Oxidation of steroids

Abstract

The present invention relates to a method of oxidizing ring-opening steroids using a mutant cytochrome P450 family 102A subfamily member (CYP102A) enzyme. The invention also relates to a mutant CYP102A enzyme, a polynucleotide encoding the enzyme, a cell expressing the enzyme, a transgenic animal or plant comprising the enzyme and a compound prepared by the method.

Description

Technical Field

[0001] The present disclosure relates to a method for oxidizing steroids and a polypeptide for use in the method. Background Art

[0002] Vitamin D (calcitriol) is a hormone that binds to nuclear receptors, which activate and regulate the expression of numerous genes. Among its many physiological roles, vitamin D is crucial for maintaining blood calcium levels. Although its effects are mainly related to skeletal effects by regulating calcium homeostasis, vitamin D is also associated with biological functions such as the immune system, potassium homeostasis, and anti-cancer regulatory properties. Vitamin D has been approved for use as a conventional drug. On the other hand, its low natural abundance and lack of a viable synthetic route mean that the more readily available precursor vitamin D 3 is used as a supplement / precursor. Vitamin D 3 is converted to 25-(OH)-D in the liver by the cytochrome P450 enzyme CYP27A1 3 , which is the circulating form of vitamin D ( Figure 1 ). Subsequently, 25-(OH)-D 3 is converted to 1,25-dihydroxyvitamin D 3 (calcitriol) in the kidney by the enzyme CYP27B1, which is the active form of vitamin D.

[0003] 25-(OH)-D 3 is a reliable indicator of vitamin D 3 levels in the blood. It has been shown that maintaining this substance in the serum at the recommended level (30–60 ng / mL) is crucial for maintaining vitamin D-related functions and overall health.

[0004] Vitamin D 3 has two main sources: ultraviolet light and food. However, each source alone is usually insufficient. Ultraviolet light acts on 7-dehydrocholesterol under the skin to form previtamin D 3 , which is thermally isomerized to form vitamin D 3 . This process depends on sunlight exposure and is seasonally and geographically dependent. Except for fatty fish, few foods contain large amounts of vitamin D 3 . Therefore, adding vitamin D precursors to foods has attracted great interest.

[0005] Vitamin D 3 is routinely supplied in the form of human vitamin D supplements. It is recommended for some populations, especially in winter when sunlight exposure is less; and in clinics, it is also used for patients with impaired immune and organ functions. Vitamin D 3Another major application is in animal feed to enhance weight gain, health, and productivity in the livestock industry. However, the low solubility and low bioavailability of vitamin D 3 in gastrointestinal fluids reduce the efficacy of oral vitamin D 3 supplements.

[0006] It is recognized that the circulating form of vitamin D, 25-(OH)-D 3 is more effective in most applications. In fact, 25-(OH)-D 3 is an approved drug and is a more effective animal feed compared to vitamin D 3 . In addition, studies have confirmed an association between 25-(OH)-D 3 and cancer prevention. The potential application of nanoencapsulation of active vitamin D 3 metabolites such as 25-(OH)-D 3 in chemotherapy has been proposed. Recent reports suggest a possible correlation between patients with vitamin D deficiency and severe cases of coronavirus disease 2019 (Covid-19). Due to the anti-inflammatory properties and immune system regulatory functions of vitamin D 3 , vitamin D 3 supplements have been proposed as an additional treatment strategy. Other studies indicating an association between the severity of coronavirus disease 2019 and phosphate levels in the blood further support the hypothesis that the phosphate metabolism regulator vitamin D 3 can play a role in the progression of the disease.

[0007] Despite substantial evidence demonstrating the higher potency of 25-(OH)-D 3 in various applications, its widespread use is limited by difficulties in production. The chemical process for synthesizing 25-(OH)-D 3 from vitamin D 3 results in low yields due to the need to oxidize an unactivated aliphatic C-H bond at the C25 position. This step is difficult to achieve through chemical system catalysis. Current industrial production of 25-(OH)-D 3 starts with 5,7,24-cholestatrienol, which is produced by fermentation using a mutant strain of Saccharomyces cerevisiae. The trienol is chemically oxidized to 24,25-epoxide, reduced to 25-alcohol, and then photochemically converted to 25-(OH)-D 3 and other products. 25-(OH)-D 3 is recovered by crystallization.

[0008] Vitamin D 2(Ergocalciferol or calciferol) and vitamin D 3 differ in that vitamin D2 has a C22-C23 double bond and a C28 methyl group ( Figure 2 ). Vitamin D 2 is produced by ultraviolet irradiation of ergosterol and is present in certain plants and mushrooms, while vitamin D 3 is obtained by isolation from animals. The rigidity of the double bond and the altered steric hindrance effect introduced by the methyl group can affect P450 substrate recognition, as demonstrated by the fact that vitamin D 2 is oxidized to its circulating form, 25-hydroxyvitamin D 2 , by the liver P450 enzyme CYP2R1, which is different from the P450 enzyme (CYP27A1) for vitamin D3. Similarly, the CYP2R1 enzyme cannot oxidize vitamin D 3 . However, both 25-hydroxyvitamin-D 2 and -D 3 are oxidized to their respective active forms by CYP27B1 in the kidney. The active form of vitamin D 2 , 1,25-dihydroxyvitamin D 2 (ercalcitriol), has a binding affinity similar to that of the vitamin D receptor and the active form of vitamin D 3 , calcitriol.

