JP5781325B2 - Method for producing 16α-hydroxylated steroid compound - Google Patents

Description

  The present invention relates to a method for producing a steroid compound in which the 16α-position is hydroxylated using an enzyme.

Cytochrome P450 (hereinafter referred to as “P450”) is widely conserved in all eukaryotes and all species except some prokaryotes such as E. coli. P450 is the largest superfamily of enzymes in which 12,456 P450 genes have been identified so far (Non-patent Document 1). P450 was discovered in 1958 by Martin Klingenberg and David Garfinkel as a chromoprotein showing a cytochrome-like carbon monoxide binding spectrum from rat and porcine liver microsomal fractions (Non-patent Documents 2 and 3). Later, in 1962, Omura and Sato et al. Reported that when the pigment was solubilized from the liver microsome fraction, a new cytochrome different from cytochrome b ₅ showing an absorption maximum near 420 nm was obtained. In this report, the carbon monoxide bond type was named P-450, and the unbonded state was named P-420 (Non-patent Document 4). However, in 1963, the following year, the group of Estabrook et al. Discovered that the above-mentioned chromoprotein considered to be cytochrome performs hydroxylation of C21 of 17-Hydroxyprogesterone (Non-patent Document 5). As a result, P450 was found to be a monooxygenase that adds a single oxygen atom to the substrate. However, since the name "cytochrome" has already been generalized, it is still called cytochrome P450 (usually abbreviated as P450 or CYP). Thereafter, the mechanism of oxygen activation by P450 was presented by the group of Omura et al., And research on P450 as a monooxygenase began (Non-Patent Document 6).

P450 is composed of about 500 amino acid residues in eukaryotes and about 400 amino acids in prokaryotes. Since P450 is a heme protein having one protoheme molecule at the active center, both eukaryotic and prokaryotic organisms exhibit a red color. The intracellular localization of P450 is different in eukaryotes and prokaryotes, with the former localized in the membrane fraction and the latter in the cytoplasmic fraction. All the three-dimensional structures of P450 hold a common prism shape with a side of about 60 mm and a thickness of about 30 mm. The primary structure has several common regions, including Phe-XX-Gly-X-Arg / His-X-Cys-X-Gly (each about 50-60 amino acid residues before the carboxyl terminus). The amino acid sequence of the heme-binding region of SEQ ID NOs: 12 and 13) is a sequence conserved in all P450s, and is used for identifying the P450 gene from genomic information. From these commonality, P450 is considered to be derived from the same ancestor (Non-patent Document 7). The classification of P450 is based on the similarity of the amino acid sequence, not the function, because the catalytic function is diverse in terms of the substrate and catalytic reactions such as secondary metabolites, biosynthesis of steroid hormones, foreign body metabolism, and hydrocarbon utilization. being classified. As a classification method, CYP indicating cytochrome P450 is added to the prefix, followed by classification in the order of family number, subfamily number, and gene number. In principle, when the amino acid sequences match 40% or more, they are classified into the same family, and when they match 55% or more, they are classified into the same subfamily. Gene numbers are assigned in the order of discovery. Named new P450s will be posted on Dr. Nelson's website (http://drnelson.uthsc.edu/CytochromeP450.html). Thus, P450s are classified into families and subfamilies based on amino acid sequence homology, but P450s that are classified into the same subfamily rarely have the same substrate specificity. This is easily predicted because the homology criterion for amino acid sequences classified into the same subfamily is as low as 55%, and it is not possible to predict P450 substrates or P450 conversion products with such classification alone. Have difficulty.

P450 catalyzes the hydroxylation of CH bonds of chemically very stable compounds such as hydrocarbons and aromatic compounds. However, the catalytic reaction of P450 is not limited to the hydroxylation reaction. Catalytic reactions of P450 currently known include epoxidation, O-demethylation, sulfoxidation, oxidative allyl transfer (Non-Patent Document 8), and oxidative Baeyer-Villiger reaction (Non-Patent Document) in addition to hydroxylation. It catalyzes various reactions such as 9) (Non-patent Document 9). Furthermore, the size of the compound as a substrate can be catalyzed from small molecules such as 2-bromophenol (M _r 173) to very large compounds such as Cyclosporin A (M _r 1202), The wide range of substrate adaptability is characteristic.

In order for P450 to function as a monooxygenase, an electron transfer protein that normally transfers electrons derived from NAD (P) H to P450 is required. P450 derived from bacteria such as actinomycetes is usually ferredoxin reductase (ferredoxin reductase) and ferredoxin (Fe-S small protein) (collectively these two proteins are electron transfer proteins) Or a redox partner protein) (Non-patent Document 10). Even when conducting functional analysis of P450, electron transfer proteins are required, but because P450 has a strict compatibility with these electron transfer proteins and P450 itself is an unstable enzyme, Even when a new P450 gene was obtained, it was often difficult to express its function.

The analysis of the catalytic function of P450 requires a complicated reaction system as described above, and the functional analysis is often difficult (Non-patent Document 11). As a countermeasure, functional expression systems for baker's yeast ( Saccharomyces cerevisiae ) are widely used in functional analysis studies of P450 (especially P450 derived from eukaryotes). The functional expression system of baker's yeast performs a substrate catalytic reaction test using NADPH-P450 reductase (which corresponds to an electron transfer protein) originally present in yeast. Yeast P450 reductase has a relatively high adaptability to P450 (referred to as P450 belonging to class II) present in eukaryotic microsomes (Non-patent Document 11), so electron transfer proteins are not examined. In many cases, results can be obtained. However, there are some problems in the yeast function expression system. The first problem is that baker's yeast possesses three types of P450s, CYP51, CYP56, and CYP61, and it is necessary to consider the effects of these P450s. Therefore, when introducing a P450 gene of a heterologous organism and conducting functional expression analysis, it is necessary to verify the presence or absence of the effect. The second problem is that P450 derived from prokaryotes and P450 functioning in eukaryotic mitochondria (referred to as P450 belonging to class I) (Non-patent Document 11) and NADPH-P450 reductase present in yeast. There is a compatibility problem. Although yeast NADPH-P450 reductase is highly adaptable to P450 belonging to class II, there are many problems with compatibility with P450 belonging to class I.

On the other hand, in the case of a functional expression system of the P450 gene using Escherichia coli , there are many advantages of the E. coli functional analysis system because it does not have P450 in addition to simple genetic manipulation such as transformation. However, the E. coli function analysis system has a problem in selecting an electron transfer protein to P450. Since E. coli does not possess P450, it usually does not also possess an electron transfer protein corresponding to P450. Therefore, a highly versatile electron transfer protein that can substitute for an electron transfer protein consisting of NAD (P) H-iron sulfur protein reductase (ferredoxin reductase) and iron sulfur protein (ferredoxin), which is often found in bacteria, has been demanded. (Non-Patent Document 11).

P450RhF (CYP116) derived from Rhodococcus genus NCIMB 9784 is a reductase domain (reductase domain, reductase peptide, electron) consisting of ferredoxin reductase (FMN as coenzyme) and ferredoxin (Fe-S small protein) It is a fusion protein in which the transfer protein part (C-terminal side) is linked to the P450 body protein (N-terminal side) via a linker sequence, and it has a rare structure different from general P450. (Non-Patent Document 12). Based on the reductase domain containing the linker sequence of P450RhF, Nozaki et al. Produced an E. coli functional expression vector pRED with an Nde I site and an EcoR I site as the cloning site for the P450 gene to be analyzed (patent) Document 1 and Non-Patent Document 13). As a pRED base vector, pET21a (manufactured by Novagen), which controls the expression of a transgene with a T7 lac promoter (hereinafter referred to as T7 promoter), was used. The structure of the pRED vector is shown in FIG. By using the pRED vector, P450 genes derived from several bacteria (P450cam (CYP101), P450Bzo (CYP203), P450balk (CYP153), CYP110E1)) are expressed as artificial fusion proteins in E. coli and function as monooxygenases. It was shown that it can be expressed (Patent Literature 1, Patent Literature 2, Patent Literature 3, Non-Patent Literature 13 and Non-Patent Literature 14). The greatest advantage of this E. coli functional expression system is that P450 derived from bacteria, whose functional expression has been difficult until now, can be expressed universally and efficiently. Therefore, it was considered suitable for functional analysis of P450 derived from different organisms.

At present, the genome of the Streptomyces genus has been decoded and its genome sequence information has been released ( Streptomyces avermitilis ); http: //www.ls.kitasato-u .ac.jp /, Streptomyces griseus ; http://streptomyces.nih.go.jp/griseus/, Streptomyces coelicolor A3 (2)) ( Streptomyces coelicolor A3 (2)); http://www.sanger.ac.uk/resources/downloads/bacteria/streptomyces-coelicolor.html, Streptomyces scabiei ; http://www.sanger.ac.uk/resources/downloads/ bacteria / streptomyces-scabies.html, Streptomyces bingchenggensis BCW-1; http://gib.genes.nig.ac.jp/single/main.php?spid=Sbin_BCW1). The genus Streptomyces, which produces various secondary metabolites including antibiotics, has more P450 genes (15 to 34 molecular species) than general bacteria. From the published genome information, 22 families and 92 molecular species of P450 gene sequences were confirmed. For example, the CYP154 family is stored in all five species for which the above-described genome analysis has been completed, and is stored in one to three molecular species.

  The substrate compound of P450 derived from the genus Streptomyces is almost unknown except for the molecular species contained in the biosynthetic cluster of secondary metabolites.

The CYP154 family is widely conserved, mainly in the genus Streptomyces. So far, the three-dimensional structure of two molecular species has been clarified, but the catalytic reaction of CYP154 derived from the genus Streptomyces is unknown (Non-patent Documents 15 and 16). However, it has been previously reported that CYP154 derived from Nocardia Farcinica IFM strain 10152 ( Nocardia farcinica ) introduces a hydroxyl group at the 16α position of testosterone (Non-patent Document 17), but the substrate concentration of testosterone is as low as 0.1 mM. Among them, the conversion rate is as low as about 50%, and it can be said that the production level of the conversion product has not been reached. In addition, in the functional analysis of CYP154H1 derived from Thermobifida fusca , conversion examples using ethylbenzene, n-propylbenzene, styrene, indole, thioanisole and other sfides as substrates are reported ( Non-patent document 18).

As mentioned above, the P450 family group has a very wide substrate adaptability (substrate specificity), and the catalytic reaction is so diverse that it is difficult to identify the substrate. Even if the P450 gene is confirmed on the genome, the function can be estimated. There are few examples. Therefore, the research of P450 so far has been progressed in metabolite analysis, and it is derived from animals such as humans whose metabolism is relatively easy to estimate, or plants where metabolite analysis is also actively conducted. Research has been conducted mainly on P450. On the other hand, many P450 gene sequences have been confirmed for bacterial-derived P450s due to the progress of genome analysis, but functional analysis research examples are compared to P450s for animals and plants, partly because there is very little information on metabolite analysis. Extremely few. As an example of bacteria-derived P450 that has been successfully analyzed for its function, actinomycete-derived P450 includes those involved in biosynthesis of antibiotics such as penicillin, erythromycin, and vermectin. Moreover, in bacteria using terpenes such as camphor, cineol and terpineol as a carbon source, P450 plays an important role in assimilation of these. Thus, it is expected that there are many P450 molecular species involved in metabolism to compounds that are difficult to synthesize using organic chemical synthesis in P450 derived from bacteria, and identification of P450 having a novel catalytic function In addition, elucidation of the catalytic reaction and utilization in industry are desired.

  Steroids are compounds in which cyclopentahydrophenanthrene is the basic skeleton and some or all of the carbons are hydrogenated. Steroidal compounds usually have methyl groups at the 10th and 13th carbons, and various compound groups in which various substituents such as hydroxyl groups, oxo groups, and alkyl groups are bonded to carbons at various positions. It is. In steroid compounds, squalene biosynthesized via the mevalonate pathway in most living organisms forms various steroid skeletons by various cyclases, and is modified in multiple steps by enzymes such as P450. As a result, various steroid compounds are biosynthesized. Steroid compounds are important components of cell membranes along with neutral lipids, proteins, and saccharides. In addition, bile acids contained in bile, steroids important for biological maintenance such as sex hormones, glucocortinoids, and mineral cortinoids. It is widely used in living organisms as hormones and insect transformation hormones. For example, progestogens and estrogen derivatives are widely used as contraceptives, and testosterone is used as a growth promoter. Antiandrogens are used as a treatment for prostate cancer. Cortisone, cortisol, prednisolone and prednisone and their derivatives are used as therapeutic agents for skin diseases, rheumatism, allergic diseases and kidney diseases. Furthermore, it is known that steroid compounds in which the 16α-position of the D ring of the steroid compound is hydroxylated have various functions and uses. For example, 16α-hydroxy-dehydroepiandrosterone is a corticosteroid hormone that exists only in the fetus and is an important precursor underlying placental estriol, and it measures 16α-hydroxy-dehydroepiandrosterone. It is a useful compound that can be used as an indicator of fetal adrenal-liver system function. Estrone, which is hydroxylated at the 16α position of estrone, is secreted in a large amount from the placenta in the late stage of pregnancy, suppresses the uterine excitatory action of estrone and estradiol, and has physiologically significant significance for fetal-placental function, In particular, urinary estriol levels are best measured clinically as indicators of fetal, placental, and maternal system function. 16α-hydroxy-Δ4-androstene-3,17-dione and 16α-testosterone have androgenic action. Furthermore, 16α-hydroxyprogesterone is useful as a raw material for the synthesis of allopregnan-3,6,20-trione and pregnane-3,11,20-trione. 16α-hydroxy-11-deoxycorticosterone is useful as a raw material for the synthesis of Δ4-androstene-3,16-dione.

