KR20060008195A - Cytochrome P450 Enzyme and Genes Encoding It - Google Patents

Abstract

The present invention is a cytochrome P450 enzyme (CYP105P2) having a function of introducing a hydroxyl group to an aromatic ring compound, a gene encoding the enzyme, a recombinant vector containing the gene, transformed with the recombinant vector The present invention relates to a method for introducing a hydroxy group into an aromatic ring compound characterized by using the microorganism and the enzyme or the transformed microorganism.

The CYP105P2 enzyme according to the present invention has a function of introducing a hydroxy group into an aromatic ring compound and particularly introducing a pentahydroxy group into a benzene ring compound, which is useful for preparing an aromatic compound into which a hydroxy group is introduced.

Cytochrome P450, 7-ethoxycoumarin, cis-5,6,7,8,9-pentahydroxycinnamic acid, cinnamic acid, cis-5,6,7,8,9-pentahydroxycinnamic acid

Description

Cytochrome P450 Enzyme and the Gene Encoding the Same}             

1 is a schematic diagram of cloning a 5.5 kb conserved operon region of S. peucetius and S. avermitilis and CYP105P2 of S. peucetius into a pET32a vector.

Figure 2 compares the amino acid sequence of CYP105D6 (PikC) of CYP105P1 (PetD) and S. venezulae of CYP105P2, S. avermitilis in S. peucetius. Gray boxes represent unique sequences for each species, and black boxes represent conserved amino acid sequences.

Figure 3 is a SDS-PAGE picture of CYP105P2 mass-expressed in E. coli. Lane 1 is cell lysate of Escherichia coli BL21 (DE3) / pNP105P2, and lane 2 is molecular weight marker.

Figure 4 shows the conversion mechanism of the substrate by CYP105P2 (7-ethoxycoumarin → 7-hydroxycoumarin → cis-5,6,7,8,9-pentahydroxycinnamic acid) according to the present invention.

5 is an HPLC result showing that 7-ethoxycoumarin is converted to cis-5,6,7,8,9-pentahydroxycinnamic acid by CYP105P2 according to the present invention.

6 is an HPLC result showing that cinnamic acid is converted to cis-5,6,7,8,9-pentahydroxycinnamic acid by CYP105P2 according to the present invention.

7 is an HPLC result showing that salicylic acid is converted to pentahydroxybenzoic acid by CYP105P2 according to the present invention.

8 is an HPLC result showing that benzoic acid is converted to pentahydroxybenzoic acid by CYP105P2 according to the present invention.

9 shows the substrate binding of CYP105P2 and the secretion activity of the reaction product.

10 is a result of ¹ H-NMR analysis of pentahydroxycinnamic acid.

11 is a result of DEPT-NMR analysis of pentahydroxycinnamic acid.

12 is a result of ¹³ C-NMR analysis of pentahydroxycinnamic acid.

Fig. 13 shows the results of FAB-MASS analysis of pentahydroxycinnamic acid.

Fig. 14 is a schematic diagram showing the conversion mechanism of the substrate by CYP105P2.

The present invention provides a cytochrome P450 enzyme (CYP105P2) having a function of introducing a hydroxy group into an aromatic ring compound, a gene encoding the enzyme, a recombinant vector containing the gene, and the recombinant The present invention relates to a method for introducing a hydroxyl group to an aromatic ring compound characterized by using the microorganism transformed with the vector, the enzyme or the transformed microorganism.

Cytochrome P450 (CYP) is a protein family found in protozoa, plants, animals, fungi and microorganisms, and CYPs have been found in 45 species of Streptomyces. In contrast to eukaryotic CYP attached to cell membranes, CYP in Streptomyces is expressed in aqueous solution, and only a few of them are known. CYP is not essential for the basic metabolism of most prokaryotic bacteria and is involved in the breakdown of carbohydrates, terpenes, etc., providing a source of carbon or energy to bacteria (Omura, T., Biochem. Biophys. Res. Commun ., 266: 690, 1999).

In addition, it is involved in the oxidation, peroxidation and reduction of various products produced intracellularly, such as steroids, bile acids, fatty acids, prostaglandins, leukotrienes, biogenic amines and secondary metabolites.