[0009] The P450 enzymes CYP27A1 and CYP21B1 catalyze the oxidation of vitamin D 3 in the physiological order of C25 first and then C1 to generate the active form of vitamin D. The liver enzyme CYP2R1 is responsible for hydroxylating vitamin D 2 at the C25 position to produce 25-hydroxyvitamin D 2 , and CYP27A1 does not catalyze this reaction. These membrane-bound enzymes have low stability and activity. It is difficult to express them in heterologous organisms, which makes them unsuitable for large-scale production.

[0010] Currently, many microorganisms have been screened for vitamin D 3 oxidation, and the isolation of new strains with such activity continues. In recent reports on strain isolation and identification, the activities and conversions have been reviewed (Schmitz et al., Chembiochem 2021, 22, 2266-2274; Tang et al., Appl. Microbiol. Biotechnol. 2020, 104, 765-774). The earliest reported was a Streptomyces strain that can hydroxylate vitamin D 3Oxidation to form human metabolites (Sasaki et al., Appl. Environ. Microbiol. 1991, 57, 2841-2846). Amycolata autotrophica FERM BP-1573 converts vitamin D 3 Converted to 25-(OH)-D 3 (Sasaki et al., Appl. Environ. Microbiol. 1992, 38, 152-157; Takeda et al., J. Ferment. Bioeng. 1994, 78, 380-382). Pseudonorcadia autotrophica ID9302 also performed the reaction (Kang et al., Biotechnol. Bioproc. Eng. 2006, 11, 408-413), with a low initial yield, but after screening and selecting mutants produced by ultraviolet irradiation and optimizing process parameters, the yield was as high as 356 mg / L, corresponding to a space-time yield (STY) of 2.97 mg / L / h (Kang et al., Biotechnol. Lett. 2015, 37, 1985-1904). A more recent isolate, Pseudonorcadia autotrophica CGMCC5098, exhibited 639 mg / L of 25-(OH)-D in a 120-h fermentation. 3 The high yield of vitamin D was 5.33 mg / L / h (Luo et al., Biocatal. Biotransform. 2017, 35, 11-18). 3 Produces up to 830 mg / L of 25-(OH)-D 3 , STY was 25.9 mg / L / h (Tang et al., Appl. Microbiol Biotechnol. 2020, 104, 765-774). The new bacterial isolate Kutzneria albida converts vitamin D 2 and D 3 Oxidation to 25-hydroxy derivatives, the yields were 13.7 mg / L and 70.4 mg / L, respectively, with a maximum STY of 1.47 mg / L / h (Schmitz et al., Chembiochem 2021, 22, 2266-2274). These absolute mass yields or space-time yields are not sufficient for industrial production.

[0011] CYP105A1 from Streptomyces griseolus has 55% amino acid sequence identity with CYP105A2 responsible for oxidizing vitamin D by Amycolatopsis autotrophica 3 The recombinant form was engineered to enhance the C25 oxidation activity of vitamin D 2 and D 3 However, its production efficiency and yield were still low (Hayashi et al., Biochemistry, 2008, 47, 11964 - 11972; Yasutake et al., J. Biol. Chem. 2010, 285, 31193 - 31201; Yasuda et al., Biochem. Biophys. Res. Commun. 2017, 486, 336 - 341). Abdulmughni et al. characterized CYP109A2 in Bacillus megaterium as a vitamin D 3 hydroxylase (Abdulmughni et al., FEBS J. 2017, 284, 3881 - 3894; J. Biotechnol. 2017, 243, 38 - 47), and expressed its engineered variant in Bacillus megaterium to produce 282.7 mg / L of 25-(OH)-D 3 , with a STY of 5.90 mg / L / h (Abdulmughni et al., J. Biotechnol. 2021, 325, 355 - 359). CYP107BR1 (Vdh) from Pseudonocardia autotrophica was engineered to enhance its C25 oxidation activity of vitamin D 3 and the variant was expressed in Rhodococcus erythropolis, while using nisin to increase cell membrane permeability, resulting in a 25-(OH)-D 3 yield of up to 573 mg / L (Yasutake et al., Chembiochem 2013, 14, 2284 - 2291). However, the product concentration, reaction rate, and space - time yield of these systems are not high enough for the economically viable industrial production of 25-(OH)-D 2 and 25-(OH)-D 3 .

[0012] Therefore, new methods are needed to selectively catalyze the oxidation of vitamin D 2 and D 3 respectively to generate 25-(OH)-D 2 and 25-(OH)-D 3 . SUMMARY OF THE INVENTION

[0013] The present inventors have unexpectedly discovered that CYP102A enzymes can be mutated to enable selective oxidation of secosteroid substrates. The mutant CYP102A enzymes are capable of catalyzing reactions and have high product selectivity (i.e., little, if any, of the unwanted product is produced), high conversion rates (the percentage of substrate converted to product), and high turnover numbers. In contrast, the wild-type enzyme did not exhibit any detectable oxidation of secosteroid substrates.