  Thus, steroid compounds hydroxylated at the 16α position have various useful applications such as standard products for clinical examination and metabolite measurement, creation of seed / lead compounds in drug discovery, and synthetic intermediates. However, the structure of steroid compounds hydroxylated at the 16α position is diverse and complicated, and therefore chemical synthesis requires many steps and is often difficult to synthesize. For this reason, development of a simple and efficient method for producing a steroid compound hydroxylated at the 16α position has been desired.

International Publication WO2006 / 051729 JP 2009-5687 A JP 2010-220609 A

David R. Nelson Progress in tracing the evolutionary paths of cytochrome P450. Biochimica et Biophysica Acta 1814: 14-18 (2011) Klingenberg, M. Pigments of Rat Liver Microsomes. Arch. Biochem. Biophys. 75: 376-386 (1958) Garfinkel, David. Studies on pig liver Microsomes. I. enzymic and pigment composition of different microsomal fraction. Arch. Biochem. Biophys. 77: 493-509 (1958) Omura, T. and Sato, R. A new cytochrome in liver Microsomes. J. Biol. Chem. 237: 1375-1376 (1962) Estabrook, R.W., Cooper, D.Y. and Rosenthal, O. The light reversible carbon monoxide inhibition of the steroid C21-hydroxylase system of the adrenal cortex. Biochem. Z. 338: 741-755 (1963) Omura, T., Sato, R., Cooper, D.Y., Rosenthal, O. and Estabrook, R.W.Function of cytochrome P-450 of Microsomes. Fed. Proc. 24: 1181-1189 (1965) Degtyarenko, K.N., Archakov, A.I.Molecular evolution of P450 superfamily and P450-containing monooxygenase systems.FEBS. Lett. 332: 1-8 (1993) Sawada, Y., Kinoshita, K., Akashi, T., Aoki, T. and Ayabe, S. Key amino acid residues require for aryl migration catalysed by the cytochrome P450 2-hydroxyisoflavanone synthase.Plant J. 31: 555-564 (2002) Kim, TW, Hwang, JY, Kim, YS, Joo, SH, Chang, SC, Lee, JS, Takatsuto, S. and Kim, SK Arabidopsis CYP85A2, a cytochrome P450, Mediates the Baeyer-Villiger oxidation of castasterone to brassinolide in brassinosteroid biosynthesis. Plant Cell 17: 2397-2412 (2005) Isin, E.M.Gugerich, F.P.Complex reactions catalyzed by cytochrome P450 enzymes.Biochim. Biophys, Acta. 1770: 314-329 (2007) F. Hannemann et al, Cytochrome P450 systems--biological variations of electron transport chains. Biochim. Biophys. Acta, 1770: 330-344 (2007) G. A. Roberts et al, Identification of a New Class of Cytochrome P450 from a Rhodococcus sp. J. Bacteriol. 184: 3898-3908 (2002) M. Nodate et al, Functional expression system for cytochrome P450 genes using the reductase domain of self-sufficient P450RhF from Rhodococcus sp.NCIMB 9784.Appl.Microbiol.Biotechnol., 71: 455-462 (2006) T. Otomatsu et al, Bioconversion of aromatic compounds by Escherichia coli that expresses cytochrome P450 CYP153A13a gene isolated from an alkane-assimilating marine bacterium Alcanivorax borkumensis. Journal of Molecular Catalysis B: Enzymatic, 66: 234-240 (2010) Rarissa m, podust et al, Comparison of the 1.85 Å structure of CYP154A1 from Streptomyces coelicolor A3 (2) with the closely related CYP154C1 and CYPs from antibiotic biosynthetic pathways.Protein Science., 13: 255-268 (2004) Rarissa m, podust et al, The 1.92-Å structure of Streptomyces coelicolor A3 (2) CYP154C1. The Journal of Biological Chemistry., 278 (14): 12214-12221 (2003) H. Agematu et al, Hydroxylation of Testosterone by Bacterial Cytochromes P450 Using the Escherichia coli Expression System. Biosci. Biotechnol. Biochem., 70 (1): 307-311 (2006) Anett Schallmey et al, Characterization of cytochrome P450 monooxygenase CYP154H1 from the thermophilic soil bacterium Thermobifida fusca. Appl. Microbiol. Biotechnol., 89: 1475-1485 (2011)

  The present invention is an efficient use of a protein belonging to cytochrome P450 family 154 (CYP154) or a living cell such as Escherichia coli synthesized with a protein belonging to CYP154, which is located at the D ring 16α position of a steroid compound having a substituent. The main object is to provide a method for introducing a hydroxyl group selectively and stereoselectively.

The inventors conducted intensive research to solve the above-mentioned problems, and conducted a search for P450 having a novel catalytic function as described below for the purpose of applying P450 derived from bacteria to bioconversion technology.
27 P450 genes derived from the Streptomyces griseus NBRC 13350 strain, whose genome information has already been made public, were obtained as subjects for research. And the functional analysis of these P450 genes was performed using the pRED vector (FIG. 1, patent document 1, nonpatent literature 13 and nonpatent literature 14).

As described above, the pRED vector has a structure in which P450RhF (Non-patent Document 12) possessed by Rhodococcus sp. NCIMB 9784 is different from general P450 (NAD (P) H-iron sulfur protein reductase (ferredoxin reductase)) The P450 reductase end (C-terminal side) consisting of the region and the iron sulfur protein (ferredoxin) region has a structure in which it is fused with the P450 protein as a single polypeptide chain via a linker sequence) Was made. Based on pET21a (manufactured by Novagen) that controls the expression of the transgene by the T7 promoter, the pRED vector was given a reductase end containing the P450RhF linker sequence and an Nde I and Eco RI site as the cloning site for the P450 gene. It is a functional expression vector for E. coli of heterologous P450 (FIG. 1). By linking several P450 genes derived from different bacteria to this pRED vector and analyzing the function in E. coli, the versatility of the vector has already been confirmed (Patent Document 1, Patent Document 2, Patent Document 3, Non-Patent Document Patent Document 13 and Non-Patent Document 14).

  As a result, when a P450 belonging to CYP154 (CYP154), for example, a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 (SGR 1085 (CYP154)) is used in the process of searching for P450 having a novel catalytic function, It was found that a hydroxyl group can be introduced in a position-stereoselective manner at the D ring 16α position of various steroidal compounds having benzene. The product also contained new compounds such as 16α-hydroxy-4-pregnene-3, 11, 20-trione, 16α-hydroxyadrenosterone. It was a completely unexpected finding that P450 derived from actinomycetes that do not biosynthesize steroid compounds is responsible for such catalytic reactions.

That is, the present inventors have obtained the following knowledge.
(I) By making a protein belonging to CYP154 or an artificial fusion protein containing the same function as a monooxygenase and acting on a steroid compound having a substituent, the steroid compound is regioselectively and stereotactically located at the D ring 16α position. A hydroxyl group can be selectively introduced efficiently.
(ii) For example, the above reaction may be carried out by using a protein belonging to CYP154 or a fusion protein containing the protein and a protein (electron transfer protein; redox partner) that transmits electrons to the protein belonging to CYP154 as necessary (particularly in the former case). A recombinant vector in which a gene encoding a protein belonging to CYP154 is inserted into a conventional pRED vector. If a transformant obtained by transforming Escherichia coli (Escherichia coli) is used, the hydroxyl group introduction reaction is likely to proceed.
(Iii) The above reaction can be performed using living cells such as E. coli that express a protein belonging to CYP154 or a fusion protein containing the same, so that it is not necessary to add expensive NADPH or NADH. It is not necessary to purify and use the enzyme during the reaction, 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 produced inexpensively, easily and 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 steroid compounds of the following items.
Item 1. A feature is that a protein belonging to the cytochrome P450 family 154 (CYP154) or a fusion protein containing the protein functions as a monooxygenase and acts on a steroid compound having a substituent to introduce a hydroxyl group into the D ring 16α position of the steroid skeleton. A method for producing a steroid compound in which the 16α-position is hydroxylated.
Item 2. By coexisting a transformant introduced with a gene encoding a protein belonging to CYP154 or a fusion protein containing the same and a steroid compound having a substituent, the protein belonging to CYP154 or a fusion protein containing the same is obtained. Item 2. The production method according to Item 1, which acts on a steroid compound having a substituent.
Item 3. A steroid compound having a substituent is represented by the following general formula (1):

(In the above formula, R ^{1 to} R ⁶ , -A-A'-, and -B-B'-B ''-are the following (A) or (B);
(A) R ¹ is a hydroxyl group, an oxo group or an acetoxy group,
R ² is hydrogen, a hydroxyl group, an acetoxy group or an oxo group,
R ³ is a hydroxyl group, acetyl group, hydroxymethylcarbonyl group, acetoxymethylcarbonyl group, 2-hydroxyethyl group, 1,2-dihydroxyethyl group or hydroxymethylcarbonyl group,
R ⁴ is hydrogen or a methyl group,
-A-A'is, -CH ₂ -CH ₂ - or a -CH = CH-,
-B-B'-B '' - _{is, -CH 2 -CH-CH 2 -} , - CH 2 -C = CH -, - CH = C-CH 2 - or -CH = a C-CHF-,
R ⁵ is hydrogen, a methyl group, a hydroxymethyl group, or a formyl group,
R ⁶ is a methyl group, a hydroxymethyl group, or a formyl group, or
(B) R ³ and R ⁴ together are an oxo group,
R ¹ is a hydroxyl group, an oxo group or an acetoxy group,
R ² is hydrogen, a hydroxyl group or an oxo group,
-A-A'is, -CH ₂ -CH ₂ - or a -CH = CH-,
-B-B'-B '' - _{is, -CH 2 -CH-CH 2 -} , - CH 2 -C = CH -, - CH = C-CH 2 - or -CH = a C-CHF-,
R ⁵ is hydrogen, a methyl group, a hydroxymethyl group, or a formyl group,
Item 3. The production method according to Item 1 or 2, wherein R ⁶ is a steroid compound represented by a methyl group, a hydroxymethyl group, or a formyl group.
Item 4. Item 4. The production method according to any one of Items 1 to 3, wherein the protein belonging to CYP154 is the following (a) or (b):
(A) a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 (b) an amino acid sequence of SEQ ID NO: 1, wherein one or a plurality of amino acid sequences are deleted, substituted or added, and a cytochrome P450 monooxygenase A polypeptide that functions as Item 5. The production method according to any one of Items 1 to 4, wherein the fusion protein is a fusion protein of a protein belonging to CYP154 and an electron transfer protein.
Item 6. The fusion protein is a fusion protein of a protein belonging to CYP154 and a reductase peptide contained in cytochrome P450 monooxygenase P450RhF derived from Rhodococcus NCIMB 9784, which is an electron transfer protein, or a reductase peptide having an equivalent function. The manufacturing method in any one of claim | item 1 -5.
Item 7. Item 7. The production method according to Item 5 or 6, wherein the electron transfer protein is the following polypeptide (c) or (d).
(c) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2
(d) a polypeptide 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; The following (h) to (5) encode a reductase peptide via a linker on the 3 ′ end side of any one of the following DNAs (e) to (g) encoding a protein belonging to CYP154: Item 8. The production method according to any one of Items 2 to 7, wherein the gene encoding the fusion protein linked to any of the DNAs of (j) is introduced into E. coli by a recombinant plasmid inserted into an E. coli vector. .
(e) DNA consisting of the nucleotide sequence of SEQ ID NO: 3
(f) DNA encoding a polypeptide that hybridizes with a DNA comprising a nucleotide sequence complementary to the DNA of SEQ ID NO: 3 under stringent conditions and functions as a cytochrome P450 monooxygenase
(g) DNA encoding a polypeptide consisting of an amino acid sequence in which one or more amino acid sequences are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 1 and which functions as cytochrome P450 monooxygenase
(h) DNA consisting of the base sequence of SEQ ID NO: 4
(i) a DNA that hybridizes with a DNA comprising a nucleotide sequence complementary to the DNA of SEQ ID NO: 4 under a stringent condition and encodes a polypeptide having a reductase activity
(j) a DNA encoding a polypeptide consisting of 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 a reductase activity
In the present specification, a protein belonging to cytochrome P450 family 154 (CYP154) is referred to as a protein belonging to CYP154.

  When a hydroxyl group is introduced regioselectively and stereoselectively into the D ring 16α position of a steroid compound by chemical synthesis, a multi-step reaction, that is, introduction of a protecting group or elimination reaction may be required. Conditions, expensive or dangerous reagents, waste liquid treatment, etc. may be required. In addition, there are problems caused by column chromatography purification and by-product generation in multiple steps. 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 using an inexpensive commercially available raw material. In addition, the reaction can be carried out using living cells such as E. coli that produce proteins belonging to CYP154, so there is no need to add expensive NADPH or NADH or to purify the enzyme. There is an excellent advantage of being easy. 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, steroid compounds useful as metabolite analyzes and pharmaceuticals can be produced easily and efficiently in large quantities under mild conditions.

