JP5324129B2 - Method for producing aromatic compound by CYP153 - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide methods for (1) introducing hydroxy group(s) into a substituent-bearing aromatic compound, (2) condensing two or more phenolic hydroxy-containing aromatic compounds via a carbon-carbon bond or ether linkage, and (3) converting a substituent-bearing cyclic olefin into the corresponding aromatic compound, in high efficiency using living cells in which CYP153 is synthesized. <P>SOLUTION: The method for producing an aromatic compound is provided with a process of making a protein belonging to CYP153 of cytochrome P450 or a fused protein containing the same function as a monooxygenase, and (1) introducing hydroxy group(s) into a substituent-bearing aromatic compound by making the monooxygenase act thereon, and a step of (2) condensing two or more phenolic hydroxy-containing aromatic compounds via a carbon-carbon bond or ether linkage or a step of (3) converting a substituent-bearing cyclic olefin into the corresponding aromatic compound. <P>COPYRIGHT: (C)2009,JPO&amp;INPIT

Description

  The present invention relates to a method for producing an aromatic compound using an enzyme.

  Cytochrome P450 (hereinafter referred to as “P450”) monooxygenase is a monooxygenase that is distributed in a wide range of species from bacteria to plants and mammals. P450s derived from bacteria, including actinomycetes, usually transfer electrons from NAD (P) H to P450. Protein) [These two proteins are collectively referred to as an electron transfer protein, or a redox partner protein. ] Is required. However, P450 has a strict affinity with these electron transfer proteins, and P450 itself is an unstable enzyme that tends to be inactivated. Therefore, even if a new P450 gene was obtained, its functional expression was difficult.

  Rhodococcus genus NCIMB9784 P450RhF has a reductase domain (reductase domain, reductase peptide, redox partner protein) consisting of ferredoxin reductase part (FMN as coenzyme) and ferredoxin part (Fe-S small protein) A group at the University of Edinburgh showed that the part; the C-terminal side has an unusual structure called a single polypeptide linked to the P450 body protein part (N-terminal side) via a linker sequence ( Non-patent document 1). Nozaki et al. Produced a functional expression vector pRED for E. coli with NdeI and EcoRI sites as cloning sites for the P450 gene to be analyzed using the reductase end containing the linker sequence of P450RhF (non-patent literature). 2). As a base vector for Escherichia coli, pET21a (Novagen) controlled by a T7 promoter is used. The structure of the pRED vector is shown in FIG. When this pRED vector was used, it was shown that several P450 genes (P450cam, P450Bzo, P450balk) derived from bacteria are functionally expressed in E. coli (Non-patent Document 2).

  P450balk is a P450 belonging to family 153 (CYP153) derived from Alcanivorax borkumensis SK2 strain, a marine bacterium that assimilates alkanes derived from petroleum. In 3), the number CYP153A13a is assigned. As for the CYP153 family, CYP153A1 was first discovered in the Acinetobacter calcoaceticus EB104 strain (Non-patent Document 4). Since then, P450 belonging to the CYP153 family has been discovered in several types of microorganisms capable of assimilating alkanes (Non-patent Document 3: the aforementioned Dr. Nelson's website). Regarding the functions of the CYP153 family, the PYP can be expressed by expressing the CYP153A1 gene using Pseudomonas putida, which lost alkane (octane) assimilation ability, as the host, and observing the growth state in a medium containing octane. There has been a report suggesting that it is a monooxygenase that introduces a hydroxyl group at the end of octane (Non-patent Document 5).

  Until recently, there was no report directly observing that P450 had an activity to introduce a hydroxyl group at the terminal of alkane, including P450 belonging to the CYP153 family (hereinafter simply referred to as CYP153). Since 2005, Noboru et al. (Non-Patent Document 2), Kubota et al. (Non-Patent Document 6) of Marine Biotechnology Research Institute, Ltd., and Fujii et al. It was observed that hydroxyl groups were directly attached to the ends of alkanes having 6 to 8 carbon atoms by the transformed Escherichia coli. The method of Nobiru et al. And Kubota et al. Of the Marine Biotechnology Research Institute Co., Ltd. uses a pRED vector containing P450RhF, and the method of Mercian Co., Ltd. Fujii et al. Is a Puchidaare derived from Pseudomonas putida. Co-expression of doxin reductase and putidaredoxin gene in E. coli. In addition, Kubota et al. Revealed that CYP153 can introduce a hydroxyl group at the end of n-butylbenzene in the aforementioned literature. Fujii et al. Have also observed in the above-mentioned literature that a hydroxyl group is also attached to the other end of n-octanol produced using n-octane as a substrate.

Here, Patent Document 1 discloses a sequence of a gene encoding a protein having monooxygenase activity derived from bacteria collected from various environments, and a linker molecule on the C-terminal side of the P450 body protein portion encoded by these genes. A fusion type P450 monooxygenase to which a peptide having a function similar to that of the reductase domain of Rhodococcus NCIMB9784 is added. This fusion protein acts on alkanes, alkenes, cyclic hydrocarbons, alkanes and alkenes containing aromatic rings in the molecule, introduces hydrogen into the terminal methyl group, and catalyzes the epoxidation of terminal olefins.
Thus, it has recently been clarified that CYP153 has an enzyme activity that can specifically hydroxylate a relatively long end of an alkane having usually 6 to 8 carbon atoms. However, it can be said that functional expression of the CYP153 gene is a difficult technique, as can be seen from the fact that direct conversion of alkane in recombinant Escherichia coli was finally achieved after 2005.

  P450BM3 (CYP102A1) derived from the bacterium Bacillus megaterium has an unusual structure as a bacterium-derived P450. That is, this enzyme consists of a P450 body protein part (N-terminal side) and a NADPH-P450 reductase part (C-terminal side) containing FAD and FMN as coenzymes in the molecule through a linker sequence. It has a connected structure as a polypeptide. Because of its structure, the gene was functionally expressed in E. coli as rare as P450. Therefore, many studies have been conducted on P450BM3. P450BM3 was originally a monooxygenase that hydroxylates the side chain of fatty acids, but by molecular evolution engineering, 2-octanol, 3-octanol and 4-octanol are produced from n-octane and 2-hexanol from n-hexane. And 3-hexanol (Non-patent Document 8). Furthermore, it was shown that the mutant strain P450BM3 (F87V) in which the 87th phenylalanine (Phe) was changed to valine (Val) in P450BM3 was mutated so as to use an aromatic compound as a substrate. That is, E. coli expressing P450BM3 (F87V) was able to perform a monooxygenase reaction using naphthalene or some three-membered hydrocarbons as substrates (Non-patent Document 9). For example, 1-naphthol and 2-naphthol were synthesized from naphthalene, 9-fluorenol was synthesized from fluorene, and 9-anthracenemethanol was synthesized from 9-methylanthracene. Furthermore, it has been clarified that P450BM3 (F87V) can introduce a hydroxyl group at the p-position of various phenol-substituted products (Non-patent Document 10). For example, synthesize hydroquinone from phenol, synthesize 2-methylhydroquinone from o-cresol, synthesize 2-methoxyhydroquinone from guaiacol (o-methoxyphenol), synthesize 2-bromohydroquinone from o-bromophenol, 2,6-Dichlorohydroquinone could be synthesized from 2,6-dichlorophenol. Furthermore, P450BM3 (F87V) was able to synthesize 4-hydroxy-β-ionone using β-ionone as a substrate (Non-patent Document 11). Thus, P450BM3 (F87V) was found to be a very multifunctional enzyme with respect to substrate affinity (substrate specificity). Nobuchi et al. Of Marine Biotechnology Research Institute, Ltd. found a protein gene fused with PPIase (peptidylprolyl cis-trans isomerase) derived from archaea having chaperone-like function at the N-terminus of P450BM3 (F87V). When E. coli is expressed in E. coli, it has been shown that E. coli expressing the P450BM3 (F87V) gene that is not fused with PPIase exhibits several times stronger conversion activity in terms of indigo synthesis from indole and improves thermal stability ( Patent Document 2). Note that. This plasmid for fusion protein gene expression of PPIase derived from archaea and P450BM3 (F87V) was named pFusionP450.

  On the other hand, as other reactions using P450, a condensation reaction via an aromatic compound having a hydroxyl group on an aromatic ring via a carbon-carbon bond and a reaction via an ether bond are known. For example, P450mel condensation using 1,3,6,8-tetrahydronaphthalene as a substrate via a carbon-carbon bond (Non-patent Document 12), CYP158A2 condensation via a carbon-carbon bond using a flaviolin as a substrate (non-patent document 12) Patent Document 13), condensation of an aromatic ring by forming an ether bond in the molecule of vancomycin by P450 (Non-Patent Document 14) and the like are known. In addition to P450, 4-hydroxybiphenyl and 3-chloro-4-hydroxybiphenyl may be condensed via a carbon-carbon bond by laccase having a function of oxidizing phenol (Non-patent Document 15), and 2,4 It is known that -dichlorophenol condenses by carbon-carbon bond or ether bond by laccase (Non-patent Document 16).

  However, in the conversion example using these enzymes, it is necessary to take out an unstable enzyme protein from the microbial cells and carry out the reaction, which is not preferable for producing a large amount of the target compound.

  In chemical synthesis, condensation of aromatic compounds having a hydroxyl group on an aromatic ring via a carbon-carbon bond is performed using a reaction using vanadyl acetylacetonate (VO (acac) 2) as a catalyst (Non-patent Document 17) or trivalent iron. A reaction using ferric chloride or the like is known (Non-patent Document 18). However, the reaction using vanadyl acetylacetonate can condense various 2-naphthol derivatives or phenol derivatives, but requires an expensive catalyst for the product, and the reaction does not proceed in aqueous systems, which is a harmful halogen system. There was a problem of requiring a solvent. The reaction using ferric chloride can condense a 2-naphthol derivative in an aqueous system, but trivalent iron is not a catalytic amount, and preferably 1 equivalent or more is required, resulting in poor catalytic efficiency. was there. Therefore, there is a need for a method that can produce various aromatic compounds safely, simply, efficiently, and in large quantities without using harmful substances.

Furthermore, CYP19, also called P450arom or aromatase, is known to be an enzyme involved in the biosynthesis system of steroid hormones that aromatize androstenedione in vivo to give estrone and aromatize testosterone to give estradiol. (Non-patent documents 19 and 20). However, CYP153 has never been reported to catalyze such a reaction.
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  The present invention efficiently uses (1) a method for introducing a hydroxyl group into an aromatic compound having a substituent, and (2) an aromatic compound having two or more phenolic hydroxyl groups is carbonized using living cells synthesized with CYP153. The main object is to provide a method of condensing via a carbon bond or an ether bond, and (3) a method of aromatizing a cyclic olefin having a substituent.

In order to solve the above-mentioned problems, the present inventors have conducted intensive research and obtained the following knowledge.
(i) By allowing a protein belonging to CYP153 or a fusion protein containing the protein to function as a monooxygenase and acting on an aromatic compound having a substituent, a hydroxyl group can be efficiently introduced into the aromatic compound.
(ii) A protein belonging to CYP153 or a fusion protein containing the same is allowed to function as a monooxygenase and act on two or more aromatic compounds having a phenolic hydroxyl group, whereby two or more aromatic rings have carbon-carbon bonds or ether bonds. Can be condensed.
(iii) The cyclic olefin of the compound can be aromatized by allowing a protein belonging to CYP153 or a fusion protein containing the protein to function as a monooxygenase and acting on a cyclic olefin having a substituent.
(iv) The reactions (i) to (iii) are carried out by using a protein belonging to CYP153 or a fusion protein containing the protein, and a protein that transmits electrons to CYP153 as necessary (particularly in the former case) (electron transfer protein; It can be performed by allowing a transformant introduced with a recombinant vector that expresses a redox partner protein) to act on an aromatic compound, etc., but it is considerably weaker than the T7 promoter used by conventional pRED vectors If a transformant obtained by transforming E. coli (Escherichia coli) with a recombinant vector in which the CYP153 gene is inserted into a pUC vector using the considered lac promoter is used, it has the conventional pFusionP450, which is a highly functional plasmid. The hydroxyl group introduction reaction is easier to proceed than in E. coli.
(v) The above reactions (i) to (iii) can be performed using living cells that express a protein belonging to CYP153 or a fusion protein containing the same. There is no need to use it, and after the reaction, the cells can be easily removed by centrifugation or the like, so that the product can be easily purified. For this reason, the target compound can be easily produced in large quantities by an enzymatic method.

The present invention has been completed based on the above findings, and provides an efficient method for producing the novel aromatic compounds of the following items and a recombinant vector.
Item 1. A process of introducing a hydroxyl group into an aromatic ring or a carbon atom of a substituent by allowing a protein belonging to the cytochrome P450 family 153 (CYP153) or a fusion protein containing the protein to function as a monooxygenase and acting on an aromatic compound having a substituent A process for producing an aromatic compound, comprising:
Item 2. A protein belonging to the cytochrome P450 family 153 (CYP153) or a fusion protein containing the protein is allowed to function as a monooxygenase and act on the aromatic compound 2 or more having a phenolic hydroxyl group, so that the aromatic ring 2 or more is a carbon-carbon bond or A method for producing an aromatic compound, comprising a step of condensing through an ether bond.
Item 3. A fragrance comprising a step of aromatizing a cyclic olefin by allowing a protein belonging to cytochrome P450 family 153 (CYP153) or a fusion protein containing the protein to function as a monooxygenase and acting on a cyclic olefin having a substituent. Group compound production method.

Item 4. Item 4. The production method according to any one of Items 1 to 3, wherein CYP153 is the following (a) or (b).
(A) Polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 (b) In the amino acid sequence of SEQ ID NO: 1, consisting of an amino acid sequence in which one or several amino acid sequences are deleted, substituted or added, and as monooxygenase P450 Functioning polypeptide term 5. Item 4. The production method according to any one of Items 1 to 3, wherein CYP153 is the following (o) or (p).
(o) a polypeptide comprising the amino acid sequence of SEQ ID NO: 12
(p) a polypeptide comprising an amino acid sequence in which one or several amino acid sequences are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 12, and functioning as monooxygenase P450

Item 6. Item 6. The method according to any one of Items 1, 4, and 5, wherein the aromatic compound having a substituent has 1 to 3 benzene rings.
Item 7. Item 7. The method according to any one of Items 1 and 4 to 6, wherein a hydroxyl group is introduced into a carbon atom of an aromatic ring of an aromatic compound having a substituent.
Item 8. Item 7. The production method according to any one of Items 1 and 4 to 6, wherein the substituent of the aromatic compound having a substituent is an alkyl group having 1 to 4 carbon atoms, and a hydroxyl group is introduced into a carbon atom of the alkyl group.
Item 9. Item 7. The production method according to any one of Items 1 and 4 to 6, wherein the substituent of the aromatic compound having a substituent is an alkoxy group having 1 to 2 carbon atoms, and a hydroxyl group is introduced by dealkylation of the alkoxy group.

Item 10. Item 6. The production method according to any one of Items 2, 4, and 5, wherein the aromatic compound having a phenolic hydroxyl group has 1 to 3 benzene rings.
Item 11. Item 6. The production method according to any one of Items 3 to 5, wherein the cyclic olefin having a substituent has a substituent bonded to the cyclic olefin, and aromatizes the cyclic olefin.

Item 12. By coexisting a transformant introduced with a gene encoding a protein belonging to CYP153 or a fusion protein containing the same and an aromatic compound having a substituent, the protein belonging to CYP153 or a fusion protein containing the same is obtained. Item 10. The production method according to any one of Items 1 and 4 to 9, which acts on an aromatic compound having a substituent.
Item 13. By coexisting a transformant introduced with a gene encoding a protein belonging to CYP153 or a fusion protein containing the same and an aromatic compound having a phenolic hydroxyl group, a protein belonging to CYP153 or a fusion protein containing the same is obtained. Item 11. The production method according to any one of Items 2, 4 to 5 and 10, wherein the method acts on an aromatic compound having a phenolic hydroxyl group.
Item 14. By coexisting a transformant introduced with a gene encoding a protein belonging to CYP153 or a fusion protein containing the same and a cyclic olefin having a substituent, the protein belonging to CYP153 or a fusion protein containing the same is converted into the circular form. Item 12. The production method according to any one of Items 3 to 5 and 11, which acts on an olefin.

Item 15. Item 15. The production method according to any one of Items 12 to 14, wherein the transformant is a gene into which a gene encoding a fusion protein of CYP153 and a redox partner protein is introduced.
Item 16. A transformant is a gene encoding a fusion protein of CYP153 and a reductase peptide contained in cytochrome P450 monooxygenase P450RhF derived from Rhodococcus sp. Item 16. The production method according to Item 15, wherein a recombinant vector inserted into a pUC vector so as to utilize a translation signal of lac promoter and LacZ is introduced.
Item 17. Item 17. The method according to Item 15 or 16, wherein the redox partner protein is the following peptide (c) or (d):
(c) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 2
(d) a peptide comprising an amino acid sequence in which one or more amino acids are added, deleted or substituted in the amino acid sequence described in SEQ ID NO: 2 and having reductase activity

Item 18. The recombinant vector contains the following DNA (g) or (h) encoding a reductase peptide via a linker on the 3 ′ end side of the following (e) or (f) DNA encoding CYP153. Item 18. The production method according to Item 16 or 17, which is a recombinant vector in which the linked fusion protein expression cassette is inserted into a pUC vector so as to use the lac promoter and LacZ translation signal.
(e) DNA consisting of the nucleotide sequence of SEQ ID NO: 3
(f) DNA that hybridizes with the DNA of SEQ ID NO: 3 under stringent conditions and encodes a protein having monooxygenase activity
(g) DNA consisting of the nucleotide sequence of SEQ ID NO: 4
(h) a DNA that hybridizes with the DNA of SEQ ID NO: 4 under a stringent condition and encodes a protein having reductase activity

Item 19. The recombinant vector contains the following (g) or (h) DNA encoding a reductase peptide via a linker on the 3 ′ end side of the following (w) or (x) DNA encoding CYP153. Item 18. The production method according to Item 16 or 17, which is a recombinant vector in which the linked fusion protein expression cassette is inserted into a pET vector so as to receive transcription of the T7 promoter.
(w) DNA consisting of the base sequence of SEQ ID NO: 16
(x) DNA that hybridizes with the DNA of SEQ ID NO: 16 under a stringent condition and encodes a protein having monooxygenase activity
(g) DNA consisting of the nucleotide sequence of SEQ ID NO: 4
(h) a DNA that hybridizes with the DNA of SEQ ID NO: 4 under a stringent condition and encodes a protein having reductase activity

Item 20. Fusion protein expression in which the following (g) or (h) DNA encoding a reductase peptide is linked to the 3 ′ end of the following (e) or (f) DNA encoding CYP153 via a linker A recombinant vector in which the cassette is inserted into a pUC vector so as to utilize the translation signal of the lac promoter and LacZ.
(e) DNA consisting of the nucleotide sequence of SEQ ID NO: 3
(f) DNA that hybridizes with the DNA of SEQ ID NO: 3 under stringent conditions and encodes a protein having monooxygenase activity
(g) DNA consisting of the nucleotide sequence of SEQ ID NO: 4
(h) a DNA that hybridizes with the DNA of SEQ ID NO: 4 under a stringent condition and encodes a protein having reductase activity

Item 21. Fusion protein expression in which the following (g) or (h) DNA encoding a reductase peptide is linked to the 3 ′ end of the following (w) or (x) DNA encoding CYP153 via a linker A recombinant vector in which a cassette is inserted into a pET vector so as to receive transcription of a T7 promoter.
(w) DNA consisting of the base sequence of SEQ ID NO: 16
(x) DNA that hybridizes with the DNA of SEQ ID NO: 16 under a stringent condition and encodes a protein having monooxygenase activity
(g) DNA consisting of the nucleotide sequence of SEQ ID NO: 4
(h) a DNA that hybridizes with the DNA of SEQ ID NO: 4 under a stringent condition and encodes a protein having reductase activity

The method for producing an aromatic compound according to the present invention comprises a protein belonging to the cytochrome P450 family 153 (CYP153) or a fusion protein containing the protein, and functioning as a monooxygenase. Or (2) condensation of two or more aromatic rings of a compound having a phenolic hydroxyl group via a carbon-carbon bond or an ether bond, or (3) aromatization of a cyclic olefin.
Conventionally, a reaction in which a protein belonging to CYP153 functions as a monooxygenase to introduce a hydroxyl group into an aliphatic compound has been known. The present invention makes it possible for the first time to introduce a hydroxyl group into an aromatic ring of an aromatic compound having a substituent using a monooxygenase belonging to CYP153, or to introduce a hydroxyl group into a substituent on the aromatic ring of an aromatic compound. became. The hydroxyl group-introducing reaction according to the present invention includes, in addition to the above reaction, a reaction that results in the generation of a hydroxyl group by dealkylation of an alkoxy group.