[0014] Accordingly, the present inventors have made an important contribution to the art by providing an efficient method that utilizes engineered variants of cytochrome P450 BM3 to directly oxidize vitamin D 3 (cholecalciferol or colecalciferol) to produce 25-hydroxyvitamin D 3 [25-(OH)-D 3 , calcifediol or calcidiol], and to directly oxidize vitamin D 2 (ergocalciferol or calciferol) to produce 25-hydroxyvitamin D 2 [25-(OH)-D 2 .

[0015] Accordingly, the present invention provides a method for oxidizing a secosteroid, comprising the step of contacting the secosteroid with a mutant CYP102A (a member of the cytochrome P450 family 102A subfamily) enzyme, wherein the CYP102A enzyme comprises a heme monooxygenase domain containing a P450 fold, and the mutant CYP102A enzyme comprises substitutions at one or more positions in the polypeptide chain of the wild-type CYP102A enzyme, thereby enhancing the monooxygenase activity and / or altering the product selectivity of the mutant enzyme.

[0016] The present invention also provides a mutant CYP102A (a member of the cytochrome P450 family 102A subfamily) enzyme, wherein the CYP102A enzyme comprises a heme monooxygenase domain containing a P450 fold, and the mutant CYP102A enzyme comprises substitutions at one or more positions corresponding to amino acid residue positions 435, 82, 184, 260, and / or 72 of SEQ ID NO:2 in the polypeptide chain of the wild-type CYP102A enzyme, thereby enhancing the monooxygenase activity and / or altering the product selectivity of the mutant enzyme.

[0017] The present invention also provides a polynucleotide comprising a sequence encoding the enzyme of the present invention; or a cell expressing the enzyme of the present invention. The present invention also provides a transgenic animal or plant comprising the cell of the present invention.

[0018] The present invention also provides the following compounds: (1S,Z)-3-(2-((1R,3aS,7aR,E)-7a-methyl-1-((2R)-5-(2-methyloxiran-2-yl)pentan-2-yl)octahydro-4H-inden-4-ylidene)ethylidene)-4-methylenecyclohexan-1-ol (i.e., 25,26-epoxyvitamin D 3 ), and (3S,6R,E)-6-((1R,3aS,7aR,E)-4-((Z)-2-((S)-5-hydroxy-2-methylenecyclohexylidene)ethylidene)-7a-methyloctahydro-1H-inden-1-yl)-2-methylhept-4-ene-2,3-diol (i.e., 25,28-dihydroxyvitamin D 2 ). BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 - Biosynthesis of vitamin D (calcitriol) by successive oxidation of vitamin D by P450 enzymes. 3

[0020] Figure 2 - Biosynthesis of ercalcitriol by successive oxidation of vitamin D by P450 enzymes. 2

[0021] Figure 3 - Hydroxylation reaction of cholecalciferol (vitamin D 3 ) catalyzed by a selected cytochrome CYP102A1 variant.

[0022] Figure 4 - Hydroxylation reaction of calciferol (vitamin D 2 ) using a second-generation CYP102A1 variant.

[0023] BRIEF DESCRIPTION OF THE SEQUENCE LISTING

[0024] SEQ ID NO:1 - CYP102A1 nucleotide sequence.

[0025] SEQ ID NO:2 - CYP102A1 amino acid sequence.

[0026] SEQ ID NO:3 (CYP102A1, residues 1 - 470).

[0027] SEQ ID NO:4 (CYP102A2, P450 domain corresponding to 1 - 470 of CYP102A1).

[0028] SEQ ID NO:5 (CYP102A3, P450 domain corresponding to 1 - 470 of CYP102A1).

[0029] SEQ ID NO:6 (CYP102A4, P450 domain corresponding to 1 - 470 of CYP102A1).

[0030] SEQ ID NO:7 (CYP102A5, P450 domain corresponding to 1 - 470 of CYP102A1).

[0031] SEQ ID NO:8 (CYP102A6, P450 domain corresponding to 1 - 470 of CYP102A1).

[0032] SEQ ID NO:9 (CYP102A7, P450 domain corresponding to 1 - 470 of CYP102A1).

[0033] SEQ ID NO:10 (CYP102A8, P450 domain corresponding to 1 - 470 of CYP102A1).

[0034] SEQ ID NO:11 (CYP102A9, P450 domain corresponding to 1 - 470 of CYP102A1).

[0035] SEQ ID NO:12 (CYP102A10, P450 domain corresponding to 1 - 470 of CYP102A1).

[0036] SEQ ID NO:13 (CYP102A11, P450 domain corresponding to 1 - 470 of CYP102A1).

[0037] SEQ ID NO:14 (CYP102A12, P450 domain corresponding to 1 - 470 of CYP102A1).

[0038] SEQ ID NO:15 (CYP102A13, P450 domain corresponding to 1 - 470 of CYP102A1).

[0039] SEQ ID NO:16 (CYP102A14, P450 domain corresponding to 1 - 470 of CYP102A1).

[0040] SEQ ID NO:17 (CYP102A15, the P450 domain corresponding to residues 1 - 470 of CYP102A1).