The main role of CYP is to single oxidize several substrates. This reaction requires oxygen molecules and NADPH or NADH, and most bacterial CYPs get the electrons they need from NADH. They bind oxygen atoms to allyl positions, double bonds or inactivated CH bonds. CYP encodes an enzyme comprising heme-thiorate and is primarily located in the biosynthetic gene cluster of macrolide antibiotics, catalyzing the stereo- and site-specific oxidation of the precursor resulting in structural diversity of the macrolide. (Lamb, DC et al ., Biochem. Biophys. Res. Commun ., 307: 610, 2003).

As an example, CYP107A1 from Streptomyces erythraea is responsible for the hydroxylation of 6-deoxyerythronolide B to erythronolide B in the biosynthesis of erythromycin. (Weber, JM et al. , Science , 252: 114, 1991).

Most of the Streptomyces genus CYP450 produces 7-hydroxycoumarin or 6,7-dihydroxycoumarin from 7-ethoxycoumarin and benzopyrene There have been reports of oxidizing (benzo (α) pyrene). However, no bacterial P450 has been reported that adds two or more hydroxy groups to the benzene ring.

Thus, the present inventors found a 5.5kb gene cluster in S. peucetius , which has similarities with the gene sites responsible for the biosynthetic pathway of the antibiotics filipin and polyene in S. avermitilis , After identifying the gene encoding the CYP450 in the cluster, it was confirmed that the protein encoded by the gene has a function of introducing a hydroxyl group to the aromatic ring compound to complete the present invention.

An object of the present invention is to provide a CYP105P2 enzyme and its gene having a function of introducing a hydroxyl group into an aromatic ring compound.

Another object of the present invention is to provide a recombinant vector comprising the gene and a microorganism transformed with the recombinant vector. Still another object of the present invention is to provide a method for introducing a hydroxyl group to an aromatic ring compound characterized by using the enzyme or the transformed microorganism.

In order to achieve the above object, the present invention provides a CYP105P2 enzyme having an amino acid sequence of SEQ ID NO: 2. The enzyme may be characterized by having a function of introducing a hydroxy group into the aromatic ring compound, in particular, may be characterized by having a function of introducing a pentahydroxy group into the benzene ring.

The present invention also provides a gene encoding the CYP105P2 enzyme. The CYP105P2 gene may be characterized by having a nucleotide sequence of SEQ ID NO: 1, and may be characterized by being derived from Streptomyces peucetius .

The present invention also provides a recombinant vector comprising the gene and a microorganism transformed with the recombinant vector.

The present invention also provides a method for producing the CYP105P2 enzyme, which is characterized by culturing the transformed microorganism.

The present invention also provides a method for introducing a hydroxyl group to an aromatic ring compound, characterized in that using the CYP105P2 enzyme, the transformed microorganism or its lysate.

In the present invention, the aromatic ring compound is 7-hydroxycoumarin (7-hydroxycoumarin), 7-ethoxycoumarin (7-ethoxycoumarin) or cinnamic acid (cinnamic acid), obtained by introducing a hydroxyl group to the aromatic ring compound The compound may be cis-5,6,7,8,9-pentahydroxycinnamic acid (cis-5,6,7,8,9-pentahydroxycinnamic acid).

In the present invention, the aromatic ring compound is a benzene ring compound, it may be characterized in that pentahydroxy groups (pentahydroxy groups) are introduced into the benzene ring compound, the benzene ring compound is benzoic acid (benzoic acid) or salicylic acid, and the compound obtained by introducing a pentahydroxy group into the benzene ring compound may be characterized as pentahydroxybenzoic acid.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and the scope of the present invention is not to be construed as being limited by these examples.

Example 1 Construction of Cosmid Library

S. peucetius ATCC 27952 was inoculated in TSB medium and incubated at 28 ° C. for 2 days. The cells were collected by centrifugation, and chromosomal DNA was isolated by Sambrook's method. The isolated chromosomal DNA of S. peucetius was cut and fragmented with restriction enzyme Sau 3AI, and a gene library of chromosomal DNA was constructed using pSuperCos1 vector (Stratagene, USA).

Recombinant DNA was packaged in vitro using the Gigapack III XL packaging extract (Stratagene Inc., USA). Chromosomal DNA was identified by screening with dnrF of SEQ ID NOs: 3 and 4 and dpsY of SEQ ID NOs: 5 and 6 to identify the cosmid comprising the sequence.