FIG. 1 is a diagram showing the structure of a P450 function expression vector pRED. FIG. 2 is a diagram showing a phylogenetic tree of P450 derived from Streptomyces griseus NBRC 13350 strain. FIG. 3 is a diagram showing a phylogenetic tree of the actinomycete-derived CYP154 family. FIG. 4 shows the nucleotide sequence inserted into the pHSG396 plasmid vector.

Hereinafter, the present invention will be described in detail.
According to the method for producing a steroid compound hydroxylated at the 16α position of the present invention, a protein belonging to CYP154, which is one family of cytochrome P450, or a fusion protein containing the same is made to function as a monooxygenase, and a steroid compound raw material having a substituent ( This is a method for producing an oxide of a steroid compound having a substituent by acting on a substrate. More specifically, in the method for producing a steroid compound hydroxylated at the 16α position according to the present invention, a protein belonging to the family 154 of cytochrome P450 (CYP154) or a fusion protein containing the same is functioned as a monooxygenase and substituted. A hydroxyl group is introduced into the 16α position of the D ring of the steroid skeleton by acting on a steroid compound having a group. In this way, a steroid compound in which the 16α position is hydroxylated is produced. As the raw material (substrate) in the production method of the present invention, steroid compounds having various substituents can be used.

Steroid compound having a substituent The steroid compound having a substituent used as a raw material in the production method of the present invention is not particularly limited, but preferably has no substituent at the 16-position of the steroid skeleton (cyclopentahydrophenanthrene skeleton). .

  For the raw material (substrate) of the production method of the present invention, for example, a steroid compound having a substituent represented by the following general formula (1) is suitably used.

In the general formula (1), R ^{1 to} R ⁶ , -A-A'-, and -BB'-B ''-are the following (A) or (B).
(A) R ¹ is a hydroxyl group, an oxo group or an acetoxy group,
R ² is hydrogen, a hydroxyl group, an acetoxy group or an oxo group,
R ³ is a hydroxyl group, acetyl group, hydroxymethylcarbonyl group, acetoxymethylcarbonyl group, 2-hydroxyethyl group, 1,2-dihydroxyethyl group or hydroxymethylcarbonyl group,
R ⁴ is hydrogen or a methyl group,
-A-A'is, -CH ₂ -CH ₂ - or a -CH = CH-,
-B-B'-B '' - _{is, -CH 2 -CH-CH 2 -} , - CH 2 -C = CH -, - CH = C-CH 2 - or -CH = a C-CHF-,
R ⁵ is hydrogen, a methyl group, a hydroxymethyl group, or a formyl group,
R ⁶ is a methyl group, a hydroxymethyl group, or a formyl group.

(B) R ³ and R ⁴ together are an oxo group,
R ¹ is a hydroxyl group, an oxo group or an acetoxy group,
R ² is hydrogen, a hydroxyl group or an oxo group,
-A-A'is, -CH ₂ -CH ₂ - or a -CH = CH-,
-B-B'-B '' - _{is, -CH 2 -CH-CH 2 -} , - CH 2 -C = CH -, - CH = C-CH 2 - or -CH = a C-CHF-,
R ⁵ is hydrogen, a methyl group, a hydroxymethyl group, or a formyl group,
R ⁶ is a methyl group, a hydroxymethyl group, or a formyl group.

Among them, as the steroid compound having a substituent in the present invention, in the general formula (1), R ^{1 to} R ⁶ , -AA ′, and —BB′-B ″ — ), R ^{1 to} R ⁶ , -A-A'-, and -B-B'-B ''-are each preferably the following.
R ¹ is preferably a hydroxyl group or an oxo group. R ² is preferably hydrogen, a hydroxyl group, or an oxo group. R ³ is preferably a hydroxyl group, an acetyl group, a hydroxymethylcarbonyl group, an acetoxymethylcarbonyl group, or a hydroxymethylcarbonyl group. R ⁴ is preferably hydrogen or a methyl group. R ⁵ is preferably a methyl group. R ⁶ is preferably a methyl group. —A—A′— is preferably —CH ₂ —CH ₂ — or —CH═CH—. -B-B'-B '' - _{is, -CH 2 -CH-CH 2 -} , - CH 2 -C = CH-, or -CH = C-CH ₂ - is preferably.

In the general formula (I), when R ^{1 to} R ² , R ^{5 to} R ⁶ , -A-A'-, and -B-B'-B ''-are the above (B), R ^1- R ² , R ^{5 to} R ⁶ , -A-A'-, and -BB'-B ''-are each preferably as follows.
R ¹ is preferably a hydroxyl group or an oxo group. R ² is preferably hydrogen or an oxo group. R ⁵ is preferably a methyl group. R ⁶ is preferably a methyl group. —A—A′— is preferably —CH ₂ —CH ₂ — or —CH═CH—. -B-B'-B '' - _{is, -CH 2 -CH-CH 2 -} , - CH 2 -C = CH-, or -CH = C-CH ₂ - is preferably.

  In the production method of the present invention, the steroid compound having a substituent represented by the above general formula (I) as a raw material (substrate) is, for example, 1,4-androstadiene-3,17-dione (1,4 -Androstadiene-3,17-dione), 11-Dehydrocorticosterone, 11α-Hydroxyprogesterone, 11α-Hydroxyprogesterone acetate, 11β, 21-dihydroxy- 3,20-oxo-5β-pregnan-18-al (11β, 21-Dihydroxy-3,20-oxo-5β-pregnan-18-al), 11β, 21-dihydroxy-5β-pregnan-3,20-dione (11β, 21-Dihydroxy-5β-pregnane-3,20-dione), 11β-hydroxyandroste-4-ene-3,17-dione (11β-Hydroxyandrost-4-ene-3,17-dione), 11β -Hydroxyprogesterone (11β-Hydroxyprogesterone), 17α-Methylandrostan-17β-ol-3-one (17α-Methylandrostan-17β-ol-3-one), 18-Hydroxyprogesterone 18-Hydroxycorticosterone, 19-Hydroxyandrost-4-ene-3,17-dione, 19-Hydroxytestosterone, 19-Nortestosterone, 19-Oxoandrost-4-ene-3,17-dione, 19-Oxotestosterone 1-Dehydro-17α-methyltestosterone, 20α-Hydroxy-4-pregnen-3-one, 21-Hydroxy-5β- Pregnane-3,11,20-trione (21-Hydroxy-5β-pregnane-3,11,20-trione), 21-hydroxypregnenolone (21-Hydroxypregnenolone), 3α, 11β, 21-trihydroxy-20-oxo- 5β-pregnan-18-al (3α, 11β, 21-Trihydroxy-20-oxo-5β-pregnan-18-al), 3α, 20α, 21-trihydroxy-5β-pregnan-11-one (3α, 20α, 21-Trihydroxy-5β-pregnane-11 -one), 3α, 21-dihydroxy-5β-pregnane-11,20-dione (3α, 21-Dihydroxy-5β-pregnane-11,20-dione), 3α-hydroxy-5α-pregnane-20-one (3α -Hydroxy-5α-pregnan-20-one), 3α-hydroxy-5β-androstan-17-one (3α-Hydroxy-5β-androstan-17-one), 3α-hydroxy-5β-pregnan-20-one ( 3α-Hydroxy-5β-pregnane-20-one), 3β, 17β-dihydroxy-5-androstene, 4-pregnene-3,11,20-trione (4-Pregnane -3,11,20-trione), 5α-Androstane-3,17-dione (5α-Androstane-3,17-dione), 5α-Dihydrodeoxycorticosterone, 5α-pregnane-20α -All-3-one (5α-Pregnan-20α-ol-3-one), 5α-Pregnane-3,20-dione (5α-Pregnane-3,20-dione), 5α-Pregnane-3α, 20α-diol (5α-Pregnane-3α, 20α-diol), 5β-Androstane-3,17-dione (5β-Androstane-3,17-dione), 5β-Dihydrotes Sterone (5β-Dihydrotestosterone), 5β-Pregnane-3,20-dione (5β-Pregnane-3,20-dione), 5β-Pregnane-3α, 20α-diol (5β-Pregnane-3α, 20α-diol), 6α -Fluorotestosterone (6α-Fluorotestosterone), Adrenosterone (Adrenosterone), Aldosterone (Aldosterone), Androstane-3α, 17β-diol (Androstan-3α, 17β-diol), Androsterone (Androsterone), Corticosterone (Corticosterone) ), Corticosterone 21-acetate, Dehydroepiandrosterone, Dehydroepiandrosterone acetate, Deoxycorticosterone, Deoxycorticosterone acetate , Dihydrotestosterone, methylandrostenediol, methyl Stosterone (Methyltestosterone), Pregnanediol, Pregnenolone, Pregnenolone acetate, Progesterone, Stanolone, Testosterone, Tetrahydrocorticosterone-Δ4- Examples include, but are not limited to, 3,17-dione (Δ4-Androstene-3,17-dione) and the like.

  In the production method of the present invention, a commercially available steroid compound having a substituent serving as a substrate (raw material compound) may be used, or produced and used by a conventionally known and commonly used method. Also good.

In the production method of the present invention, a hydroxyl group is regioselectively and stereoselectively introduced into the 16α-position of the steroid skeleton D ring of the steroid compound having the above substituent.
The introduction of a hydroxyl group at the 16α position of the steroid skeleton D ring of the steroid compound having a substituent means that a hydroxyl group is directly introduced into the carbon atom at the 16α position of the D ring constituting the steroid compound.

Bioconversion reaction in the production method of the bioconversion reaction present invention (hereinafter sometimes abbreviated as "reaction".) Is a suitable solution or solvent, substrate (starting compound) with a protein belonging to the CYP154 (hereinafter, " It may be abbreviated as “CYP154 protein”.) Or a fusion protein containing the protein may be reacted with a redox (redox) partner protein (electron transfer protein) as necessary. As the electron transfer protein, ferredoxin reductase and ferredoxin are preferable. CYP154 protein can also be used after being isolated from a biological material. Examples of the isolated CYP154 protein include crushed materials, extracts, and purified products of biological materials such as bacterial cells. The isolated CYP154 may be immobilized on a suitable carrier. In this case, it is preferable that ferredoxin reductase and ferredoxin, which are electron transfer proteins that catalyze an oxidation reaction, coexist in the reaction system. When a protein (fused protein) in which CYP154 protein is fused with an electron transfer protein such as the reductase end of P450RhF (cytochrome P450 monooxygenase derived from Rhodococcus genus NCIMB 9784) is reacted with a substrate separately, ferredoxin It is not necessary to coexist with reductase or ferredoxin. By reacting the CYP154 protein and the electron transfer protein together, the CYP154 protein functions more efficiently as a monooxygenase.

  In addition, since P450 family proteins are unstable enzymes, the reaction in the present invention is preferably carried out by contacting or coexisting a CYP154 protein-producing cell and a substrate in the reaction solution. This reaction may be performed by adding a substrate to the culture medium of cells that produce CYP154 protein, for example, by allowing cells that produce CYP154 protein and the substrate to coexist in a buffer solution. For example, by stirring or shaking a cell producing CYP154 protein in a buffer solution (buffer) in the presence of a steroid compound having a substituent as a substrate, a steroid compound hydroxylated at the 16α position can be efficiently obtained. Can be manufactured.

The CYP154 protein and the cells that produce it will be described in detail later.
Hereinafter, a method using cells that produce CYP154 protein will be described. In the following, a method using cells that produce CYP154 protein will be described as an example, but the same applies to the case of using cells that produce a fusion protein containing CYP154 protein.
The reaction can be carried out either by a batch system such as a batch reaction or a fed-batch (semi-batch) reaction; The substrate can be added in one batch or continuously.

  The reaction conditions may be appropriately set according to the type of cells used (cells such as microorganisms producing CYP154 protein) and the substrate (raw compound). For example, in the case of batch reaction, the substrate concentration in the reaction solution is about 0. 1 to 10 mM is preferred, and about 0.5 to 3 mM is more preferred. As the reaction solution, for example, a normal liquid medium containing a carbon source, a nitrogen source, a mineral source or the like usually used for culturing microorganisms, or a buffer solution can be suitably used. Preferably, a buffer solution such as a phosphate buffer is used. In addition, it is preferable to carry out the reaction in a buffer solution using a microorganism that produces CYP154 protein previously cultured in a normal medium or the like. The buffer solution is not particularly limited as long as the effects of the present invention are exhibited, and a phosphate buffer, a Tris buffer, or the like can be used. In the case of a water-insoluble and liquid substrate, the substrate can be overlaid on a reaction solution containing cells that produce CYP154 protein. The pH of the reaction solution is preferably about 5-9, more preferably about 6-8. In addition, the reaction is usually performed at about 20 to 40 ° C, preferably about 20 to 30 ° C. The reaction time is usually about 12 to 48 hours. During the reaction, the reaction solution is preferably stirred or shaken appropriately. In the case of fed-batch (semi-batch) reaction or continuous reaction, the conditions may be set according to the batch reaction conditions. Within the above range, the conversion rate from the substrate to the target product hydroxylated at the 16α position is high, and purification becomes easy. The produced steroid compound hydroxylated at the 16α position usually accumulates in the reaction solution.

