KR20210019291A - Bioconversion method for cleavage of C-C bond in steroid using CYP154C8 - Google Patents

Abstract

The present invention provides a bioconversion method for steroids, comprising a step of cleaving a C-C bond of carbons 17 and 20 of steroids by using CYP154C8. According to the present invention, a bioconversion method for cleavage of a C-C bond in steroids using a CYP154C8 enzyme bioconverts existing steroids, thereby being useful as a drug development method which can be applied to a wide range of fields including preservatives, pharmaceutical compounds, pesticides, biofuels, bioremediations, and biodegradations in a pharmaceutical field related to organic synthesis.

Description

Bioconversion method for cleavage of C-C bond in steroid using CYP154C8 enzyme

The present invention relates to a bioconversion method for cleaving carbon-carbon bonds of steroids using CYP154C8 enzyme, and more particularly, cleaving carbon-carbon bonds at positions C17 and C20 of steroids using CYP154C8 enzymes in the presence of NADPH or an oxidizing agent. It relates to a method of biotransformation.

Cytochrome P450 (CYP) enzyme is hydroxylation, epoxidation, dealkylation, deamination, dehydrogenation, dehydration, dehydrogenation. It is a superfamily of hemoproteins well known to catalyze various reactions such as demethylation and cleavage of carbon (C)-carbon (C) bonds. CYP enzymes play an important role in a variety of chemical processes, including drug metabolism, steroid biosynthesis, and detoxification of xenobiotics. Most CYP enzymes require a redox partner protein that can transfer two electrons from NADPH or NADH to reduce the oxygen atom from O ₂ to H ₂ O. In bacteria, CYP was initially thought to be primarily involved in the catabolism of exogenous substrates, which are primarily sources of energy. However, it has been reported that CYP also plays an important role in secondary metabolism. Substrates for a large number of CYPs in bacteria are unknown, but CYP has various roles within the substrate (Kelly SL et al., Philos Trans R Soc Lond B Biol Sci. 2013, 368, 20120476).

CC bond cleavage reactions are not particularly common for CYP enzymes derived from bacterial sources. Some of the well-known reactions, including CC bond cleavage reactions, are CYP51 [lanosterol 14α-demethylase], CYP11 (cholesterol side chain cleavage), CYP17 (C17α-C20ase) and CYP19 [aromatase. )]. OleT (CY152L1), BSβ (cyp152A1), CYP51 and P450 _BioI (CYP107H1) are the only known CYPs isolated from bacterial sources that catalyze CC bond cleavage reactions with fatty acids.

Recently, two non-specific peroxygenases from the mushrooms Marusmius rotula (MroUPO) and Marasmius wettsteinii (MweUPO) are corticosteroids (cortisone, 11- It has been reported to catalyze the selective deacylation of deoxycortisol (11-deoxycortisol and prednisone) [Ullrich R, et al., J Inorg Biochem 183, 8493]. Enzymatic cleavage of CC bonds has been shown to be a sequential oxidation reaction in the immediate vicinity, and is generally promoted by a single CYP. These reactions produce unstable intermediates, making it difficult to investigate and characterize the mechanisms involved.

The hydroxylation reaction catalyzed by CYP uses a Fe ⁴⁺ -oxo intermediate, also known as compound I, but ferric peroxide in a CYP-catalyzed CC cleavage reaction including 17A1, 19A1, and 51A1. (ferric peroxide, FeO ₂ ^-) vs. (versus) spread tocopheryl was under a debate which has been a long-time interest in participation ^{(perferryl, FeO 3+, compound ⅰ} ). Recently, there has been a study on OleT peroxide-dependent CYP, which is known to catalyze the cleavage of CC bonds to fatty acids for the synthesis of alkenes, and the CYP compound I is a reaction that does not involve oxygen rebound. It has been shown to be able to catalyze [Grant JL, et al., Proc Natl Acad Sci USA 2016, 113, 10049-10054; Grant JL, et al., J Am Chem Soc 2015, 137, 4940-4943].

The role of CYP-catalytic C-C bond cleavage has attracted much attention due to its potential application in organic synthesis. These challenging reactions are currently of considerable interest due to a wide range of applications including preservatives, pharmaceutical compounds, pesticides, utilization of main chemicals as biofuels, bioremediation and biodegradation. More importantly, although the lyase activity in biological systems is widely known, the mechanism has not been clearly identified to date.

Japanese patent registration JP5800040 (2015.09.04.) US Patent Publication US2,902,410 A (1959.09.01.)

Bilash Dangi, et al., ChemBioChem 2018, 19, 1066-1077 Fatbardha Varfaj, et al., Drug Metab Dispos 2014, 42, 828-838 Job L. Grant, et al., PNAS 2016, 113(36), 10049-10054

The inventors of the present invention identified CYP154C8, the first bacterial CYP that catalyzes the CC bond cleavage of steroid substrates (see FIG. 1) such as prednisone, prednisolone, cortisone, and hydrocortisone. I did. Until now, the CC bond cleavage reaction with steroids as a substrate has not been reported for any microbial CYP. CYP154C8 has previously been reported to synthesize mono-hydroxylated products of steroid substrates in reactions reconstituted with NADH and alternative oxidation-reduction partners, putidaredoxin and putidaredoxin reductase. The reaction here was supported by NDADPH, spinach ferredoxin, and ferredoxin reductase, and in vitro reactions were carried out with oxygen substitutes diacetoxyiodobenzene (PDA) and hydrogen peroxide (H ₂ O ₂ ), respectively. .

Accordingly, an object of the present invention is to provide a method for bioconversion of steroids using CYP154C8 enzyme that catalyzes carbon-carbon bond cleavage in the presence of NADPH or specific oxidizing agents.

According to an aspect of the present invention, there is provided a method for bioconversion of a steroid comprising the step of cleaving the C-C bond of carbon 17 and carbon 20 of the steroid using CYP154C8.

In one embodiment, the steroid may be one or more selected from the group consisting of prednisone, prednisolone, cortisone and hydrocortisone.

In one embodiment, the cleavage may be performed in the presence of a NADPH-dependent system using redox proteins ferredoxin (Fdx) and ferredoxin reductase (Fdr). The Fdx may be 0.1 to 100 μM, and the Fdr may be 0.01 to 10 U. In addition, the NADPH may be 10 to 1000 μM, and the NADPH dependent system may include glucose 6-phosphate, glucose 6-phosphate dehydrogenase, and catalase.