Furthermore, according to the present invention, when a protein belonging to CYP153 functions as a monooxygenase and acts on a compound having a phenolic hydroxyl group, it condenses two or more aromatic rings via a carbon-carbon bond or an ether bond, and a substituent. For the first time, it has become possible to aromatize cyclic olefins in cyclic olefins having the following.
When (1) introducing a hydroxyl group into an aromatic compound by chemical synthesis, (2) when two or more aromatic rings are condensed via a carbon-carbon bond or an ether bond, or (3) when a cyclic olefin is aromatized In some cases, multiple steps are required, and introduction or elimination reaction of a protecting group may be required. Moreover, severe conditions of high temperature and high pressure, expensive or dangerous reagents, waste liquid treatment, etc. may be required. In this respect, since the method of the present invention is a reaction using an enzyme, the reaction can be advanced specifically in one step. Furthermore, since the reaction is carried out using living cells that produce CYP153, there is an excellent advantage that the enzyme is not necessary to be purified, and it is easy to mass-produce the conversion product. In addition, since the reaction is performed with an enzyme, the reaction can be performed under mild conditions, and it is a very environmentally friendly production method that does not require the use of a reagent that adversely affects the environment.
Therefore, according to the present invention, various aromatic compounds useful as pharmaceuticals, agricultural chemicals, dyes, and synthetic intermediates thereof can be easily and efficiently produced in large quantities under mild conditions.

Hereinafter, the present invention will be described in detail.
The method for producing an aromatic compound of the present invention is a method for producing an aromatic compound by causing a protein belonging to CYP153 or a fusion protein containing these to function as a monooxygenase and acting on a raw material (substrate). As the raw material (substrate) in the production method of the present invention, (1) an aromatic compound having a substituent, (2) two or more aromatic compounds having a phenolic hydroxyl group, or (3) a cyclic olefin having a substituent is used. Hereinafter, a method for producing an aromatic compound using an aromatic compound having a substituent as a raw material (also referred to as production method 1), a method for producing an aromatic compound using two or more aromatic compounds having a phenolic hydroxyl group (manufacturing method) 2) and a method for producing an aromatic compound using a cyclic olefin having a substituent as a raw material (also referred to as production method 3).

Manufacturing method 1
In the production method 1 of the present invention, an aromatic compound having a substituent is allowed to function as a monooxygenase from a protein belonging to CYP153 or a fusion protein containing the same, and this is allowed to act so that a hydroxyl group is attached to a carbon atom of the aromatic ring or substituent. The process of introducing. Thereby, the aromatic compound which has a hydroxyl group can be manufactured.
The aromatic compound having a hydroxyl group obtained by the production method 1 of the present invention has an affinity for water by hydrogen bonding and is rich in reactivity, so that it can be suitably used as a pharmaceutical, agrochemical, dye, synthetic intermediate or the like. Is.

Aromatic compound having a substituent An aromatic compound having a substituent is used as a raw material of the production method 1 of the present invention. As such an aromatic compound, an aromatic compound having 1 to 3 aromatic rings is preferable. Among them, a compound having 1 to 3 benzene rings, for example, a compound having a ring structure formed only with an aromatic hydrocarbon such as benzene, naphthalene, biphenyl, anthracene, phenanthrene and the like is preferable. Further, it may be an aromatic compound containing a heterocyclic ring, and examples of such a compound include compounds in which a benzene ring and a heterocyclic ring are condensed such as indole, quinoline, anthraquinone, pyridylbenzene, As the aromatic compound containing a heterocyclic ring, an aromatic compound containing nitrogen in the benzene ring is preferable. In addition, examples of the aromatic compound in which the aromatic ring is not directly bonded include those in which the aromatic ring and the aromatic ring are bonded via an oxygen atom, a sulfur atom, an amino group, or a carbonyl group, such as diphenyl ether and benzophenone. However, an aromatic compound bonded through an oxygen atom is preferable.

The type of the substituent is not particularly limited, but for example, an alkyl group having 1 to 4 carbon atoms (particularly, a methyl group, an ethyl group, a normal propyl group, an isopropyl group, an isobutyl group), a saturated or unsaturated 5- to 7-membered ring Hydrocarbon group (especially cyclohexenyl group), phenyl group, naphthyl group, phenoxy group, pyridyl group, phenylalkyl group having 7 to 9 carbon atoms, alkoxy group having 1 to 2 carbon atoms, halogen atom, hydroxyl group, amino group, substitution Amino group, protected amino group, aldehyde group, formyl group, carbonyl group, carboxyl group, protected carboxyl group, carboxylalkyl group, protected carboxyl group, alkyl group acyl group, acyl group (especially acetyl group), cyano group, oxo group, A nitro group, a sulfo group, an azo group, an azide group, etc. are mentioned. Of these, a methyl group, an ethyl group, an isopropyl group, an isobutyl group, an alkoxy group having 1 to 2 carbon atoms, a halogen atom, and a hydroxyl group are preferable.
The number of substituents varies depending on the type of ring of the aromatic compound. For example, in the case of a benzene ring, 1 to 6 can be used, but 1 to 3 is preferable. In the case of naphthalene, 1 to 8 can be used, but 1 to 4 is preferable. In the case of biphenyl, 1 to 10 is possible, but 1 to 4 is preferable. In the case of anthracene, 1 to 10 is possible, but 1 to 4 is preferable. In the case of phenanthrene, 1 to 10 can be used, but 1 to 4 is preferable.

Hydroxyl Introduction Reaction In the production method 1 of the present invention, a hydroxyl group is introduced into the aromatic ring of the aromatic compound having the substituent or a carbon atom of the substituent.
To introduce a hydroxyl group into a carbon atom of an aromatic ring of an aromatic compound having a substituent means to introduce a hydroxyl group into a carbon atom constituting the aromatic ring.
In the reaction for introducing a hydroxyl group into the carbon atom of the substituent of the aromatic compound having the substituent, the substituent of the aromatic compound having a substituent is an alkyl group having 1 to 4 carbon atoms, and the carbon of the alkyl group It is preferable to introduce a hydroxyl group into the atom. When the substituent of the aromatic compound having the above substituent is an alkoxy group, the reaction for introducing a hydroxyl group by dealkylation of the alkoxy group is also the carbon of the substituent of the aromatic compound having a substituent in the present invention. Included in the reaction of introducing a hydroxyl group into an atom. In this case, it is preferable that the substituent of the aromatic compound which has a substituent is a C1-C2 alkoxy group.

Manufacturing method 2
In the production method 2 of the present invention, a protein belonging to CYP153 or a fusion protein containing the protein is allowed to function as a monooxygenase, and is allowed to act on an aromatic compound 2 or more having a phenolic hydroxyl group, whereby an aromatic ring 2 or more is carbon-carbon bonded Or the process of condensing through an ether bond is included. Thereby, a condensate in which two or more aromatic rings in the raw material (substrate) compound are condensed via a carbon-carbon bond or an ether bond is produced.

Aromatic compound having a phenolic hydroxyl group As the aromatic compound 2 or more having a phenolic hydroxyl group, which is the substrate of the production method 2 of the present invention, two or more molecules of the same compound or a mixture of two or more different compounds are used. When an aromatic compound having two or more phenolic hydroxyl groups is used, a hybrid type condensation product can be obtained. Preferably, an aromatic compound having 1 to 3, more preferably 1 to 2 phenolic hydroxyl groups is used.

The aromatic compound in Production Method 2 is the same as the aromatic compound in Production Method 1 described above, and an aromatic compound having 1 to 3 benzene rings is preferable.
The number of phenolic hydroxyl groups in the aromatic compound that is a substrate varies depending on the type of ring of the aromatic compound. preferable. In the case of naphthalene, 1 to 7 can be used, but 1 to 4 is preferable. In the case of biphenyl, 1-9 are possible, but 1-4 are preferred. The aromatic compound having a phenolic hydroxyl group may have a substituent other than the phenolic hydroxyl group, but preferably has no substituent at the o-position or p-position of the phenolic hydroxyl group. More preferably, it has no substituent at the -position and p-position. Moreover, when it has a substituent in o-position and / or p-position, it is preferable that a substituent is a halogen atom.

Condensation reaction In the production method of the present invention 2, a condensate in which two or more aromatic rings are condensed via a carbon-carbon bond or an ether bond is formed. The condensation reaction in the present invention is not particularly limited. Specifically, for example, when phenol is used as an aromatic compound having a phenolic hydroxyl group, 4,4′-dihydroxybiphenyl is obtained as a condensation product via a carbon-carbon bond. can get. When 4-bromophenol is used as an aromatic compound having a phenolic hydroxyl group, 4,4′-dihydroxybiphenyl is converted as a condensation product via a carbon-carbon bond, and 2- (4 -Bromophenoxy) -4-bromophenol is obtained respectively.

Manufacturing method 3
Production method 3 of the present invention includes a step of aromatizing a cyclic olefin of the compound by causing a protein belonging to CYP153 or a fusion protein containing the protein to function as a monooxygenase and acting on a cyclic olefin having a substituent. . Thereby, the compound which has an aromatic ring, ie, an aromatic compound, can be manufactured.

Cyclic Olefin Having Substituents As the raw material (substrate) in the production method 3 of the present invention, those having a substituent bonded to the cyclic olefin are preferable. As the substituent, a group formed by removing a hydrogen atom from an aromatic compound having a benzene ring or a substituted benzene ring 1 to 3, a group formed by removing a hydrogen atom from a heterocyclic compound, or The above-mentioned alkyl group, acyl group, ester group, cyano group, nitro group, ether group and the like are preferable. Examples of the group formed by removing a hydrogen atom from a benzene ring or an aromatic compound having a benzene ring 1 to 3 having a substituent or a heterocyclic compound include a phenyl group, a naphthyl group, a biphenyl group, and an anthracenylene group. , A phenanthrene group, an indolyl group, a pyrrolidino group, a piperidino group, a morpholino group, and a group in which a substituent is bonded to these groups.
As the cyclic olefin, for example, a ring having 3 or more carbon atoms, preferably 5 to 8 carbon atoms having one or more unsaturated bonds, specifically, cyclopentene, cyclopentadiene, cyclohexene, cyclohexadiene, cycloheptene, cycloheptadiene. , Cyclooctene, cyclooctadiene and the like. Of these, cyclohexene and cyclohexadiene are more preferable.

Aromatization reaction of cyclic olefin having a substituent In the production method 3 of the present invention, the cyclic olefin is aromatized to become an aromatic ring. Specifically, for example, when 1-phenyl-1-cyclohexene is used as the cyclic olefin having a substituent in the production method 3 of the present invention, the cyclohexene ring in the compound is aromatized to produce biphenyl.

In the production methods 1 to 3, the aromatic compound having a substituent, the aromatic compound having a phenolic hydroxyl group and the cyclic olefin having a substituent, which are raw materials (substrates), may be commercially available, It may be produced and used by a conventionally known and commonly used method.
The production methods 1 to 3 of the present invention will be further described below.

Reaction The reactions in the production methods 1 to 3 of the present invention are carried out in a suitable solution or solvent in a protein belonging to CYP153 isolated from a raw material compound and a biological material or a fusion protein containing this protein (hereinafter abbreviated as “CYP153”). Can be reacted with ferredoxin reductase and ferredoxin, which are redox (redox) partner proteins (electron transfer proteins) as necessary. Examples of isolated CYP153 include crushed materials, extracts, and purified products of biological materials such as bacterial cells. The isolated CYP153 may be immobilized on a suitable carrier. In this case, it is necessary that ferredoxin reductase and ferredoxin, which are electron transfer proteins that catalyze the oxidation reaction, coexist in the reaction system. When a protein (fusion protein) in which CYP153 protein is fused with a redox partner protein such as the reductase end of P450RhF is reacted with a raw material compound, it is not necessary to coexist with ferredoxin reductase or ferredoxin. CYP153 functions as a monooxygenase by reacting CYP153 and a redox partner protein together.
In addition, since P450 family proteins are unstable enzymes, the reaction of the present invention is preferably performed by contacting or coexisting a cell compound producing CYP153 and a raw material compound. This reaction may be performed by adding a raw material compound to a culture solution of cells that produce CYP153, for example, by allowing cells that produce CYP153 and the raw material compound to coexist in a buffer.

CYP153 and the cells that produce it will be described in detail later.
Hereinafter, a method using cells that produce CYP153 will be described. The reaction can be performed by a batch system such as a batch reaction or a fed-batch (semi-batch) reaction, or a continuous reaction system such as a perfusion reaction. The substrate can be added all at once or continuously.
The reaction conditions vary depending on the microorganism used and the type of raw material compound. For example, in the case of batch reaction, the concentration of the raw material compound in the reaction solution is preferably about 0.1 to 10 mM, more preferably about 0.5 to 3 mM. In the case of a water-insoluble and liquid raw material compound, it can be layered. The pH of the reaction solution is preferably about 5-9, more preferably about 6-8. Moreover, reaction may be normally performed at about 20-30 degreeC for about 12-24 hours. In the case of fed-batch (semi-batch) reaction or continuous reaction, the conditions may be set according to the batch reaction conditions. If it is the said range, the conversion rate of a raw material compound is high, and refinement | purification will become easy.

The aromatic compound produced in the reaction product reaction can be purified by conventional methods. For example, if necessary, it is subjected to treatment such as centrifugation and filtration to remove suspensions such as bacterial cells, and then extracted with a general extraction solvent, for example, an organic solvent such as ethyl acetate, chloroform, methanol, etc. Can be removed under reduced pressure and purified by subjecting to a treatment such as vacuum distillation, chromatography, ion exchange resin, or adsorbent resin.

Examples of the protein belonging to the monooxygenase CYP153 or the fusion protein monooxygenase CYP153 including the same include , for example, Rhodococcus genus, Acinetobacter genus, Alcanivorax genus, Candida Genus, Bacillus, Mycobacterium, Pseudomonas, Gordonia, Smaragdicoccus, Caulobacter or Streptomyces ) And the like.
Specifically, for example, CYP153A1 derived from Acinetobacter calcoaceticus (accession number AJ311718 registered on Nelson's website (http://drnelson.utmem.edu/CytochromeP450.html)), CYP153A2 (same accessor) Session No. AE005680), CYP153A13a derived from Arcaniborax volcummensis (same accession number (AY505118); SEQ ID NO: 1), CYP153 derived from Gordonia (SEQ ID NO: 12), CYP153A2 derived from Chorodobacter crescentus (SEQ ID NO: 11) and the like. In addition, CYP153A (A) (SEQ ID NO: 9), CYP153A (B) (SEQ ID NO: 10), etc. obtained from the metagenome can also be used.

Among these, (a) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 is preferable. In addition, (b) one or several amino acids in the amino acid sequence of SEQ ID NO: 1 (several amino acids are, for example, 20, preferably 10 and so on) deleted, substituted or added A polypeptide comprising a sequence and functioning as monooxygenase P450 can also be preferably used.
The polypeptide of SEQ ID NO: 1 can be isolated from a culture of Alcanivorax borkumensis strain SK2. Alternatively, it can be chemically synthesized based on SEQ ID NO: 1.

Regarding the method for obtaining the polypeptide of (b) based on the polypeptide of (a), modifications that do not lose the biological function include, for example, polar, charge, soluble, hydrophilic, The amino acid can be substituted with an amino acid having properties similar to those of the amino acid before substitution in terms of property / hydrophobicity. For example, glycine, alanine, valine, leucine, isoleucine, proline are classified as nonpolar amino acids; serine, threonine, cysteine, methionine, asparagine, glutamine are classified as polar amino acids; phenylalanine, tyrosine, tryptophan Lysine, arginine and histidine are classified as basic amino acids; aspartic acid and glutamic acid are classified as acidic amino acids. Accordingly, substitution can be made by selecting from the same group of amino acids.
Monooxygenase activity means the activity of reducing oxygen molecules to make one molecule of water and catalyzing the reaction of introducing the remaining oxygen atoms into various low-molecular organic compounds to convert them into oxidation products. The presence of monooxygenase P450 activity can be confirmed by the absorption spectrum changing when carbon monoxide is bubbled through the polypeptide in a reduced state and a difference spectrum (CO difference spectrum) having a maximum at 450 nm appearing.

As the protein belonging to monooxygenase CYP153 in the present invention, a polypeptide selected from the group consisting of the following (i) to (p) can also be suitably used.
(i) a polypeptide comprising the amino acid sequence of SEQ ID NO: 9
(j) a polypeptide comprising an amino acid sequence in which one or several amino acid sequences are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 9 and functioning as monooxygenase P450
(k) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 10
(l) a polypeptide comprising an amino acid sequence in which one or several amino acid sequences are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 10 and functioning as monooxygenase P450
(m) a polypeptide comprising the amino acid sequence of SEQ ID NO: 11
(n) a polypeptide comprising an amino acid sequence in which one or several amino acid sequences are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 11, and functioning as monooxygenase P450
(o) a polypeptide comprising the amino acid sequence of SEQ ID NO: 12
(p) a polypeptide comprising an amino acid sequence in which one or several amino acid sequences are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 12, and functioning as monooxygenase P450

  The polypeptides of SEQ ID NOs: 9 and 10 are metagenome-derived polypeptides. The polypeptide of SEQ ID NO: 11 can be isolated from a culture of Caulobacter crescentus NCIMB9789. The polypeptide of SEQ ID NO: 12 can be isolated from a culture of Gordonia sp. I72 (MBIC 01536). These polypeptides can also be chemically synthesized based on the sequences of SEQ ID NOs: 9 to 12, respectively.