[0041] SEQ ID NO:18 (CYP102A16, the P450 domain corresponding to residues 1 - 470 of CYP102A1).

[0042] SEQ ID NO:19 (CYP102A25, the P450 domain corresponding to residues 1 - 470 of CYP102A1).

[0043] SEQ ID NO:20 (CYP102A26, the P450 domain corresponding to residues 1 - 470 of CYP102A1).

[0044] SEQ ID NO:21 (Krac9955 (also known as CYP102A18), the P450 domain corresponding to residues 1 - 470 of CYP102A1).

[0045] SEQ ID NO:22 (Krac0936 (also known as CYP102A18), the P450 domain corresponding to residues 1 - 470 of CYP102A1).

[0046] SEQ ID NO:23 (CYP102B1, the P450 domain corresponding to residues 1 - 470 of CYP102A1). Detailed Description of the Invention

[0047] The present invention relates to a method for oxidative ring - opening of steroids. The method comprises the step of contacting the ring - opened steroid with a mutant CYP102A (a member of the cytochrome P450 family 102A sub - family) enzyme, wherein the CYP102A enzyme comprises a heme - monooxygenase domain having a P450 fold, and the mutant CYP102A enzyme comprises substitutions at one or more positions in the polypeptide chain of the wild - type CYP102A enzyme, thereby enhancing the monooxygenase activity of the mutant enzyme and / or altering its product selectivity.

[0048] An open-ring steroid is a steroid with a broken ring, for example, the cycloalkyl ring in a traditional steroid is broken. The open-ring steroid can be a 9,10-open-ring steroid, that is, an open-ring steroid obtained by breaking the bond between C9 and C10 atoms in steroid B ring. Preferably, the open-ring steroid contains a carbon chain with at least 5 carbon atoms at C17, more preferably, contains a carbon chain with at least 6 carbon atoms at C17, and most preferably, contains a carbon chain with 6 carbon atoms at C17 and is substituted by a methyl group at the fifth carbon atom of this carbon chain. The open-ring steroid containing a carbon chain with at least 5 carbon atoms at C17 is preferably a 9,10-open-ring steroid. In this article, the fifth carbon atom of the carbon chain at C17 is called "C25" (see, for example Figures 1-4 ).

[0049] The open-ring steroid can be vitamin D. The vitamin D can be selected from vitamin D 2 (ergocalciferol), vitamin D 3 (cholecalciferol), vitamin D 4 (22,23-dihydroergocalciferol), vitamin D 5 (sitocalciferol). Vitamin D 1 is a mixture of vitamin D 2 and lumisterol rather than a pure compound, and the term "vitamin D 1 " is no longer used. The vitamin D 2 can be present in the mixture of vitamin D 2 and lumisterol. The method of the present invention can oxidize C25 of the open-ring steroid (such as vitamin D). Preferably, the vitamin D is vitamin D 2 or vitamin D 3 .

[0050] This method is generally used to oxidize vitamin D at C25. This method is generally selectively used to oxidize vitamin D at C25 rather than at other positions of vitamin D, for example, oxidize at any other position of vitamin D. This method can be selectively used to oxidize vitamin D at C25 rather than oxidize at C20, C21, C22, C23, C24, C26 and / or C27 of vitamin D. Compared with the product obtained by oxidizing at another position of vitamin D (such as the product obtained by oxidizing at any other position of vitamin D), this method can produce at least 5 times the product obtained by oxidizing at C25. Compared with the product obtained by oxidizing at another position of vitamin D (such as compared with the product obtained by oxidizing at any other position of vitamin D), this method can produce at least 10 times or at least 100 times the oxidation product at C25.

[0051] This method is generally carried out in the presence of a mutant CYP102A enzyme, a substrate, and the native cofactor of the wild-type enzyme, and the native cofactor is generally dioxygen and a reducing agent, such as NADPH. This method can be carried out by using an enzyme (such as dehydrogenase) and its cosubstrate to regenerate NADPH from NADP+ while the cosubstrate of the dehydrogenase is oxidized. This method can regenerate the NADPH cofactor by an electrochemical method known in the art. Alternatively, this method can be carried out in the presence of hydrogen peroxide, for example, wherein the heme monooxygenase domain contains a mutation that allows activation of the heme in the absence of the reductase domain, such as the T268E mutation of SEQ ID NO:2.

[0052] This method can be carried out in the presence of an additional monooxygenase to oxidize the product produced by the mutant CYP102A enzyme at an additional site. The additional site can be at the 1st carbon of the oxidative ring-opened steroid produced by the mutant CYP102A enzyme. The additional monooxygenase can oxidize 25-hydroxy-vitamin D 3 to 1,25-dihydroxyvitamin D 3 . The additional monooxygenase can oxidize 25-hydroxy-vitamin D 2 to 1,25-dihydroxyvitamin D 2 . The additional monooxygenase can be a CYP27A enzyme (such as CYP27A1) and / or a CYP27R enzyme (such as CYP27R1).