In one embodiment, the cleavage may be performed in the presence of an oxidizing agent of diacetoxyiodobenzene or H ₂ O ₂ . The diacetoxyiodobenzene may be 0.1 to 50 mM. In addition, the H ₂ O ₂ may be 1 to 100 mM.

In one embodiment, the CYP154C8 may be 0.1 to 100 μM.

In one embodiment, the bioconversion method may be performed at pH 4 to 10.

In one embodiment, the bioconversion method may be carried out at 20 to 40 °C.

According to the present invention, the bioconversion method of cleaving carbon-carbon bonds of steroids using the CYP154C8 enzyme in the presence of NADPH or diacetoxyiodobenzene or H ₂ O ₂ oxidizing agent is the first bacterial CYP450 catalyzing CC bond cleavage. It was found to be a biotransformation method using enzymes. In addition, in the system using an oxidizing agent of NADPH or diacetoxyiodobenzene or H ₂ O ₂ in addition to the mono-hydroxylated product identified in the conventional NADH-dependent system using CYP154C8, the di-hydroxylated product, di- Various distributions of oxidation products, CC bond cleavage products, and hydroxylation products of CC bond cleavage products for the hydroxylated product were shown.

Therefore, the bioconversion method for the CC bond cleavage of steroids using the CYP154C8 enzyme of the present invention bioconverts the steroids, and includes a wide range of preservatives, pharmaceutical compounds, pesticides, biofuels, biological worms, and biodegradation in the pharmaceutical field related to organic synthesis. It can be usefully used as a drug development method that can be applied to the field.

1 is a diagram showing the chemical structure of a steroid substrate used in an in vitro reaction catalyzed by CYP154C8.
Figure 2 is an HPLC chromatogram for a reaction mixture of hydrocortisone (a), prednisone (b), cortisone (c) and prednisolone (d) catalyzed by CYP154C8 in a NADPH system. HC, PD, CS and PL represent the substrate peaks of hydrocortisone, prednisone, cortisone and prednisolone, respectively. P1 and P3 are mono-hydroxylated product peaks; P4 corresponds to the exact mass of the dihydroxylated product of each substrate; In inset (a) P7 represents the exact mass of the likely 11,17-dihydroxy-3-oxoandrost-4-ene-17-carboxylic acid ([M + H] ⁺ 349.2010); In insets (a), (b) and (c), P5 represents the peak of the likely oxidation product of hydroxylated hydrocortisone. P5 in the inset (d) and P6 in the insets (a), (b) and (c) represent the peaks of the CC bond cleavage product.
3 is an LC-MS spectrum and UV-absorbance of a reaction mixture of the substrates hydrocortisone (a), cortisone (b), prednisone (c) and prednisolone (d).
Figure 4 shows the ¹ H NMR spectrum and atomic chemical shift of the CC bond cleavage product (1-dehydroadrenosterone, 1-dehydroadrenosterone) obtained by the catalytic action of CYP154C8. For the same product, FIG. 4b is a ¹³ C NMR spectrum, FIG. 4c is a correlated spectroscopy (COSY), and FIG. 4d is a heteronuclear single-quantum correlation-distortionless enhancement. by polarization transfer, HSQC-DEPT) + heteronuclear multiple-bond correlation (HMBC), Figure 4e is a rotating-frame overhauser effect spectroscopy (ROESY), Figure 4f is a polarity shift Anti-distortion enhanced by -heteronuclear single-quantum correlation-distortionless enhancement by polarization transfer (HSQC-DEPT) and Figure 4g shows a heteronuclear multiple-bond correlation (HMBC).
Figure 5 shows the ¹ H NMR spectrum and atomic chemical shift of the major prednisone hydroxylated products (21-hydroxyprogesterone, 21-hydroxyprogesterone) and free aldehyde form products obtained by the catalysis of CYP154C8. For the same product, FIG. 5B shows a ¹³ C NMR spectrum, FIG. 5C shows COSY, FIG. 5D shows HSQC-DEPT+HMBC, FIG. 5E shows ROESY, FIG. 5F shows HSQC-DEPT, and FIG. 5G shows HMBC.
Fig. 6 is a chromatogram of a reaction mixture of 21-hydroxyprednisone (insertion figure I) and 21-hydroxyprednisone (insertion figure II) as standard substances measured by HPLC. P1 is di-hydroxyprednisone obtained by hydroxylation of 21-hydroxyprednisone. P3 is a possible oxidation product of di-hydroxyprednisone with an exact mass of [M + H] ⁺ 389.1958. P4 represents the peak of 1-dihydroadrenosterone (CC bond cleavage product), and P2 represents the exact mass of hydroxy,1-dihydroadrenosterone ([M + H] ⁺ 315.1591).
7 is an HPLC elution profile of a Cortisone reaction mixture at different time intervals (15-240 minutes) catalyzed by CYP154C8 in a NADPH system. The peaks in the black box indicate possible oxidation-like products for the hydroxylated product. In addition, the peak indicated by the arrow indicates the production of adrenosterone obtained by the CC bond cleavage reaction. P3 was identified as the di-hydroxylated product of cortisone obtained by sequential hydroxylation of possible 21-hydroxycortisone (P1). P2 represents the exact mass value of the monohydroxylated product (possibly 16-hydroxylated product); P4 represents the exact mass value of the hydroxylated product of adrenosterone.
8 is an HPLC chromatogram for the reaction mixture of prednisone (a), prednisolone (b), hydrocortisone (c) and cortisone (d) catalyzed by CYP154C8 in a H ₂ O ₂ system. Insets I and II show HPLC chromatograms of reactions performed in the presence and absence of CYP154C8, respectively. P1 represents the exact mass value of the monohydroxylated product of each substrate; P2 was identified as the CC bond cleavage product of each substrate; P3 exhibited an exact mass value similar to the 17-carboxylic acid of each substrate; P4 and P5 represent the exact mass values for the possible oxidation products of the hydroxylated product.
FIG. 9 is an HPLC chromatography of prednisone (a), cortisone (b), hydrocortisone (c) and prednisolone (d) reaction mixture catalyzed by CYP154C8 in a reaction supported by diacetoxyiodobenzne (PIDA). It's grams. Insets I and II show reaction chromatograms performed in the presence and absence of CYP154C, respectively. For the substrate prednisone, peak P1 is 21-hydroxyprednisone (identified by NMR), and for other substrates, peak P1 is the 21-hydroxylation product of each substrate based on the peak retention pattern. Another peak P2 for prednisone was identified as 1-dihydroadrenosterone (based on NMR structural analysis). Likewise, the product P2 of prednisolone, cortisone and hydrocortisone was identified as a possible CC bond cleavage product based on LC-MS analysis and peak retention pattern for peak P2.
FIG. 10 is an HPLC chromatogram for the standard adrenosterone (Inset I) and Cortisone reaction mixture (Inset II) supported by the H ₂ O ₂ system. The peaks indicated by arrows indicate the exact retention time of the adrenosterone peak in the chromatogram indicating the formation of adrenosterone in the cortisone reaction mixture. This was further confirmed through comparison of LC-MS analysis.
11 is a diagram showing a possible sequence of reactions that may be involved in the formation of 1-dihydroadrenosterone (17-ketosteroid) from the substrate prednisone. (a) The reaction sequence is the formation of the initial hydroxyl group at the C21 position (I, prednisone 21-geminal-diol) and the carboxylic acid (II, prednisone 21-oic acid) at the C21 position, and finally 1-dihydroadrenose. It proceeds in the order of removing the side chain to form theron (III). (b) Another possible sequence of reactions of the product of 1-dihydroadrenosterone (17-ketosteroid) is involved in the initial hydroxylation at position C21 (I) and finally forming 1-dihydroadrenosterone. Hazardous CC bond cleavage reaction, or prednisone directly through CC bond cleavage, 1-dihydroadrenosterone (17-ketosteroid) can be formed.
Figure 12 is a possible related to the formation of oxidation-like products with masses of [M + H] ⁺ 373.1644 (P Ib) and [M + H] ⁺ 389.1958 (P IIb) of mono- and di-hydroxylated prednisone This is a picture showing the reaction sequence. The 21-hydroxyprednisone (P I) obtained by the hydroxylation of prednisone is equilibrated with the corresponding free aldehyde at C21 (P Ia). Similarly, di-hydroxyprednisone (PII) obtained by hydroxylation of 21-hydroxyprednisone can equilibrate with the corresponding aldehyde (PIIa) found in 21-hydroxyprednisone. The aldehyde form in the reaction can be subsequently hydroxylated to form oxidation, perhaps as the product of mono- and di-hydroxylated prednisone.
13 is a diagram showing an overview of the prednisone reaction catalyzed by CYP154C8. Initially, prednisone (A) was found to be hydroxylated at two different positions. 21-hydroxyprednisone (B) was identified as the main product in equilibrium with the corresponding free aldehyde (confirmed by NMR). 21-hydroxyprednisone was sequentially hydroxylated to give dihydroxyprednisone (F). Accurate mass values for oxidation-like products, such as the product of mono- and di-hydroxyprednisone, were detected in the trace. The same reaction mixture showed the formation of 1-dihydroadrenosterone (C) by CC bond cleavage confirmed by NMR, which could possibly be hydroxylated at the C16α-position (D). Oxidation-like products of mono- and di-hydroxyprednisone are oxidized at C-21 to produce prednisone-21-carboxylic acid (J, [M + H] ⁺ 373.1644) and hydroxyprednisone-21-carboxylic acid (I , [M + H] ⁺ 389.1958), or 21-hydroxyprednisone (B) and 21-geminal-diol in the form of dihydroxyprednisone (F) are sequentially hydroxylated In the absence of H ₂ O, which can form oxidation-like products (L) and (H) of each of the products in the form of aldehydes in the reaction mixture to form aldehydes at C21 (K and G in the form of 21-geminal-diol, respectively). It is in equilibrium with form.