  A method of obtaining the polypeptide of (j) based on the polypeptide of (i) above, a method of obtaining the polypeptide of (l) based on the polypeptide of (k), and a polypeptide of (n) based on the polypeptide of (m). The method for obtaining the peptide and the method for obtaining the polypeptide (p) based on the polypeptide (o) are the same as the method for obtaining the polypeptide (b) based on the polypeptide (a).

Examples of the fusion protein containing CYP153 include a fusion protein of CYP153 and an electron transfer protein (redox (redox) partner protein) and a fusion protein with a secretory signal. And a reductase peptide (redox partner protein) contained in cytochrome P450 monooxygenase or a fusion protein with a peptide having reductase activity equivalent thereto. Specifically, this fusion protein is a fusion protein of CYP153 and a reductase peptide having the functions of ferredoxin reductase and ferredoxin.
In particular, a fusion protein with a reductase peptide (domain) contained in cytochrome P450 monooxygenase P450RhF derived from Rhodococcus sp. NCIMB9784 or a peptide having a reductase activity equivalent thereto is preferred. The reductase peptide (domain) contained in cytochrome P450 monooxygenase derived from Rhodococcus sp. NCIMB9784 is a peptide consisting of the amino acid sequence of (c) SEQ ID NO: 2. Further, (d) a peptide having an amino acid sequence in which one or a plurality of amino acids are added, deleted or substituted in SEQ ID NO: 2 and having reductase activity can also be preferably used. Having reductase activity can be confirmed by preparing a fusion protein of a known P450 with a known substrate and a reductase peptide and confirming whether or not a reaction product is actually produced.

The peptide of SEQ ID NO: 2 can be obtained by isolation from a culture of Rhodococcus NCIMB9784 strain. It can also be obtained by chemical synthesis. The method for obtaining the peptide (d) by modifying the sequence while retaining the biological activity of the peptide (c) is the same as the method for obtaining the polypeptide (b) based on the polypeptide (a) described above. .
A linker peptide can be interposed between the CYP153 monooxygenase and the reductase peptide. Usually, a reductase peptide may be bound to the C-terminal side of P450 monooxygenase via a linker peptide. The length of the linker peptide can be about 6 to 26 aa, preferably about 16 aa, in order to efficiently proceed the reaction between CYP153 and the reductase peptide.

  These proteins belonging to the monooxygenase CYP153 or a fusion protein containing the protein may be extracted from microorganisms existing in nature, or may be chemically synthesized based on the amino acid sequence. Moreover, you may isolate and refine | purify from the transformant of this invention mentioned later.

Recombinant vector
The cell producing CYP153 or a fusion protein containing the same is preferably a transformant obtained by transforming a host with a recombinant vector containing DNA encoding them. Thus, the reaction can be efficiently performed by using a transformant that highly expresses CYP153.
This recombinant vector can be obtained by ligating DNA encoding a protein belonging to the monooxygenase CYP153 of the present invention or a fusion protein containing the same to an appropriate vector DNA. The vector is not particularly limited as long as it can replicate in the host, and a known vector can be selected from a wide range depending on the host. For example, a phage, a phagemid vector, a plasmid vector, etc. are mentioned. Examples of plasmid DNA include plasmids derived from Escherichia coli (Escherichia coli), plasmids derived from Bacillus subtilis, and plasmids derived from yeast. Phage DNA includes λ phage and the like. Furthermore, animal viruses such as retroviruses and vaccinia viruses, and insect virus vectors such as baculoviruses and togaviruses can also be used.
Since bacterial cells are easy to grow and react easily, bacterial-derived vectors are preferred, and Escherichia coli-derived vectors are more preferred. As a vector derived from Escherichia coli, a pUC vector and a pET vector are preferable.

In order to highly express a protein belonging to monooxygenase CYP153 or a fusion protein containing the protein, selection of a promoter and a translation signal is important. The promoter is not particularly limited as long as it can function in the host, and examples thereof include lac promoter, lacUV5 promoter, trp promoter, tac promoter, T7 promoter and the like. Among them, CYP153 can be highly expressed by using lac promoter or T7 promoter. Any translation signal may be used as long as it can function in the host, and examples thereof include a LacZ translation signal and an Lpp translation signal. Among them, a LacZ translation signal is preferable in that CYP153 can be highly expressed.
Most preferably, the recombinant vector has a reductase domain of cytochrome P450RhF derived from Rhodococcus sp. NCIMB9784 strain or a function equivalent thereto via a linker at the 3 ′ end of DNA encoding a protein belonging to monooxygenase CYP153. This is a CYP153 gene functional expression plasmid in which a fusion protein expression cassette linked with DNA encoding a reductase peptide is inserted into a pUC vector so as to utilize a lac promoter and a LacZ translation signal.

DNA encoding a protein belonging to CYP153 includes (e) DNA consisting of the nucleotide sequence of SEQ ID NO: 3 or (f) DNA which consists of the nucleotide sequence of SEQ ID NO: 3 under stringent conditions, and monooxygenase A DNA encoding a protein that functions as P450 is preferred. In addition, as a DNA encoding a cytochrome P450 reductase domain derived from Rhodococcus sp. NCIMB9784 or a reductase peptide having a function equivalent to this, (g) DNA consisting of the nucleotide sequence of SEQ ID NO: 4, or (h) sequence A DNA that hybridizes with a DNA comprising the nucleotide sequence of No. 4 under stringent conditions and encodes a protein having reductase activity is preferred.
The DNA comprising the nucleotide sequence of SEQ ID NO: 3 uses, for example, hybridization using a probe designed based on SEQ ID NO: 3 or a primer designed based on SEQ ID NO: 3 from the genomic DNA library of Alcanibolax Volcumensis. Thus, it can be obtained by PCR using the genomic DNA of Arcaniborax volcumensis as a template. It can also be obtained by chemical synthesis. DNA consisting of the base sequence of SEQ ID NO: 4 can be obtained in the same manner as the DNA of SEQ ID NO: 3, using, for example, genomic DNA of Rhodococcus bacteria or a library thereof. It can also be chemically synthesized. Further, as a method of obtaining the gene of (f) or (h) by modifying the DNA of (e) or (g), for example, a method of chemically synthesizing based on the DNA sequence of (e) or (g), ( General methods such as a method of irradiating the DNA of e) or (g) with γ-rays and a method of performing error-prone PCR using the DNA of (e) or (g) as a template can be mentioned.

  A fusion protein expression cassette wherein the DNA of (g) or (h) encoding a reductase peptide is linked to the 3 ′ end of the DNA of (e) or (f) encoding CYP153 via a linker. A recombinant vector inserted into a pUC vector so as to utilize the translation signal of the lac promoter and LacZ is also one aspect of the present invention.

As the DNA encoding a protein belonging to CYP153, the following DNAs (q) to (x) can also be preferably used.
(q) DNA consisting of the nucleotide sequence of SEQ ID NO: 13
(r) DNA that hybridizes with the DNA of SEQ ID NO: 13 under a stringent condition and encodes a protein having monooxygenase activity
(s) DNA consisting of the nucleotide sequence of SEQ ID NO: 14
(t) DNA that hybridizes with the DNA of SEQ ID NO: 14 under a stringent condition and encodes a protein having monooxygenase activity
(u) DNA consisting of the nucleotide sequence of SEQ ID NO: 15
(v) DNA that hybridizes with the DNA of SEQ ID NO: 15 under stringent conditions and encodes a protein having monooxygenase activity
(w) DNA consisting of the base sequence of SEQ ID NO: 16
(x) DNA that hybridizes with the DNA of SEQ ID NO: 16 under a stringent condition and encodes a protein having monooxygenase activity

As a recombinant vector in the present invention, the following (g) or (h) encoding a reductase peptide via a linker on the 3 ′ end side of any one of DNAs (q) to (x) encoding CYP153 It is also possible to use a recombinant vector in which a fusion protein expression cassette ligated with the DNA of) is inserted into a pET vector so as to receive transcription of the T7 promoter.
Fusion protein expression in which the following (g) or (h) DNA encoding a reductase peptide is linked to the 3 ′ end of the above (w) or (x) DNA encoding CYP153 via a linker A recombinant vector in which the cassette is inserted into a pET vector so as to receive transcription of the T7 promoter is also one aspect of the present invention.

The nucleotide sequences of SEQ ID NOs: 13 to 16 are DNA sequences encoding the polypeptides of SEQ ID NOs: 9 to 12, respectively.
DNA consisting of the nucleotide sequence of SEQ ID NO: 13 or 14 can be obtained by the method described in Non-Patent Document 6.
As in the case of SEQ ID NO: 13 or SEQ ID NO: 14, the DNA consisting of the base sequence of SEQ ID NO: 15 or SEQ ID NO: 16 is designed based on the common sequence region of CYP153, and the central part of the P450 gene is designed. A partial length sequence can be obtained by amplifying by PCR. Next, the full-length sequence of the P450 gene can be obtained using the inverse PCR method or Takara Bio's in vitro cloning kit, and the full-length CYP153 gene can be isolated based on this information. These DNAs can also be chemically synthesized.

  In the present invention, a DNA that hybridizes with a certain DNA under stringent conditions is, for example, Molecular Cloning: A Laboratory Manual (edited by Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989). Year). In the present invention, “stringent” conditions include 42 × 6 in a solution of 6 × SSC (standard saline citrate; 1 × SSC = 0.15M NaCl, 0.015M sodium citrate), 0.5% SDS and 50% formamide. An example of the condition is that it is not desorbed from the polynucleotide when heated at 68 ° C. for 30 minutes in a solution of 0.1 × SSC and 0.5% SDS after heating at ° C. overnight.

  The method for obtaining the DNA of (f) or (h) by modifying the DNA of (e) or (g) is as described above. (q) DNA modification method (r) to obtain (r) DNA, (s) DNA modification method (t) to obtain DNA (u) DNA modification method (v) to obtain DNA (v) The method of obtaining the DNA of (x) by modifying the DNA of (w) is the same as the method of obtaining the DNA of (f) or (h) by modifying the DNA of (e) or (g) described above.

  In the present invention, a DNA that hybridizes under stringent conditions with a certain DNA, for example, a DNA consisting of the base sequence of SEQ ID NO: 3, is a DNA consisting of a base sequence complementary to the DNA consisting of the base sequence of SEQ ID NO: 3. It has preferably about 90% or more sequence homology, more preferably about 95% or more sequence homology, and particularly preferably about 98% or more sequence homology.

Transformant The transformant of the present invention can be obtained by introducing the above recombinant vector into a host. The method for introducing the recombinant vector is not particularly limited, and for example, a method using calcium ions may be used as long as the method is introduced into bacteria [Cohen et a1 .: Proc. Nat1. Acad. Sci., USA, 69, 2110]. , 1972], electroporation method, and the like. In addition, for example, electroporation method [Becker, DM et a1 .: Methods Enzymo1., 194,182, 1990], spheroplast Method [Hinnen, A. et a1 .: Proc. Nat1. Acad. Sci., USA, 75, 1929, 1978], lithium acetate method [Itoh, H .: J. Bacterio1., 153, 163, 983] etc. In addition, if it is a method for introduction into animal cells, for example, electroporation method, calcium phosphate method, lipofection method and the like, and if it is a method for introduction into insect cells, for example, calcium phosphate method, lipofection method, electroporation The law is used.

  The host may be selected according to the vector. Eukaryotic cells such as animals and yeasts, or prokaryotic cells such as actinomycetes and eubacteria may be used, but eubacterial cells are preferred because they are easy to grow and can easily undergo a conversion reaction. Examples of eubacterial cells include bacteria belonging to the genus Escherichia such as Escherichia coli, Bacillus genus such as Bacillus subtilis, and Pseudomonas putida such as Pseudomonas putida. Among them, Escherichia coli is preferable.

EXAMPLES Hereinafter, the present invention will be described in detail based on examples, but the present invention is not limited to these examples.

Example 1
(1) Preparation of plasmids pUCRED and pSTVRED
pUC vectors that use the lac promoter and LacZ translation signal (copy number is about 100 per cell), and pSTV28 vectors that use the same promoter and translation signal (copy number is about 20 to 30 per cell) PUCRED and pSTVRED, which are pRED improved vectors, were prepared from the above materials.
Specifically, PRED vector (Ltd. Marine Biotechnology Institute) was double-digested with Eco RI and Hin dIII and reductase end of P450RhF agarose gel electrophoresis (reductase domain) 1.0 kb fragment containing the ( 1) was cut from the gel.
Next, pUC18 [ampicillin (Ap) resistance; Takara Bio Inc.] was double-digested with Eco RI and Hin dIII, and to that subjected to CIAP-treated, and the 1.0-kb- Eco RI- Hin dIII fragment The mixture was mixed and subjected to ligation reaction to obtain the desired plasmid pUCRED.
Furthermore, pSTV28 [chloramphenicol (Cm) resistance; Takara Bio Inc.] was double-digested with Eco RI and Hin dIII, and to that subjected to CIAP treatment, the 1.0-kb- Eco RI- Hin dIII fragment And ligation reaction was performed to obtain the target plasmid pSTVRED.

(2) Evaluation of pUCRED and pSTVRED
MoxA is a P450 belonging to CYP105 and is known to be a highly functional P450 with a wide range of substrate adaptability from analysis by Mercian (H. Agematsu et al, Biosci. Biotechnol. Biochem., 70: 307). -311 (2006)). Using this MoxA and P450Balk (CYP153A13a; M. Nodate et al, Appl. Microbiol. Biotechnol., 71: 455-462 (2006)), a P450 gene derived from the Alkanibolax volcumensis SK2 strain, PUCRED and pSTVRED, which were the modified pRED vectors prepared in (1), were evaluated.

<Production of pUCRED-MoxA and pSTVRED-MoxA>
pUCRED and pSTVRED were each treated with the restriction enzyme EcoRI at 37 ° C. for 5 hours. After the treatment, gel extraction was performed using QIAquick Gel Extraction Kit (manufactured by QIAGEN). This was designated as vector DNA.
Next, using PCR, first, 12.5 μL of 2 × PrimeSTAR MaxPremix (Takara Bio), 1.0 μL forward primer: MoxA-EcoRIF (10 pmol / μL), 1.0 μL reverse primer: MoxA-EcoRIR (10 pmol / μL), 0.5 μL of pRED-MoxA plasmid (≈20 ng) and 10.0 μL of sterilized water were mixed, and PCR reaction was performed at 98 ° C. for 10 seconds, 60 ° C. for 5 seconds, and 72 ° C. for 7 seconds. The MoxA gene was amplified by performing 32 cycles per second. Thereafter, 1 μL of the reaction completion solution was used for electrophoresis on a 1% agarose gel to confirm amplification (1,220 bp). The pRED-MoxA plasmid is a fusion protein of the MoxA gene (H. Agematsu et al, Biosci. Biotechnol. Biochem., 70: 307-311 (2006)) with the RhF reductase terminal portion removed. The pRED vector (M. Nodate et al, Appl. Microbiol. Biotechnol., 71: 455-462 (2006)) was inserted into the Nde I- Eco RI site. Next, DNA was purified using Minelute Reaction Cleanup Kit (manufactured by QIAGEN) and treated with restriction enzyme Eco RI at 37 ° C. for 5 hours. Next, gel extraction was performed using QIAquick Gel Extraction Kit (manufactured by QIAGEN). This was used as insert DNA.

<Expression in E. coli>
Next, vector DNA and insert DNA were ligated at 16 ° C for 15 minutes using Ligation-Convenience Kit (Nippon Gene) and mixed with E. coli competent cells (Ecos competent E.coli DH5α, Nippon Gene) on ice water. It was. Thereafter, the cells were transformed by treatment at 42 ° C. for 30 seconds, and spread on LB plates containing 100 μg / mL ampicillin (Ap) or 25 μg / mL chloramphenicol (Cm). This was cultured at 37 ° C. for 16 hours to form colonies. Next, the colonies were inoculated into 3 mL of LB medium containing Ap or Cm, and cultured with shaking at 30 ° C. for 16 hours. Then, plasmid extraction was performed using QIAprep Spin Miniprep Kit (QIAGEN). Thereafter, 1 μL of the extracted plasmid was treated with the restriction enzyme Bam HI at 37 ° C. for 2 hours, and the entire amount of the treatment solution was electrophoresed on a 1% agarose gel to confirm that the insert DNA was inserted and the direction was correct. did. These were designated as pUCRED-MoxA and pSTVRED-MoxA, respectively.

(3) Preparation of pUCRED-Balk and pSTVRED-Balk and expression in E. coli <Preparation of pUCRED-Balk and pSTVRED-Balk>
pUCRED and pSTVRED were each treated with the restriction enzyme Eco RI at 37 ° C. for 5 hours. After the treatment, gel extraction was performed using QIAquick Gel Extraction Kit (manufactured by QIAGEN). These were used as vector DNA.
Next, using PCR method, 12.5 μL 2 × PrimeSTAR MaxPremix, 1.0 μL forward primer: Balk-MfeIF (10 pmol / μL), 1.0 μL reverse primer: Balk-MfeIR (10 pmol / μL), 0.5 μL PBalk-Red plasmid (see M. Nodate et al, Appl. Microbiol. Biotechnol., 71: 455-462 (2006)) (about 20 ng) and 10.0 μL of sterilized water were mixed to prepare a PCR reaction. The P450balk gene was amplified by 32 cycles of 10 seconds at 60 ° C, 5 seconds at 60 ° C, and 7 seconds at 72 ° C. Thereafter, 1 μL of the reaction completion solution was used for electrophoresis on a 1% agarose gel to confirm amplification (1,410 bp). Then, DNA was purified using Minelute Reaction Cleanup Kit (manufactured by QIAGEN) and treated with restriction enzyme Mfe I at 37 ° C. for 5 hours. Next, gel extraction was performed using QIAquick Gel Extraction Kit (manufactured by QIAGEN). This was used as insert DNA.

<Expression in E. coli>
Then, vector DNA and insert DNA are ligated for 15 minutes at 16 ° C using Ligation-Convenience Kit (Nippon Gene) and mixed with E. coli competent cell (Ecos competent E. coli DH5α (Nippon Gene)) on ice water. Combined. Thereafter, the cells were transformed by treatment at 42 ° C. for 30 seconds and spread on LB plates containing Ap or Cm. This was cultured at 37 ° C. for 16 hours to form colonies. Next, the colonies were inoculated into 3 mL of LB medium containing Ap or Cm, and cultured with shaking at 30 ° C. for 16 hours. Then, plasmid extraction was performed using QIAprep Spin Miniprep Kit (manufactured by QIAGEN). Thereafter, 1 μL of the extracted plasmid was treated with the restriction enzyme Bam HI at 37 ° C. for 2 hours, and the entire amount of the treatment solution was electrophoresed on a 1% agarose gel to confirm that the insert DNA was inserted and the direction was correct. did. These were designated as pUCRED-Balk and pSTVRED-Balk, respectively.