[0053] In this method, the concentration of the mutant CYP102A enzyme is generally 10 -8 to 10 -2 M, preferably 10 -7 to 10 -4 M. Generally, this method is carried out at a temperature and / or pH at which the enzyme can function, for example, when the enzyme has at least 20%, 50%, 80% or higher peak activity. Usually, the pH is between 3 and 11, such as 5 to 9 or 6 to 8, preferably 7 to 7.8 or 7.4. Usually, the temperature is 10°C to 90°C, such as 25°C to 75°C or 30°C to 60°C.

[0054] This method can be carried out when the ratio of the initial substrate to the enzyme is at least 100:1, such as at least 500:1, at least 1000:1, at least 5000:1 or at least 10000:1. This method can be carried out when the ratio of the initial substrate to the enzyme is from 100:1 to 100000:1, such as between 500:1 - 20000:1.

[0055] Mutant CYP102A enzyme

[0056] The mutant CYP102A enzyme comprises a heme monooxygenase domain having a P450 fold.

[0057] The enhanced monooxygenase activity and / or altered product selectivity can be evaluated relative to the corresponding wild-type CYP102A enzyme. The enhanced monooxygenase activity and / or altered product selectivity can be evaluated relative to SEQ ID NO:2. The enhanced monooxygenase activity and / or altered product selectivity can be evaluated relative to the starting CYP102A enzyme prior to mutation.

[0058] A monooxygenase is an enzyme that incorporates a single oxygen atom into a substrate. Enhanced monooxygenase activity refers to an increase in the rate at which the monooxygenase catalyzes the incorporation of a single oxygen atom into a substrate. The enhanced monooxygenase activity can be an enhancement in incorporating a single oxygen atom into a ring-opened steroid, more preferably into vitamin D, more preferably at the C25 of vitamin D, and most preferably into vitamin D 3 or vitamin D 2 at the C25. The enhanced monooxygenase activity can be an enhancement in the oxidation of vitamin D 3 to 25-hydroxy-vitamin D 3 The enhanced monooxygenase activity can be an enhancement in the oxidation of vitamin D 2 to 25-hydroxy-vitamin D 3

[0059] The enhanced monooxygenase activity can be characterized by an increase in substrate conversion rate or an increase in the turnover number of one or more ring-opened steroids used for oxidation. The increase in substrate conversion rate or turnover number may or may not be present in all ring-opened steroids used by the CYP102A enzyme. The mutant CYP102A enzyme typically exhibits a substrate conversion rate that is at least 10%, 20%, 50%, 100%, 500%, 1000%, 2000%, 5000%, or 10000% higher than that of the wild-type enzyme. The mutant CYP102A enzyme may also have a turnover number that is at least 50%, 100%, 150%, 500%, 1000%, 2000%, 5000%, or 10000% higher than that of the wild-type enzyme.

[0060] The mutant CYP102A enzyme exhibits altered substrate specificity, which enables it to preferentially utilize ring-opened steroids that cannot be oxidized by the wild-type enzyme and known mutants.

[0061] ​The mutant CYP102A enzyme exhibits altered product selectivity, that is, new products that are not formed at all by the wild-type enzyme become the major or dominant products. Further altered characteristics of the mutant enzyme and further altered characteristics of the oxidation process catalyzed thereby are described below. Generally, the new products are characterized by a single hydroxyl group at C21-C27 of the ring-opened steroid, such as at C25 of the steroid, preferably at C25 of the ring-opened steroid, more preferably at C25 of vitamin D, and most preferably at C25 of vitamin D 3 or vitamin D 2 at C25.

[0062] The mutant enzyme has altered substrate selectivity. Altered substrate selectivity refers to the modification of the wild-type CYP102A enzyme to increase or decrease the binding affinity for a compound at the enzyme active site. For example, the CYP102A enzyme can be modified to increase the binding affinity for a steroid (preferably a ring-opened steroid, more preferably vitamin D, and most preferably vitamin D 3 or vitamin D 2 ) at the enzyme active site. The CYP102A enzyme can be modified to decrease the binding affinity for an undesired substrate (such as the natural substrate of the enzyme), a fatty acid, or a steroid that is not a 9,10-ring-opened steroid at the enzyme active site.

[0063] The heme monooxygenase domain containing the P450 fold may contain a sequence having at least 39.8% identity with the amino acid residues at positions 1-456 of SEQ ID NO:2. Preferably, the heme monooxygenase domain containing the P450 fold may contain a sequence having at least 40%, 41.6%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% identity with the amino acid residues at positions 1-456 of SEQ ID NO:2. More preferably, the heme monooxygenase domain containing the P450 fold may contain a sequence having at least 80%, 85%, 90%, 95%, 97%, or 99% identity with the amino acid residues at positions 1-456 of SEQ ID NO:2. The heme monooxygenase domain containing the P450 fold may contain the amino acid residues at positions 1-456 of SEQ ID NO:2.

[0064] The amino acid residues 1-456 of SEQ ID NO:2 contain the complete P450 fold of the monooxygenase domain. It has been shown that the monooxygenase domain can be considered as the amino acid residues 1-470 of SEQ ID NO:2 (see Peterson et al., Steroids, 1992, 62, 117-123). Thus, in some embodiments, the heme monooxygenase domain containing the P450 fold may contain a sequence having at least 39.8% identity to the amino acid residues 1-470 of SEQ ID NO:2. Preferably, the heme monooxygenase domain containing the P450 fold may contain a sequence having at least 40%, 41.6%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to the amino acid residues 1-470 of SEQ ID NO:2. More preferably, the heme monooxygenase domain containing the P450 fold may contain a sequence having at least 80%, 85%, 90%, 95%, 95%, 97% or 99% identity to the amino acid residues 1-470 of SEQ ID NO:2. The heme monooxygenase domain containing the P450 fold may contain the amino acid residues 1-470 of SEQ ID NO:2.