In the present specification, the term "bioconversion method" refers to the production of new products through biotechnology such as microbial fermentation and enzyme treatment, or by replacing existing chemicals synthesized and produced by existing chemical synthesis processes. It means technology, and is also called bioconversion, biotransformation, biosynthesis, and bio-catalysis.

The present invention provides a method for bioconversion of a steroid comprising the step of cleaving a carbon (C)-carbon (C) bond at positions 17 and 20 of the steroid using CYP154C8. CYP154C8 is an enzyme that cleaves the C-C bond of a substrate having a hydroxyl group at the C17 and C21 positions through multi-step hydroxylation, and further hydroxylates the product obtained therefrom. Substrates that can be used in the reaction are represented by the following Chemical Formulas 1 and 2 (wherein X is a hydroxy group represented by -OH or a keto group represented by =O at the carbon 11 position).

[Formula 1]

[Formula 2]

The steroid may be selected from the group consisting of prednisone, prednisolone, cortisone, and hydrocortisone, which are substrates having hydroxy groups at C17 and C21 positions, as a substrate (see Fig. 1). ), for example, the CC bond cleavage reaction process of prednisone can be represented as follows.

The reaction process is the formation of prednisone 21-geminal-diol (I) in which a hydroxy group is added to the C21 position of prednisone, and then prednisone 21-oic acid carboxylated at the C21 position. After the formation of acid, Ⅱ), the side chain bonded to carbon-carbon at the C17 and C20 positions is finally cleaved to form 1-dihydroadrenosterone (17-ketosteroid, Ⅲ). .

Another reaction process is the formation of prednisone 21-geminal-diol (I) with a hydroxyl group added at the C21 position, followed by a CC bond cleavage reaction to finally form 1-dihydroadrenosterone. Or, prednisone can form 1-dihydroadrenosterone (17-ketosteroid) through CC bond cleavage.

From the above reaction process, a product (17-ketosteroid) having the structures of Chemical Formulas 3 and 4 can be obtained (where X at the carbon 11 position is a hydroxy group represented by -OH or a keto group represented by =O). . A compound with a substrate of Formula 1 can produce a product of Formula 3, and a compound with a substrate of Formula 2 may produce a product of Formula 4, and in the case of prednisolone, a substrate, 11-hydroxyboldione In the case of cortisone, adrenosterone may be produced, and in the case of hydrocortisone, 11-hydroxyandrostenedione may be produced.

[Formula 3]

[Formula 4]

In one embodiment, the cleavage may be performed in the presence of a NADPH-dependent system using redox proteins ferredoxin (Fdx) and ferredoxin reductase (Fdr). The Fdx is 0.1 to 100 μM, preferably 1 to 20 μM, most preferably 4 to 8 μM, and Fdr is 0.01 to 10 U, preferably 0.03 to 5 U, most preferably 0.05 to 3 U. Can be used. The NADPH may be used in 10 to 1000 μM, preferably 50 to 700 μM, and most preferably 100 to 500 μM. In addition, the system may include glucose 6-phosphate, glucose 6-phosphate dehydrogenase and catalase.