  For example, when a transformant obtained by introducing the above gene into Escherichia coli described later is used as a microorganism that produces CYP154 protein, the reaction in the presence of the substrate is preferably performed under aerobic conditions.

Reaction product (conversion product)
The steroid compound having a hydroxyl group at the 16α position generated by the bioconversion reaction can be purified by a conventional method. For example, if necessary, a treatment such as centrifugation or filtration is performed to remove suspensions such as bacterial cells, and then extraction is performed with a common extraction solvent, for example, an organic solvent such as ethyl acetate, chloroform, or methanol. The organic solvent can then be removed under reduced pressure and purified by treatment with vacuum distillation, chromatography, ion exchange resin, adsorbent resin or the like.

A protein belonging to the cytochrome P450 family 154 (CYP154) or a fusion protein containing the same The protein belonging to the cytochrome P450 family 154 (CYP154) in the present invention is a protein having monooxygenase activity. Preferably, the protein belonging to CYP154 in the present invention is a protein having monooxygenase activity in which a monooxygen atom is selectively added to the D ring 16α position of the steroid skeleton.
Examples of the protein belonging to CYP154 include CYP154 protein produced by bacteria belonging to the genus Streptomyces , Nocardia , Thermobifida , Nocardiopsis, and the like. Can be mentioned.

Specifically, for example, CYP154 derived from Streptomyces griseus NBRC 13350 (CYP154; DNA sequence is registered under NCBI under accession number AP009493; the amino acid sequence of CYP154 is represented by SEQ ID NO: 1 CYP154H derived from Nocardia farcinica IFM 10152 (DNA sequence is registered in NCBI under accession number AP006618), CYP154H derived from Streptomyces roseosporus NRRL 15998 (The DNA sequence is registered with the accession number ZP_04697453 in NCBI) and the like are preferably used.

  Among these, as the CYP154 protein in the present invention, (a) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 is preferable. In addition, (b) one or more amino acids in the amino acid sequence of SEQ ID NO: 1 (the plurality is, for example, about 2 to 20, preferably about 2 to 10, more preferably about 2 to 7, more preferably about 2-5, particularly preferably about 2-3, most preferably about 2, and so on.) The amino acid sequence is deleted, substituted or added, and as cytochrome P450 monooxygenase A functional polypeptide can also be preferably used.

The polypeptide of SEQ ID NO: 1 is a CYP154 derived from Streptomyces griseus (Streptomyces griseus) NBRC 13350 strain can be isolated from cultures of Streptomyces griseus (Streptomyces griseus) NBRC 13350 strain. 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, polarity, charge, solubility, hydrophilicity from the viewpoint of maintaining the structure of the resulting protein. 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, and proline are classified as nonpolar amino acids; serine, threonine, cysteine, methionine, asparagine, and glutamine are classified as polar amino acids; phenylalanine, tyrosine, and tryptophan are aromatic side chains. 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.

  In the present specification, a polypeptide that functions as cytochrome P450 means that a difference spectrum (CO difference spectrum) having a maximum at 450 nm changes when absorption of carbon monoxide is reduced in solution in a solution and carbon monoxide is passed through the polypeptide. Means to appear. In addition, the polypeptide that functions as a cytochrome P450 monooxygenase in the present invention preferably has a monooxygenase activity of selectively adding a single oxygen atom to the D ring 16α position of the steroid skeleton, more preferably, When the electron transfer protein described later is present together with the polypeptide, it exhibits a monooxygenase activity of adding a monooxygen atom to the D ring 16α position of the steroid skeleton.

  Examples of the fusion protein containing CYP154 protein include a fusion protein of CYP154 protein and an electron transfer protein (redox (redox) partner protein) and a fusion protein with a secretion signal. A fusion protein of a protein and an electron transfer protein is preferred. A typical example of such a protein is a fusion protein of CYP154 protein and a reductase peptide (electron transfer protein) contained in cytochrome P450 monooxygenase or a peptide having a reductase activity equivalent thereto. Specifically, the fusion protein is preferably a fusion protein of CYP154 protein and ferredoxin reductase and a reductase peptide having the function of ferredoxin. Having a reductase activity equivalent to the reductase peptide contained in cytochrome P450 monooxygenase means having a functionally equivalent activity to the reductase activity of the reductase peptide.

Among them, as the electron transfer protein, a reductase peptide (domain) contained in cytochrome P450 monooxygenase P450RhF derived from Rhodococcus NCIMB 9784 strain or a peptide having reductase activity equivalent thereto is more preferable.
That is, the fusion protein containing CYP154 protein has reductase peptide (domain) contained in CYP154 protein and cytochrome P450 monooxygenase P450RhF derived from Rhodococcus NCIMB 9784, which is an electron transfer protein, or equivalent reductase activity. A fusion protein with a peptide is preferred. The reductase peptide (domain) contained in cytochrome P450 monooxygenase P450RhF derived from Rhodococcus sp. NCIMB 9784 is a polypeptide consisting of (c) the amino acid sequence of SEQ ID NO: 2. In the present invention, the peptide having reductase activity equivalent to the reductase peptide contained in the cytochrome P450 monooxygenase P450RhF derived from Rhodococcus NCIMB 9784 is (1) in the amino acid sequence described in SEQ ID NO: 2, A polypeptide having an amino acid sequence in which a plurality of amino acids are added, deleted or substituted and having reductase activity can also be preferably used. Whether a polypeptide has reductase activity can be confirmed by preparing a fusion protein of the polypeptide with a known P450 having a known substrate and confirming whether or not a reaction product is actually produced.

  The polypeptide of SEQ ID NO: 2 is obtained by isolation from a culture of Rhodococcus NCIMB 9784 strain. It can also be obtained by chemical synthesis. For example, the method of obtaining the polypeptide of (d) by modifying the sequence while retaining the biological activity of the polypeptide of (c) is the method of obtaining the polypeptide of (b) based on the polypeptide of (a) described above. It is the same.

  A linker peptide can be interposed between the CYP154 protein and the reductase peptide. Usually, a reductase peptide may be bound to the C-terminal side of the CYP154 protein via a linker peptide. The length of the linker peptide is preferably about 6 to 60 amino acids, and more preferably about 14 to 40 amino acids, in order to efficiently proceed the reaction between the CYP154 protein and the reductase peptide. The sequence of the linker peptide is not particularly limited, but for example, VLHRHQPVTIGEPAAR (SEQ ID NO: 5) and the like are preferable.

  These proteins belonging to the monooxygenase CYP154 or a fusion protein containing the protein may be extracted from microorganisms or the like existing in nature, or may be chemically synthesized based on the amino acid sequence. The fusion protein is usually artificially produced by a genetic engineering technique. Moreover, you may isolate and refine | purify from the transformant of this invention mentioned later. The purification method is not particularly limited, and an extract is prepared from a transformant of Escherichia coli by a known method, and the extract may be purified using a known method such as a column.

Recombinant vector
The cell producing the CYP154 protein or a fusion protein containing the same is preferably a transformant obtained by transforming a host with a recombinant vector containing a DNA encoding them. That is, in the present invention, a protein that belongs to CYP154 or a fusion containing the same, by coexisting a transformant introduced with a gene encoding a protein belonging to CYP154 or a fusion protein containing the same and a steroid compound having a substituent. It is preferable that a type protein acts on a steroid compound having the substituent. Thus, by using a transformant that highly expresses CYP154 protein, the reaction can be carried out efficiently.

  The above recombinant vector is obtained by ligating DNA encoding a protein belonging to CYP154 having monooxygenase activity in the present invention or a fusion protein containing the same using a known genetic engineering technique. be able to. The vector is not particularly limited as long as it can be replicated in the host and can express the target protein 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 the vector derived from E. coli, a pUC vector, a pTTQ vector, and a pET vector are preferable. Of these, the pET vector is preferable.

  In order to highly express a protein belonging to CYP154 or a fusion protein containing the same in a transformant, 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, CYP154 protein can be highly expressed by using lac promoter or T7 promoter. More preferably, the T7 promoter is used. Any translation signal may be used as long as it can function in the host, and examples thereof include a translation signal of a pUC vector (LacZ) and a translation signal of a pET vector. Among them, the translation signal of the pET vector is preferable in that CYP154 protein can be highly expressed.

  The most preferable recombinant vector is, for example, reduction of cytochrome P450 monooxygenase P450RhF derived from Rhodococcus NCIMB 9784 via a linker at the 3 ′ end of DNA encoding a protein belonging to CYP154 protein having monooxygenase activity. A fusion protein expression cassette ligated with DNA encoding an enzyme peptide (domain) or a reductase peptide having the same function was inserted into a pET vector so as to use the T7 promoter and the signal of the pET vector. This is a plasmid for expressing CYP154 gene function.

DNA encoding a protein belonging to CYP154 includes (e) DNA consisting of the base sequence of SEQ ID NO: 3 and (f) DNA consisting of the base sequence of SEQ ID NO: 3 (DNA of SEQ ID NO: 3) complementary to the base sequence. DNA encoding a polypeptide that functions as a cytochrome P450 monooxygenase, and (g) the amino acid sequence of SEQ ID NO: 1 has one or more amino acid sequences deleted or substituted Alternatively, a DNA encoding a polypeptide consisting of an added amino acid sequence and functioning as a cytochrome P450 monooxygenase is preferred. The DNA encoding the reductase peptide (domain) of cytochrome P450 monooxygenase P450RhF derived from Rhodococcus sp. NCIMB 9784 or a reductase peptide having a function equivalent to this (h) consists of the base sequence of SEQ ID NO: 4. DNA, (i) DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to DNA consisting of the base sequence of SEQ ID NO: 4 and encodes a protein having reductase activity, (j) SEQ ID NO: In the amino acid sequence described in 2, DNA or the like that encodes a polypeptide having an amino acid sequence in which one or a plurality of amino acids are added, deleted or substituted and having reductase activity is preferable.
The base sequence of SEQ ID NO: 3 is a DNA sequence encoding the polypeptide of SEQ ID NO: 1, and the base sequence of SEQ ID NO: 4 is a DNA sequence encoding the polypeptide of SEQ ID NO: 2.

DNA consisting of the base sequence of SEQ ID NO: 3 is, for example, hybridized using a probe designed based on SEQ ID NO: 3 from the genomic DNA library of Streptomyces griseus NBRC 13350 strain, The primers designed based on the above can be obtained by PCR or the like using the genomic DNA of Streptomyces griseus NBRC 13350 strain as a template. It can also be obtained by chemical synthesis. The 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. Examples of the method of obtaining the gene (f) or (g) by modifying the DNA of (e) and the method of obtaining the gene of (i) or (j) by modifying the DNA of (h) include: Method of chemical synthesis based on DNA sequence of e) or (h), method of irradiating γ-ray to DNA of (e) or (h), error using DNA of (e) or (h) as template General methods such as a method for performing prone PCR can be mentioned.

  The DNA of any one of (h) to (j) that encodes the reductase peptide is linked to the 3 ′ end of any of the DNAs of (e) to (g) that encode the CYP154 protein via a linker. A recombinant vector in which the linked fusion protein expression cassette is inserted into the pET vector so as to utilize the translation signal of the T7 promoter and the pET vector is one of the preferred embodiments of the present invention.

  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 6 × SSC (standard saline citrate; 1 × SSC = 0.15 M NaCl, 0.015 M Sodium citrate), 0.5% SDS and 50% formamide in a solution at 42 ° C. overnight. An example of the condition is that it is not detached from the polynucleotide when it is washed in a solution of 0.1 × SSC, 0.5% SDS at 68 ° C. for 30 minutes after warming. More preferable `` stringent conditions '' preferably means that hybridization occurs when homology of about 90% or more, more preferably about 95% or more, particularly preferably about 98% or more exists between sequences. means. Such “stringent conditions” are described in the above-mentioned Molecular Cloning, particularly in Section 11.45 “Conditions for Hybridization of Oligonucleotide Probes”, and the conditions described herein can be used.

  In the present invention, a DNA that hybridizes under stringent conditions with a certain DNA, for example, a DNA consisting of a base sequence complementary to the base sequence of SEQ ID NO: 3, is a 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 in the present invention is preferably one into which a gene encoding a fusion protein of CYP154 protein and electron transfer protein is introduced. The transformant is one of the above (h) to (j) encoding a reductase peptide via a linker on the 3 ′ end side of any one of the DNAs (e) to (g) encoding the CYP154 protein. It is more preferable to introduce a recombinant plasmid obtained by inserting a gene encoding a fusion protein linked with any DNA into the above-described vector into a host. The gene encoding CYP154 protein is more preferably the gene of (e) above. More preferably, the gene encoding the reductase peptide is the gene of (h) above. Further, in a preferred embodiment, the above (h) to (3) encoding a reductase peptide via a linker on the 3 ′ end side of any one of the DNAs (e) to (g) encoding a protein belonging to CYP154. A transformant obtained by introducing into E. coli a recombinant plasmid in which a gene encoding a fusion protein linked with any of the DNAs of (j) is inserted into an E. coli vector is used.
As a recombinant plasmid, the DNA of any one of the above (h) to (j) encoding a reductase peptide via a linker on the 3 ′ end side of any one of the above DNAs (e) to (g) Particularly preferred is a recombinant vector in which a fusion protein expression cassette linked to is inserted into a pET vector so as to utilize the translation signal of the T7 promoter and the pET vector.