SEQ ID NO: 5'-AGG TTT GAG GTG GCC TTG ACG-3 '

SEQ ID NO: 5'-TCC GCG TCA GTT CGC CGG AGG-3 '

SEQ ID NO: 5'-GGA CTG CCG GTG TGC TGT GGT-3 '

SEQ ID NO: 5'CCG GAA CGT TCA TTC GTC GAC-3 '

Example 2. S. peucetius  Sequence sequencing and analysis of the genome

In order to determine the nucleotide sequence of the cosmid constructed in Example 1, S. peucetius chromosome DNA was first sonicated to construct a shotgun library. The nucleotide sequence of the whole genome of S. peucetius was determined using various cosmids constructed in Example 1 and 2-4 kb genome fragments prepared by the shotgun method. All non-overlapping fragments were organized using PHRED (Ewing, B. and Green, P., Genome Res., 8: 186, 1998) and PHRAP (http://www.phrap.org).

The entire genome sequence was interpreted using GenBank's BLAST search to identify the most similar sequences in other organisms using NCBI's recent non-redundant protein database.

Example 3 Identification and Analysis of CYPs

The genome database of S. peucetius was first built by the inventors (http://203.247.223.80). Cytochrome P450 was first screened using GXXXCXG, a marker of heme binding domain. Open reading frames (ORFs) of all CYPs were obtained using Glimmer 2.0 (http://www.tigr.org/software/glimmer/) and interpreted using BLAST. The ORF with the GXXXCXG motif was secondary screened with ORF with the highly conserved threonine at the site expected to be I-helix, and with the EXXR motif conserved in K-helix. A search was performed using a gene containing all three motifs as a query for BLAST search. The amino acid sequence of each gene was predicted using the GeneDoc program.

The CYP DNA sequence (CYP105P2) of S. peucetius screened by the above method was registered under accession number AJ605540 in the EMBL GenBank nucleotide sequence database (SEQ ID NO: 1).

The nucleotide sequence of CYP105P2 of the present invention clusters with modular PKS and dehydrogenase in S. avermitilis , and has 89% homology with CYP105P1 (PteC), which is involved in biosynthesis of antifungal polyene and filipin. (FIG. 1). In S. peucetius , the same PKS and dehydrogenase homologues were also arranged with CYP105P2.

CYP105P2 has an ATG start codon and a TGA stop codon and is located in the forward position with the ribosome-attachment site, with a clear I-helix ( ²³⁷ AAHDT ²⁴¹ ), K-helix ( ²⁷⁶ ELLR ²⁷⁹ ) and heme-attached motifs ( ^341). FGFGAHQCIG ³⁵⁰ ) and it can be seen that it is cytochrome P450 (FIG. 2). The amino acid sequence converted by the nucleotide sequence of CYP105P2 is shown in SEQ ID NO: 2.

Example 4 Construction and Transformation of Recombinant Plasmids

The CYP105P2 gene was amplified by PCR in the genome of S. peucetius using a primer of SEQ ID NO: 7 designed to contain an Nde I cleavage site and a primer of SEQ ID NO: 8 designed to include a Kpn I cleavage site. PCR conditions used were denatured at 94 ° C., annealed at 65 ° C., extended at 74 ° C., 2.5 units of Taq-DNA polymerase, 10% dimethylsulfoxide, 2.5 pmol of the primer, 0.2 mM dNTP and 0.1 μg of genome DNA was dissolved in titration buffer and used at 50 μl.

SEQ ID NO: 7' -AGA CATATG TCCCAGCCCACCG-3

SEQ ID NO: 5-TCA GGTACC AAGGAGCACCGTCGG-3

As a result of PCR, 1200 bp CYP105P2 was amplified, and the amplified CYP105P2 (1200 bp) was cloned into the Nde I and Kpn I sites of the pET32a expression vector (Novagen, USA) to prepare plasmid pNP105P2. The prepared pNP105P2 was introduced into E. coli BL21 (DE3) to obtain transformed E. coli BL21 (DE3) / pNP105P2.