In one embodiment, the cleavage may be performed in the presence of an oxidizing agent of diacetoxyiodobenzene or H ₂ O ₂ . The diacetoxyiodobenzene may be used in an amount of 0.1 to 50 mM, preferably 0.3 to 20 mM, most preferably 0.5 to 5 mM, and the H ₂ O ₂ is 1 to 100 mM, preferably 10 to 100 mM, most preferably 60 to 100 mM may be used.

The CYP154C8 may be used in an amount of 0.1 to 100 μM, preferably 0.5 to 20 μM, and most preferably 1 to 6 μM.

In addition, the bioconversion method can be carried out at pH 4 to 10, preferably at pH 6 to 8.5, most preferably at pH 7 to 8, and at 20 to 40°C, preferably at 25 to 35°C, most preferably Can be carried out at a reaction temperature of 28 ~ 32 ℃.

The bioconversion method for carbon-to-carbon bond cleavage of steroids using the CYP154C8 enzyme of the present invention can cleave the CC bond compared to the conventional bioconversion method using the CYP154C8 enzyme, thereby increasing the distribution of the product. It can be usefully used as a drug development method that can replace.

Hereinafter, the present invention will be described in more detail through examples. However, the following examples are for illustrating the present invention, and the scope of the present invention is not limited thereto.

< Example >

1. Materials and methods

(1) Chemical substances and reagents

Prednisone, cortisone, and hydrocortisone steroid substrates were purchased from Sigma Aldrich (Yonjin, Korea). Prednisolone and 17α-hydroxyprogesterone were purchased from TCI (Tokyo Chemical Industry Co., Ltd, Tokyo, Japan). Isopropyl from Duchefa Biochemie Co. (Netherlands) 1- thio -β- _D - galacto-pyrano seed _{(Isopropyl 1-thio-β- D} -galactopyranoside, IPTG) and 1,4-dithiothreitol (1,4- dithiothreitol, DTT) was obtained. Ampicillin (Amp), α-aminolevulinic acid (ALA), NADPH, H ₂ O ₂ , Diacetoxyiodobenzene (PIDA), catalase, formate dehydrogena (Formate dehydrogenase), glucose-6-phosphate (glucose 6-phosphate), spinach ferredoxin (Fdx) and spinach ferredoxin reductase (Fdr) were purchased from Sigma-Aldrich. Restriction enzymes were obtained from Takara Clontech (Japan), T4 DNA ligase (ligase), DNA polymerase and dNTP were obtained from Takara Bio (Shiga, Japan). High performance liquid chromatography (HPLC) grade acetonitrile and water were purchased from Fisher Scientific (MA, USA).

(2) Expression and purification of CYP154C8

The heterologous expression and purification of CYP154C8 was previously described [B. Dangi, et al., ChemBioChem 2018, 19, 1066-1077. Streptomyces sp. CYP154C8 encoding sequence (1,266 bp, 421 amino acids, GenBank accession number MF398962, see Table 1) was amplified from the genomic DNA of W2233-SM. PCR primers used for gene amplification were designed as 5'- GAATTC ATG AAC GGT CAG TCA GCG A-3'(EcoRI) forward and 5'- AAGCTT TCA GCT GCC GTG GAG CA-3' (HindIII) reverse primers. . Each of the underscore characters represents an endonuclease restriction enzyme site.

The obtained PCR product was cloned into a pMD20-T vector using E. coli XL1-Blue, and the nucleotide sequence was confirmed by automated sequencing (Macrogen, Korea). In addition, a pET32aCYP154C8 construct was prepared by linking the gene product to the pET32a(+) vector. The construct generated by encoding the N-terminal His _6- tag protein under the control of the T7 promoter was transformed into Ec o li Bl21 (DE3) and cultured in LB agar containing 100 μg/ml ampicillin. I did. A single colony was selected from the cultured LB agar plate and incubated overnight at 37°C. 1 ml of the overnight culture medium was added to 100 ml of the LB medium to which 100 μg/ml ampicillin was added, and at 37°C until the cell density reached about 0.6 ~ 0.8 at OD ₆₀₀ , at 180 rpm in an orbital shaker. Incubated by stirring. The culture was induced with 0.5 mM IPTG, and 1 mM 5-aminolevulinic acid hydrochloride (5-ALA) and 0.5 mM FeCl ₃ were added to promote heme synthesis. Cells were incubated at 20° C. for 48 hours, the pellet was obtained by centrifugation at 3,500 rpm for 20 minutes at 4° C., and 50 mM Tris-HCl buffer containing 10% glycerol, 100 mM NaCl, and 1 mM DTT ( pH 7.4) twice.

The washed pellet was harvested, sonicated, and dissolved by centrifugation at 4° C. at 24650 g for 40 minutes. The soluble protein extract was mixed on ice with TALON His-tag resin (resin) for 60 minutes and stirred, and the soluble fraction of the protein was purified by Ni ²⁺ affinity chromatography. The protein-binding resin was pre-equilibrated with 2 times the equilibration buffer (potassium phosphate, pH 7.4). The bound proteins were eluted with an elution buffer (elution buffer, potassium phosphate (pH 7.4), 10% glycerol, 100 mM NaCl) containing 20, 100, and 250 mM imidazole. Fractions containing the eluted protein were concentrated using Amicon centrifugal filters (Millipore) having a molecular weight cut-off (MWCO) of 30 kDa.

(3) Enzyme reaction test using CYP154C8

The enzyme concentration was determined based on the difference in the CO-reduction absorbance spectra, a method widely used to determine the concentration of CYP [Omura T et al., J Biol Chem 1962, 239, 2370-2378].

The in vitro enzymatic activity of CYP154C8 was individually evaluated in the presence of NADPH, diacetoxyiodobenzne (PIDA) and H ₂ O ₂ . The reaction supported by NADPH was 6 μM Fdx and 0.1 U Fdr as an oxidation-reduction partner derived from spinach, 10 mM glucose-6-phosphate and 1 U glucose-6-phosphate dehydrogenase for NADPH regeneration, 100 μg· Ml ^-1 catalase, 1 mM MgCl ₂ and 250 μM NADPH. Other reactions were supported by 2 mM PIDA and 40 mM H ₂ O ₂ as separate reactions.