<Primer sequence>
The base sequences of the aforementioned primers are as follows.
Balk-MfeIF 5'-acatcaattggATGTCAACGAGTTCAAGTA-3 '(SEQ ID NO: 5)
Balk-MfeIR 5'-cagaccaattgTTTTTTAGCCGTCAACTTA-3 '(SEQ ID NO: 6)
MoxA-EcoRIF 5'-catggaattcgATGACGAAGAACGTCGCCG-3 '(SEQ ID NO: 7)
MoxA-EcoRIR 5'-atcagaattcCCAGGTGACCGGGAGTTCGT-3 '(SEQ ID NO: 8)

(4) Protein expression induction Each constructed plasmid and pRED-MoxA and pBalk-Red as control plasmids are mixed with E. coli competent cells (BL21 (DE3) Singles Competent Cells (Novagen)) on ice water. It was. Then, transform by treating at 42 ° C for 30 seconds, pUCRED-Balk, pUCRED-MoxA recombinants on LB (Ap) plate, pSTVRED-Balk, pSTVRED-MoxA recombinants on LB (Cm) Each was spread on the plate. These were cultured at 37 ° C. for 16 hours to form colonies. Next, pUCRED-Balk and pUCRED-MoxA recombinant colonies were inoculated into 3 mL of LB (Ap) medium, and pSTVRED-Balk and pSTVRED-MoxA recombinant colonies into 3 mL of LB (Cm) medium. And cultured with shaking at 30 ° C. for 16 hours.
Thereafter, protein synthesis induction was performed using O / N Express Autoinduction System 1 (Novagen). First, pUCRED-Balk and pUCRED-MoxA recombinants were cultured in a 300 mL Erlenmeyer flask with 0.5 mL cultured cells, 1 mL OnEx Solution I, 2.5 mL OnEx Solution II, 50 μL OnEx Solution III, 50 mL. LB medium, 100 μg / mL FeSO ₄ .7H ₂ O, 80 μg / mL 5-aminolevulinic acid, and 100 μg / mL Ap were mixed and cultured at 28 ° C. for 24 hours. On the other hand, recombinants of pSTVRED-Balk and pSTVRED-MoxA were subjected to shaking culture under the same conditions as described above except that 25 μg / mL Cm was added instead of 100 μg / mL Ap.
Next, the cells subjected to protein synthesis induction were centrifuged (8000 rpm, 4 ° C., 10 minutes), and the supernatant was discarded. Thereafter, 5 mL of CV3 buffer (5% glycerol, 50 mM phosphate buffer) was added to the precipitate and resuspended. Of these, 2 mL was used for CO difference spectrum measurement described later, and 1.5 mL was used for the conversion test.

(5) CO difference spectrum measurement <Measurement method>
Transfer 2 mL of the bacterial solution obtained in (4) suspended in CV3 buffer (50 mM potassium phosphate buffer containing 5% glycerol, pH 7.4) to a 15 mL Falcon tube, and add 100 μL Bug Buster 10 × Protein Extraction Reagent (Novagen), 1 μL Benzonase Nuclease (Novagen) and 2 mM DTT were added, and the mixture was mixed by inverting at room temperature for 20 minutes. Thereafter, centrifugation (8000 rpm, 10 minutes, 4 ° C.) was performed, and the supernatant was transferred to a new 15 mL falcon tube on ice water. This was divided into two equal parts, 1 mg as a blank, and 5 mg of sodium hydrosulfite (2 mL) was added and dissolved. The other was CO bubbled, and 5 mg of sodium hydrosulfite was added and dissolved. Using a spectrophotometer (DU640 type, manufactured by BECKMAN), the difference spectra of these samples were measured over an absorption wavelength of 350 nm to 600 nm.

<Result>
Functional expression of pRED-MoxA, pUCRED-MoxA and pSTVRED-MoxA recombinants
As a result of measuring the CO difference spectrum, a peak at an absorption wavelength of 450 nm was confirmed in the recombinant of pUCRED-MoxA and the recombinant of pRED-MoxA (see FIG. 2). In addition, the pUCRED-MoxA recombinant had a peak value about 2.5 times larger at the absorption wavelength of 450 nm than the pRED-MoxA recombinant (FIGS. 2A and B). In the recombinant of pSTVRED-MoxA, a peak at an absorption wavelength of 450 nm could not be confirmed (FIG. 2C).

Functional expression of pBalk-Red, pUCRED-Balk and pSTVRED-Balk recombinants
As a result of measuring the CO difference spectrum, a peak at an absorption wavelength of 450 nm was confirmed in all recombinants (see FIG. 3). In addition, the pUCRED-Balk recombinant had a peak value at an absorption wavelength of 450 nm that was about 2.7 times greater than the pBalk-Red recombinant (FIGS. 3A and B). When the pBalk-Red recombinant and the pSTVRED-Balk recombinant were compared, there was almost no difference in the peak value at an absorption wavelength of 450 nm (FIGS. 3A and C).

<Conclusion>
Although pUCRED and pSTVRED share the same transcriptional and translational signals, that is, the promoter of lac and the SD (Shine-Dalgarno) sequence of LacZ and the N-terminal sequence, there is a large difference in the degree of functional expression. The number of copies of the former per E. coli cell is about 3 to 5 times that of the latter, which means that the former copy number was just suitable for efficient functional expression of the P450 gene in the case of the relevant transcription and translation signals. I think that the. Moreover, the level of functional expression of the P450 gene when using pUCRED is several times better than when using pRED, which has a multiple copy of the same level as pUCRED and several times stronger than pUCRED. It was.

(6) Recombinant E. coli having co-cultured modified P450 of E. coli transformant and substrate for conversion rate measurement coli (BL21), that is, E. coli prepared in (3). E. coli (pUCRED-Balk / BL21) or E. coli prepared by the method described in JP-A-2005-278637. Escherichia coli (pFusion-102 / BL21 (DE3)) in LB medium (1% tryptone, 0.5% yeast extract, 1% NaCl) containing 100 μg / mL ampicillin (Ap) for 16 hours at 30 ° C until mid-log phase Liquid culture was performed, the suspension was suspended so that the final concentration of glycerol was about 8%, and placed in a deep freezer at −70 to −80 ° C. to obtain a glycerol stock. Further, as a control, Escherichia coli (BL21 strain) having only an Ap resistant vector such as pUCRED was cultured in the same manner to prepare a glycerol-preserved strain. The recombinant vector pFusion-102 is described under the name pFusionP450 in JP-A-2005-278637.
E. In starting the substrate conversion reaction by co-culture with Escherichia coli (pUCRED-Balk / BL21 or pFusion-102 / BL21 (DE3)), first, E. The E. coli transformant was scraped with a platinum loop, suspended in 3 mL of LB medium containing 100 μg / mL Ap, and cultured at 100 rpm at 30 ° C. for about 16 hours (preculture). Next, add 2.5 mL of this preculture to 250 mL of LB medium containing 100 μg / mL Ap, pUCRED-Balk / BL21 for 7 hours at 100 rpm and 30 ° C, and 6 for pFusion-102 / BL21 (DE3). Cultured for a long time (main culture). To this main culture solution, a final concentration of 0.5 mM isopropyl-1-thio-β-galactopyranoside (IPTG), a final concentration of 80 mg / mL 5-aminolevulinic acid (5-ALA) and 0.1 mM ammonium iron sulfate (II ), And cultured at 100 rpm at 20 ° C. for about 19 hours. This was centrifuged at 8000 rpm for 8 minutes to collect only the cells, and then suspended in 250 mL of CV-3 medium containing 5% glycerol, 5 mM proline, 1 mM EDTA, and 0.2 mM dithiothreitol (DTT). Using 0.5 mL of the turbid solution, 1 mM (final concentration) substrate (starting material compound) was added, and co-culture was performed at 840 rpm and 28 ° C. for 24 hours. As the substrate, 2-bromophenol, 4-methylbiphenyl, 2-methylnaphthalene, or 1-methoxynaphthalene was used. In addition, the substrate dissolved in a small amount of DMSO or ethanol in advance was added to the medium.

(7) HPLC analysis of conversion products
E. Products obtained by reaction of E. coli (pUCRED-Balk / BL21 or pFusion-102 / BL21 (DE3)) with the various substrates described above were analyzed by HPLC.
Specifically, E.I. E. coli (pUCRED-Balk / BL21 or pFusion-102 / BL21 (DE3)) and various substrates are used for co-culture as described in (6). Equal volume of ethyl acetate is added to 0.5 mL of a mixed culture of Escherichia coli (pUCRED-Balk / BL21 or pFusion-102 / BL21 (DE3)) and these substrates, then vortexed and centrifuged at 15,000 rpm for 15 minutes. The ethyl acetate layer was collected.
10 μL of this ethyl acetate layer was subjected to HPLC under the following analysis conditions to determine the conversion rate of the product.
The detection was performed using a Photodiode array detector (Waters, 2996), and absorption at 200 to 500 nm was detected.

<Analysis conditions>
Column: Waters Xterra MS C18 5 μm ID 4.6 mm x 100 mm
Mobile phase: A solution = 5% -CH ₃ CN / H ₂ O (20 mM phosphoric acid), B solution = 95% -CH ₃ CN / H ₂ O (20 mM phosphoric acid)
0 to 3 minutes: Solution A 100%
3 → 28 minutes: Solution A 100% → Solution B 100% (linear gradient)
28 → 43 minutes: B solution 100%
Flow rate: 1 mL / min Temperature: 30 ° C
The substrate and its retention time, product retention time, and conversion are shown in Table 1. The compound numbers in the following table are numbers in the structure determination described later.

Example 2
The structure of the product of Example 1 (compounds 1 to 7) was determined.
<E. Co-culture of Escherichia coli transformant and substrate>
In the following procedure, E. The co-culture of the Escherichia coli transformant and the substrate was performed. As a substrate, 2-bromophenol, 4-methylbiphenyl, 2-methylnaphthalene, or 1-methoxynaphthalene was used.
E. In starting the substrate conversion reaction by co-culture with Escherichia coli (pUCRED-Balk / BL21 or pFusion-102 / BL21 (DE3)), the E. coli strain from the glycerol-preserved strain described in (6) The E. coli transformant was scraped with a platinum loop, suspended in 21 mL of LB medium containing 100 μg / mL Ap, and cultured at 100 rpm at 30 ° C. for about 16 hours (preculture). Next, 20 mL of this preculture solution is placed in 2000 mL of LB medium containing 100 μg / mL Ap, and at 100 rpm and 30 ° C., 7 hours 30 minutes in (I) described later, and 7 hours 40 in (II). Min, (III) for 5 hours and 20 minutes, (IV) for 6 hours, and (V) for 4 hours and 45 minutes (main culture). To this main culture solution, a final concentration of 0.5 mM isopropyl-1-thio-β-galactopyranoside (IPTG), a final concentration of 80 mg / mL 5-aminolevulinic acid (5-ALA) and 0.1 mM ammonium iron sulfate (II ), And cultured at 100 rpm at 20 ° C. for about 19 hours. This was centrifuged at 8000 rpm for 8 minutes to collect only bacterial cells, and then suspended in 200 mL of CV-3 medium containing 5% glycerol, 5 mM proline, 1 mM EDTA, and 0.2 mM dithiothreitol (DTT). The solution became turbid, and 1.5 mM (final concentration) substrate was added, and co-culture was performed at 200 rpm and 28 ° C. for 48 hours. In addition, the substrate dissolved in a small amount of DMSO or ethanol in advance was added to the medium.

<Purification and identification of products obtained by co-culture>
E. E. coli (pUCRED-Balk / BL21 or pFusion-102 / BL21 (DE3)) and various substrates were used for co-culture according to the procedure described in (7). Repeat the operation of adding 2,000 mL of a mixed culture solution of E. coli (pUCRED-Balk / BL21 or pFusion-102 / BL21 (DE3)) and these substrates, and then separating the solution by adding twice the amount of ethyl acetate. Was recovered.
The obtained ethyl acetate layer was concentrated under reduced pressure to obtain a crude product. The crude product was subjected to thin layer chromatography (TLC) using silica gel [Slicagel 60 F254 (manufactured by Merck)], and after confirming the conversion product, silica gel chromatography [ID 10 × 200 mm, Silicagel 60 ( Merck)) to obtain a purified product.

(I) Identification of conversion product of 2-bromophenol When the crude extract (233.2 mg) subjected to the 2-bromophenol conversion experiment using E. coli (pUCRED-Balk / BL21) was subjected to TLC (developing solvent: hexane / ethyl acetate = 2/1), a product with an Rf value of 0.4 was obtained. Turned out to be generated. This compound was purified by silica gel chromatography (eluent: hexane / ethyl acetate = 2/1) to obtain Compound 1 (3.7 mg). The molecular formula of Compound 1 was determined to be C ₆ H ₅ Br O ₂ from HRAPCI-MS [m / z 186.94036 (MH) ⁻ , calcd .: 186.93947]. Furthermore, Compound 1 was identified as 2-bromobenzene-1,4-diol (CAS No.583-69-7) by NMR analysis ( ¹ H, ¹³ C, ¹ H- ¹ H DQF COZY, HMQC, HMBC).
¹ H NMR (400 MHz, CDCl ₃ ): δ 6.60 (1H, dd J = 2.7, 8.9 Hz), 6.72 (1H, d J = 8.9 Hz), 6.89 (1H, d J = 2.7 Hz), ¹³ C NMR (100 MHz, CDCl ₃ ): δ110.7, 116.4, 117.8, 120.3, 148.2, 152.0

(II) Identification of conversion product of 4-methylbiphenyl When the crude extract (197.9 mg) subjected to the 4-methylbiphenyl conversion experiment using E. coli (pUCRED-Balk / BL21) was subjected to TLC (developing solvent: hexane / ethyl acetate = 2/1), a product with an Rf value of 0.4 was obtained. Turned out to be generated. This compound was purified by silica gel chromatography (eluent: hexane / ethyl acetate = 2/1) to give Compound 2 (6.1 mg).
The molecular formula of Compound ₂ was determined as C ₁₃ H ₁₂ O ₂ from HREI-MS [m / z 184.08821 (M ⁺ ), calcd .: 184.08882]. Furthermore, Compound 2 was identified as 4-hydroxymethylbiphenyl (CAS-3597-91-9) by NMR analysis ( ¹ H, ¹³ C, ¹ H- ¹ H DQF COZY, HMQC, HMBC).
¹ H NMR (400 MHz, CDCl ₃ ): δ4.67 (2H, s), 7.27 (1H, dd J = 7.4 Hz, 7.4 Hz), 7.37 (2H, dd, J = 7.4, 7.9 Hz), 7.37 ( 2H, d, J = 7.9 Hz), 7.52 (2H, d, J = 7.9 Hz), 7.52 (2H, d, J = 7.9 Hz), ¹³ C NMR (100 MHz, CDCl ₃ ): δ65.1, 127.1 , 127.3, 127.4, 128.8, 139.9, 140.7, 140.8

(III) Identification of conversion product of 2-methylnaphthalene The crude extract (217.6 mg), which was converted from 2-methylnaphthalene using E. coli (pUCRED-Balk / BL21), was subjected to TLC (developing solvent: hexane / ethyl acetate = 5/1). Turned out to be generated. This compound was purified by silica gel chromatography (eluent: hexane / ethyl acetate = 2/1) to obtain Compound 3 (0.5 mg).
The molecular formula of Compound 3 was determined to be C ₁₁ H ₁₀ O from HREI-MS [m / z 158.07278 (M ⁺ ), calcd .: 158.07316]. Furthermore, Compound 3 was identified as 2-hydroxymethylnaphthalene (CAS-1592-38-7) by NMR analysis ( ¹ H, ¹³ C, ¹ H- ¹ H DQF COZY, HMQC, HMBC).
¹ H NMR (400 MHz, CDCl ₃ ): δ 4.87 (2H, s), 7.47 (1H, s), 7.47 (1H, d J = 8.5 Hz), 7.50 (2H), 7.84 (2H), 7.85 ( 1H, d J = 8.5 Hz), ¹³ C NMR (100 MHz, CDCl ₃ ): δ 65.5, 125.2, 125.5, 125.9, 126.2, 127.7, 127.9, 128.4, 133.0, 133.4, 138.3

(IV) Identification of conversion product of 1-methoxynaphthalene The crude extract (255.7 mg), which was converted from 1-methoxynaphthalene using E. coli (pUCRED-Balk / BL21), was subjected to TLC (developing solvent: hexane / ethyl acetate = 5/1). Turned out to be generated. This compound was purified by silica gel chromatography (eluent: hexane / ethyl acetate = 5/1) to obtain Compound 4 (2.5 mg). The molecular formula of Compound 4 was determined as C ₁₀ H ₈ O from HRESI-MS [m / z 143.0491 (MH) ⁻ , calcd .: 143.0497]. Further NMR analysis ^{(1 H, 13 C, 1} H- 1 H DQF COSY, HMQC, HMBC) Compound 4 was identified as 1-naphthol (CAS No.90-15-3) by.
¹ H NMR (400 MHz, CDCl ₃ ): δ6.82 (1H, d, J = 7.3 Hz), 7.31 (1H, dd, J = 7.3, 8.3 Hz), 7.44 (1H, d, J = 8.3), 7.49 (2H), 7.81 (1H, dd, J = 2.3, 7.3 Hz), 8.17 (1H, dd, J = 2.4 Hz, 7.3 Hz), ¹³ C NMR (100 MHz, CDCl ₃ ): δ108.6, 120.7, 121.5, 124.4, 125.8, 125.8, 126.4, 127.7, 134.6, 151.4

(V) Identification of conversion product of 2-methylnaphthalene The crude extract (162.8 mg), which was subjected to the conversion experiment of 2-methylnaphthalene using E. coli (pFusion-102 / BL21 (DE3)), was subjected to TLC (developing solvent: hexane / ethyl acetate = 5/1) to give an Rf value. It was found that 0.40, 0.33, and 0.30 products were produced. These compounds were purified by silica gel chromatography (eluent: hexane / ethyl acetate = 5/1) to obtain Compound 5 (5.0 mg), Compound 6 (1.5 mg), and Compound 7 (1.5 mg). The molecular formula of Compound 5 was determined as C ₁₁ H ₁₀ O from HRESI-MS [m / z 157.06481 (MH) ⁻ , calcd .: 157.06534]. Further, Compound 5 was identified as 1-hydroxy-2-methylnaphthalene (CAS No. 856081-23-7) by NMR analysis ( ¹ H, ¹³ C, ¹ H- ¹ H DQF COZY, HMQC, HMBC).
¹ H NMR (400 MHz, CDCl ₃ ): δ2.41 (3H, s), 5.06 (1H, s), 7.24 (1H, d, J = 8.2 Hz), 7.38 (1H, d, J = 8.2 Hz) , 7.42 (1H, dd, J = 7.8Hz, 8.0Hz), 7.47 (1H, dd, J = 8.0Hz, 8.1Hz), 7.77 (1H, d, J = 7.8Hz), 8.12 (1H, d, J = 8.1 Hz), ¹³ C NMR (100 MHz, CDCl ₃ ): δ15.6, 116.2, 120.2, 120.8, 124.2, 125.3, 125.4, 127.6, 129.0, 133.4, 148.5

The molecular formula of Compound 6 was determined as C ₁₁ H ₁₀ O from HRESI-MS [157.06501 (MH) ⁻ , calcd .: 157.06534]. Furthermore, Compound 6 was identified as 8-hydroxy-2-methylnaphthalene (CAS No. 354589-50-7) by NMR analysis ( ¹ H, ¹³ C, ¹ H- ¹ H DQF COZY, HMQC, HMBC).
¹ H NMR (400 MHz, CDCl ₃ ): δ2.53 (3H, s), 6.78 (1H, d, J = 7.5 Hz), 7.25 (1H, dd, J = 7.5 Hz, 8.4 Hz), 7.32 (1H , d, J = 8.4Hz), 7.40 (1H, d, J = 8.4Hz), 7.71 (1H, d, J = 8.4Hz), 7.91 (s, 1H), ¹³ C NMR (100 MHz, CDCl ₃ ) : Δ21.9, 108.7, 120.4, 120.5, 124.4, 125.4, 127.5, 129.1, 133.0, 135.0, 150.8

The molecular formula of Compound 7 was determined as C ₁₁ H ₁₀ O from HRESI-MS [m / z 157.06576 (MH) ⁻ , calcd. 157.06534]. Further, Compound 7 was identified as 5-hydroxy-2-methylnaphthalene (CAS No. 24894-78-8) by NMR analysis ( ¹ H, ¹³ C, ¹ H- ¹ H DQF COZY, HMQC, HMBC).
¹ H NMR (400 MHz, CDCl ₃ ): δ2.51 (3H, s), 6.74 (1H, d, J = 7.7 Hz), 7.29 (1H, dd, J = 7.7 Hz, 8.4 Hz), 7.32 (1H , d, J = 8.6Hz), 7.34 (1H, d, J = 8.4Hz), 7.58 (1H, s), 8.06 (d, J = 8.6Hz, 1H), ¹³ C NMR (100 MHz, CDCl ₃ ) : Δ21.6, 107.8, 120.1, 121.3, 122.5, 126.7, 127.6, 128.6, 135.0, 136.1, 151.4

<Conclusion>
Since the compounds 1 to 7 were formed, pUCRED-Balk (CYP153) not only hydroxylates the already known normal alkane terminus, but also hydroxylated aromatic rings, hydroxylated hydroxyl groups, and dehydrated aromatic methyls. It became clear that it had an alkylating function. Further, it was revealed that pUCRED-Balk (CYP153) has a higher conversion ability of the substance than pFusion-102, which was known to efficiently convert 2-bromophenol to compound 1.