Example 5 Expression of CYP105P2 in Escherichia Coli

Recombinant CYP105P2 expressed by Escherichia coli BL21 (DE3) / pNP105P2 was expressed in water-soluble form, and mass expression was induced by 0.4 mM IPTG. 3 ml LB medium containing 3 μl / ml ampicillin was inoculated with E. coli BL21 (DE3) transformed with plasmid pNP105P2 and incubated overnight. Transfer the culture medium to 50 ml of new LB medium containing 50 μg of ampicillin and incubate at 37 ° C. at 600 nm until the OD value is 0.6. Then, 0.4 mM of IPTG is added and incubated at 20 ° C. for 37 hours to obtain CYP105P2. Expression was induced. Cells were obtained by centrifugation, and then washed twice with cooled 50 mM Tris / HCl (pH 7.5) solution. Cells were disrupted by ultrasonication, centrifuged, and a supernatant sample was taken and electrophoresed. Electrophoresis using a standard molecular weight marker (Novagen Co., USA) revealed that the expressed CYO105P2 had a molecular weight of about 44 kDa (FIG. 3).

Example 6. Determination of Activity of CYP105P2 Enzyme

To measure the activity of cytochrome P450 (CYP105P2), 100 μl of reaction solution [5 mM Tris / HCl (pH7.5), 1 mM substrate (7-ethoxycoumarin or 7-hydroxycoumarin), 1.25 mM NADH And 30 μl of bacterial extract and 50 μl of distilled water] were reacted at 37 ° C. for 24 hours. Thereafter, 1 ml of ethyl acetate was added to stop the reaction, followed by centrifugation at 4 DEG C and 33,172 x g for 20 minutes to remove the supernatant, and the pellet was dissolved in DMSO.

Similarly, in order to observe the biochemical conversion in the in vivo system, E. coli BL21 (DE3) / pNP105P2 was incubated in the same manner as in Example 3 to induce the expression of CYP105P2, after 10 minutes of 1 mM in 50 ml culture Substrates were added and then incubated at 20 ° C. for 48 hours. After the reaction was stopped by adding 1 ml of ethyl acetate to 100 µl of the culture supernatant, the above treatment was performed.

In order to separate the reaction product from the reaction solution, a column packed with silica gel 60 (70-230 mesh ASTM) was used. The column chromatography was able to obtain several fractions using hexane and ethyl acetate as the developing solvent, TLC was performed using a 3: 2 / hexane: ethyl acetate solvent as a mobile phase. In addition, reverse phase HPLC was performed using a Mightysil, RP-18 column to analyze the biochemical conversion of the substrate at 254 nm. The substrate and the product were separated using a methanol: acetonitrile: water (3.5: 3: 3.5) solvent as a mobile phase at a flow rate of 1 ml / min.

HPLC analysis showed that the same reaction product cis-5,6,7,8,9-pentahydroxycinnamic acid was produced from 7-ethoxycoumarin and 7-hydroxycoumarin in the in vitro system (FIGS. 4 & 5). ). Many documents have reported deethylation of 7-ethoxycoumarin, but no 7-hydroxycoumarin was found in the in vivo assay using 7-ethoxycoumarin as a substrate (Sarialani FS et al ., Appl. Environ.Micrbiol , 46: 468, 1983). However, in vitro analysis showed the formation of 7-hydroxycoumarin by deethylation.

On the other hand, the two substrates (7-ethoxycoumarin and 7-hydroxycoumarin) was found to bind effectively with the enzyme when reacted for more than 4 hours at 37 ℃. Therefore, no pronounced reaction product formation was observed within 4 hours, and the substrate was completely converted when reacted for 24 hours. In addition, NADH played an important role in CYP105P2 enzyme activity. In other words, NADH was essential for CYP105P2 to completely convert lipids.

The same reaction was carried out using cinnamic acid as a substrate, resulting in three different products, the main of which was cis-5,6,7,8,9- the same as the reaction product of 7-ethoxycoumarin. It was pentahydroxycinnamic acid (FIG. 6).

In addition, when the same reaction was performed using benzoic acid and salicylic acid as a substrate, it was confirmed that pentahydroxybenzoic acid is produced (FIGS. 7 and 8).

Finally, cytochrome P450 (CYP105P2) according to the present invention converts 7-ethoxycoumarin, 7-hydroxycoumarin and cinnamic acid to cis-5,6,7,8,9-pentahydroxycinnamic acid, It was confirmed that it has the activity of converting benzoic acid and salicylic acid into pentahydroxybenzoic acid.