All in vitro conversion reaction tests were performed in a final volume of 0.5 ml in a potassium phosphate buffer (pH 7.4) containing 3 μM CYP154C8 and 0.5 mM substrate (steroid). The reaction was carried out at 30° C. for 2 hours using a shaking reaction chamber (500 rpm). The reaction mixture was extracted twice with 0.5 ml ethyl acetate and dried for analysis purposes.

(4) Product purification and analysis method

The products 21-hydroxyprednisone and 1-dehydroandrosterone were obtained by in vitro reaction of the substrates prednisone, PIDA and H ₂ O ₂ , respectively. The reaction was carried out separately in a volume of 10 ml. Both products were purified from a total reaction volume of 200 ml.

The reaction mixture was initially analyzed by HPLC (Shimadzu, Kyoto, Japan). The sample was separated and passed through a Mightysil reverse phase C ₁₈ GP column (4.6 × 250 mm, 5 μm; Kanto Chemical, Tokyo, Japan). Water (A) and acetonitrile (B) were used as mobile phases for separation. The reaction mixture was a gradient of acetonitrile (B) of 15% for 0-10 minutes, 50% for 10-20 minutes, 70% for 20-25 minutes, and 15% for 25-40 minutes at a flow rate of 1 ml/min. It was analyzed using the system. After HPLC analysis, the samples were analyzed using liquid chromatography-mass spectroscopy (LC-MS). The target peak for confirming the modified position was purified by preparative HPLC using a Mightysil reverse phase C ₁₈ GP column (4.6 × 150 mm, 5 μm; Kanto Chemical, Tokyo, Japan), and then the purified product was D ₆ ]DMSO And analyzed by NMR spectroscopy on a 900 MHz Unity INOVA spectrometer (Varian). One-dimensional NMR spectra ( ¹ H NMR and ¹³ C NMR) and 2D NMR (heteronuclear multiple bond correlation, correlation spectroscopy, ROESY and HSQC) were used to describe the appropriate structure.

2. Results

(1) In vitro reaction analysis

The in vitro reaction catalyzed by CYP154C8 was carried out separately in three different systems. The reaction of the control was performed individually without CYP enzyme supported by three different systems containing Fdx-Fdr-NADPH, H ₂ O ₂ and PIDA. No product formation was observed in any system. Previously, CYP154C8 was shown to hydroxylate prednisone, cortisone, hydrocortisone and prednisolone at two different sites in a reaction supported by NADH and its redox partners Pdr and Pdx. Reported (B. Dangi, et al., ChemBioChem 2018, 19, 1066-1077).

However, in another CYP154C8 reaction supported by NADPH and spinach oxidation/reduction partners Fdr and Fdx, additional product peaks appeared in addition to the two monohydroxylated products observed in reactions supported by NADH (see FIG. 2). LC-MS analysis of all reactions with four substrates (prednisone, cortisone, hydrocortisone and prednisolone) revealed two monohydroxylated products (P1 and P3) that were also observed in the NADH system. In addition, at least one dehydroxylated product (P4), possibly a CC bond cleavage product (P6, predrisone, cortisone and hydrocortisone; and P5, prednisolone) and possibly a hydroxylated product (P5). The oxidation product of was detected. The formation of a C-C bond cleavage product [17-ketosteroid] in the reaction mixture was estimated based on LC-MS analysis (see Fig. 3) and peak retention patterns.

(2) Characterization of the reaction product of prednisone

Based on the similarity of all four substrate reaction mixtures, the reaction product of the substrate prednisone was characterized. The HPLC chromatogram for the prednisone reaction mixture (see Figure 2, inset (b)) supported by the NADPH system shows two conventional mono-hydroxylated products characterized by comparison of LC-MS and HPLC data ( P1 and P3) are shown (data not shown). LC-MS analysis showed that P4 was a di-hydroxylated product ([M + H] ⁺ 391.1750) and P6 was the correct mass (m/z ⁺ [M + H] ⁺ 299.1634); The mass was exactly similar to the mass of the CC bond cleavage product at C17 of prednisone. These observations were further confirmed by structural analysis based on NMR ( ¹ H, ¹³ C and 2D NMR), which was found to be 1-dehydroadrenosterone generated due to CC bond cleavage (Fig. 4A to See Figure 4g).

Similarly, structural analysis of another major mono-hydroxylated product peak (P1) by NMR indicated the formation of 21-hydroxyprednisone as a result of the hydroxylation at the C21 position of prednisone. Done. The same NMR sample also showed another low concentration product identified in the form of dehydrated ketoaldehyde (see FIGS. 5A-5G). These aldehyde containing products were observed in the form of geminal-diols in the absence of water. P2 was identified as a possible hydroxylated product of the CC bond cleavage product (P6) with an exact mass of [M + H] ⁺ 315.1591, which is precisely 1-dehydroadrenosterone ([M + H] ⁺ 315.1591). Another product peak (P5) identified as a trace showed the correct mass of [M + H] ⁺ 389.1958, a possible oxidation-like product obtained from the reaction, di-hydroxylated prednisone, P4). One or more possible oxidation-like products of dihydroxylated prednisone were detected in trace amounts of LC-MS analysis, which was not clearly observed in the HPLC chromatogram.

(3) Analysis of reaction products of substrates other than prednisone

The reaction with another substrate, hydrocortisone, showed a clear conversion to an oxidation-like product for hydroxyhydrocortisone with an exact mass value of 377.1968 (see Fig. 2), and the calculated mass value is the oxidation of hydroxyhydrocortisone. Product (C ₂₁ H ₂₉ O ₆ ⁺ ) [M + H] ⁺ 377.1953. Another peak (P7) observed in the hydrocortisone reaction showed a major mass of [M + H] ⁺ 349.2009, which is a possible 11,17-dihydroxy-3-oxoandrost-4-ene-17-car It was very similar to the mass of the acid (11,17-dihydroxy-3-oxoandrost-4-ene-17-carboxylic acid, [M + H] ⁺ 349.2010). This product (17-carboxylic acid for each substrate) was also detected in small amounts in LC-MS (data not shown) for prednisone and cortisone, m/z ⁺ [M + H] ^{+ of} 345.1697 for prednisone. Possibly with 17-hydroxy-3,11-dioxo-1,4-diene-17-carboxylic acid (17-hydroxy-3,11-dioxo-1,4-diene-17-carboxylic acid) And a possible 17-hydroxy-3,11-dioxoandrost-4-ene-17-carboxylic acid (17-hydroxy-3, with m/z ⁺ [M + H] ⁺ of 347.1847 for cortisone) 11-dioxoandrost-4-ene-17-carboxylic acid) respectively.