[0065] The heme monooxygenase domain containing the P450 fold may contain a sequence having at least 39.8% identity to SEQ ID NO:3, such as the sequences listed in SEQ ID NOs: 3-22. Preferably, the heme monooxygenase domain containing the P450 fold may contain a sequence having at least 40%, 41.6%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 95%, 97% or 99% identity to SEQ ID NO:3. More preferably, the heme monooxygenase domain containing the P450 fold may contain a sequence having at least 80%, 85%, 90%, 95%, 95%, 97% or 99% identity to SEQ ID NO:3. The heme monooxygenase domain containing the P450 fold may contain SEQ ID NO:3.

[0066] Preferably, the mutant CYP102A enzyme is a fusion of the heme monooxygenase domain and the reductase domain.

[0067] Reductase domains generally transfer one or more electrons from a reductant (e.g., NADH or NADPH) to the heme of a monooxygenase domain containing a P450 fold. The reductase domain can contain one or more cofactors to transfer one or more electrons from the reductant to the heme, such as FAD and FMN, ferredoxin, or flavodoxin.

[0068] The reductase domain can comprise or consist of a naturally occurring reductase, or can comprise or consist of a domain having at least 40% identity with a naturally occurring reductase, such as at least 80%, 85%, 90%, 95%, 95%, 97%, or 99% identity. The reductase domain can comprise or consist of the reductase domain of any electron transfer chain found in a naturally occurring P450 system.

[0069] The reductase domain can be a biflavin electron transfer domain that contains both FAD and FMN cofactors in a single polypeptide chain. The reductase domain can comprise or consist of a cytochrome P450 reductase (CPR) domain, such as a prokaryotic CPR domain or a eukaryotic CPR domain.

[0070] The reductase domain can comprise a flavin-dependent reductase domain, such as a Pseudomonas flavodoxin reductase. The reductase domain can comprise an FAD-containing reductase domain, such as a prokaryotic FAD-containing reductase domain, and ferredoxin or flavodoxin. The reductase domain can comprise or consist of an electron transfer flavoprotein capable of mediating electron transfer from a reductant (e.g., NADPH, NADH, or FADH) to the heme of a monooxygenase domain containing a P450 fold. The electron transfer flavoprotein can be a naturally occurring electron transfer flavoprotein, or a protein having at least 40% identity with a naturally occurring electron transfer flavoprotein, such as at least 80%, 85%, 90%, 95%, 95%, 97%, or 99% identity. The electron transfer flavoprotein is generally the flavoprotein of any electron transfer chain found in a naturally occurring P450 enzyme system. The electron transfer flavoprotein is generally a 2Fe-2S flavoprotein, such as Pseudomonas flavodoxin, or flavodoxin.

[0071] The reductase domain may comprise a sequence having at least 40% identity with the amino acid residues at positions 471 - 1048 of SEQ ID NO:2. Preferably, the reductase domain may comprise a sequence having at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 95%, 97% or 99% identity with the amino acid residues at positions 471 - 1048 of SEQ ID NO:2. More preferably, the reductase domain may comprise a sequence having at least 80%, 85%, 90%, 95%, 95%, 97% or 99% identity with the amino acid residues at positions 471 - 1048 of SEQ ID NO:2. The reductase domain may comprise the amino acid residues at positions 471 - 1048 of SEQ ID NO:2.

[0072] It should be understood that the reductase domain of CYP102A1 mediates the transfer of two electrons from NADPH to the heme in the P450 monooxygenase domain to activate dioxygen and generate the ferryl compound I species that oxidizes the substrate C - H bond. The amino acid substitutions discussed herein generally alter the substrate binding orientation, exposing a specific target C - H bond to the ferryl species, resulting in higher activity and selectivity. These substitutions do not affect the mechanism of ferryl species generation. Thus, the ferryl species of mutant CYP102A enzyme variants with the desired product selectivity can be generated using the pathway and substrate oxidation can be achieved in the absence of the reductase domain or NADPH or oxygen. An example is the conversion of CYP102A1 to a peroxygenase by the T268E mutation, such that the heme with an iron center in the Fe(III) state can be converted to the ferryl species by hydrogen peroxide, thereby achieving substrate oxidation - without the reductase domain, NADPH, and dioxygen (Shoji et al., Catal. Sci. Technol. 2016, 6, 5806–5811). Other substitutions, such as T268D and T268H, also enable the CYP102A1 enzyme to function as a peroxygenase. Adding the T268E mutation to the heme domain of the mutant CYP102A enzymes disclosed herein with open - ring steroid oxidation activity will provide an alternative system for oxidizing vitamin D 2 to 25 - hydroxyvitamin D 2 and oxidizing vitamin D 3 to 25 - hydroxyvitamin D 3 .