Example 7. Substrate Binding Spectrum of CYP105P2 Enzyme

Cytochrome P450 contained in bacterial, cytoplasmic and membrane fractions was quantified by Omura's method using extinction coefficient (91 mM / cm) using a spectrophotometer (Omura, T. et al ., Biochem. Biophys. Res. Commun ., 266: 690, 1999). Substrate binding spectra were obtained by splitting CYP105P2 expressed in the cytoplasm into a sample cuvette and measuring it by the split cell method. Both the control cuvettes and the sample cuvettes were measured solution [glycerol: 50 mM Tris / HCl (pH 7.5) = 1: 8, 0.5 mM to 5 mM substrate (7-ethoxycoumarin, erythromycin, orolemycin) and Chloramphenicol)]. 100 μl of CYP105P2 cell lysate was added to the sample cuvette and the spectra were measured at 350 nm and 500 nm. The binding spectrum can be used to analyze the role of CYP105P2 in drug metabolism.

Enzyme determination using Orlandomycin and erythromycin was performed using 100 μl of reaction solution [200 mM Tris / HCl (pH7.5), 5 mM substrate, 1.25 mM NADH, 5 μl, perredoxin, 5 μl of peredoxin reductase and 30 μl CYP105P2] was used.

Biological conversion of the substrate was measured by TLC and LC-Mass. TLC was developed with EtOAC: MeOH: Ammonia (1: 2: 0.1) solution.

As a result, 2.5 mM Orlandomycin and chloramphenicol were metabolized by 138 μg / ml CYP105P2 (FIG. 9). 2.7 mM 7-ethoxycoumarin and 1.5 mM erythromycin were also metabolized by the same concentration (138 μg / ml) of CYP105P2.

Example 8. Structural Analysis of Reaction Products

NMR and mass spectroscopy were performed to elucidate the structures of cis-5,6,7,8,9-pentahydroxycinnamic acid and pentahydroxybenzoic acid. In addition, high resolution FAB-MASS (fast atom bonbarment mass spectroscopy) was performed, and IR spectra (Bio-Rad model FT3000MX FT-IR) were obtained from pure samples of KBr disks. All NMR data was recorded as DMSO-d ₆ . The ¹ H-NMR of the sample in DMSO-d ₆ was ppm5.5 (d, 1H-H2), 7.4 (d, 1H, H-3) (FIG. 10), indicating the presence of two carbon atoms and two protons. Indicates. In addition, the presence of two hydrogen atoms was confirmed by DEPT-NMR (FIG. 11). ¹³ C-NMR in DMSO-d ₆ confirmed the number of carbon atoms and their positions [C (1) 165.2 ppm, C (2) 100.9 ppm, C (3) 142.9 ppm, C (5) (9) 138.4 ppm, C (7) 129.1 ppm, C (6) (8) 126.7 ppm] (FIG. 12). The results indicate that nine carbon atoms are present in the sample. FAB-MASS spectrum showed the fragmentation pattern of the reaction product component as shown in FIG. In the FT-IR spectrum, C = C double bonds (1415 cm ^-1 ), phenolic hydroxy groups, carboxyl groups (2955-3554 cm ^-1 wide band) and carbonyl groups (1563 cm ^-1 ) were identified. .

As a result, it was confirmed that the substance produced by the conversion of 7-ethoxycoumarin and 7-hydroxycoumarin by CYP105P2 was cis-5,6,7,8,9-pentahydroxycinnamic acid. That is, as shown in Figure 14, it was confirmed that 7-ethoxycoumarin is finally converted to cis-5,6,7,8,9-pentahydroxycinnamic acid via 7-hydroxycoumarin.

Having described the specific part of the present invention in detail, it is obvious to those skilled in the art that such a specific description is only a preferred embodiment, thereby not limiting the scope of the present invention. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

The present invention provides a cytochrome P450 enzyme (CYP105P2) having a function of introducing a hydroxyl group into an aromatic ring compound, a gene encoding the enzyme, a recombinant vector containing the gene, a microorganism transformed with the recombinant vector, the enzyme Or there is an effect of providing a method for introducing a hydroxyl group to the aromatic ring compound, characterized in that using the transformed microorganism.