  The transformant can be obtained by introducing the above recombinant vector into a host. The method for introducing the recombinant vector is not particularly limited, and any method using calcium ions can 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. Examples of the method for introduction into yeast include the electroporation method (Becker, DM et al .: Methods Enzymo1., 194, 182, 1990), and the spheroplast method (Hinnen, A. et al .: Proc. Nat1). Acad. Sci., USA, 75, 1929, 1978), lithium acetate method (Itoh, H .: J. Bacterio 1, 153, 163, 983) and the like. Moreover, if it is a method to introduce into animal cells, for example, electroporation method, calcium phosphate method, lipofection method, etc. If it is a method to introduce into insect cells, for example, calcium phosphate method, lipofection method, electroporation method Etc. are 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 easily undergo a conversion reaction. Examples of eubacterial cells include bacteria belonging to the genus Escherichia such as Escherichia coli , Bacillus subtilis such as Bacillus subtilis , and Pseudomonas putida such as Pseudomonas putida. Among them, Escherichia coli is preferable.

  A protein belonging to CYP154 produced by the transformant or a fusion type containing the same produced by allowing the obtained transformant and a steroid compound having a substituent as a substrate to coexist in the reaction solution as described above. A protein acts on a steroid compound having the substituent, and a target steroid compound in which the 16α-position is hydroxylated can be produced.

As an example, in the case of a transformant obtained by introducing the above gene into Escherichia coli, for example, it is preferable to first culture the Escherichia coli transformant under aerobic conditions to increase the cell concentration. By culturing under aerobic conditions, it is possible to cultivate to a high microorganism concentration in a short time. A known method is used as a means for culturing. Next, it is preferable to carry out the reaction in a buffer solution or the like in the presence of the steroid compound having a substituent as a substrate and the E. coli transformant.
In the case of performing inducible expression of the introduced gene in the Escherichia coli transformant, it is usually preferable to perform inducible expression by adding IPTG or the like after increasing the cell concentration. In addition, when E. coli is used as a host to induce and express heme protein P450, specifically a protein belonging to CYP154 or a fusion protein containing this, 5-aminolevulinic acid and iron chloride ( II) Divalent iron salts such as (FeCl ₂ ), iron sulfate (II) (FeSO ₄ ), and ammonium iron sulfate (Fe (NH ₄ ) ₂ (SO ₄ ) ₂ ) are each brought to a final concentration of about 10 to 300 mM. It is preferable to add to the culture medium or reaction solution. Inducible expression may be carried out simultaneously with the culture in the presence of the substrate.

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

[Example 1]
(1) Phylogenetic analysis of P450 derived from Streptomyces Genus Streptomyces avermitilis (http://www.ls.kitasato-u.ac.jp/), Streptomyces griseus (http://streptomyces.nih.go.jp/griseus/), Streptomyces coelicolor A3 (2) (http://www.sanger.ac.uk/resources/downloads/bacteria/streptomyces-coelicolor.html, Streptomyces scabiei (http://www.sanger.ac.uk/resources/downloads/bacteria/streptomyces-scabies.html, Streptomyces bingchenggensis BCW-1 (http://gib.genes.nig.ac.jp/single/main. We searched and collected amino acid sequence information of P450 gene in the genome of php? spid = Sbin_BCW1) Blast ( http://blast.ncbi.nlm.nih.gov/Blast.cgi ) To classify each CYP154 into subfamilies.

Table 1 shows the results of system analysis. From Table 1, for example, Streptomyces griseus IFO13350 has three CYP154. From the results shown in Table 1, it was found that the CYP154 family group is widely distributed as the main molecular species in the genus Streptomyces.

(2) Phylogenetic position of CYP154
The amino acid sequence information of CYP154 present in the following bacterial genomes published from P450 Engineering Database (http://www.cyped.uni-stuttgart.de/) was searched and collected. The obtained amino acid sequence information was subjected to homology search using Blast (http://blast.ncbi.nlm.nih.gov/Blast.cgi).

Streptomyces griseus NBRC13350
Streptomyces albus J1074
Streptomyces roseosporus NPRL 15998
Streptomyces flavogriseus ATCC 33331
Streptomyces sp. Mg1
Streptomyces ghanaensis ATCC 14672
Streptomyces griseoflavus Tu4000
Streptomyces sp. E14
Streptomyces coelicolor A3 (2)
Streptomyces lividans TK24
Streptomyces ambofaciens ATCC 23877
Streptomyces avermitilis MA-4680
Nocardia farcinica IFM 10152
Streptomyces hygroscopicus ATCC 53653
Nocardiopsis dassonvillei DSM 43111
Thermobifida fusca YX
Catenulispora acidiphila DMS 44928
Streptomyces clavuligerus ATCC 27064
Streptomyces violaceusniger Tu 4113
Streptomyces hygroscopicus ATCC 53653
Streptomyces bingchenggensis BCW-1
Streptomyces flaveolus FJ 809786
Thermomonospora curvata DSM 43183
Streptomyces ghanaensis ATCC 14672
Frankia sp. EUN1f
Streptomyces viridochromogenes DSM 40736
Streptomyces scabiei 87.22
Streptomyces pristinaespiralis ATCC25486
Streptomyces fradiae AF 145049
Streptomyces rishinensis EU 147298
Streptomyces rochei pSLA2-L
Streptomyces tsukubaensis GU 067679
Streptomyces peucetius ATCC 27952
Streptomyces roseum DSM 43021
Salinispora tropica CNB-440
Salinispora arenicola CNS-205

  2 and 3 show the system analysis results. 2 and 3 represent the homology of CYP154. From the results shown in FIGS. 2 and 3, it was found that the CYP154 family group is widely distributed mainly in the genus Streptomyces.

[Example 2]
Preparation of plasmid for functional expression of CYP154 gene derived from Streptomyces griseus NBRC 13350 strain in E. coli Next, plasmid pRED-CYP154 for functional expression of CYP154 gene derived from Streptomyces griseus NBRC 13350 strain was prepared.
As described above, the present inventors constructed a functional expression system of the P450 gene in Escherichia coli (Patent Document 1 and Non-Patent Document 13). This pRED vector was used for the functional expression analysis of the CYP154 gene derived from Streptomyces griseus NBRC 13350, which performs functional analysis in this study.

(1) Streptomyces Griseus NBRC 13350-derived plastid for CYP154 gene base sequence confirmation and PCR-amplified CYP154 gene base sequence confirmation To confirm the base sequence, blue-and-white selection using α complementation was performed. A possible plasmid vector for Escherichia coli, pHSG396 (chloramphenicol resistance; manufactured by TaKaRa Bio) was used as a material. In order to facilitate subsequent subcloning of the P450 gene, a multi-cloning site was newly added to construct a pHSG396NMS vector with improved versatility. After that, the CYP154 gene amplified by PCR from the genome of Streptomyces griseus NBRC 13350 strain (excluding the stop codon) was inserted into this pHSG396NMS to prepare the pHSG396NMS-CYP154 plasmid and confirm the base sequence of the CYP154 gene Went. The base sequence of the CYP154 gene linked (inserted) to pHSG396NMS is the base sequence represented by SEQ ID NO: 3 with the termination codon (TAG) removed (1,251 bp). In addition, the start codon GTG was changed to ATG, which is an easy start codon in E. coli.

1) Preparation of plasmid pHSG396NMS plasmid vector for P450 gene base sequence confirmation
After double digestion with pHSG396 plasmid vector (TakaraBio Inc.) and Hin dIII and Hin cII, over agarose electrophoresis to cut out a DNA fragment from the gel, the gel extracted using the QIAquick Gel Extraction Kit (QIAGEN Inc.) Performed (2,218 bp). Then Hin dIII- Nde I- Not I- Mfe I (Mun I) - Spe I- design synthetic DNA to have Hin cII sites which were annealed so that the duplex (0.5 μg) (sequence 4) is mixed with pHSG396 vector (0.2 μg) cleaved with Hin dIII- Hin cII, and mixed with Ligation high solution (manufactured by Toyobo) at a 1: 1 ratio, and ligation reaction at 16 ° C. for 45 minutes. The synthetic double-stranded DNA was ligated to the pHSG396 vector. Then, 1/10 volume of the ligation reaction solution is mixed in E. coli competent cells (ECOS Competent E. coli DH5α (Nippon Gene)) in ice water, left in ice water for 5 minutes, and then treated at 42 ° C for 45 seconds. And transformed. Next, an SOC medium (2% tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl ₂ , 10 mM MgSO ₄ , 20 mM glucose) was added in an amount twice that of the bacterial cell solution, 37 ° C., Incubated for 30 minutes. Then 30 μg / ml chloramphenicol, 30 μl 100 mM IPTG (isopropyl-β-thiogalactopyranoside) and 30 μl 20% X-Gal (5-bromo-4-chloro- LB medium plate coated with 3-indolyl-β-D-galactopyranoside) -DMSO (Dimethyl sulfoxide) solution (1% Bacto-tryptone, 0.5% Bacto-yeast extract, 1% NaCl, 0.02% 5 N NaOH, The bacterial cell solution incubated in 1.5% agarose was applied and cultured at 37 ° C. for 16 hours. After 16 hours of culture, to extract the plasmid, white colonies were picked and LB medium containing 30 μg / ml chloramphenicol (1% Bacto-tryptone, 0.5% Bacto-yeast extract, 1% NaCl, 0.02% 5 N NaOH) and cultured under shaking at 37 ° C. for 16 hours under the condition of 170 rpm (constant temperature shaking incubator). Plasmids were extracted from the cultured transformed Escherichia coli using QIAprep Spin Miniprep Kit (manufactured by QIAGEN). The extracted plasmid was subjected to a sequencing reaction using a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) using primers designed from the base sequence outside the multicloning site. Sequencing reaction conditions are 1 μl Ready reaction premix, 3.5 μl 5 × BigDye sequencing Buffer, primer (3.5 μl forward primer: pHSG396NMS-Fo (1 μmol / μl) or 3.5 μl reverse primer: pHSG396NMS-Rv (1 μmol / μl)), 200 ng of plasmid DNA and sterilized distilled water to 20 μl, and after heating at 96 ° C for 1 minute as a sequencing reaction, 96 ° C for 10 seconds, 50 ° C for 5 seconds, And 30 cycles of 4 minutes at 60 ° C. Thereafter, the model 3730 DNA analyzer (Applied Biosystems) was used to confirm the base sequence of the insert, and the P450 gene base sequence confirmation plasmid was designated as pHSG396NMS (2,347 bp).

<Base sequence of sequencing primer>
The base sequences of the primers used for sequence confirmation are as follows.
pHSG396NMS-Fo: 5'- AGTCACGACGTTGTA -3 '(SEQ ID NO: 6)
pHSG396NMS-Rv: 5'-CAGGAAACAGCTATGAC-3 '(SEQ ID NO: 7)

<PHSG396 insertion base sequence>
Aforementioned pHSG396 the inserted Hin dIII- Nde I- Not I- Mfe I (Mun I) - Spe I- synthetic double-stranded DNA of the Hin cII is as shown in FIG. The base sequence of the constructed pHSG396NMS is shown in SEQ ID NO: 8. The sequence shown in the upper part of FIG. 4 was inserted at the 1187th to 1219th positions in the base sequence of SEQ ID NO: 8.

2) Construction of plasmid pHSG396NMS-CYP154 for nucleotide sequence confirmation of CYP154 gene derived from Streptomyces griseus NBRC 13350 <Extraction of genomic DNA of Streptomyces griseus NBRC 13350>
Streptomyces griseus NBRC strain 13350 (approx. 1 g) added with 300 μl STE Buffer (100 mM NaCl, 10 mM Tris · HCl (pH 8.0), 1 mM EDTA (pH 8.0)) and suspended Thereafter, the mixture was centrifuged at 8,000 rpm for 15 minutes at 4 ° C., and the supernatant was removed. The bacterial cells were resuspended in 300 μl of STE Buffer, and DNase was inactivated by incubation at 68 ° C. for 15 minutes. The supernatant was removed by centrifuging at 8,000 rpm for 15 minutes at 4 ° C. 600 μl of lysis buffer (50 mM glucose, 25 mM Tris · HCl (pH 8.0), 10 mM) containing lysozyme at a final concentration of 5 mg / ml and 60 μl RNase A (10 mg / ml) EDTA (pH 8.0)) was added, resuspended, and incubated at 37 ° C. for 30 minutes. 12 μl of Proteinase K (20 mg / ml) (TaKaRa Bio) was added, mixed, and further incubated at 37 ° C. for 10 minutes. 6 mg of N-Lauroylsarcosine · Na was added, and after mixing, the cells were incubated at 37 ° C. for 16 hours to lyse the cells. Add 600 μl of phenol / chloroform (TE Buffer (10 mM Tris · HCl (pH 8.0), 1 mM EDTA (pH 8.0)) saturated phenol: (chloroform: isoamyl alcohol = 24: 1) = 1: 1) The mixture was mixed for 5 minutes and then centrifuged at 9,000 rpm for 15 minutes at 10 ° C. The upper layer was recovered, 400 μl of phenol / chloroform was added, mixed for 5 minutes, and then centrifuged at 10 ° C. for 15 minutes at 9,000 rpm. The upper layer was collected again, 400 μl of phenol / chloroform was added, mixed for 5 minutes, and then centrifuged at 9,000 rpm for 15 minutes at 10 ° C. Collect the upper layer, add 1/10 volume of 3 M sodium acetate, 1/10 volume of 125 mM EDTA, and 3 volumes of 100% ethanol, and centrifuge at 4 ° C for 20 minutes at 10,000 rpm. Excluded. 500 μl of 70% ethanol was added, centrifuged at 10,000 rpm for 10 minutes at 4 ° C., and the supernatant was removed well. 200 μl of TE Buffer was added, and the precipitated genomic DNA was dissolved at 4 ° C. for 24 hours.