Similar products have been reported in small amounts by two nonspecific peroxygenases characterized by lyase reactions using similar substrates [Ullrich R, et al., J Inorg Biochem 183, 84-93]. CYP154C8 is known to sequentially oxidize steroids. From now on, it was confirmed that the di-hydroxylated product (P3) of prednisone was obtained due to the sequential hydroxylation of 21-hydroxyprednisone by CYP154C8. When 21-hydroxyprednisone is used instead of prednisone as a substrate, further hydroxylated product (P1, m/z ⁺ [M + H] ⁺ 391.1752), di-hydroxylated product of prednisone (P1) Potential oxidation products for (P3, m/z ⁺ [M + H] ⁺ 389.1958), CC cleavage (P4, 1-dihydroadrenosterone) and possible hydroxylation of 1-dihydroadrenosterone The formation of product (P2) was observed (see Fig. 6). At least two possible oxidation products of di-hydroxylated prednisone were identified in LC-MS, but showed very low conversions as observed in the prednisone reaction mixture.

(4) Conversion in the NADPH system over time for cortisone

The effect of time on product formation was investigated by performing a time-dependent reaction of cortisone supported by the NADPH system. As shown in FIG. 7, as the reaction time increased, the hydroxylated product increased. More importantly, the formation of 21-hydroxylated product (P1) increased with time, which was further hydroxylated to give di-hydroxylated product (P3). In addition, with the increase of the time interval, an increase in the peak (arrow) of the C-C bond cleavage product was confirmed, and a low conversion rate of the probable hydroxylated product peak (P4) to the C-C bond cleavage product was also identified. A number of other products have also been observed with very low conversion rates with the correct mass of possible oxidation-like products of the hydroxylated product (data not shown).

(5) Oxidant H ₂ O ₂ CYP154C8 system evaluation using

CYP154C8 is therefore showed the catalytic function in vitro as H ₂ O ₂ and PIDA, the in vitro reaction was performed in the presence of two oxidant (H ₂ O ₂ and PIDA). The reaction mixture supported by H ₂ O ₂ of prednisone showed little production of 21-hydroxyprednisone, which was found to be a major product in the NADPH system (see Fig. 8A). Another mono-hydroxylated product (P1, possibly 16α-hydroxyprednisone), a product with a mass of [M + H] ⁺ 345.1697 (P2, possible 17-hydroxy-3,11-dioxo- 1,4-diene-17-carboxylic acid), possible oxidation-like products for hydroxyprednisone (P3 and P4) and 1-dihydroadrenosterone (P5) were identified. P2-like products were observed prominently in other substrates with H ₂ O ₂ systems. Similar product formation patterns were observed with prednisolone (FIG. 8b ), cortisone (FIG. 8c) and hydrocortisone (FIG. 8d ).

(6) Evaluation of CYP154C8 system using oxidizer PIDA

The reaction supported by PIDA confirmed the major conversion to 21-hydroxyprednisone (P1), and the second product (P2) was the CC bond cleavage product (1-dihydroa) based on HPLC chromatogram and LC-MS. Drenosterone) (see Fig. 9A). In addition, peak P2 was observed in trace amounts even in the presence of other substrates (see FIGS. 9B to 9D).

(7) Confirmation of C-C bond cuts

All reactions supported by H ₂ O ₂ and PIDA showed the formation of a CC lytic product confirmed by LC-MS analysis (data not shown). The CC bond cleavage product of cortisone was confirmed as adrenosterone by HPLC comparison with an actual standard (see FIG. 10). Since the conversion rate of the remaining products was low, structural analysis by NMR could not be performed. Because CYP154C8 hydroxylates steroids deficient in C11 and C21 functional groups (hydroxy or carbonyl) at the C16α position, a possible position for the hydroxylation of other mono-hydroxylated products obtained in the reaction may be at the C16 position. Is assumed. Prior literature [B. Dangi, et al., ChemBioChem 2018, 19, 1066-1077], P1 was found to be the 21-hydroxy product of prednisone, and the structural analysis showed that product peaks of that type (P1 of cortisone, hydrocortisone and prednisolone) were found. The corresponding 21-hydroxy (21-identical-diol, 21-geminal-diol) product is shown for each substrate.

Other substrates, prednisolone, cortisone, hydrocortisone, also formed products in a similar pattern to prednisone, individually supported by NADPH, H ₂ O ₂ and PIDA. The reaction supported by NADPH showed a much higher conversion rate by CYP154C8 compared to the NADH system, indicating the effect of spinach oxidation/reduction partners compared to Pdx and Pdr (see Table 2).

Assuming that the absorbance properties of the product and the substrate are similar, the product was quantified by correlating the peak area of the product and the peak area to which the product and the substrate are bound. Unlike the reactions supported by NADH, the NADPH system is a modified product in which the 21-hydroxy product of each substrate (which is further hydroxylated to form a dihydroxylated product) is found to have a predominant conversion rate (high selectivity). The profile is shown. The mono-hydroxylated product (P3; possible 16α-hydroxylated) showed low conversion (low selectivity). The hydrocortisone reaction mixture exhibited a selectivity of about 90% over the possible 21-hydroxycortisone. The conventional NADH-dependent system showed nearly similar conversions for the two products (21-hydroxy and possibly 16α hydroxy products) with the substrates cortisone, prednisone and prednisolone; On the other hand, the selectivity to 21-hydroxyhydrocortisone was lower than that of 16α-hydroxyhydrocortisone, which is possible through the substrate hydrocortisone.

(8) Discussion

CYP154C8 is the first bacterial CYP known to catalyze the C-C bond cleavage reaction of hydroxy acetyl and hydroxy functional steroids at C17. Prior literature [B. Dangi, et al., ChemBioChem 2018, 19, 1066-1077], when CYP154C8 is reconstituted with NADH and alternative redox partners including Pdx and Pdr, detection of cleavage products is not obtained and two mono- The formation of the hydroxylated product was clearly confirmed. However, NADPH and spinach replacement oxidation and reduction partners Fdx and Fdr have been shown to affect the distribution of the product as well as the catalytic efficiency by CYP154C8. In the present invention, in addition to the hydroxylated product, C-C bond cleavage products were detected in all systems used. These results indicate that the choice of an oxidation-reduction partner can change its type and selectivity in the formation of a product by CYP. More importantly, alternative redox partner proteins can influence the full characterization of CYP.