The CYP105P2 enzyme according to the present invention converts 7-ethoxycoumarins, 7-hydroxycoumarins and gentic acid to cis-5,6,7,8,9-pentahydroxycinnamic acid, or benzoic acid and salicylic acid to pentahydride. It is useful to convert to oxybenzoic acid or to introduce hydroxy groups into other aromatic ring compounds. In addition, through the modification of all or part of the gene sequence provided in the present invention, it is possible to increase productivity of existing Philippines, polyene, or the like, or to prepare variants of the Philippines, polyene, and the like.

<110> GENECHEM INC. <120> Cytochrome P450 Enzyme and The Gene Encoding the Same <130> P04-155 <150> KR10-2004-0057844 <151> 2004-07-23 <160> 8 <170> KopatentIn 1.71 <210> 1 <211> 1200 <212> DNA <213> Streptomyces peucetius <400> 1 atgtcccagc ccaccgccgg cgcgcccgcc gcacccaagg cccgcagctg cccgttcctg 60 ccgccggacg gcattgccga catccgggcg gccgcaccgg tgacccgggc caccttcacc 120 agcggccacg aggcgtggct ggtgaccggc tacgagcagg tgcgcgccgt gctgcgcgac 180 ccgtcgttca gcgtcggtgt accccacgcg ctgcacacac aggacggcgt cgtcacgcag 240 aagcccggcc gcggcagcct gctgtggcag gacgccccgg agcacaccga cgaccgcaag 300 ctgctcgcca aagagttcac cgtccggcgc atgcaggcgc tccgtccgaa catccagcgc 360 atcgtcgacg aacacctcga cgccatcgag gcccggggcg ggccggtcga cctggtgaag 420 accttcgcca accctgtgcc gtccatggtg atctccgacc tcttcggcgt cccggccgaa 480 cggcgggcgg agttccagga gatcgccgag gcgatgatgc gggtcgacca ggacgccgcc 540 gccacggagg ccgccggcat gcggctcggc ggactgctct accagctcgt ccaggagcgc 600 cgggccaacc ccggcgacga tctgatctcg gcgctgatca ccaccgagga ccccgacggc 660 gtcatcgacg acatgttcct catgaacgcg gccggaaccc tgctgatcgc agcccacgac 720 accaccgcct gcatgatcgg cctcggtacg gcgctgttgc tcgaccgccc cgaccagctg 780 gccctgctcc aaaaggaccc gtcactgatc ggcaacgcgg tcgaggagct gctgcgctac 840 ctcaccatcg gccagttcgg cgccgagcgc gtcgccaccc aggacgggga gatcggcggc 900 gtgcgcatcg ccaagggcga gcaggtcgtc acgcacctcc tgtccgccga cttcgacccc 960 gccttcgtgg aggacccgga gcgcttcgac atcacccgcc gccccgcgcc gcacctggcc 1020 ttcggcttcg gcgcacacca gtgcataggc cagcaactcg cccggatcga gctccagatc 1080 gtcttcggga ccctcttccg gcgcttcccg accctgcgcc tggccaagcc cgtggaggaa 1140 ctccgcttcc gcaacgacat ggtcttctac ggcgtgcacg agctgcccgt cacctggtga 1200                                                                         1200 <210> 2 <211> 399 <212> PRT <213> Streptomyces peucetius <400> 2 Met Ser Gln Pro Thr Ala Gly Ala Pro Ala Ala Pro Lys Ala Arg Ser   1 5 10 15 Cys Pro Phe Leu Pro Pro Asp Gly Ile Ala Asp Ile Arg Ala Ala Ala              20 25 30 Pro Val Thr Arg Ala Thr Phe Thr Ser Gly His Glu Ala Trp Leu Val          35 40 45 Thr Gly Tyr Glu Gln Val Arg Ala Val Leu Arg Asp Pro Ser Phe Ser      50 55 60 Val Gly Val Pro His Ala Leu His Thr Gln Asp Gly Val Val Thr Gln  65 70 75 80 Lys Pro Gly Arg Gly Ser Leu Leu Trp Gln Asp Ala Pro Glu His Thr                  85 90 95 Asp Asp Arg Lys Leu Leu Ala Lys Glu Phe Thr Val Arg Arg Met Gln             100 105 110 Ala Leu Arg Pro Asn Ile Gln Arg Ile Val Asp Glu His Leu Asp Ala         115 120 125 Ile Glu Ala Arg Gly Gly Pro Val Asp Leu Val Lys Thr Phe Ala Asn     130 135 140 Pro Val Pro Ser Met Val Ile Ser Asp Leu Phe Gly Val Pro Ala Glu 145 150 155 160 Arg Arg Ala Glu Phe Gln Glu Ile Ala Glu Ala Met Met Arg Val Asp                 165 170 175 Gln Asp Ala Ala Ala Thr Glu Ala Ala Gly Met Arg Leu Gly Gly Leu             180 185 190 Leu Tyr Gln Leu Val Gln Glu Arg Arg Ala Asn Pro Gly Asp Asp Leu         195 200 205 Ile Ser Ala Leu Ile Thr Thr Glu Asp Pro Asp Gly Val Ile Asp Asp     210 215 220 Met Phe Leu Met Asn Ala Ala Gly Thr Leu Leu Ile Ala Ala His Asp 225 230 235 240 Thr Thr Ala Cys Met Ile Gly Leu Gly Thr Ala Leu Leu Leu Asp Arg                 245 250 255 Pro Asp Gln Leu Ala Leu Leu Gln Lys Asp Pro Ser Leu Ile Gly Asn             260 265 270 Ala Val Glu Glu Leu Leu Arg Tyr Leu Thr Ile Gly Gln Phe Gly Ala         275 280 285 Glu Arg Val Ala Thr Gln Asp Gly Glu Ile Gly Gly Val Arg Ile Ala     290 295 300 Lys Gly Glu Gln Val Val Thr His Leu Leu Ser Ala Asp Phe Asp Pro 305 310 315 320 Ala Phe Val Glu Asp Pro Glu Arg Phe Asp Ile Thr Arg Arg Pro Ala                 325 330 335 Pro His Leu Ala Phe Gly Phe Gly Ala His Gln Cys Ile Gly Gln Gln             340 345 350 Leu Ala Arg Ile Glu Leu Gln Ile Val Phe Gly Thr Leu Phe Arg Arg         355 360 365 Phe Pro Thr Leu Arg Leu Ala Lys Pro Val Glu Glu Leu Arg Phe Arg     370 375 380 Asn Asp Met Val Phe Tyr Gly Val His Glu Leu Pro Val Thr Trp 385 390 395 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Probe <400> 3 aggtttgagg tggccttgac g 21 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Probe <400> 4 tccgcgtcag ttcgccggag g 21 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Probe <400> 5 ggactgccgg tgtgctgtgg t 21 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Probe <400> 6 ccggaacgtt cattcgtcga c 21 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 7 agacatatgt cccagcccac cg 22 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 8 tcaggtacca aggagcaccg tcgg 24  