<Production of pHSG396NMS-CYP154>
The pHSG396NMS plasmid vector was double-digested with the restriction enzymes Nde I and Eco RI at 37 ° C. for 4 hours. Thereafter, 1.5% agarose electrophoresis was performed, pHSG396NMS cleaved with Nde I and Eco RI was excised, and gel extraction was performed using QIAquick Gel Extraction Kit. This DNA was used as vector DNA.

Next, CYP154 gene derived from Streptomyces griseus NBRC 13350 strain was amplified using PCR. For PCR amplification, a primer designed to give an Nde I sequence to the N-terminal side of the CYP154 gene and an Eco RI sequence to the C-terminal side for ligation to the pHSG396NMS plasmid vector was used. In addition, for expression as a chimeric protein with P450 reductase described later, the C-terminal primer was designed to exclude the stop codon. PCR amplification reaction was performed using 25 μl 2 × PrimeSTAR MaxPremix (TaKaRa Bio), 1 μl forward primer: SGR1085_F (10 μmol / μl), 1 μl reverse primer: SGR1085_R (10 μmol / μl), 0.5 μl Streptomyces griseus NBRC 13350 genomic DNA (25 ng), 2.5 μl DMSO (dimethyl sulfoxide), and 20 μl sterile distilled water were mixed and heated at 98 ° C. for 2 minutes as a PCR reaction. Second, 5 cycles of 55 ° C. for 10 seconds and 72 ° C. for 15 seconds. Thereafter, 30 cycles of 98 ° C. for 10 seconds, 62 ° C. for 5 seconds, and 72 ° C. for 15 seconds were performed to amplify the CYP154 gene. After completion of the PCR amplification reaction, 1.5% agarose electrophoresis was performed using 2 μl of the reaction solution, and it was confirmed that a PCR amplification product having a size of CYP154 gene (1,266 bp) was obtained. After confirming the amplification of the CYP154 gene, the PCR amplification product was purified using MinElute PCR Purification Kit (QIAGEN). The purified PCR amplification product was subjected to 1.5% agarose electrophoresis, a band having a CYP154 gene size was excised, and gel extraction was performed using the QIAquick Gel Extraction Kit. The extracted PCR amplification product was subjected to double digestion with restriction enzymes Nde I and Eco RI at 37 ° C. for 4 hours. Thereafter, 1.5% agarose electrophoresis was performed, and gel extraction was performed using QIAquick Gel Extraction Kit. This PCR amplification product was used as insert DNA.

Subsequently, the vector DNA and the insert DNA were mixed at a molar ratio of 3: 1, and an equal amount of Ligation Convenience Kit (manufactured by Nippon Gene) was added and mixed, and then ligated at 16 ° C. for 15 minutes. In ice water, 2.5 μl of the ligation reaction solution was mixed with 25 μl of E. coli competent cell (ECOS Competent E. coli DH5α). This was left in ice water for 5 minutes and then treated at 42 ° C. for 45 seconds to transform competent cells. Thereafter, 25 μl of SOC medium was added to the obtained transformant, and the mixture was allowed to stand at 37 ° C. for 30 minutes, and then contained 30 μg / ml chloramphenicol, 30 μl 100 mM IPTG and 30 μl 20 A LB plate coated with% X-Gal-DMSO solution was spread. Thereafter, the cells were cultured at 37 ° C. for 16 hours to form colonies. Next, the resulting white colonies were picked and plasmid inserts were confirmed using the colony direct PCR method. Colony direct PCR is performed using 0.125 μl SpeedSTAR HS DNA polymelase (TaKaRa Bio), 2.5 μl 10 × Fast Buffer I (TaKaRa Bio), 0.7 μl forward primer: SGR1085_F (10 pmol / μl), 0.7 μl Reverse primers: SGR1085_R (10 pmol / μl), 1.25 μl DMSO, 2 μl dNTP Mixture (2.5 mM each), and 17.725 μl of sterile distilled water in a PCR reaction solution. The PCR reaction for 30 seconds, 64 ° C. for 10 seconds, and 72 ° C. for 10 seconds was repeated 30 cycles. After the reaction, 3 μl of the PCR reaction solution was subjected to 1.5% agarose electrophoresis to select colonies into which the insert DNA had been inserted. The colonies with confirmed inserts are then inoculated into 10 ml of LB medium containing 30 μg / ml chloramphenicol and shaken under conditions of 170 rpm (constant temperature shaking incubator) at 37 ° C for 16 hours. Cultured. Plasmids were extracted from the cultured transformed E. coli using the QIAprep Spin Miniprep Kit. The extracted plasmid was subjected to a sequencing reaction using BigDye Terminator v3.1 Cycle Sequencing Kit using primers designed from the base sequence outside the multicloning site, and the base sequence of the insert DNA was confirmed. Since the GC content of the CYP154 gene is high, 5% DMSO was added to the reaction solution and the sequencing reaction was performed in the same manner as in the preparation of pHSG396NMS. The plasmid in which insertion of the CYP154 gene was confirmed was designated as pHSG396NMS-CYP154.

<Base sequence of primers used for PCR amplification of CYP154 gene>
The base sequences of the aforementioned primers are as follows.
SGR1085_F: 5'-TACCATATGAACTGCCCGCACACTGC-3 '(SEQ ID NO: 9)
SGR1085_R: 5'-TACGAATTCTCAGCCCAGGAGGACCG-3 '(SEQ ID NO: 10)
The amino acid sequence of CYP154 (417 amino acids) is shown in SEQ ID NO: 1.

(2) Preparation of pRED-CYP154 plasmid for functional expression of CYP154 gene derived from Streptomyces griseus NBRC 13350
The pRED vector (SEQ ID NO: 11) was subjected to double digestion with restriction enzymes Nde I and Eco RI at 37 ° C. for 3 hours. After the treatment, 0.8% agarose electrophoresis was performed, the band was cut out, and gel extraction was performed using QIAquick Gel Extraction Kit. This was used as a vector DNA for enzyme function expression. The plasmid pHSG396NMS-CYP154 was double digested with the restriction enzymes Nde I and Eco RI at 37 ° C. for 3 hours. After restriction enzyme treatment, it was subjected to 0.8% agarose electrophoresis, and gel extraction was performed using QIAquick Gel Extraction Kit. This was used as insert DNA. Subsequently, the vector DNA and the insert DNA were mixed at a molar ratio of 3: 1, mixed with an equal amount of Ligation Convenience Kit, and then ligated at 16 ° C. for 15 minutes. In ice water, 2.5 μl of the ligation reaction solution was added to 25 μl of E. coli competent cells (ECOS Competent E. coli DH5α), left in ice water for 5 minutes, and then transformed by treatment at 42 ° C. for 45 seconds. Thereafter, 25 μl of SOC medium was added and spread on an LB plate containing 100 μg / ml ampicillin. This was cultured at 37 ° C. for 16 hours to form colonies. Next, the resulting colonies were picked, and insertion of the insert sequence into the plasmid was confirmed using the colony direct PCR method. The conditions for colony direct PCR were as follows: 0.125 μl SpeedSTAR HS DNA polymelase, 2.5 μl 10 × Fast Buffer I, 0.7 μl forward primer: SGR1085_F (10 pmol / μl), 0.7 μl reverse primer: SGR1085_R ( 10 pmol / μl), 1.25 μl DMSO, 2 μl dNTP Mixture (2.5 mM each), and 17.725 μl sterile distilled water, and mix as a PCR reaction at 98 ° C for 5 seconds, 64 ° C for 10 seconds, and 30 cycles of 10 seconds at 72 ° C were performed. After the reaction, 3 μl of the PCR reaction solution was subjected to 1.5% agarose electrophoresis to confirm insertion of the insert sequence. Thereafter, in order to extract the plasmid, the colonies whose inserts were confirmed were inoculated into 10 ml of LB medium containing 100 μg / ml ampicillin and cultured with shaking at 37 ° C. for 16 hours. Plasmids were extracted from the cultured transformed Escherichia coli using QIAprep Spin Miniprep Kit. The target CYP154 function expression plasmid (named CYP154-Red) is 7,631 bp in size. As a result of the insert check, 1,251 bp amplification of the inserted CYP154 gene was confirmed, confirming the production of CYP154-Red did.

[Example 3]
Substrate screening CYP154 derived from Streptomyces griseus NBRC 13350 strain has not been analyzed for functions, and the function of Streptomyces in the metabolic pathway was unknown. Therefore, it was necessary to perform substrate screening. In order to efficiently perform substrate screening and find the catalytic function of CYP154, the present inventors made 96-well plates containing various substrate candidate substances as organic low-molecular compounds for screening. The compounds to be converted were searched for, and the converted substrate compounds were subjected to conversion tests of analogs thereof to identify the reaction catalyzed by CYP154 and the substrates capable of being catalyzed.

(1) Introduction of CYP154-Red into E. coli of BLR (DE3) strain and confirmation of functional expression of the transgene Before the substrate screening, the CYP154-Red plasmid prepared in Example 2 was expressed in E. coli BLR (DE3). Then, it was confirmed whether or not an active form of a fusion protein (hereinafter referred to as CYP154-Red) of CYP154 and P450RhF reductase domain encoded thereby was produced. The details are shown below. P450RhF is a cytochrome P450 monooxygenase derived from Rhodococcus NCIMB 9784.

0.5 μl of CYP154-Red plasmid (about 200 ng / μl) was added to 5 μl of BLR (DE3) E. coli competent cell (Novagen) and left in ice water for 30 minutes. Thereafter, the mixture was heated at 42 ° C. for 45 seconds, cooled in ice water for 3 minutes, and 25 μl of SOC medium was added to the transformation solution. The obtained medium containing the transformant was spread on an LB plate containing 100 μg / ml carbenicillin and then cultured at 37 ° C. for 16 hours. Three colonies each were transferred to a 50 ml tube containing 5 ml of 2 × YT medium containing carbenicillin (final concentration 100 μg / ml), and incubated at 37 ° C for 4 hours at 300 rpm (reciprocating shaker incubator). Under the conditions, shaking culture was performed to prepare a preculture solution. Subsequently, it contains carbenicillin (final concentration 100 μg / ml), 5-aminolevulinic acid (5-ALA) (final concentration 80 μg / ml), Fe (NH ₄ ) ₂ (SO ₄ ) ₂ (final concentration 0.1 mM) 50 ml of 2 × YT medium was prepared in three 500 ml baffled Erlenmeyer flasks, and 50 μl of the preculture was transplanted. The cells were cultured at 37 ° C. for 3 hours at 150 rpm (rotary incubator) until the OD ₆₀₀ reached about 0.8. Thereafter, IPTG (final concentration 0.05 mM) was added, and the culture was continued for 21 hours under the conditions of 20 ° C. and 150 rpm (rotary incubator) to express the transgene to obtain a main culture solution.

After completion of the culture, OD ₆₀₀ was measured, the culture solution was transferred to a 50 ml centrifuge tube, and centrifuged at 5,000 rpm, 4 ° C. for 20 minutes. After removing the culture supernatant, the wet cell weight is measured, and 1/5 volume of phosphate buffer (50 mM sodium phosphate buffer, glycerol (final concentration 10%), pH 7.2) is used. In addition to the cells, the cells were completely resuspended with vortex. Dispense 2 ml of each suspension into three 15 ml Falcon tubes, 100 μl Bug Buster 10 × Protein Extraction Reagent (Novagen), 1 μl Benzonase Nuclease (Novagen), and Lysozyme (final concentration 200 μg / ml) was added, and the cells were lysed and disrupted by gently rotating and mixing at room temperature for 20 minutes. Centrifugation was performed at 15,000 rpm and 4 ° C. for 20 minutes, and the soluble fraction of the supernatant was recovered. A small amount of dithionate was added to the collected soluble fraction, and the mixture was incubated in ice water for 5 minutes, and then transferred to a half-volume 1.5 ml Eppendorf tube. Using a spectrophotometer, the blank was measured with a sample that was not bubbled with carbon monoxide at intervals of 0.5 nm from the absorption wavelength of 400 nm to 500 nm, and then the absorption spectrum of the other sample that was bubbled with carbon monoxide was measured. (N = 3).