CYP154C8 has been known to sequentially hydroxylate steroid substrates (progesterone, androstenedione, testosterone and nandrolone). When CYP154C8 is partnered with an appropriate and efficient redox partner protein, it has been found to catalyze the sequential oxidation of steroid substrates including prednisone, cortisone, hydrocortisone and cortisone. In the presence of NADPH and FAD and spinach redox partner protein comprising Fdx, catalytic efficiency of CYP154C8 was higher compared to the reaction that is supported by the redox partner protein comprising a Pdx and Pdr of NADH and Pseudomonas putida origin . These results indicate that the spinach Fdx and Fdr systems are more effective as redox donors for CYP154C8 compared to the Pdx and Pdr redox systems. However, there have been many reports of the role of oxidation-reduction partners on the catalytic activity and product distribution of CYP enzymes. Moreover, it cannot be denied its role as an effector for two different systems in the distribution of various products catalyzed by CYP154C8. CYP154C8 exhibits higher selectivity for 21-hydroxylated products for substrates containing functional groups (hydroxy or carbonyl) at C11 and C21 positions, but these enzymes are substrates lacking functional groups from both positions. Catalyzes the position- and stereospecific hydroxylation at C16α of.

The exact mechanism of the CC bond cleavage product (17-ketosteroid) by CYP154C8 is not clearly identified, but most of the reactants catalyzed by CYP involve successive hydroxylation that produces the required function and promotes CC bond cleavage. . It is believed that a similar reaction pattern was involved in the formation of C-C bond cleavage products catalyzed by CYP154C8 (see Fig. 11). The generation of a C-C bond cleavage product in the reaction using 21-hydroxyprednisone as a substrate instead of prednisone indicated that such a product (21-hydroxyprednisone) is involved in the formation of the C-C bond cleavage product.

Recently, it has been reported that non-specific peroxygenase catalyzes the C-C bond cleavage reaction of similar steroids in multiple stages. The two peroxygenases catalyze the hydroxylation at the C21 position of the substrates cortisone, prednisone and 11-deoxycortisol, which in turn leads to the cleavage of the CC bond to form a 17-ketosteroid. Convert to acid. The sequence of reactions for the formation of C-C bond cleavage products by CYP154C8 may be similar to these two non-specific peroxynogenases. However, with an increase in the reaction time catalyzed by CYP154C8, no change was observed in the formation of the C-C bond cleavage product, as opposed to the pattern of the reaction product by peroxygenase. While an increase in the di-hydroxylated product from 21-hydroxyprednisone was observed with an increase in reaction time, the sequential hydroxylation of this product (17-ketosteroid) was observed in trace amounts. This may indicate that CYP154C8 is the main function of hydroxylation. However, 21-hydroxylation of steroid substrates with hydroxy acetyl and hydroxy functional groups at C17 can proceed as a CC bond cleavage reaction, but compared to the formation of dihydroxylated products when the reaction is supported by the NADPH system. Therefore, it can have low selectivity. The formation of possible oxides, such as hydroxylated products detected in trace amounts, was not clear.

NMR structural analysis of the prednisone mono-hydroxylated product (main product) showed hydroxylation at the C21 position (21-geminal-diol), and the same sample showed prednisone 21-aldehyde formation but was confirmed at low concentration. . The formation of aldehydes with the same geminal-diol was observed under dehydration conditions. Products comprising 21-aldehydes in the reaction can undergo further hydroxylation to form probable oxidation-like products. Similarly, the di-hydroxylated product of prednisone may exist in the form of 21-aldehyde, and the di-hydroxylated product was obtained as a result of the further hydroxylation of 21-hydroxyprednisone, and thus, sequentially hydroxyated by CYP154C8. It can be oxylated to form the respective oxidation-like products for dihydroxylated prednisone and similar substrates in the reaction mixture (see Figure 12). Since keto groups and epoxides can be formed from hydroxy groups, the possibility of formation of oxidation products is thought to be due to the presence of double bonds or the formation of keto or epoxides (Khatri Y, et al., FEBS Lett 2016, 590, 4638-4648; Chiang JY, Front Biosci 1998, 3, d176-d193).

17-hydroxy-3,11-dioxo-1,4-diene-17-carboxylic acid (17-carboxyl) of prednisone, identified as trace using the NADPH system, but clearly observed with oxygen substitutes. It has not yet been clear to form acid)-like products and similar products for each substrate. The possibility of formation of such a product may be due to the C-C bond cleavage between C20 and C21. Here, CYP154C8 may be involved in the cleavage of steroids having hydroxyacetyl and hydroxy functional groups at C17 by cleaving the C-C bond at different positions. Thus, the formation of the 17-hydroxy-3,11-dioxo-1,4-diene-17-carboxylic acid-like product of prednisone and the like product on other substrates is caused by 1-dihydroadrenosterone and other substrates. It may be independent of the formation of a similar product to

Steroids are ubiquitous growth substrates in microorganisms, and bacterial hydroxylase plays an important role in the cleavage of steroids. For example, CYP125A1, CYP142A1 and CYP124A1 contribute to the survival of Mycobacterium tuberculosis and the spread of infection by cholesterol cleavage by oxyfunctionalization, which forms terminal alcohols at C27 and then oxidizes them to aldehydes and carboxylic acids. It plays an essential role. This reaction was found to be essential for the survival and infectiousness of Mycobacterium tuberculosis . The reaction leading to the formation of di-hydroxyprednisone from 21-hydroxyprednisone was found to be much higher than the CC bond cleavage product (1-dihydroadrenosterone) in the reaction supported by NADPH. Although it is not clear that dihydroxyprednisone is involved in the formation of the CC bond cleavage product, the pattern of product formation indicates that the hydroxylated product does not completely induce the CC bond cleavage. CYP17A1, a widely studied mammalian CYP, is known to catalyze the 17α-hydroxylation of progesterone and pregnenolone, and both hydroxylated products are cleaved at the C17-C20 bond. Androstenedione and dehydroepiandrosterone are formed, respectively. Recently, CYP17A1 was found to convert progesterone to 16α-hydroxyprogesterone as a minor product, and 16α-hydroxylating activity was observed in 17α-hydroxypregnenolone and 17α-hydroxyprogesterone substrates. Dehydroepiandrostenedione and androstenedione products formed by the lyase activity of CYP17A1 were further hydroxylated at the 16α-position. Similarly, zebrafish CYP17A1 and CYP17A2 were 17α,21-(OH) ₂ progesterone, 16,17α-(OH) ₂ progesterone, 6β,16,17α-(OH) ₃ progesterone under high concentration and increased reaction time. , 17α,21-(OH) ₂ pregnenolone and 16,17α-(OH) ₂ pregnenolone.