Claims (15)

CYP105P2 enzyme having the amino acid sequence of SEQ ID NO. The CYP105P2 enzyme according to claim 1, which has a function of introducing a hydroxyl group into the aromatic ring compound. 3. The CYP105P2 enzyme according to claim 2, which has a function of introducing a pentahydroxy group into the benzene ring. The gene encoding the CYP105P2 enzyme of claim 1. The gene according to claim 4, which has a nucleotide sequence of SEQ ID NO: 1. The method of claim 4, characterized in that derived from Streptomyces peucetius gene. Recombinant vector containing the gene of claim 4. A microorganism transformed with the recombinant vector of claim 7. The transformed microorganism according to claim 8, wherein the microorganism is Escherichia coli. A method for producing the CYP105P2 enzyme according to claim 1, wherein the microorganism of claim 8 is cultured. A method of introducing a hydroxy group into an aromatic ring compound using the CYP105P2 enzyme of claim 1, the transformed microorganism of claim 8, or a lysate thereof. The compound according to claim 11, wherein the aromatic ring compound is 7-hydroxycoumarin, 7-ethoxycoumarin or cinnamic acid, and the compound obtained by introducing a hydroxy group into the aromatic ring compound is cis-5,6,7,8, 9-pentahydroxycinnamic acid. 12. The method of claim 11, wherein the aromatic ring compound is a benzene ring compound and a pentahydroxy group is introduced into the benzene ring compound. The method of claim 13, wherein the benzene ring compound is benzoic acid or salicylic acid, and the compound obtained by introducing a pentahydroxy group into the benzene ring compound is pentahydroxybenzoic acid. 12. The method of claim 11, wherein NADH is added.

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