[0073] Thus, in some aspects, the mutant CYP102A enzyme does not contain a reductase domain. In such aspects, the process is carried out in the presence of a mechanism for generating the high-valent iron species in the heme oxygenase domain, such as in the presence of hydrogen peroxide, or in the presence of a separate polypeptide encoding the reductase domain described herein. The mutant CYP102A enzyme may additionally contain the T268E / T268D / T268H substitution in SEQ ID NO:2, or a corresponding substitution at one or more positions corresponding to amino acid residue position 268 of SEQ ID NO:2 in the polypeptide chain of the wild-type CYP102A enzyme. The substitution may be an additional substitution in addition to any other substitutions discussed herein.

[0074] The mutant CYP102A enzyme can be a mutant enzyme selected from CYP102A1, CYP102A2, CYP102A3, CYP102A4, CYP102A5, CYP102A6, CYP102A7, CYP102A8, CYP102A9, CYP102A10, CYP102A11, CYP102A12, CYP102A13, CYP102A14, CYP102A15, CYP102A16, CYP102A25, CYP102A26, Krac9955, and Krac0936, as shown in SEQ ID NOs: 3-22, respectively. Preferably, the mutant CYP102A enzyme is a mutant CYP102A1 enzyme. The CYP102A1 enzyme can be a natural or artificial homolog of CYP102A1, such as having at least 40% amino acid identity with SEQ ID NO:2. More preferably, the mutant CYP102A1 enzyme contains a sequence having at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 95%, 97%, or 99% identity with SEQ ID NO:2. The homolog typically contains an amino acid sequence corresponding (i.e., homologous or identical) to the amino acid sequence in the heme oxygenase domain of CYP102A1 (represented by amino acid positions 1-456 of SEQ ID NO:2; or in some embodiments represented by amino acid positions 1-470 of SEQ ID NO:2).

[0075] The CYP102A1 enzyme may comprise (or consist of) a sequence having at least 40% identity to SEQ ID NO:2 (the sequence of CYP102A1). The CYP102A1 enzyme may have any specified percentage identity when compared to amino acid residues 1-456 of SEQ ID NO:2. The CYP102A1 enzyme may have any specified percentage identity when compared to amino acid residues 1-470 of SEQ ID NO:2. The homologous sequences may represent mutant portions of the CYP102A1 sequence, and / or may exist in the form of the full-length fusions disclosed herein.

[0076] Homology can be measured using known methods. For example, the UWGCG software package provides the BESTFIT program which can be used to calculate homology (e.g., using it under its default settings) (Devereux et al., Nucleic Acids Res. 1984, 12, 387-395). The PILEUP and BLAST algorithms can be used to calculate homology or to align sequences (usually under their default settings), e.g., as described in Altschul S.F. (J. Mol. Evol. 1993, 36, 290-300) and Altschul, S.F. et al. (J. Mol. Biol. 1990, 215, 403-410).

[0077] Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ).

[0078] Typically, compared to amino acid residues 1-456 of SEQ ID NO:2, the mutant CYP102A enzyme may contain at least 2, 5, 10, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200 or 220 mutations (each mutation can be a substitution, insertion or deletion). Compared to amino acid residues 1-456 of SEQ ID NO:2, the mutant CYP102A enzyme may contain at most 2, 5, 10, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200 or 220 mutations (each mutation can be a substitution, insertion or deletion). When compared to SEQ ID NO:2, the mutant CYP102A enzyme may contain at least 2, 5, 10, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200 or 220 mutations (each mutation can be a substitution, insertion or deletion). When compared to SEQ ID NO:2, the mutant CYP102A enzyme may contain at most 2, 5, 10, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200 or 220 mutations (each mutation can be a substitution, insertion or deletion).

[0079] When compared to amino acid residues 1-470 of SEQ ID NO:2, the mutant CYP102A enzyme may contain at least 2, 5, 10, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200 or 220 mutations (each mutation can be a substitution, insertion or deletion). When compared to amino acid residues 1-470 of SEQ ID NO:2, the mutant CYP102A enzyme may contain at most 2, 5, 10, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200 or 220 mutations (each mutation can be a substitution, insertion or deletion).

[0080] The enzyme activity of the CYP102A enzyme of the present invention is typically measured in vitro using any of the substrates or test conditions mentioned herein, and is typically expressed as a conversion rate (Conv; percentage of substrate converted to product) and / or preferably as a turnover number (TON; turnover number of the enzyme for producing oxidized ring-opened steroids). The conversion rate and / or turnover number are preferably when the ring-opened steroid is vitamin D 2 or vitamin D 3 and / or when the product is 25-hydroxyvitamin D 2 or 25-hydroxyvitamin D 3Measurement is carried out at this time. This conversion rate is usually measured when the ratio of substrate to enzyme is, for example, 500:1 or 1000:1. The mutant CYP102A enzyme (for example, when used in the process of the present invention) may have a conversion rate of at least 10%, such as at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80% or higher. The mutant CYP102A enzyme may have a turnover number of at least 50, such as at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700 or at least 800 or higher. The mutant CYP102A enzyme may have a conversion rate of at least 10% and a turnover number of at least 50.