CYP154C8 actively demonstrated the conversion of steroids in the presence of oxygen substitutes H ₂ O ₂ and PIDA. Two oxidizing agents supported the reaction, indicating a distinct formation of the CC bond cleavage product. H ₂ O ₂ is one of the efficient ways to use CYP for industrial use due to its low availability, and not all CYPs can function in the presence of H ₂ O ₂ . However, oxidative cleavage of heme by peroxide is an important problem. Surprisingly, CYP154C8 exhibited an activity in which no product was observed at a concentration of 10 mM or less in the presence of a high concentration of H ₂ O ₂ . The CC bond cleavage product obtained using an oxidizing agent such as PIDA indicates that compound I(FeO ³⁺ ) is likely to be the cause of the CC bond cleavage reaction catalyzed by CYP154C8. However, since the reaction with H ₂ O ₂ also represents the lyase product, the participation of the compound O (Fe ^{2 +} O ₂ H) in the lyase reaction cannot be ignored. The role of compound I in the formation of lyase products with oxidants has been interesting and controversial. Recently, CYP17A1 using the oxidant iodosobenzene formed a lyase product, providing evidence that the Compound I reaction may participate in the CC bond cleavage reaction. Both systems clearly showed the formation of CC bond cleavage products, but the reaction supported by H ₂ O ₂ showed that the 21-hydroxylated product of each substrate had very low conversion or no conversion compared to PIDA. Confirmed. The exact reason for this variable product distribution is not clear, but it is clear that the two systems produce two different reactive oxygen species that are involved in the oxidation of the substrate. Thus, it can be assumed that these reactions may be mechanically different reactions initiated by the two systems.

The difference in the regioselectivity of oxidation products in the catalytic reaction by CYP2D6 is due to the distinct chemical mechanisms of the different systems used, the reaction by NADPH-P450 reductase, cumene hydroperoxide, and iodosobenzene. This was observed when individually supported. Similarly, in another reaction catalyzed by CYP2B1 and supported separately by the P450/NADPH/O ₂ ^- and P450/PhIO systems, the reactive oxygen species of the two systems are ferryl-oxo-species ), but the mechanical differences observed between the two systems were most likely to be related to the chemical differences between the two systems. Initially, compound I was considered to be the only important intermediate associated with catalytically active oxidizing agents. However, in subsequent experiments, the presence of another oxygen species [hydroperoxy-iron species (FeIIIO ₂ H), which can function as an active oxidizing agent capable of forming altered products compared to compound I]. Provided evidence.

In conclusion, CYP154C8 is a multi-functional enzyme that catalyzes the multistage hydroxylation of various steroids, C-C bond cleavage of substrates with hydroxy groups at C17 and C21 positions, and the possible oxidation of hydroxylated products (see Figure 13). The reaction carried out in the presence of the NADPH system suggests that CYP154C8 catalyzes the hydroxyation at the C21 position of all substrates with hydroxy or carbonyl groups at the C11 and C21 positions. This reaction produced the 21-geminal-diol product of each substrate. The 21-geminal-diol product was sequentially hydroxylated to give a di-hydroxylated product. The di-hydroxylated product of prednisone, as well as the 21-geminal-diol, may further oxidize the two products, or the aldehyde form will be further hydroxylated to form a trace of the oxidation product. I hypothesized that it could be. 21-geminal-diol is also involved in the formation of the CC bond cleavage product (1-dehydroadrenosterone), although the exact reaction involved in the formation of the CC bond cleavage product was not clearly identified. The CC bond cleavage product was further possibly hydroxylated at the C16α position.

(9) Summary

The present inventors reported CYP154C8, the first bacterial cytochrome P450 that catalyzes the C-C bond cleavage reaction of steroids. When the reaction by CYP154C8 is supported by NADPH and spinach redox partners ferredoxin and ferredoxin reductase, previously reported putidaredoxin and putidaredoxin supported by NADH. Major changes in product distribution by CYP154C8 were observed compared to the oxidation-reduction reactions containing putidaredoxin reductase. Structural analysis of the NMR-based reaction product showed that 21-hydroxyprednisone is the main product of prednisone, while the other product was 1-dihydroadre obtained by CC bond cleavage. It was confirmed that it was rosterone (1-dehydroadrenosterone). Similar product formation patterns were observed with cortisone, hydrocortisone and prednisolone. In addition, the reaction catalyzed by CYP154C8 in the presence of an oxygen substitute significantly indicated the formation of C-C bond cleavage products.

Claims (12)

A method for bioconversion of a steroid comprising the step of cleaving the C-C bond of carbon 17 and carbon 20 of the steroid using CYP154C8. The method of claim 1, wherein the steroid is at least one selected from the group consisting of prednisone, prednisolone, cortisone and hydrocortisone. The method of claim 1, wherein the cleavage is performed in the presence of a NADPH-dependent system using redox proteins ferredoxin (Fdx) and ferredoxin reductase (Fdr). The method of claim 3, wherein the Fdx is 0.1 to 100 μM, and the Fdr is 0.01 to 10 U. The method of claim 3, wherein the NADPH is 10 to 1000 μM. The method of claim 3, wherein the NADPH dependent system comprises glucose 6-phosphate, glucose 6-phosphate dehydrogenase and catalase. The method of claim 1, wherein the cleavage is carried out in the presence of an oxidizing agent of diacetoxyiodobenzene or H ₂ O ₂ . The method of claim 7, wherein the diacetoxyiodobenzene is 0.1 to 50 mM. 8. The method of claim 7, wherein the H ₂ O ₂ is 1 to 100 mM. The method of claim 1, wherein the CYP154C8 is 0.1 to 100 μM. The method of claim 1, wherein the bioconversion method is carried out at a pH of 4 to 10. The method of claim 1, wherein the bioconversion method is performed at 20 to 40°C.

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