Example 3
(1) Co-culture of E. coli transformant and substrate for conversion rate measurement Recombinant E. coli having the modified P450 prepared in Example 1 Escherichia coli (BL21), that is, pUCRED-Balk / BL21 in LB medium (1% tryptone, 0.5% yeast extract, 1% NaCl) containing 100 μg / mL ampicillin (Ap) for 16 hours at 30 ° C. until mid-log phase Liquid culture was performed, the suspension was suspended so that the final concentration of glycerol was about 8%, and placed in a deep freezer at −70 to −80 ° C. to obtain a glycerol stock. Further, as a control, Escherichia coli (BL21 strain) having only an Ap resistant vector such as pUCRED was cultured in the same manner to prepare a glycerol-preserved strain.
E. In starting the substrate conversion reaction by co-culture with Escherichia coli (pUCRED-Balk / BL21), first, from the above glycerol-preserved strain, The E. coli transformant was scraped with a platinum loop, suspended in 3 mL of LB medium containing 100 μg / mL Ap, and cultured at 100 rpm at 30 ° C. for about 16 hours (preculture). Next, 2.5 mL of this preculture was put into 250 mL of LB medium containing 100 μg / mL Ap, and cultured at 100 rpm and 30 ° C. for 6 hours (main culture). To this main culture solution, a final concentration of 0.5 mM isopropyl-1-thio-β-galactopyranoside (IPTG), a final concentration of 80 mg / mL 5-aminolevulinic acid (5-ALA) and 0.1 mM ammonium iron sulfate (II ), And cultured at 100 rpm at 20 ° C. for about 19 hours. This was centrifuged at 8000 rpm for 8 minutes to collect only the cells, and then suspended in 250 mL of CV-3 medium containing 5% glycerol, 5 mM proline, 1 mM EDTA, and 0.2 mM dithiothreitol (DTT). Using 0.5 mL of the turbid solution, 1 mM (final concentration) substrate (starting material compound) was added, and co-culture was performed at 1700 rpm, 28 ° C. for 24 hours. The substrates used are shown in Table 2. In addition, the substrate dissolved in a small amount of DMSO or ethanol in advance was added to the medium.

(2) HPLC analysis of conversion products
E. The co-cultivation described in (1) was performed using E. coli (pUCRED-Balk / BL21) and various substrates shown in Table 2. Then E. An equal amount of ethyl acetate was added to 0.5 mL of a mixed culture solution of Escherichia coli (pUCRED-Balk / BL21) and various substrates, and then vortexed and centrifuged at 15,000 rpm for 15 minutes to recover the ethyl acetate layer.
10 μL of this ethyl acetate layer was subjected to HPLC to determine the conversion rate of the product. The HPLC analysis conditions were as follows: Photodiode array detector (manufactured by Shimadzu Corporation, LC-20A system) was used instead of Photodiode array detector (Waters, 2996) for detection. The conditions are the same as those described in Example 1 (7). The substrates and their retention times, product retention times, and conversions are shown in Tables 2 and 3. The compound numbers in the table are the numbers in the structure determination described later.

Example 4
(1) Preparation of transformants Each of four types of genes encoding CYP153 described in SEQ ID NOs: 13 to 16 was designated as Nodate et al. (M. Nodate et al, Appl. Microbiol. Biotechnol., 71: 455-462 ( 2006)) and the method of Kubota et al. (M. Kubota et al, Biosci. Biotechnol. Biochem., 69: 2421-2430 (2005)) were inserted into the pRED vector to prepare four types of recombinant vectors. . In addition, about the preparation method of such a vector, it describes also in WO2006 / 016432, A vector can also be prepared according to description of this gazette. Furthermore, E. coli (BL21 (DE3) was transformed with these recombinant vectors, and four types of transformants, namely pP02-RED / BL21 (DE3), pN03-RED / BL21 (DE3), p153A2-RED / BL21 (DE3) and pgoaBAC-RED / BL21 (DE3) were prepared, and the following conversion test was performed using E. coli pUCRED-Balk / BL21 prepared in Example 1 and the above four types of transformants. .

(2) Co-culture and HPLC analysis of the resulting conversion product The four transformants or pUCRED-Balk / BL21 prepared in (1) above and the substrates shown in Table 4 (2-bromophenol, 4-bromophenol, Using 4-methylbiphenyl, 2-methylnaphthalene or 1-methoxynaphthalene), co-culture was performed in the same manner as in Example 1, (1).
An equal amount of ethyl acetate was added to 0.5 mL of a mixed culture solution of each transformant and these substrates, and then vortexed and centrifuged at 15,000 rpm for 15 minutes to recover the ethyl acetate layer.
10 μL of this ethyl acetate layer was subjected to HPLC to determine the conversion rate of the product. The HPLC analysis conditions were as follows: Photodiode array detector (manufactured by Shimadzu Corporation, LC-20A system) was used instead of Photodiode array detector (Waters, 2996) for detection. The conditions are the same as those described in Example 1 (7). Table 4 shows product retention times and conversion rates.

Example 5
Comparison with reaction using inorganic catalyst
The condensation reaction of the aromatic compound by P450 was compared with the condensation reaction using ferric chloride. The condensation reaction of the aromatic compound by P450 was performed in the same manner as in Example 3. The reaction conditions using ferric chloride were set as follows according to Ding et al. (K. Ding et al., Terrahedron, 52; 1005-1009 (1996)).
Add 0.503 mmol of 0.703 mol / L ferric chloride aqueous solution (2.0 equivalents) to 0.52 mmol of raw material (4-phenoxyphenol; 88.5 mg, 2-naphthol; 75.0 mg), 48 hours at 25 ° C or 24 hours at 50 ° C Shake. The reaction mixture was extracted with 1.5 mL of ethyl acetate, and the organic layer was washed with 0.75 mL of water, and analyzed by HPLC under the same conditions as (2) of Example 3. The results are shown in Table 5.

Example 6
The structure of the products of Examples 3-5 (Compounds 8-50) was determined.

(1) E.E. co-culture of Escherichia coli transformants and substrates
E. In starting the substrate conversion reaction by co-cultivation with E. coli (pUCRED-Balk / BL21) or the four types of transformants prepared in Example 5, (1) of Example 3 or (2) of Example 4 From the glycerol stocks described in E. The E. coli transformant was scraped with a platinum loop, suspended in 21 mL of LB medium containing 100 μg / mL Ap, and cultured at 100 rpm at 30 ° C. for about 16 hours (preculture). Next, 20 mL of this preculture was put into 2000 mL of LB medium containing 100 μg / mL Ap, and cultured at 120 rpm and 30 ° C. for 6 hours (main culture). To this main culture solution, a final concentration of 0.5 mM isopropyl-1-thio-β-galactopyranoside (IPTG), a final concentration of 80 mg / mL 5-aminolevulinic acid (5-ALA) and 0.1 mM ammonium iron sulfate (II ), And cultured at 100 rpm at 20 ° C. for about 19 hours. This was centrifuged at 8000 rpm for 8 minutes to collect only the cells, and then suspended in 400 mL of CV-3 medium containing 5% glycerol, 5 mM proline, 1 mM EDTA, and 0.2 mM dithiothreitol (DTT). The solution became turbid, and 1.0 mM (final concentration) substrate was added, and co-culture was performed at 160 rpm and 28 ° C. for 24 hours. In addition, the substrate dissolved in a small amount of DMSO or ethanol in advance was added to the medium. However, for 1-phenylcyclohexene, 1.98 g was added in multiple layers.

(2) Purification and identification of products obtained by co-culture . The coculture was performed according to the procedure described in (1) using E. coli (pUCRED-Balk / BL21) or the four types of transformants prepared in Example 4 and various substrates. Then E. The procedure of adding 2,000 mL of a mixed culture solution of E. coli (pUCRED-Balk / BL21) or the four types of transformants prepared in Example 4 and these substrates to each other by adding an equal volume of ethyl acetate was repeated twice. The ethyl acetate layer was recovered.
The obtained ethyl acetate layer was concentrated under reduced pressure to obtain a crude product. The crude product was subjected to thin layer chromatography (TLC) using silica gel [Slicagel 60 F254 (manufactured by Merck)], and after confirming the conversion product, silica gel chromatography [ID 12 × 250 mm, Silicagel 60 ( Merck)) to obtain a purified product.

Identification of conversion products of 3-bromophenol When the crude extract (195.5 mg) subjected to the conversion experiment of 3-bromophenol by E. coli (pUCRED-Balk / BL21) was subjected to TLC (developing solvent: hexane / ethyl acetate = 5/1), a product having an Rf value of 0.28 Turned out to be generated. This compound was purified by silica gel chromatography (eluent: hexane / ethyl acetate = 2/1) to obtain Compound 8 (22.7 mg). The molecular formula of Compound 8 was determined as C ₆ H ₅ O ₂ Br from HRAPCI-MS [m / z 186.93878 (MH) ⁻ , calcd .: 186.93947]. Further, NMR analysis ( ¹ H, ¹³ C, ¹ H- ¹ H gDQF COZY, gHSQC, gHMBC) identified Compound 8 as 2-bromobenzene-1,4-diol (CAS No.583-69-7).
¹ H NMR (500 MHz, CDCl ₃ ): δ5.10 (2H, brS), 6.73 (1H, dd J = 2.9, 8.8 Hz), 6.89 (1H, d J = 8.8 Hz), 6.99 (1H, d J = 2.9 Hz), ¹³ C NMR (125 MHz, CDCl ₃ ): δ 109.9, 116.3, 116.4, 118.6, 146.5, 149.5

Identification of 2-chlorophenol conversion products When the crude extract (51.0 mg) subjected to the 2-chlorophenol conversion experiment using E. coli (pUCRED-Balk / BL21) was subjected to TLC (developing solvent: hexane / ethyl acetate = 5/1), a product with an Rf value of 0.23 was obtained. Turned out to be generated. This compound was purified by silica gel chromatography (eluent: hexane / ethyl acetate = 5/1) to obtain Compound 9 (18.2 mg). The molecular formula of Compound 9 was determined as C ₆ H ₅ O ₂ Cl from HRAPCI-MS [m / z 142.98928 (MH) ⁻ , calcd .: 142.98998]. Furthermore, Compound 9 was identified as 2-chlorobenzene-1,4-diol by NMR analysis ( ¹ H, ¹³ C, ¹ H- ¹ H gDQF COZY, gHSQC, gHMBC).
¹ H NMR (500 MHz, CDCl ₃ ): δ 6.67 (1H, dd J = 2.9, 8.8 Hz), 6.85 (1H, d J = 2.9 Hz), 6.89 (1H, d J = 8.8 Hz), ¹³ C NMR (125 MHz, CDCl ₃ ): δ115.5, 115.8, 116.6, 119.9, 145.5, 149.4

Identification of conversion products of 2-iodophenol When the crude extract (148.9 mg) subjected to the conversion experiment of 2-iodophenol using E. coli (pUCRED-Balk / BL21) was subjected to TLC (developing solvent: hexane / ethyl acetate = 2/1), a product having an Rf value of 0.25 Turned out to be generated. This compound was purified by silica gel chromatography (eluent: hexane / ethyl acetate = 2/1) to obtain Compound 10 (42.1 mg). The molecular formula of compound 10 was determined as C ₆ H ₅ O ₂ I from HRAPCI-MS [m / z 234.92563 (MH) ⁻ , calcd .: 234.92560]. Further, NMR analysis ( ¹ H, ¹³ C, ¹ H- ¹ H gDQF COZY, gHSQC, gHMBC) identified Compound 10 as 2-iodobenzene 1,4-diol.
¹ H NMR (500 MHz, MeOH-d4): δ6.63 (1H, dd J = 2.7, 8.7 Hz), 6.67 (1H, d J = 8.7 Hz), 7.12 (1H, d J = 2.7 Hz), ¹³ C NMR (125 MHz, MeOH-d4): δ 83.3, 114.9, 116.1, 125.1, 149.9, 150.9

Identification of 2-acetylphenol conversion products When the crude extract (111.8 mg) subjected to the conversion experiment of 2-acetylphenol by E. coli (pUCRED-Balk / BL21) was subjected to TLC (developing solvent: hexane / ethyl acetate = 2/1), an Rf value of 0.30 product was obtained. It was found that it was generated. This compound was purified by silica gel chromatography (eluent: hexane / ethyl acetate = 2/1) to obtain Compound 11 (4.3 mg). The molecular formula of Compound 11 was determined as C ₈ H ₈ O ₃ from HRAPCI-MS [m / z 151.03883 (MH) ⁻ , calcd .: 151.03952]. Furthermore, Compound 11 was identified as 1- (2,5-dihydroxyphenyl) ethanone by NMR analysis ( ¹ H, ¹³ C, ¹ H- ¹ H gDQF COZY, gHSQC, gHMBC).
¹ H NMR (500 MHz, MeOH-d4): δ2.58 (3H, s), 6.78 (1H, d J = 8.9 Hz), 7.00 (1H, dd J = 2.9 Hz, 8.9 Hz), 7.22 (1H, d J = 2.9 Hz), ¹³ C NMR (125 MHz, MeOH-d4): δ 25.8, 115.2, 118.4, 119.6, 124.7, 149.5, 155.4, 204.7

Identification of phenol conversion products When the crude extract (93.9 mg) subjected to the phenol conversion experiment using E. coli (pUCRED-Balk / BL21) was subjected to TLC (developing solvent: chloroform / methanol = 19/2), a product with an Rf value of 0.23 and an Rf value of 0.25 Turned out to be generated. This compound was purified by silica gel chromatography (eluent: chloroform / methanol = 19/2) to obtain Compound 12 (5.0 mg) having an Rf value of 0.23 and Compound 13 (2.4 mg) having an Rf value of 0.25. The molecular formula of Compound 12 was determined as C ₆ H ₆ O ₂ from HRAPCI-MS [m / z 109.02857 (MH) ⁻ , calcd .: 109.02895]. Furthermore, Compound 12 was identified as hydroquinone by NMR analysis ( ¹ H, ¹³ C, ¹ H- ¹ H gDQF COZY, gHSQC, gHMBC).
¹ H NMR (500 MHz, MeOH-d4): δ6.61 (4H, s), ¹³ C NMR (125 MHz, MeOH-d4): δ116.8, 151.3

The molecular formula of Compound 13 was determined as C ₁₂ H ₁₀ O ₂ from HRAPCI-MS [m / z 185.05973 (MH) ⁻ , calcd .: 185.06025]. Further, NMR analysis ( ¹ H, ¹³ C, ¹ H- ¹ H gDQF COZY, gHSQC, gHMBC) identified Compound 13 as 4,4′-dihydroxybiphenyl.
¹ H NMR (500 MHz, MeOH-d4): δ 6.80 (4H, d, J = 8.8 Hz), 7.35 (4H, d, J = 8.8 Hz), ¹³ C NMR (125 MHz, MeOH-d4): δ 115.3, 127.3, 132.8, 156.2

Identification of conversion products of 3-hydroxybiphenyl When the crude extract (83.5 mg) subjected to the conversion experiment of 3-hydroxybiphenyl by E. coli (pUCRED-Balk / BL21) was subjected to TLC (developing solvent: hexane / ethyl acetate = 2/1), the Rf value was 0.32. It was found that a product with an Rf value of 0.53 was produced. This compound was purified by silica gel chromatography (eluent: hexane / ethyl acetate = 3/1) to obtain Compound 14 (11.7 mg) having an Rf value of 0.32 and Compound 15 (2.3 mg) having an Rf value of 0.47. The molecular formula of Compound 14 was determined to be C ₁₂ H ₁₀ O ₂ from HRAPCI-MS [m / z 185.06060 (MH) ⁻ , calcd .: 185.06025]. Further, NMR analysis ( ¹ H, ¹³ C, ¹ H- ¹ H gDQF COZY, gHSQC, gHMBC) identified Compound 14 as 2-phenylbenzene-1,4-diol.
¹ H NMR (500 MHz, CDCl ₃ ): δ 6.83 (1H, d J = 8.2 Hz), 6.94 (1H, dd J = 2.2 Hz, 8.2 Hz), 7.04 (1H, d J = 2.2 Hz), 7.24 ( 1H, m), 7.35 (1H, m), 7.50 (1H, m), ¹³ C NMR (125 MHz, CDCl ₃ ): δ113.9, 115.5, 118.3, 126.2, 126.3, 128.5, 133.4, 141.4, 145.0, 145.4