The OD ₆₀₀ and the wet cell weight of the three main cultures were OD ₆₀₀ = 3.09 for the first, wet cell weight = 446.8 mg, the second for OD ₆₀₀ = 3.03, wet cell weight = 446.1 mg, and 3 This time, OD ₆₀₀ = 3.04, wet cell weight = 448.2 mg. As a result of measuring the CO difference spectrum, an active CYP154 fusion protein was expressed because the P450 450, which was confirmed to bind CO to the heme domain, was detected in all three samples. It was confirmed. Also based on the method of Omura and Sato (T. Omura and R. Sato, The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature. J. Biol. Chem. 239: 2370-2378, 1964). The concentration of P450 (CYP154) in the main broth calculated from the CO difference spectrum was 474.3 μg / L for the first, 651.1 μg / L for the second, and 447.9 μg / L for the third. It was found that about 524.4 μg of active CYP154 fusion protein was expressed in 1 L of the main culture.

(2) Bioconversion method using Escherichia coli BLR (DE3) retaining CYP154-Red In substrate screening, a suspension of E. coli BLR (DE3) strain introduced with plasmid CYP154-Red is used as a unique compound substrate for screening. In addition, the reaction product is added to the plate, and then the conversion product is extracted from the reaction solution, followed by HPLC (High Performance Liquid Chromatography) -PDA (PhotoDiode Array Detector) analysis for bioconversion of the entire substrate. I checked my profile. The details are shown below.

(a) Introduction of CYP154-Red into E. coli of BLR (DE3) strain
0.5 μl of CYP154-Red plasmid (about 200 ng / μl) was added to 5 μl of E. coli BLR (DE3) strain competent cells and left in ice water for 30 minutes. Thereafter, the mixture was heated at 42 ° C. for 45 seconds, cooled in ice water for 3 minutes, and 25 μl of SOC medium was added to the transformation solution. The obtained medium containing the transformant was spread on an LB plate containing 100 μg / ml carbenicillin and then cultured at 37 ° C. for 16 hours. Three colonies each were transferred to a 50 ml test tube containing 5 ml of 2 × YT medium containing 100 μg / ml carbenicillin (final concentration 100 μg / ml), and 37 rpm, 4 hours, 300 rpm (reciprocating The mixture was shaken under the conditions of a shaking incubator to prepare a preculture solution. Subsequently, it contains carbenicillin (final concentration 100 μg / ml), 5-aminolevulinic acid (5-ALA) (final concentration 80 μg / ml), Fe (NH ₄ ) ₂ (SO ₄ ) ₂ (final concentration 0.1 mM) 50 ml of 2 × YT medium was prepared in two 500 ml baffled Erlenmeyer flasks, and 50 μl of the preculture was transplanted. The cells were cultured at 37 ° C. for 3 hours at 150 rpm (rotary incubator) until the OD ₆₀₀ reached about 0.8. Thereafter, and IPTG (final concentration 0.05 mM) was added, and the culture was continued for 21 hours under the conditions of 20 ° C. and 150 rpm (rotary incubator) to express the transgene, and used as the main culture solution.

(b) Substrate conversion test (bioconversion reaction)
After completion of the culture, OD ₆₀₀ was measured, the culture solution was transferred to a 50 ml centrifuge tube, and centrifuged at 5,000 rpm, 4 ° C. for 20 minutes. After removing the culture supernatant, the wet cell weight is measured, and 1/5 volume of phosphate buffer (50 mM sodium phosphate buffer, glycerol (final concentration 10%), pH 7.2) is used. In addition to the cells, the cells were completely resuspended with vortex. Next, add 5 μl of various 100 mM substrate candidate-DMSO solutions to each well of a 96-well deep well plate (PP-MASTER BLOCK 2ML, 128,0 / 85 MM 96WELL STERIL, Greiner bio-one). Each was added, and 500 μl of each cell suspension was dispensed. Then, seal the well with a sealing mat (Flexible Sealing Mat for 2.2 ml Deep Well Plate, manufactured by IWAKI) or a CO ₂ permeable sheet (BREATHseal, manufactured by Greiner bio-one), and at 1,500 rpm (product) The conversion test was conducted under the conditions of the name MS 3 basic (manufactured by IKA).

(c) HPLC-PDA analysis of conversion product After completion of the conversion test, the solution in each well was pipetted out into a 2.0 ml Eppendorf tube containing 200 μl of saturated saline and 500 μl of ethyl acetate. After vortexing for 10 minutes, the mixture was centrifuged at 15,000 rpm, 25 ° C. for 15 minutes to recover the ethyl acetate layer. After adding 500 μl of ethyl acetate again to the aqueous layer and vortexing for 10 minutes, the mixture was centrifuged at 15,000 rpm, 25 ° C. for 15 minutes to recover the ethyl acetate layer. The combined ethyl acetate layer was concentrated under reduced pressure, and then redissolved in 200 μl of ethyl acetate to prepare an HPLC analysis sample. HPLC analysis conditions were as follows.

HPLC system: Waters e2695 Separation Module (manufactured by waters)
Detector: Waters2998 Photodiode array detector (PDA) detector (manufactured by waters)
Column: Waters Xterra MS C18 5μm ID4.6 mm x 100 mm (made by waters)
Mobile phase: 0-3 min: 5% CH ₃ CN / H ₂ O (0.1% TFA)
3-38 min: 5% CH ₃ CN / H ₂ O (0.1% TFA)
→ 100% CH ₃ CN / H ₂ O (0.1% TFA) (Linear gradient)
38-43 min: 100% CH ₃ CN / H ₂ O (0.1% TFA)
Column temperature: 35 ° C
Sample temperature: 20 ℃
Flow rate: 1 ml / min
Detection wavelength range: PDA (200-450 nm)
Analytical sample: 10 μl
The conversion rate was calculated as the ratio of the area of the product to the total area of the product and the substrate from the peak area value of each of the substrate extracted by λmax and the conversion product.

(3) Results of bioconversion using E. coli BLR (DE3) carrying CYP154-Red
As a result of HPLC-PDA analysis, 1,4-androstadiene-3,17-dione (1,4-Androstadiene-3,17-dione), 11α-hydroxyprogesterone (11α-Hydroxyprogesterone), 1-dehydro-17α- Methyltestosterone (1-Dehydro-17α-methyltestosterone), 4-Pregnane-3,11,20-trione (4-Pregnane-3,11,20-trione), Adrenosterone (Adrenosterone), Corticosterone (Corticosterone) , Dehydroepiandrosterone, deoxycorticosterone, methyltestosterone, pregnenolone, progesterone, testosterone and Δ4-androstene-3,17-dione (Δ4) It was revealed that the bioconversion reaction proceeds for steroidal compounds with substituents such as -Androstene-3,17-dione). The bioconversion results are shown in Table 2. On the other hand, it is reported that CYP154H1 derived from Thermobifida fusca can be converted (Non-patent Document 18) Ethylbenzene, n-Propylbenzene, thioanisole and Thioanisole. A bioconversion reaction was performed using Indole, but the reaction did not proceed at all. At this time, in the experiment using ethylbenzene (Ethylbenzene) and normal propylbenzene (n-Propylbenzene), detection was performed using gas chromatography.

[Example 4]
Structure analysis of conversion product A part of the substrate that was found to be converted in the substrate screening of Example 3 was subjected to a scaled-up substrate conversion test. The resulting conversion product was analyzed by MS (Mass Spectrometry) and NMR. Structural analysis was performed by (Nuclear Magnetic Resonance Spectroscopy).

(1) Scaled-up substrate conversion test (bioconversion reaction)
Three colonies of E. coli BLR (DE3) strain into which CYP154-Red was introduced were suspended in a 50 ml glass test tube to which 5 ml of 2 × YT medium containing carbenicillin (final concentration 100 μg / ml) was added. The culture solution was shaken at 37 ° C., 300 rpm for 4 hours until the OD ₆₀₀ reached about 0.8 (reciprocating shake incubator), and used as a preculture solution. Carbenicillin (final concentration 100 μg / ml), glycerol (final concentration 0.4%), 5-ALA (final concentration 80 μg / ml), Fe (NH ₄ ) ₂ (SO ₄ ) ₂ (final concentration 0.1 mM) Two 5 L baffled Erlenmeyer flasks containing 1 L of TB medium were prepared, and 1 ml each of the preculture was transplanted. After culturing at 37 ° C and 150 rpm until the OD ₆₀₀ reaches about 0.8 (rotary incubator), add IPTG (final concentration 0.05 mM) and 10 μl of antifoam SI (manufactured by Wako Pure Chemical Industries, Ltd.) The culture was continued at 20 ° C. and 150 rpm for 20 hours to express the transgene. After completion of the culture, the culture solution was transferred to a 500 ml centrifuge tube and centrifuged at 6,000 rpm, 4 ° C. for 20 minutes. After removing the supernatant, the cells were resuspended in 400 ml phosphate buffer (50 mM sodium phosphate buffer, glycerol (final concentration 10%), pH 7.2). Next, the whole amount of the cell suspension was transferred to a 5 L baffled Erlenmeyer flask, and DMSO-dissolved substrate (final concentration 1 mM) was added to prepare a conversion test solution. The conversion test solution was subjected to a conversion test under conditions of 150 rpm at 25 ° C. for 48 hours with the mouth of the flask sealed with a CO ₂ permeable sheet (BREATHseal, manufactured by Greiner bio-one).

(2) Method for structural analysis of conversion products
(a) Purification of Conversion Product After adding 200 ml of saturated saline to the conversion test solution, 600 ml of ethyl acetate was added, and then extracted with stirring at room temperature for 20 minutes using a rotor and a stirrer. The tube was transferred to an organic solvent-resistant centrifuge tube and centrifuged at 8,000 rpm, 30 ° C. for 20 minutes. After recovering the ethyl acetate layer, an equal amount of ethyl acetate was added to the aqueous layer, and extraction, centrifugation, and recovery of the ethyl acetate layer were again performed in the same manner. The collected ethyl acetate layers were combined and dehydrated with anhydrous sodium sulfate, and then subjected to silica gel column chromatography (ID 10 × 200 mm, Silica Gel 60 (Merck)), and then concentrated under reduced pressure to obtain a crude product. . The crude product was subjected to preparative HPLC, and the conversion product was collected. The fraction containing the target product was subjected to ice-cooling of the liquid obtained by distilling off acetonitrile under reduced pressure, followed by centrifugation (0 ° C., 3500 rpm, 15 min), and the supernatant was removed. Furthermore, distilled water is added to the residue, ice-cooled, centrifuged (0 ° C, 3500 rpm, 15 min), and the operation of removing the supernatant is repeated 3 times. did. Preparative HPLC was performed under the following conditions.

HPLC system: Waters 600 Separation Module (manufactured by waters)
Detector: Waters996 Photodiode array detector (PDA) detector (made by waters)
Column: DOCOSIL-B ID 20 mm x 250 mm (made by Senshu Kagaku) + DOCOSIL-B ID 10 mm x 30 mm (made by Senshu Kagaku)
Mobile phase: 0-5 min: 27% CH ₃ CN / H ₂ O (0.1% TFA)
5-35 min: 27% CH ₃ CN / H ₂ O (0.1% TFA)
→ 73% CH ₃ CN / H ₂ O (0.1% TFA) (Linear gradient)
36-50 min: 100% CH ₃ CN / H ₂ O (0.1% TFA)
Column temperature: Room temperature Flow rate: 5 ml / min
Detection wavelength range: PDA (200-500 nm)
Analytical sample: 150 μl

(b) Structure identification by conversion product MS and NMR The structure of the purified product was analyzed by LC-ESI-TOF-MS (JMS-T100LC JEOL) and NMR (500 MHz, JNM-A500 JEOL or 500 MHz, INOVA-500AS Varian) The spectrum data obtained by the above was analyzed and identified.

(3) Conversion product identification results
(a) Progesterone

The substrate Progesterone was detected by HPLC at a retention time of 24.2 minutes. As a result of HPLC-PDA analysis, a peak of 29.1% conversion product was detected at a retention time of 17.59 minutes. This compound was purified by preparative HPLC to give Compound 1 (67.1 mg). Compound 1 LC-ESI-TOF-MS analysis for m / z 329.21189 (MH) ^- determined from giving ion peak, the molecular formula C ₂₁ H ₃₀ O ₃ (the theoretical 329.21167 C ₂₁ H ₂₉ O ₃₎ did. As a result of further detailed NMR analysis [ ¹ H, ¹³ C, ¹ H- ¹ H COZY, gHMQC, gHMBC, NOESY], compound 1 was identified as 16α-hydroxyprogesterone (16α-Hydroxyprogesterone). The structural formula of Compound 1 (16α-hydroxyprogesterone) is shown below.

For reference, Table 3 shows the attribution data of Compound 1 by ¹ H, ¹³ C, ¹ H- ¹ H COZY, gHMQC, gHMBC, and NOESY.

