) was increased up to about 5 times compared to the addition of 1 mM in Experiment 2. The area value of the peak of paraisopropenylcatechol (II-1) was larger compared to flask culture. As a result, it was found that a higher concentration of paraisopropenylcatechol (II-1) could be obtained.

Claims

1. A vinylphenol compound represented by the following formula (I), At least one selected from the group consisting of a first bacterial body containing 4-hydroxyphenylacetate-3-monooxygenase, a treated product of the first bacterial body, a second bacterial body containing phenol-2-monooxygenase, and a treated product of the second bacterial body; A method for producing a vinylcatechol compound, comprising the step of reacting 【Chemistry 1】 [In formula (I), R represents a hydrogen atom or a methyl group, The position of the hydroxyl group is -C(R)=CH ２ The position is meta or para to the group.

2. The 4-hydroxyphenylacetate-3-monooxygenase has an amino acid sequence having 60% or more identity to the amino acid sequence of SEQ ID NO:1, The method for producing a vinylcatechol compound according to claim 1, wherein the phenol-2-monooxygenase has an amino acid sequence having 60% or more identity with the amino acid sequence of SEQ ID NO:

2.

3. R represents a methyl group; The method for producing a vinylcatechol compound according to claim 1 or 2, wherein the position of the hydroxyl group is a para position.

4. the step includes adding the vinylphenol compound all at once to a medium containing at least one selected from the group consisting of the first bacterial body, a treated product of the first bacterial body, the second bacterial body, and a treated product of the second bacterial body, and a liquid medium; The method for producing a vinylcatechol compound according to claim 1 or 2, wherein the concentration of the vinylphenol compound in the liquid medium is 1.5 mmol / L or less.

5. the step includes adding the vinylphenol compound in two or more divided portions to a medium containing at least one selected from the group consisting of the first bacterial body, a treated product of the first bacterial body, the second bacterial body, and a treated product of the second bacterial body, and a liquid medium; The method for producing a vinylcatechol compound according to claim 1 or 2, wherein the final concentration of the vinylphenol compound is more than 1.5 mmol / L relative to the liquid medium.

6. The method for producing a vinylcatechol compound according to claim 5, wherein the final concentration of the vinylphenol compound is 6.0 mmol / L or more relative to the liquid medium.

7. 3. The method for producing a vinylcatechol compound according to claim 1, wherein in the step, the vinylphenol compound is reacted with at least one selected from the group consisting of the second bacterial body and a processed product of the second bacterial body.

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