The molecular formula of Compound 15 was determined as C ₂₄ H ₁₈ O ₂ from HRAPCI-MS [m / z 337.12339 (MH) ⁻ , calcd .: 337.12285]. Furthermore, Compound 15 was identified as 5-hydroxy-2- (3-phenylphenoxy) biphenyl by NMR analysis ( ¹ H, ¹³ C, ¹ H- ¹ H gDQF COZY, gHSQC, gHMBC).
¹ H NMR (500 MHz, CDCl ₃ ): δ 6.71 (1H, ddd, J = 1.0, 2.1, 8.2 Hz), 6.82 (1H, dd, J = 2.9, 8.7 Hz), 6.90 (1H, d, J = 2.9), 6.95 (1H, dd, J = 2.1, 2.1 Hz), 6.97 (1H, d, J = 8.7 Hz), 7.13 (1H, ddd, 1.0, 2.1, 8.7 Hz), 7.22 (1H, m), 7.23 (1H, dd, 8.2, 8.7 Hz), 7.28 (2H, m), 7.30 (1H, m), 7.38 (2H, m), 7.45 (2H, m), 7.46 (2H, m), ¹³ C NMR (125 MHz, CDCl ₃ ): δ 114.7, 115.0, 115.4, 117.1, 120.2, 122.9, 126.8, 127.1, 127.4, 127.9, 128.6, 128.9, 129.6, 135.8, 138.1, 140.9, 142.8, 145.2, 154.5, 159.8

Identification of conversion products of 4-ethylbiphenyl When the crude extract (169.2 mg) subjected to the 4-ethylbiphenyl conversion experiment using E. coli (pUCRED-Balk / BL21) was subjected to TLC (developing solvent: hexane / ethyl acetate = 2/1), a product with an Rf value of 0.40 was obtained. Turned out to be generated. This compound was purified by silica gel chromatography (eluent: hexane / ethyl acetate = 2/1) to obtain Compound 16 (38.6 mg). The molecular formula of Compound 16 was determined as C ₁₄ H ₁₄ O from HREI-MS [m / z 198.10419 (M ⁺ ), calcd .: 198.10446]. Further, NMR analysis ( ¹ H, ¹³ C, ¹ H- ¹ H gDQF COZY, gHSQC, gHMBC) identified Compound 16 as 4- (2-hydroxyethyl) biphenyl.
¹ H NMR (500 MHz, CDCl ₃ ): δ 2.91 (2H, t J = 6.6 Hz), 3.90 (2H, t J = 6.6 Hz), 7.30 (2H, d J = 8.2 Hz m), 7.33 (1H, m), 7.43 (2H, m), 7.54 (1H, d J = 8.2 Hz), 7.58 (2H, m), ¹³ C NMR (125 MHz, CDCl ₃ ): δ38.8, 63.6, 127.0, 127.1, 127.3 , 128.7, 129.4, 137.5, 139.4, 140.9

Identification of conversion products of 3-methylbiphenyl When the crude extract (70.3 mg) subjected to the conversion experiment of 3-methylbiphenyl using E. coli (pUCRED-Balk / BL21) was subjected to TLC (developing solvent: hexane / ethyl acetate = 5/2), a product with an Rf value of 0.35 was obtained. Turned out to be generated. This compound was purified by silica gel chromatography (eluent: hexane / ethyl acetate = 10/1) to obtain Compound 17 (7.8 mg). The molecular formula of Compound 17 was determined as C ₁₃ H ₁₂ O from HREI-MS [m / z 184.08827 (M ⁺ ), calcd .: 184.08881]. Furthermore, Compound 17 was identified as 3-hydroxymethylbiphenyl by NMR analysis ( ¹ H, ¹³ C, ¹ H- ¹ H gDQF COZY, gHSQC, gHMBC).
¹ H NMR (500 MHz, CDCl ₃ ): δ 4.77 (2H, s), 7.35 (1H, m), 7.35 (1H, ddd, J = 1.2, 1.8, 7.6 Hz), 7.44 (1H, m), 7.44 (2H, m), 7.53 (1H, ddd, J = 1.2, 1.8, 7.7 Hz), 7.60 (1H, m), 7.60 (2H, ddd, J = 1.2, 1.3, 7.1 Hz), ¹³ C NMR (125 MHz, CDCl ₃ ): δ 65.4, 125.8, 125.9, 126.5, 127.2, 127.4, 128.8, 129.0, 140.9, 141.3, 141.6

Identification of conversion products of 4-isopropylbiphenyl When the crude extract (57.3 mg) subjected to 4-isopropylbiphenyl conversion experiment using E. coli (pUCRED-Balk / BL21) was subjected to TLC (developing solvent: hexane / ethyl acetate = 5/2), a product with an Rf value of 0.38 was obtained. Turned out to be generated. This compound was purified by silica gel chromatography (eluent: hexane / ethyl acetate = 5/1) to obtain Compound 18 (5.8 mg). The molecular formula of Compound 18 was determined as C ₁₅ H ₁₆ O from HREI-MS [m / z 212.12093 (M ⁺ ), calcd .: 212.12011]. Further NMR analysis ^{(1 H, 13 C, 1} H- 1 H gDQF COSY, gHSQC, gHMBC) Compound 18 was identified as 4- (1-hydroxy-2-propyl) biphenyl by.
¹ H NMR (500 MHz, CDCl ₃ ): δ 1.32 (3H, d, J = 7.1 Hz), 3.02 (1H, m), 3.75 (2H, d, J = 6.7 Hz), 7.32 (2H, d, J = 8.1 Hz), 7.34 (1H, m), 7.44 (2H, m), 7.57 (2H, d, J = 8.1 Hz), 7.58 (2H, m), ¹³ C NMR (125 MHz, CDCl ₃ ): δ 17.6, 42.1, 68.7, 127.0, 127.2, 127.4, 127.9, 128.8, 139.7, 140.9, 142.7

Identification of conversion products of 2- (p-tolyl) -pyridine The crude extract (106.2 mg), which was converted from 2- (p-tolyl) -pyridine using E. coli (pUCRED-Balk / BL21), was subjected to TLC (developing solvent: hexane / ethyl acetate = 1/1). It was found that a product having an Rf value of 0.26 was produced. This compound was purified by silica gel chromatography (eluent: hexane / ethyl acetate = 2/1) to give Compound 19 (40.3 mg). The molecular formula of Compound 19 was determined to be C ₁₂ H ₁₁ NO from HRESI-MS [m / z 186.09195 (M + H) ⁺ , calcd .: 186.09189]. Furthermore, Compound 19 was identified as (4- (pyridin-2-yl) phenyl) methanol by NMR analysis ( ¹ H, ¹³ C, ¹ H- ¹ H gDQF COZY, gHSQC, gHMBC).
¹ H NMR (500 MHz, CDCl ₃ ): δ 4.71 (2H, s), 7.24 (1H, ddd, J = 1.2, 4.9, 7.3 Hz), 7.42 (1H, dd, J = 2.0, 8.3 Hz), 7.69 (1H, dd, J = 1.2, 7.9 Hz), 7.75 (1H, ddd, J = 1.8, 7.3, 7.9 Hz), 7.90 (1H, dd, J = 2.0, 8.3 Hz), 8.62 (1H, dd, J = 1.8, 4.9), ¹³ C NMR (125 MHz, CDCl ₃ ): δ 64.7, 120.8, 122.1, 127.1, 127.2, 137.0, 138.4, 141.9, 149.4, 157.2

Identification of conversion products of 1,6-dimethylnaphthalene When the crude extract (80.0 mg) subjected to 1,6-dimethylnaphthalene conversion experiment using E. coli (pUCRED-Balk / BL21) was subjected to TLC (developing solvent: hexane / ethyl acetate = 3/1), the Rf value was 0.32. It was found that the product of This compound was purified by silica gel chromatography (eluent: hexane / ethyl acetate = 3/1) to obtain Compound 20 (3.6 mg). The molecular formula of Compound 20 was determined as C ₁₂ H ₁₂ O from HREI-MS [m / z 172.08915 (M ⁺ ), calcd .: 172.08881]. Furthermore, Compound 20 was identified as (1-methylnaphthalen-6-yl) methanol by NMR analysis ( ¹ H, ¹³ C, ¹ H- ¹ H gDQF COZY, gHSQC, gHMBC).
¹ H NMR (500 MHz, CDCl ₃ ): δ 2.70 (3H, s), 4.88 (2H, s), 7.31 (1H, d, J = 7.0 Hz), 7.34 (1H, dd, J = 1.8, 8.7 Hz ), 7.38 (1H, dd, J = 7.0, 8.3 Hz), 7.70 (1H, d, J = 8.3 Hz), 7.82 (1H, brs), 8.00 (1H, d, J = 8.7 Hz), ¹³ C NMR (125 MHz, CDCl ₃ ): δ 19.4, 65.5, 124.6, 125.5, 125.9, 126.1, 126.3, 126.6, 132.1, 133.5, 134.2, 137.9

Identification of conversion products of 3-phenoxytoluene When the crude extract (63.0 mg) subjected to the conversion experiment of 3-phenoxytoluene by E. coli (pUCRED-Balk / BL21) was subjected to TLC (developing solvent: hexane / ethyl acetate = 5/2), a product having an Rf value of 0.23 Turned out to be generated. This compound was purified by silica gel chromatography (eluent: hexane / ethyl acetate = 4/1) to obtain Compound 21 (27.0 mg). The molecular formula of Compound 21 was determined as C ₁₃ H ₁₂ O ₂ from HREI-MS [m / z 200.08409 (M ⁺ ), calcd .: 200.08373]. Further, NMR analysis ( ¹ H, ¹³ C, ¹ H- ¹ H gDQF COZY, gHSQC, gHMBC) identified Compound 21 as 1-phenoxy-3-hydroxymethylbenzene.
¹ H NMR (500 MHz, CDCl ₃ ): δ 4.66 (2H, s), 6.94 (1H, dd, J = 2.4, 8.06 Hz), 7.02 (2H, dd, J = 2.1, 8.4 Hz), 7.02 (1H , m) 7.10 (1H, m), 7.11 (1H, m), 7.32 (1H, m), 7.33 (2H, m), ¹³ C NMR (125 MHz, CDCl ₃ ): δ 64.9, 117.1, 117.9, 119.0 , 121.5, 123.4, 129.8, 129.9, 142.8, 156.9, 157.6

Identification of conversion products of di-p-tolyl ether When the crude extract (51.3 mg) subjected to di-p-tolyl ether conversion experiments using E. coli (pUCRED-Balk / BL21) was subjected to TLC (developing solvent: hexane / ethyl acetate = 1/1), the Rf value was 0.22. It was also found that a product having an Rf value of 0.43 was produced. This compound was purified by silica gel chromatography (eluent: hexane / ethyl acetate = 4/1 → 1/1) to obtain compound 22 (10.4 mg) having an Rf value of 0.22 and compound 23 (5.0 mg) having an Rf value of 0.43. It was. The molecular formula of Compound 22 was determined as C ₁₄ H ₁₂ O ₄ from HRAPCI-MS [m / z 243.06462 (MH) ⁻ , calcd .: 243.06573]. Furthermore, Compound 22 was identified as 4- (4- (hydroxymethyl) phenoxybenzoic acid by NMR analysis ( ¹ H, ¹³ C, ¹ H- ¹ H gDQF COZY, gHSQC, gHMBC).
¹ H NMR (500 MHz, MeOH-d4): δ 4.61 (2H, s), 6.98 (2H, d, J = 9.0 Hz), 7.05 (2H, d, J = 8.5 Hz), 7.41 (2H, d, J = 8.5 Hz), 7.99 (2H, d, J = 9.0 Hz), ¹³ C NMR (125 MHz, MeOH-d4): δ 63.4, 116.9, 119.9, 125.1, 128.8, 131.8, 138.1, 155.1, 162.3, 168.3

The molecular formula of Compound 23 was determined as C ₁₄ H ₁₂ O ₃ from HRAPCI-MS [m / z 227.07026 (MH) ⁻ , calcd .: 227.07082]. Furthermore, Compound 23 was identified as 4- (p-tolyloxy) benzoic acid by NMR analysis ( ¹ H, ¹³ C, ¹ H- ¹ H gDQF COZY, gHSQC, gHMBC).
¹ H NMR (500 MHz, CDCl ₃ ): δ 2.37 (3H, s), 6.98 (2H, d, J = 8.3 Hz), 6.98 (2H, d, J = 8.3 Hz), 7.20 (2H, d, J = 8.7 Hz), 8.05 (2H, d, J = 8.9 Hz), ¹³ C NMR (125 MHz, CDCl ₃ ): δ 20.8, 116.8, 120.3, 123.1, 130.5, 132.3, 134.4, 152.9, 163.0, 171.0

Identification of conversion products of ibuprofen methyl ester When the crude extract (163.6 mg) subjected to the conversion experiment of ibuprofen methyl ester by E. coli (pUCRED-Balk / BL21) was subjected to TLC (developing solvent: hexane / ethyl acetate = 3/1), the product with Rf value 021 was obtained. It was found that it was generated. This compound was purified by silica gel chromatography (eluent: hexane / ethyl acetate = 3/1) to obtain Compound 24 (16.1 mg). The molecular formula of Compound 24 was determined as C ₁₄ H ₂₀ O ₃ from HREI-MS [m / z 236.14198 (M ⁺ ), calcd .: 236.14124]. Furthermore, Compound 24 was identified as methyl 2- (4- (3-hydroxy-2-methylpropyl) phenyl) propionate by NMR analysis ( ¹ H, ¹³ C, ¹ H- ¹ H gDQF COZY, gHSQC, gHMBC).
¹ H NMR (500 MHz, CDCl ₃ ): δ 0.92 (3H, d, J = 6.7Hz), 1.49 (3H, d, J = 7.1 Hz), 1.93 (1H, m), 2.40, 2.59 (2H, m ), 3.50 (2H, m), 3.66 (3H, s), 3.70 (1H, q, J = 7.1 Hz), 7.12 (2H, d, J = 8.1 Hz), 7.21 (2H, d, J = 8.1 Hz) ), ¹³ C NMR (125 MHz, CDCl ₃ ): δ 16.5, 18.6, 37.7, 39.2, 45.0, 52.0, 67.6, 127.3, 129.4, 138.0, 139.5, 175.2

Identification of 4-bromophenol conversion products The crude extract (177.1 mg) subjected to the 4-bromophenol conversion experiment using E. coli (pUCRED-Balk / BL21) was subjected to TLC (developing solvent: hexane / ethyl acetate = 2/1). It was found that a product with a value of 0.51 was produced. This compound was purified by silica gel chromatography (eluent: hexane / ethyl acetate = 4/1) to obtain Compound 25 (2.4 mg) having an Rf value of 0.26 and Compound 26 (8.2 mg) having an Rf value of 0.51. The molecular formula of Compound 25 was determined as C ₁₂ H ₁₀ O ₂ from HRAPCI-MS [m / z 185.05941 (MH) ⁻ , calcd .: 185.06025]. Furthermore, Compound 25 was identified as 4,4′-dihydroxybiphenyl by NMR analysis ( ¹ H, ¹³ C, ¹ H- ¹ H gDQF COZY, gHSQC, gHMBC).
¹ H NMR (500 MHz, CDCl ₃ ): δ 6.87 (4H, d, J = 8.8 Hz), 7.38 (4H, d, J = 8.8 Hz), ¹³ C NMR (125 MHz, CDCl ₃ ): δ 115.7, 127.9, 133.1, 155.8

The molecular formula of Compound 26 was determined as C ₁₂ H ₈ Br ₂ O ₂ from HRAPCI-MS [m / z 340.88285 (MH) ⁻ , calcd .: 340.88128]. Further, NMR analysis ( ¹ H, ¹³ C, ¹ H- ¹ H gDQF COZY, gHSQC, gHMBC) identified Compound 26 as 2- (4-bromophenoxy) -4-bromophenol.
¹ H NMR (500 MHz, CDCl ₃ ): δ 6.93 (1H, d, J = 8.9 Hz), 6.93 (1H, d, J = 8.7 Hz), 6.95 (1H, d, J = 2.3 Hz), 7.16 ( 1H, dd, J = 2.3, 8.7 Hz), 7.49 (1H, d, J = 8.9 Hz), ¹³ C NMR (125 MHz, CDCl ₃ ): δ 111.8, 117.0, 117.7, 120.2, 121.1, 127.7, 133.1, 144.1, 146.4, 155.0

Identification of o-cresol conversion products The crude extract (40.8 mg) subjected to the conversion experiment of o-cresol using E. coli (pUCRED-Balk / BL21) was subjected to TLC (developing solvent: hexane / ethyl acetate = 5/2). It was found that 0.41 product was produced. This compound was purified by silica gel chromatography (eluent: hexane / ethyl acetate = 10/1 → 5/2) to obtain compound 27 (7.0 mg) having an Rf value of 0.27 and compound 28 (5.9 mg) having an Rf value of 0.41. It was. The molecular formula of Compound 27 was determined as C ₁₄ H ₁₄ O ₂ from HREI-MS [m / z 213.09109 (MH) ⁻ , calcd .: 213.09155]. Further, NMR analysis ( ¹ H, ¹³ C, ¹ H- ¹ H gDQF COZY, gHSQC, gHMBC) identified Compound 27 as 3,3′-dimethyl-4,4′-dihydroxybiphenyl.
¹ H NMR (500 MHz, CDCl ₃ ): δ 2.30 (6H, s), 6.81 (2H, d, J = 8.3 Hz, 7.25 (2H, dd, J = 2.7, 8.3 Hz), 7.30 (2H, d, J = 2.7 Hz), ¹³ C NMR (125 MHz, CDCl ₃ ): δ 15.9, 115.1, 123.9, 125.3, 129.4, 133.7, 152.9

The molecular formula of Compound 28 was determined as C ₁₄ H ₁₄ O ₂ from HREI-MS [m / z 213.09087 (MH) ⁻ , calcd .: 213.09155]. Further, NMR analysis ( ¹ H, ¹³ C, ¹ H- ¹ H gDQF COZY, gHSQC, gHMBC) identified compound 28 as 3,3′-dimethyl-4,2′-dihydroxybiphenyl.
¹ H NMR (500 MHz, CDCl ₃ ): δ 2.30 (3H, s), 2.30 (3H, s), 5.05 (1H, brs), 5.30 (1H, brs), 6.87 (1H, dd, J = 7.4, 7.6 Hz), 6.89 (1H, d, J = 8.0 Hz), 7.04 (1H, dd, J = 1.3, 7.6 Hz), 7.11 (1H, dd, J = 0.9, 7.4 Hz), 7.17 (1H, dd, J = 2.2, 8.0 Hz), ¹³ C NMR (125 MHz, CDCl ₃ ): δ 15.8, 16.2, 115.7, 120.0, 124.4, 124.9, 127.4, 127.7, 127.9, 129.4, 130.1, 131.8, 150.7, 153.7