(b) 1,4-Androstadiene-3, 17-dione

The substrate 1,4-androstadiene-3,17-dione was detected by HPLC at a retention time of 18.8 minutes. As a result of HPLC-PDA analysis, a peak of 78.4% conversion product was detected at a retention time of 15.08 minutes. This compound was purified by preparative HPLC to give Compound 2 (38.0 mg). Compound 2 with LC-ESI-TOF-MS analysis for m / z 229.16450 (MH) ^- determined from giving ion peak, the molecular formula C ₁₉ H ₂₄ O ₃ (the theoretical 229.16472 C ₁₉ H ₂₃ O ₃₎ did. As a result of further detailed NMR analysis [ ¹ H, ¹³ C, ¹ H- ¹ H gDQFCOSY, gHSQC, gHMBC, NOESY], Compound 2 was converted to 16α-hydroxy-1,4-androstadiene-3, 17-dione (16α -Hydroxy-1, 4-androstadiene-3,17-dione). The structural formula of Compound 2 (16α-hydroxy-1,4-androstadiene-3,17-dione) is shown below.

For reference, the assignment data of compound 2 by ¹ H, ¹³ C, ¹ H- ¹ H gDQFCOSY, gHSQC, gHMBC, NOESY is shown in Table 4.

(c) Δ4-Androstene-3, 17-dione

The substrate Δ4-Androstene-3, 17-dione was detected by HPLC at a retention time of 20.3 minutes. As a result of HPLC-PDA analysis, a peak of 45.9% conversion product was detected at a retention time of 16.1 minutes. This compound was purified by preparative HPLC to give Compound 3 (40.2 mg). Compound LC-ESI-TOF-MS analysis 3 to m / z 301.18119 (MH) ^- determined from giving ion peak, the molecular formula C ₁₉ H ₂₆ O ₃ (the theoretical 301.18037 C ₁₉ H ₂₅ O ₃₎ did. As a result of further detailed NMR analysis [ ¹ H, ¹³ C, ¹ H- ¹ H gDQFCOSY, gHSQC, gHMBC, NOESY], compound 3 was converted into 16α-hydroxy-Δ4-androstene-3, 17-dione (16α-Hydroxy- Δ4-androstene-3, 17-dione). The structural formula of Compound 3 (16α-hydroxy-Δ4-androstene-3,17-dione) is shown below.

For reference, Table 5 shows the attribution data of Compound 3 by ¹ H, ¹³ C, ¹ H- ¹ H gDQFCOSY, gHSQC, gHMBC, and NOESY.

(d) 4-Pregnene-3, 11, 20-trione

The substrate 4-Pregnene-3, 11, 20-trione was detected by HPLC at a retention time of 19.7 minutes. As a result of HPLC-PDA analysis, a peak of 32.8% conversion product was detected at a retention time of 14.9 minutes. This compound was purified by preparative HPLC to give Compound 4 (32.0 mg). Compound LC-ESI-TOF-MS analysis 4 to m / z 343.19125 (MH) ^- determined from giving ion peak, the molecular formula C ₂₁ H ₂₈ O ₄ and (theoretical 343.19093 C ₂₁ H ₂₇ O ₄₎ did. As a result of further detailed NMR analysis [ ¹ H, ¹³ C, ¹ H- ¹ H COZY, gHMQC, gHMBC, NOESY], compound 4 was converted to 16α-hydroxy-4-pregnene-3, 11, 20-trione (16α-Hydroxy -4-pregnene-3, 11, 20-trione). The structural formula of Compound 4 (16α-hydroxy-4-pregnene-3, 11, 20-trione) is shown below. As a result of CAS database analysis, Compound 4 was found to be a novel substance.

For reference, Table 6 shows the attribution data of Compound 4 by ¹ H, ¹³ C, ¹ H- ¹ H COZY, gHMQC, gHMBC, and NOESY.

(e) Testosterone

The substrate Testosterone was detected by HPLC at a retention time of 19.6 minutes. As a result of HPLC-PDA analysis, a peak of 95.4% conversion product was detected at a retention time of 14.8 minutes. This compound was purified by preparative HPLC to give Compound 5 (36.7 mg). Compound LC-ESI-TOF-MS analysis 5 to m / z 303.19684 (MH) ^- determined from giving ion peak, the molecular formula C ₁₉ H ₂₈ O ₃ (the theoretical 303.19602 C ₁₉ H ₂₇₈ O ₃₎ did. As a result of further detailed NMR analysis [ ¹ H, ¹³ C, ¹ H- ¹ H COZY, gHMQC, gHMBC, NOESY], Compound 5 was identified as 16α-hydroxytestosterone. The structural formula of Compound 5 (16α-hydroxytestosterone) is shown below.

For reference, the assignment data of compound 5 by ¹ H, ¹³ C, ¹ H- ¹ H COZY, gHMQC, gHMBC, NOESY is shown in Table 7.

(f) Deoxycorticosterone

The substrate Deoxycorticosterone was detected by HPLC at a retention time of 20.0 minutes. As a result of HPLC-PDA analysis, a peak of 49.4% conversion product was detected at a retention time of 15.2 minutes. This compound was purified by preparative HPLC to give Compound 6 (51.9 mg). LC-ESI-TOF-MS analysis with compound 6 in the m / z 345.20544 (MH) ^- determined from giving ion peak, the molecular formula C ₂₁ H ₃₀ O ₄ and (theoretical 345.20658 C ₂₁ H ₂₉ O ₄₎ did. As a result of further detailed NMR analysis [ ¹ H, ¹³ C, ¹ H- ¹ H COZY, gHMQC, gHMBC, NOESY], compound 6 was identified as 16α-hydroxy-deoxycorticosterone (16α-Hydroxy-deoxycorticosterone). The structural formula of Compound 6 (16α-hydroxy-deoxycorticosterone) is shown below.

For reference, the assignment data of compound 6 by ¹ H, ¹³ C, ¹ H- ¹ H COZY, gHMQC, gHMBC, NOESY is shown in Table 8.

(g) Dehydroepiandrosterone

The substrate Dehydroepiandrosterone was detected by HPLC at a retention time of 20.6 minutes. As a result of HPLC-PDA analysis, a peak of 14.1% conversion product was detected at a retention time of 16.0 minutes. This compound was purified by preparative HPLC to give Compound 7 (38.0 mg). LC-ESI-TOF-MS analysis with compound 7 in the m / z 303.19484 (MH) ^- determined from giving ion peak, the molecular formula C ₁₉ H ₂₈ O ₃ (the theoretical 303.19602 C ₁₉ H ₂₇ O ₃₎ did. As a result of further detailed NMR analysis [ ¹ H, ¹³ C, ¹ H- ¹ H COZY, gHMQC, gHMBC, NOESY], Compound 7 was identified as 16α-hydroxy-dehydroepiandrosterone. The structural formula of Compound 7 (16α-hydroxy-dehydroepiandrosterone) is shown below.

For reference, Table 9 shows the attribution data of Compound 7 by ¹ H, ¹³ C, ¹ H- ¹ H COZY, gHMQC, gHMBC, and NOESY.

(h) Adrenosterone

The substrate Adrenosterone was detected by HPLC at a retention time of 17.1 minutes. As a result of HPLC-PDA analysis, a peak of 41.4% conversion product was detected at a retention time of 13.8 minutes. This compound was purified by preparative HPLC to give Compound 8 (21.4 mg). Compound 8 LC-ESI-TOF-MS analysis for m / z 315.16045 (MH) ^- determined from giving ion peak, the molecular formula C ₁₉ H ₂₄ O ₄ and (theoretical 315.15963 C ₁₉ H ₂₄ O ₃₎ did. As a result of further detailed NMR analysis [ ¹ H, ¹³ C, ¹ H- ¹ H COZY, gHMQC, gHMBC, NOESY], Compound 8 was identified as 16α-hydroxy-adrenosterone (16α-Hydroxy-adrenosterone). The structural formula of Compound 8 (16α-hydroxy-adrenosterone) is shown below. As a result of CAS database analysis, Compound 8 was found to be a novel compound.

For reference, the assignment data of compound 8 by ¹ H, ¹³ C, ¹ H- ¹ H COZY, gHMQC, gHMBC, NOESY is shown in Table 10.

(4) Consideration of the catalytic function of CYP154 As a result of substrate screening, CYP154 derived from Streptomyces griseus NBRC 13350 was regioselectively and stereotactically located at the 16α position of the D ring of steroid compounds having various substituents. It has been found to catalyze the reaction of selective hydroxylation. In particular, the catalytic reaction by CYP154, which synthesizes difficult chemical synthetic compounds such as 16α-hydroxy-4-pregnene-3, 11, 20-trione and 16α-hydroxyadrenosterone, which are novel compounds, The possibility of catalyzing the synthesis of compounds that are not in the category of screening for seed and lead compounds in the research and development stage is expected, and the applicability of the catalytic function is expected.
The catalytic ability of CYP154, for example, converts 1 mM testosterone by about 70%, so CYP154 derived from Nocardia farcinica IFM 10152 strain converted 0.1% testosterone by about 50% (non- Compared with Patent Document 17), it becomes clear that it is more than 10 times higher and is expected to be used at the industrial production level.

  The production method of the present invention is useful in the creation of seed-lead compounds in drug discovery, preparation of metabolites, shortening of production processes that require long steps, and synthesis of compounds that are difficult to synthesize by chemical synthesis.

Claims (6)

A protein belonging to family 154 (CYP154) of cytochrome P450 functions as a monooxygenase in the presence of an electron transfer protein, or a fusion protein of a protein belonging to CYP154 and an electron transfer protein functions as a monooxygenase and has a substituent. A method for producing a steroid compound hydroxylated at the 16α position , wherein the hydroxyl group is regioselectively and stereoselectively introduced into the D ring 16α position of the steroid skeleton by acting on the steroid compound ,
The protein belonging to CYP154 is
(A) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1, or
(B) a polypeptide comprising an amino acid sequence in which 1 to 10 amino acid sequences are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 1 and functioning as a cytochrome P450 monooxygenase
And
The electron transfer protein is
(C) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2
(D) a polypeptide comprising an amino acid sequence in which 1 to 10 amino acids are added, deleted or substituted in the amino acid sequence described in SEQ ID NO: 2 and having reductase activity
The manufacturing method which is. A transformant transformed with a gene encoding a fusion protein between the protein and the electron transfer protein belonging to the CYP154, by the coexistence of a steroid compound having a substituent group, the fusion proteins, having the substituent The method according to claim 1, wherein the method is applied to a steroid compound. A steroid compound having a substituent is represented by the following general formula (1):
(In the above formula, R ^{1 to} R ⁶ , -A-A'-, and -B-B'-B ''-are the following (A) or (B);
(A) R ¹ is a hydroxyl group, an oxo group or an acetoxy group,
R ² is hydrogen, a hydroxyl group, an acetoxy group or an oxo group,
R ³ is a hydroxyl group, acetyl group, hydroxymethylcarbonyl group, acetoxymethylcarbonyl group, 2-hydroxyethyl group, 1,2-dihydroxyethyl group or hydroxymethylcarbonyl group,
R ⁴ is hydrogen or a methyl group,
-A-A'is, -CH ₂ -CH ₂ - or a -CH = CH-,
-B-B'-B '' - _{is, -CH 2 -CH-CH 2 -} , - CH 2 -C = CH -, - CH = C-CH 2 - or -CH = a C-CHF-,
R ⁵ is hydrogen, a methyl group, a hydroxymethyl group, or a formyl group,
R ⁶ is a methyl group, a hydroxymethyl group, or a formyl group, or
(B) R ³ and R ⁴ together are an oxo group,
R ¹ is a hydroxyl group, an oxo group or an acetoxy group,
R ² is hydrogen, a hydroxyl group or an oxo group,
-A-A'is, -CH ₂ -CH ₂ - or a -CH = CH-,
-B-B'-B '' - _{is, -CH 2 -CH-CH 2 -} , - CH 2 -C = CH -, - CH = C-CH 2 - or -CH = a C-CHF-,
R ⁵ is hydrogen, a methyl group, a hydroxymethyl group, or a formyl group,
The method according to claim 1 or 2, wherein R ⁶ is a steroid compound represented by a methyl group, a hydroxymethyl group, or a formyl group. The production method according to any one of claims 1 to 3, wherein the protein belonging to CYP154 is CYP154 derived from Streptomyces griseus NBRC 13350 strain. The electron transfer protein is a reductase peptides contained in cytochrome P450 monooxygenase P450RhF from Rhodococcus NCIMB 9784 strain, The method according to any one of claims 1-4. Transformants, the 3 'end of the DNA of the following encoding a protein belonging to the CYP154 (e) or (f), via the linker, the following encoding reductase peptide (h) or ( The production method according to any one of claims 2 to 5 , wherein the recombinant plasmid obtained by inserting the gene encoding the fusion protein linked with the DNA of i) into an E. coli vector is introduced into E. coli.
(E) DNA consisting of the nucleotide sequence of SEQ ID NO: 3
(F) DNA encoding a polypeptide having a sequence homology of 95% or more with the DNA comprising the nucleotide sequence of SEQ ID NO: 3 and functioning as a cytochrome P450 monooxygenase
(H) DNA consisting of the base sequence of SEQ ID NO: 4
(I) DNA encoding a polypeptide having a sequence homology of 95% or more with the base sequence of DNA consisting of the base sequence of SEQ ID NO: 4 and having reductase activity