Identification of conversion products of 2-ethylphenol When the crude extract (128.3 mg) subjected to the conversion experiment of 2-ethylphenol by E. coli (pUCRED-Balk / BL21) was subjected to TLC (developing solvent: hexane / ethyl acetate = 2/1), Rf value 0.46 and Rf It was found that a product with a value of 0.57 was produced. This compound was purified by silica gel chromatography (eluent: hexane / ethyl acetate = 2/1) to obtain Compound 29 (13.0 mg) having an Rf value of 0.46 and Compound 30 (8.1 mg) having an Rf value of 0.57. The molecular formula of Compound 29 was determined as C ₁₆ H ₁₈ O ₂ from HRAPCI-MS [m / z 241.12300 (MH) ⁻ , calcd .: 241.12285]. Further, Compound 29 was identified as 3,3′-diethyl-4,4′-dihydroxybiphenyl by NMR analysis ( ¹ H, ¹³ C, ¹ H- ¹ H gDQF COZY, gHSQC, gHMBC).
¹ H NMR (500 MHz, CDCl ₃ ): δ 1.28 (6H, t, J = 7.6 Hz), 2.69 (4H, q, J = 7.6 Hz), 4.8 (2H, brs), 6.81 (2H, d, J = 8.2), 7.25 (2H, dd, J = 2.2, 8.2 Hz), 7.32 (2H, d, J = 2.2 Hz), ¹³ C NMR (125 MHz, CDCl ₃ ): δ 14.1, 23.1, 115.4, 125.3, 130.0, 134.0, 152.4

The molecular formula of Compound 30 was determined as C ₁₆ H ₁₈ O ₂ from HRAPCI-MS [m / z 241.12342 (MH) ⁻ , calcd .: 241.12285]. Further, NMR analysis ( ¹ H, ¹³ C, ¹ H- ¹ H gDQF COZY, gHSQC, gHMBC) identified Compound 30 as 3,3′-diethyl-2,4′-dihydroxybiphenyl.
¹ H NMR (500 MHz, CDCl ₃ ): δ 1.264 (3H, t, J = 7.6 Hz), 1.266 (3H, t, J = 7.6 Hz), 2.69 (2H, q, J = 7.6 Hz), 2.71 ( 2H, q, J = 7.6 Hz), 6.87 (1H, d, J = 8.2 Hz), 6.90 (1H, m), 7.05 (1H, dd, J = 1.7, 7.6 Hz), 7.13 (1H, m), 7.16 (1H, dd, J = 2.1, 8.2 Hz), 7.22 (1H, d, J = 2.1 Hz), ¹³ C NMR (125 MHz, CDCl ₃ ): δ 13.9, 14.1, 23.0, 23.4, 115.9, 120.2, 126.8, 127.7, 127.8, 128.4, 129.6, 130.2, 130.4, 131.0, 150.2, 153.2

Identification of conversion products of 2-methoxyphenol When the crude extract (97.3 mg) subjected to the conversion experiment of 2-methoxyphenol by E. coli (pUCRED-Balk / BL21) was subjected to TLC (developing solvent: hexane / ethyl acetate = 2/1), the Rf value was 0.32. It was found that a product having an Rf value of 0.35 was produced. This compound was purified by silica gel chromatography (eluent: hexane / ethyl acetate = 9/5) to obtain Compound 31 (2.3 mg) having an Rf value of 0.32 and Compound 32 (2.7 mg) having an Rf value of 0.35. The molecular formula of Compound 31 was determined as C ₁₄ H ₁₄ O ₄ from HRAPCI-MS [m / z 245.08167 (MH) ⁻ , calcd .: 245.08138]. Further, NMR analysis ( ¹ H, ¹³ C, ¹ H- ¹ H gDQF COZY, gHSQC, gHMBC) identified compound 31 as 3,3′-dimethoxy-4,4′-dihydroxybiphenyl.
¹ H NMR (500 MHz, CDCl ₃ ): δ 3.96 (6H, s), 6.97 (2H, d, J = 8.1 Hz), 7.02 (2H, d, J = 2.0 Hz), 7.04 (2H, dd, J = 2.0, 8.1 Hz), ¹³ C NMR (125 MHz, CDCl ₃ ): δ 56.0, 109.6, 114.6, 119.8, 133.9, 144.8, 146.6

The molecular formula of Compound 32 was determined as C ₁₄ H ₁₄ O ₂ from HRAPCI-MS [m / z 245.08128 (MH) ⁻ , calcd .: 245.08138]. Further, NMR analysis ( ¹ H, ¹³ C, ¹ H- ¹ H gDQF COZY, gHSQC, gHMBC) identified Compound 32 as 3,4′-dihydroxy-2,3′-dimethoxybiphenyl.
¹ H NMR (500 MHz, MeOH-d4): δ 3.87 (3H, s), 3.89 (3H, s), 6.77 (1H, dd, J = 1.2, 8.4 Hz), 6.80 (1H, m), 6.99 ( 1H, dd, J = 2.0, 8.2 Hz), 7.19 (1H, d, J = 2.0 Hz), 8.2 (1H, dd, J = 1.2, 8.3 Hz), ¹³ C NMR (125 MHz, MeOH-d4): δ 55.2, 55.4, 109.6, 113.2, 114.7, 119.2, 122.0, 122.3, 128.5, 130.5, 143.4, 145.4, 147.2, 148.1

Identification of conversion products of 2,6-dimethylphenol When the crude extract (136.0 mg) subjected to the 2,6-dimethylphenol conversion experiment using E. coli (pUCRED-Balk / BL21) was subjected to TLC (developing solvent: hexane / ethyl acetate = 3/1), the Rf value was 0.45. It was found that a product having an Rf value of 0.47 was produced. This compound was purified by silica gel chromatography (eluent: hexane / ethyl acetate = 3/1) to obtain Compound 33 (6.2 mg) having an Rf value of 0.45 and Compound 34 (3.2 mg) having an Rf value of 0.47. The molecular formula of Compound 33 was determined as C ₁₆ H ₁₈ O ₂ from HRAPCI-MS [m / z 241.12263 (MH) ⁻ , calcd .: 241.12285]. Further, NMR analysis ( ¹ H, ¹³ C, ¹ H- ¹ H gDQF COZY, gHSQC, gHMBC) identified Compound 33 as 3,3 ′, 5,5′-tetramethyl-4,4′-dihydroxybiphenyl.
¹ H NMR (500 MHz, CDCl ₃ ): δ 2.25 (12H, s), 7.08 (4H, d, J = 0.5 Hz), ¹³ C NMR (125 MHz, CDCl ₃ ): δ 15.8, 124.6, 126.4, 133.2 , 152.0

The molecular formula of Compound 34 was determined as C ₁₆ H ₁₈ O ₂ from HRAPCI-MS [m / z 241.12305 (MH) ⁻ , calcd .: 241.12285]. Further, NMR analysis ( ¹ H, ¹³ C, ¹ H- ¹ H gDQF COZY, gHSQC, gHMBC) revealed that compound 34 was 6- (4-hydroxy-3,5-dimethylphenyl) -2,6-dimethylcyclohexa-2. It was identified as 4-dienone.
¹ H NMR (500 MHz, CDCl ₃ ): δ 1.55 (3H, s), 1.87 (3H, s), 2.20 (6H, s), 4.6 (1H, brs), 6.23 (1H, dd, J = 3.6, 9.4 Hz), 6.33 (1H, m), 6.68 (2H, s), 6.85 (1H, m), ¹³ C NMR (125 MHz, CDCl ₃ ): δ 15.7, 16.1, 23.9, 53.5, 119.4, 123.1, 126.7 , 127.0, 132.3, 138.0, 145.8, 151.4, 204.9

Identification of conversion products of 4-phenylphenol When the crude extract (137.1 mg) subjected to the 4-phenylbiphenyl conversion experiment using E. coli (pUCRED-Balk / BL21) was subjected to TLC (developing solvent: hexane / ethyl acetate = 3/1), a product with an Rf value of 0.21 was obtained. Turned out to be generated. This compound was purified by silica gel chromatography (eluent: hexane / ethyl acetate = 3/1) to obtain Compound 35 (6.0 mg). The molecular formula of Compound 35 was determined as C ₂₄ H ₁₈ O ₂ from HRAPCI-MS [m / z 337.12275 (MH) ⁻ , calcd .: 337.12285]. Further, NMR analysis ( ¹ H, ¹³ C, ¹ H- ¹ H gDQF COZY, gHSQC, gHMBC) identified Compound 35 as 2,2′-dihydroxy-5,5′-diphenylbiphenyl.
¹ H NMR (500 MHz, CDCl ₃ ): δ 5.95 (2H, brs), 7.11 (2H, d, J = 8.8 Hz), 7.32 (2H, m), 7.42 (4H, m), 7.566 (2H, d , J = 2.3 Hz), 7.568 (4H, d, J = 7.1 Hz), 7.569 (2H, m), ¹³ C NMR (125 MHz, CDCl ₃ ): δ 117.5, 124.5, 127.1, 127.3, 128.9, 129.1, 130.4, 135.2, 140.5, 152.7

Identification of conversion products of 4-phenylphenol When the crude extract (116.6 mg) subjected to 4-phenylbiphenyl conversion experiments using E. coli (pUCRED-Balk / BL21) was subjected to TLC (developing solvent: hexane / ethyl acetate = 10/1), a product with an Rf value of 0.23 was obtained. Turned out to be generated. This compound was purified by silica gel chromatography (eluent: hexane / ethyl acetate = 10/1) to obtain Compound 36 (3.0 mg). The molecular formula of Compound 36 was determined as C ₂₄ H ₁₈ O ₂ from HRAPCI-MS [m / z 337.12250 (MH) ⁻ , calcd .: 337.12285]. Further, NMR analysis ( ¹ H, ¹³ C, ¹ H- ¹ H gDQF COZY, gHSQC, gHMBC) identified Compound 36 as 2- (4-phenylphenoxy) -4-phenylphenol.
¹ H NMR (500 MHz, CDCl ₃ ): δ 5.65 (1H, brs), 7.14 (2H, d, J = 8.4 Hz), 7.15 (1H, d, J = 8.4 Hz), 7.19 (1H, d, J = 2.2 Hz), 7.28 (1H, m), 7.32 (1H, dd, J = 2.2, 8.4 Hz), 7.34 (1H, m), 7.38 (2H, m), 7.44 (2H, m), 7.57 (2H , m), 7.58 (2H, d, J = 8.4 Hz), ¹³ C NMR (125 MHz, CDCl ₃ ): δ 116.5, 117.6, 118.2, 123.5, 126.7, 126.92, 126.94, 127.2, 128.6, 128.7, 128.8, 134.3, 136.8, 140.26, 140.30, 143.6, 147.0, 156.2

Identification of conversion products of 2-chloro-4-phenylphenol The crude extract (125.5 mg) subjected to the conversion experiment of 2-chloro-4-phenylbiphenyl by E. coli (pUCRED-Balk / BL21) was subjected to TLC (developing solvent: hexane / ethyl acetate = 5/2) and Rf It was found that a product with a value of 0.67 was produced. This compound was purified by silica gel chromatography (eluent: hexane / ethyl acetate = 5/2) to obtain Compound 37 (1.9 mg). The molecular formula of Compound 37 was determined to be C ₂₄ H ₁₆ Cl ₂ O ₂ from HRAPCI-MS [m / z 405.04475 (MH) ⁻ , calcd .: 405.04491]. Further, NMR analysis ( ¹ H, ¹³ C, ¹ H- ¹ H gDQF COZY, gHSQC, gHMBC) identified Compound 37 as 2- (2-chloro-4-phenylphenoxy) -6-chloro-4-phenylphenol. .
¹ H NMR (500 MHz, CDCl ₃ ): δ 6.97 (1H, d, J = 2.0 Hz), 7.11 (1H, d, J = 8.5 Hz), 7.32 (1H, m), 7.37 (1H, m), 7.38 (2H, m), 7.39 (1H, d, J = 2.0 Hz), 7.44 (2H, m), 7.45 (2H, m), 7.46 (1H, dd, J = 2.0, 8.5 Hz), 7.55 (2H , m), 7.71 (1H, d, J = 2.0 Hz), ¹³ C NMR (125 MHz, CDCl ₃ ): δ 115.2, 120.2, 121.4, 123.5, 125.6, 126.67, 126.7, 126.9, 127.4, 127.5, 127.8, 128.9, 129.0, 134.2, 138.8, 139.0, 139.1, 142.6, 144.5, 150.9

Identification of 2-naphthol conversion products When the crude extract (96.1 mg) subjected to the conversion experiment of 2-naphthol using E. coli (pUCRED-Balk / BL21) was subjected to TLC (developing solvent: hexane / ethyl acetate = 3/1), the Rf value was 0.20 and the Rf value was It was found that a product with 0.28 and Rf value 0.52 was produced. This compound was purified by silica gel chromatography (eluent: hexane / ethyl acetate = 3/1) to give compound 38 (6.7 mg) having an Rf value of 0.20, compound 39 (0.9 mg) having an Rf value of 0.28, and a compound having an Rf value of 0.52. 40 (8.8 mg) was obtained. The molecular formula of Compound 38 was determined as C ₂₀ H ₁₄ O ₂ from HRAPCI-MS [m / z 285.09119 (MH) ⁻ , calcd .: 285.09155]. Further, NMR analysis ( ¹ H, ¹³ C, ¹ H- ¹ H gDQF COZY, gHSQC, gHMBC) identified compound 38 as 6- (2-hydroxynaphthalen-1-yl) naphthalen-2-ol.
¹ H NMR (500 MHz, MeOH-d4): δ7.14 (1H, dd, J = 2.4, 8.8 Hz), 7.22 (1H, d, J = 2.4 Hz), 7.25 (1H, d, J = 8.9 H ), 7.26 (1H, m), 7.27 (1H, m), 7.38 (1H, dd, J = 1.7, 8.4 Hz), 7.44 (1H, m), 7.74 (1H, d, J = 1.7 Hz), 7.77 (1H, m), 7.78 (1H, m), 7.79 (1H, m), 7.80 (1H, m), ¹³ C NMR (125 MHz, MeOH-d4): δ 108.7, 117.9, 118.3, 122.1, 122.6, 124.6, 126.00, 126.04, 127.8, 128.7, 128.9, 129.1, 129.4, 129.67, 129.72, 130.9, 134.4, 134.5, 155.4, 155.5

The molecular formula of Compound 39 was determined as C ₂₀ H ₁₄ O ₂ from HRAPCI-MS [m / z 285.09185 (MH) ⁻ , calcd .: 285.09155]. Further, NMR analysis ( ¹ H, ¹³ C, ¹ H- ¹ H gDQF COZY, gHSQC, gHMBC) identified Compound 39 as 1,1′-bi (2-naphthol).
¹ H NMR (500 MHz, MeOH-d4): δ 7.01 (2H, d, J = 8.5 Hz), 7.16 (2H, ddd, J = 1.3, 8.1, 8.5 Hz), 7.24 (2H, ddd, J = 1.2 , 8.1, 8.5 Hz), 7.28 (2H, d, J = 8.9 Hz), 7.82 (2H, d, J = 8.1 Hz), 7.86 (2H, d, J = 8.9 Hz), ¹³ C NMR (125 MHz, MeOH-d4): δ 114.9, 118.1, 122.7, 124.7, 125.9, 127.9, 129.3, 129.4, 134.7, 150.3

The molecular formula of Compound 40 was determined as C ₂₀ H ₁₄ O ₂ from HRAPCI-MS [m / z 285.09187 (MH) ⁻ , calcd .: 285.09155]. Furthermore, Compound 40 was identified as 1- (naphthalen-2-yloxy) -2-hydroxynaphthalene by NMR analysis ( ¹ H, ¹³ C, ¹ H- ¹ H gDQF COZY, gHSQC, gHMBC).
¹ H NMR (500 MHz, MeOH-d4): δ 6.89 (1H, d, J = 2.6 Hz), 7.29 (1H, m), 7.30 (1H, m), 7.30 (1H, m), 7.33 (1H, m), 7.33 (1H, m), 7.36 (1H, dd, J = 2.6, 8.9 Hz), 7.50 (1H, d, J = 7.9 Hz), 7.73 (1H, d, J = 8.4 Hz), 7.73 ( 1H, dd, J = 1.3, 8.4 Hz), 7.78 (1H, d, J = 7.9 hz), 7.83 (1H, d, J = 8.9 Hz), 7.83 (1H, dd, J = 1.7, 8.9 Hz), ¹³ C NMR (125 MHz, MeOH-d4): δ 108.9, 117.8, 119.0, 120.6, 123.4, 123.8, 126.3, 126.3, 126.3, 126.7, 127.5, 127.9, 129.1, 129.5, 129.7, 129.9, 134.1, 134.7, 146.8 , 156.8

Identification of conversion products of 2-methyl-1-naphthol The crude extract (12.2 mg), which was subjected to the conversion experiment of 2-methyl-1-naphthol using E. coli (pUCRED-Balk / BL21), was subjected to TLC (developing solvent: hexane / ethyl acetate = 2/1) to give an Rf value. It was found that 0.49 product was produced. This compound was purified by silica gel chromatography (eluent: hexane / ethyl acetate = 2/1) to obtain Compound 41 (6.0 mg). The molecular formula of Compound 41 was determined as C ₂₂ H ₁₈ O ₂ from HRAPCI-MS [m / z 313.12222 (MH) ⁻ , calcd .: 313.12285]. Furthermore, Compound 41 was identified as 1,1′-bi (3-methyl-4-hydroxynaphthalene) by NMR analysis ( ¹ H, ¹³ C, ¹ H- ¹ H gDQF COZY, gHSQC, gHMBC).
¹ H NMR (500 MHz, MeOH-d4): δ 2.46 (6H, s), 7.16 (2H, m), 7.19 (2H, s), 7.20 (2H, m), 7.38 (2H, m), 8.29 ( d, J = 8.5 Hz), ¹³ C NMR (125 MHz, MeOH-d4): δ 15.3, 117.6, 121.6, 124.4, 124.6, 125.8, 126.3, 130.6, 131.4, 133.1, 149.2

Identification of conversion products of 1-phenyl-1-cyclohexene The crude extract (2.3 g) obtained by overlaying 1.98 g of 1-phenyl-1-cyclohexene with E. coli (pUCRED-Balk / BL21) was converted to TLC (developing solvent: hexane / ethyl acetate = 50/1). It was found that a product with an Rf value of 0.55 was generated. This compound was purified by silica gel chromatography (eluent: hexane / ethyl acetate = 50/1) to obtain Compound 42 (39.2 mg). The molecular formula of Compound 42 was determined as C ₁₂ H ₁₀ from HRAPCI-MS [m / z 154.07868 (MH) ⁻ , calcd .: 154.07825. Furthermore, Compound 42 was identified as biphenyl by NMR analysis ( ¹ H, ¹³ C, ¹ H- ¹ H gDQF COZY, gHSQC, gHMBC).
¹ H NMR (500 MHz, CDCl ₃ ): δ 7.34 (2H, m), 7.44 (4H, m), 7.59 (4H, m), ¹³ C NMR (125 MHz, CDCl ₃ ): δ 127.1, 127.2, 128.7 , 141.2

Identification of conversion products in the presence of 2-bromophenol and 4-phenylphenol The crude extract (150.0 mg) subjected to the conversion experiment in which 2-bromophenol and 4-phenylphenol coexisted with E. coli (pUCRED-Balk / BL21) was added to TLC (developing solvent: hexane / ethyl acetate = 3/1). As a result, it was found that products having Rf values of 0.19 and 0.28 and an Rf value of 0.61 were produced. This compound was purified by silica gel chromatography (eluent: hexane / ethyl acetate = 3/1) to give compound 43 (0.7 mg) having an Rf value of 0.19, compound 44 (1.7 mg) having an Rf value of 0.28, and a compound having an Rf value of 0.61 45 (2.0 mg) was obtained. The molecular formula of Compound 43 was determined as C ₁₈ H ₁₃ BrO ₂ from HRAPCI-MS [m / z 339.00177 (MH) ⁻ , calcd .: 339.00207]. Further, NMR analysis ( ¹ H, ¹³ C, ¹ H- ¹ H gDQF COZY, gHSQC, gHMBC) identified Compound 43 as 3-bromo-4-hydroxy-2′-hydroxy-5′-phenylbiphenyl.
¹ H NMR (500 MHz, CDCl3): δ7.02 (1H, d, J = 8.3 Hz), 7.15 (1H, d, J = 8.3 Hz), 7.32 (1H, m), 7.40 (1H, dd, J = 2.1, 8.3 Hz), 7.42 (2H, m), 7.43 (1H, m), 7.48 (1H, dd, J = 2.3, 8.3 Hz), 7.56 (2H, m), 7.67 (1H, d, J = 2.1 Hz), ¹³ C NMR (125 MHz, CDCl ₃ ): δ 110.9, 116.3, 116.6, 126.7, 126.8, 126.9, 127.9, 128.8, 129.0, 130.0, 130.8, 132.7, 134.3, 140.5, 152.0, 152.1

The molecular formula of Compound 44 was determined as C ₁₈ H ₁₃ BrO ₂ from HRAPCI-MS [m / z 339.00201 (MH) ⁻ , calcd .: 339.00207]. Further, NMR analysis ( ¹ H, ¹³ C, ¹ H- ¹ H gDQF COZY, gHSQC, gHMBC) identified Compound 44 as 3-bromo-2-hydroxy-2′-hydroxy-5′-phenylbiphenyl.
¹ H NMR (500 MHz, MeOH-d4): δ 6.87 (1H, dd, J = 7.7, 7.8 Hz), 7.02 (1H, d, J = 8.42), 7.27 (1H, dd, J = 1.7, 7.7 Hz) ), 7.27 (1H, m), 7.38 (2H, m), 7.46 (1H, d, J = 2.3 Hz), 7.51 (1H, dd, J = 1.7, 7.8 Hz), 7.51 (1H, dd, J = 2.3, 8.4 Hz), 7.58 (2H, m), ¹³ C NMR (125 MHz, MeOH-d4): δ 111.8, 116.1, 121.1, 125.8, 126.4, 126.5, 127.6, 128.6, 128.7, 130.2, 130.8, 132.1, 133.5, 140.9, 151.3, 153.7

The molecular formula of Compound 45 was determined as C ₁₈ H ₁₃ BrO ₂ from HRAPCI-MS [m / z 339.00298 (MH) ⁻ , calcd .: 339.00207]. Further, NMR analysis ( ¹ H, ¹³ C, ¹ H- ¹ H gDQF COZY, gHSQC, gHMBC) identified Compound 45 as 4- (3-bromo-2-hydroxyphenoxy) biphenyl.
¹ H NMR (500 MHz, MeOH-d4): δ 6.74 (1H, dd, J = 8.1, 8.2 Hz), 6.89 (1H, dd, J = 1.5, 8.1 Hz), 7.04 (2H, d, J = 8.8 Hz), 7.29 (1H, dd, J = 1.5, 8.2 Hz), 7.30 (1H, m), 7.40 (2H, m), 7.56 (2H, m), 7.59 (2H, d, J = 8.8 Hz), ¹³ C NMR (125 MHz, MeOH-d4): δ 111.8, 116.1, 121.1, 125.8, 126.4, 126.5, 127.6, 128.6, 128.7, 130.2, 130.8, 132.1, 133.5, 140.9, 151.3, 153.7

Identification of conversion product in the presence of o-cresol and 4-phenylphenol The crude extract (390.0 mg), which was subjected to a conversion experiment in the presence of o-cresol and 4-phenylphenol with E. coli (pUCRED-Balk / BL21), was applied to TLC (developing solvent: hexane / ethyl acetate = 3/1). As a result, it was found that a product having an Rf value of 0.32 was generated. This compound was purified by silica gel chromatography (eluent: hexane / ethyl acetate = 3/1) to obtain Compound 46 (2.0 mg). The molecular formula of Compound 46 was determined as C ₁₉ H ₁₆ O ₂ from HRAPCI-MS [m / z 275.10700 (MH) ⁻ , calcd .: 275.10720]. Further, NMR analysis ( ¹ H, ¹³ C, ¹ H- ¹ H gDQF COZY, gHSQC, gHMBC) identified Compound 46 as 2-hydroxy-3-methyl-2′-hydroxy-5′-phenylbiphenyl.
¹ H NMR (500 MHz, MeOH-d4): δ 2.30 (3H, s), 6.88 (1H, dd, J = 7.3, 7.6 Hz), 7.03 (1H, d, 8.4 Hz), 7.13 (1H, d, J = 7.3 Hz), 7.13 (1H, d, J = 7.6 Hz), 7.26 (1H, m), 7.34 (2H, m), 7.47 (1H, d, J = 2.3 Hz), 7.49 (1H, dd, J = 2.3, 8.4 Hz), 7.57 (2H, m), ¹³ C NMR (125 MHz, MeOH-d4): δ 15.6, 116.1, 120.3, 126.3, 126.4, 126.5, 126.8, 126.8, 127.2, 128.6, 129.0, 130.0, 130.5, 133.8, 141.1, 152.2, 153.3

Identification of conversion products in the presence of 2,6-dimethylphenol and 4-phenylphenol The crude extract (393.2 mg), which was subjected to a conversion experiment in the presence of 2,6-dimethylphenol and 4-phenylphenol using E. coli (pUCRED-Balk / BL21), was TLC (developing solvent: hexane / ethyl acetate = 3/1 ) Was found to produce a product with an Rf value of 0.28. This compound was purified by silica gel chromatography (eluent: hexane / ethyl acetate = 3/1) to obtain Compound 47 (3.4 mg). The molecular formula of Compound 47 was determined as C ₂₀ H ₁₈ O ₂ from HRAPCI-MS [m / z 289.12281 (MH) ⁻ , calcd .: 289.12285]. Further, NMR analysis ( ¹ H, ¹³ C, ¹ H- ¹ H gDQF COZY, gHSQC, gHMBC) identified Compound 47 as 3,5-dimethyl-4-hydroxy-2′-hydroxy-5′-phenylbiphenyl.
¹ H NMR (500 MHz, MeOH-d4): δ 2.32 (6H, s), 7.04 (1H, d, J = 8.3 Hz), 7.12 (2H, s), 7.31 (1H, m), 7.40 (2H, m), 7.43 (1H, dd, J = 2.3, 8.3 Hz), 7.49 (1H, d, J = 2.3 Hz), 7.57 (2H, m), ¹³ C NMR (125 MHz, MeOH-d4): δ 16.0 , 115.8, 124.1, 126.6, 126.7, 127.3, 128.3, 128.4, 128.7, 128.8, 129.2, 133.8, 140.8, 152.1, 152.2

Identification of conversion products of 4-methoxybiphenyl When the crude extract (102.1 mg) subjected to 4-methoxybiphenyl conversion experiments using E. coli (pUCRED-Balk / BL21) was subjected to TLC (developing solvent: hexane / ethyl acetate = 4/1), a product with an Rf value of 0.33 was obtained. Turned out to be generated. This compound was purified by silica gel chromatography (eluent: hexane / ethyl acetate = 4/1) to obtain Compound 48 (7.6 mg). The molecular formula of Compound 48 was determined to be C ₁₂ H ₁₀ O from HRAPCI-MS [m / z 169.06500 (MH) ⁻ , calcd .: 169.06534]. Further, Compound 48 was identified as 4-hydroxybiphenyl by NMR analysis ( ¹ H, ¹³ C, ¹ H- ¹ H gDQF COZY, gHSQC, gHMBC).
¹ H NMR (500 MHz, CDCl ₃ ): δ 6.91 (2H, d, J = 8.7 Hz), 7.30 (1H, m), 7.41 (2H, m), 7.48 (2H, d, J = 8.7 Hz), 7.54 (2H, m), ¹³ C NMR (125 MHz, CDCl ₃ ): δ 115.6, 126.7, 126.7, 128.4, 128.7, 134.0, 140.7, 155.1

Identification of conversion products of 1-phenylnaphthalene When the crude extract (280.3 mg) subjected to the conversion experiment of 1-phenylnaphthalene with E. coli (pUCRED-Balk / BL21) was subjected to TLC (developing solvent: hexane / ethyl acetate = 5/1), the product with an Rf value of 0.30 Turned out to be generated. This compound was purified by silica gel chromatography (eluent: hexane / ethyl acetate = 5/1) to obtain Compound 49 (0.5 mg). The molecular formula of Compound 49 was determined as C ₁₆ H ₁₂ O from HRAPCI-MS [m / z 219.08203 (MH) ⁻ , calcd .: 219.08099]. Furthermore, Compound 49 was identified as 1- (4-hydroxyphenyl) naphthalene by NMR analysis ( ¹ H, ¹³ C, ¹ H- ¹ H gDQF COZY, gHSQC, gHMBC).
¹ H NMR (500 MHz, CDCl ₃ ): δ 6.96 (2H, d, J = 8.8 Hz), 7.38 (2H, d, J = 8.8 Hz), 7.40 (1H, d, J = 8.0 Hz), 7.45 ( 1H, m), 7.48 (1H, m), 7.51 (1H, dd, J = 8.0, 9.0 Hz), 7.84 (1H, d, J = 9.0 Hz), 7.90 (1H, d, J = 7.0 Hz), 7.91 (1H, d, J = 7.0 Hz), ¹³ C NMR (125 MHz, CDCl ₃ ): δ 115.1, 125.4, 125.7, 125.9, 126.0, 126.9, 127.4, 128.2, 130.1, 131.3, 133.2, 133.8, 139.8, 154.9

Identification of conversion products in the presence of 2-bromophenol and 2-naphthol The crude extract (210.0 mg) subjected to a conversion experiment in which 2-bromophenol and 2-naphthol coexisted with E. coli (pUCRED-Balk / BL21) was applied to TLC (developing solvent: hexane / ethyl acetate = 3/1). As a result, it was found that a product having an Rf value of 0.22 was produced. This compound was purified by silica gel chromatography (eluent: hexane / ethyl acetate = 3/1) to obtain Compound 50 (2.5 mg). The molecular formula of Compound 50 was determined as C ₁₆ H ₁₁ BrO ₂ from HRAPCI-MS [m / z 312.98522 (MH) ⁻ , calcd .: 312.98642]. Furthermore, Compound 50 was identified as 1- (3-bromo-4-hydroxyphenyl) -2-hydroxynaphthalene by NMR analysis ( ¹ H, ¹³ C, ¹ H- ¹ H gDQF COZY, gHSQC, gHMBC).
¹ H NMR (500 MHz, MeOH-d4): δ4.92 (2H, brs), 7.03 (1H, d, J = 8.2 Hz), 7.13 (1H, dd, J = 2.0, 8.2 Hz), 7.17 (1H , d, J = 8.9 Hz), 7.25 (1H, ddd, J = 1.5, 8.1, 8.1 Hz), 7.30 (1H, ddd, J = 1.5, 8.1, 8.5 Hz), 7.40 (1H, dd, J = 1.5 , 8.5 Hz), 7.42 (1H, d, J = 2.0 Hz), 7.73 (1H, d, J = 8.9 Hz), 7.75 (1H, dd, J = 1.5, 7.9 Hz), ¹³ C NMR (125 MHz, MeOH-d4): δ 110.7, 117.1, 119.0, 121.7, 123.8, 125.4, 127.2, 129.0, 130.0, 130.1, 130.3, 132.5, 135.5, 136.6, 152.8, 154.5

<Conclusion>
Balk (CYP153) is used in addition to the above-mentioned hydroxylation of aromatic rings, hydroxylation of aromatic alkyl (especially methyl, ethyl, isopropyl, isobutyl) groups, dealkylation, and carbon atoms of two or more aromatic rings. It has been found that it has a function of condensation and aromatization via a carbon bond or an ether bond, and is useful for the production of various aromatic compounds.
Moreover, when different types of aromatic compounds are used simultaneously as substrates, hybrid type condensation products could be obtained.
In addition, CYP153 is a novel reaction such as hydroxylation to aromatic rings, hydroxylation of aromatic methyl, dealkylation, and condensation via carbon-carbon bonds of two or more aromatic rings only with Balk (CYP153A13). It was revealed that various CYP153 catalyzed.
Furthermore, when Balk (CYP153) was used, various conversion products could be obtained as compared with the case of using ferric chloride. Compared with ferric chloride, CYP has a small amount of heme iron, but the conversion rate is high, suggesting that the efficiency of P450 redox reaction is high.

  According to the present invention, using a protein belonging to monooxygenase CYP153 or a fusion protein containing the same, (1) a reaction of introducing a hydroxyl group into an aromatic ring of an aromatic compound having a substituent, an alkyl group or the like Hydroxyl introduction reaction, dealkylation reaction, (2) condensation reaction via carbon-carbon bond or ether bond of aromatic ring in 2 or more molecules of aromatic compound having phenolic hydroxyl group, and (3) aromatization reaction It becomes possible to do. Furthermore, in the present invention, since the reaction is carried out using living cells, there is no need to purify the enzyme, and it is easy to mass-produce conversion products. Furthermore, since the production method of the present invention uses a biocatalyst derived from bacteria, it can react under mild conditions such as normal temperature and normal pressure, and does not require dangerous synthetic catalyst or waste liquid treatment. It ’s an environmentally friendly way. By using the production method of the present invention, not only the production of various aromatic compounds but also industrial applications such as the creation of new compounds at the drug discovery stage, the preparation of metabolites, and the shortening of production processes that require long steps are possible. .

FIG. 3 is a diagram showing the structure of a P450 function expression vector pRED. It is the figure which compared the CO difference spectrum between the vectors using MoxA. It is the figure which compared the CO difference spectrum between the vectors using P450balk.

Claims (14)

A process of introducing a hydroxyl group into an aromatic ring or a carbon atom of a substituent by allowing a protein belonging to the cytochrome P450 family 153 (CYP153) or a fusion protein containing the protein to function as a monooxygenase and acting on an aromatic compound having a substituent only containing aromatic compound having a substituent having 1 to 3 things or aromatic ring having one to three aromatic rings, and an aromatic compound containing nitrogen or oxygen in the aromatic ring, said substituent The group is an alkyl group having 1 to 4 carbon atoms, a saturated or unsaturated hydrocarbon group having 5 to 7 members, a phenylalkyl group having 7 to 9 carbon atoms, an alkoxy group having 1 to 2 carbon atoms, a halogen atom, a hydroxyl group, amino 1 or more selected from the group consisting of a group, an aldehyde group, a carboquinyl group, a carboxyl group, a carboxylalkyl group, an acyl group, a cyano group, and an oxo group, and the number of the substituents is 1 A method for producing an aromatic compound, characterized in that the fusion protein is a fusion protein with a reductase peptide (domain) contained in cytochrome P450 monooxygenase P450RhF derived from Rhodococcus NCIMB9784 strain . The production method according to claim 1 , wherein CYP153 is the following (a) or (b).
(A) a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 (b) in the amino acid sequence of SEQ ID NO: 1, comprising an amino acid sequence in which one amino acid sequence has been deleted, substituted or added, and functions as a monooxygenase P450 Polypeptide The production method according to claim 1 , wherein CYP153 is the following (o) or (p).
(o) a polypeptide comprising the amino acid sequence of SEQ ID NO: 12
(p) a polypeptide comprising an amino acid sequence in which one amino acid sequence is deleted, substituted or added in the amino acid sequence of SEQ ID NO: 12, and which functions as monooxygenase P450 The manufacturing method according to any one of claims 1 to 3 , wherein the aromatic compound having a substituent has 1 to 3 benzene rings. The production method according to claim 4, wherein the aromatic compound having 1 to 3 benzene rings and having a substituent is an aromatic compound bonded via an oxygen atom. The process according to any one of claims 1-5 for introducing a hydroxyl group to a carbon atom of an aromatic ring of an aromatic compound having a substituent. The manufacturing method according to any one of claims 1 to 6 , wherein the substituent of the aromatic compound having a substituent is an alkyl group having 1 to 4 carbon atoms, and a hydroxyl group is introduced into a carbon atom of the alkyl group. The production method according to any one of claims 1 to 7 , wherein the substituent of the aromatic compound having a substituent is an alkoxy group having 1 to 2 carbon atoms, and a hydroxyl group is introduced by dealkylation of the alkoxy group. By coexisting a transformant introduced with a gene encoding a protein belonging to CYP153 or a fusion protein containing the same and an aromatic compound having a substituent, the protein belonging to CYP153 or a fusion protein containing the same is obtained. The manufacturing method in any one of Claims 1-8 made to act on the aromatic compound which has a substituent. The production method according to claim 9 , wherein the transformant has a gene encoding a fusion protein of CYP153 and a redox partner protein. A transformant is a gene encoding a fusion protein of CYP153 and a reductase peptide contained in cytochrome P450 monooxygenase P450RhF derived from Rhodococcus sp. The production method according to claim 10 , wherein a recombinant vector inserted into a pUC vector so as to utilize a translation signal of lac promoter and LacZ is introduced. The production method according to claim 10 or 11 , wherein the redox partner protein is the following peptide (c) or (d).
(c) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 2
(d) a peptide comprising an amino acid sequence in which one amino acid is added, deleted or substituted in the amino acid sequence described in SEQ ID NO: 2 and having reductase activity The recombinant vector contains the following DNA (g) or (h) encoding a reductase peptide via a linker on the 3 ′ end side of the following (e) or (f) DNA encoding CYP153. The production method according to claim 11 or 12 , which is a recombinant vector in which the linked fusion protein expression cassette is inserted into a pUC vector so as to utilize the translation signal of lac promoter and LacZ.
(e) DNA consisting of the nucleotide sequence of SEQ ID NO: 3
(f) DNA that hybridizes with the DNA of SEQ ID NO: 3 under stringent conditions and encodes a protein having monooxygenase activity
(g) DNA consisting of the nucleotide sequence of SEQ ID NO: 4
(h) a DNA that hybridizes with the DNA of SEQ ID NO: 4 under a stringent condition and encodes a protein having reductase activity The recombinant vector contains the following (g) or (h) DNA encoding a reductase peptide via a linker on the 3 ′ end side of the following (w) or (x) DNA encoding CYP153. The production method according to claim 11 or 12 , which is a recombinant vector in which the linked fusion protein expression cassette is inserted into a pET vector so as to receive transcription of the T7 promoter.
(w) DNA consisting of the base sequence of SEQ ID NO: 16
(x) DNA that hybridizes with the DNA of SEQ ID NO: 16 under a stringent condition and encodes a protein having monooxygenase activity
(g) DNA consisting of the nucleotide sequence of SEQ ID NO: 4
(h) a DNA that hybridizes with the DNA of SEQ ID NO: 4 under a stringent condition and encodes a protein having reductase activity

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