[0081] The mutant CYP102A enzyme may comprise a sequence having at least 40% identity with SEQ ID NO:2. Preferably, the mutant CYP102A enzyme may comprise a sequence having at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 95%, 97% or 99% identity with SEQ ID NO:2. More preferably, the mutant CYP102A enzyme may comprise a sequence having at least 80%, 85%, 90%, 95%, 95%, 97% or 99% identity with SEQ ID NO:2. The mutant CYP102A enzyme may comprise SEQ ID NO:2.

[0082] The mutant CYP102A enzyme comprises substitutions at one or more positions in the polypeptide chain of the wild-type CYP102A enzyme, thereby enhancing the monooxygenase activity of the mutant enzyme and / or altering its product selectivity.

[0083] Preferably, the mutant CYP102A enzyme comprises substitutions at one or more positions corresponding to amino acid positions 435, 82, 184, 260 and / or 72 of SEQ ID NO:2. Generally, the mutant CYP102A enzyme may comprise 2 or more, 3 or more, 4 or more or all 5 substitutions at positions corresponding to amino acid positions 435, 82, 184, 260 and / or 72 of SEQ ID NO:2.

[0084] One or more substitutions may be selected from E435I, E435M, E435T, A82M, A82L, A82I, A82F, A82W, A184I, T260G, T260A, S72A and / or S72G in SEQ ID NO:2, or corresponding substitutions at one or more positions corresponding to amino acid residues 435, 82, 184, 260 and / or 72 of SEQ ID NO:2 in the polypeptide chain of the wild-type CYP102A enzyme. Preferably, the one or more substitutions include E435I, E435M, E435T, S72A and / or S72G in SEQ ID NO:2. More preferably, the one or more substitutions include E435I or E435M in SEQ ID NO:2.

[0085] The CYP102A enzyme may have 1, 2, 3, 4, 5 to 10, 10 to 20, 20 to 40 or more additional mutations, such as substitutions, insertions or deletions. These additional mutations may or may not enhance the monooxygenase activity of the mutant CYP102A enzyme and / or alter its product selectivity. Other mutations may be within or outside the active site. For example, these mutations may be in the second sphere, i.e., residues that affect or contact the position or orientation of one or more amino acids in the active site. Insertions are usually at the N-terminus and / or C-terminus. Thus, the enzyme may contain a short peptide of up to 20 amino acids or a full-length protein fused to one or both of its ends, e.g., to assist protein purification by affinity chromatography or immobilization on a solid matrix (such as via a histidine tag). Deletions usually involve deleting amino acids that do not participate in catalysis, such as amino acids outside the active site (so the enzyme is a mutant fragment of a naturally occurring enzyme).

[0086] Other mutations in the active site may alter the position and / or conformation of the substrate when the substrate binds to the active site. These mutations may make the site to be oxidized on the substrate more accessible to the heme group. Thus, these mutations may be substitutions of amino acids with smaller or larger, more polar or less polar side chains.

[0087] Preferably, the CYP102A enzyme further comprises one or more substitutions at one or more positions corresponding to amino acid residue positions 29, 78, 87, 178, 263, 264, 268, 328, and / or 354 of SEQ ID NO:2. The CYP102A enzyme may comprise two or more, three or more, four or more, or five or more substitutions at positions corresponding to amino acid residue positions 78, 87, 178, 263, 264, 268, 328, and / or 354 of SEQ ID NO:2. The one or more additional substitutions are optionally selected from V78F, F87A, F87V, F87I, V178F, V178W, V178L, V178I, I263G, A264G, T268S, A328G, and / or M354F, or from the corresponding substitutions at one or more positions corresponding to amino acid residue positions 78, 87, 178, 263, 264, 268, 328, and / or 354 of SEQ ID NO:2.

[0088] The CYP102A enzyme may comprise the above-mentioned substitution at position 82 of SEQ ID NO:2 or its corresponding position, and comprise the above-mentioned substitution at position 87 of SEQ ID NO:2 or its corresponding position. The CYP102A enzyme may comprise one or more further mutations at position 184 of SEQ ID NO:2 or its corresponding position, the above-mentioned substitution at position 260 of SEQ ID NO:2 or its corresponding position, the above-mentioned substitution at position 72 of SEQ ID NO:2 or its corresponding position, and / or the above-mentioned substitution at position 435 of SEQ ID NO:2 or its corresponding position. Preferably, the mutant CYP102A enzyme comprises the substitution at position 82 of SEQ ID NO:2 or its corresponding position, the substitution at position 87 of SEQ ID NO:2 or its corresponding position, and the substitution at position 435 of SEQ ID NO:2 or its corresponding position.

[0089] The CYP102A enzyme may comprise one or more of the following sets of substitutions in SEQ ID NO:2:

[0090] -A82M / F87A;

[0091] -A82M / F87I;

[0092] -A82M / F87V;

[0093] -A82M / F87S;

[0094] -A82M / F87T;

[0095] -A82M / T260G;

[0096] -A82M / A184I;

[0097] -A82M / S72A;

[0098] -A82M / E435I, E435M or E435T;

[0099] -A82M / E435I;

[0100] -A82M / E435M;

[0101] -F87A / T260G;

[0102] -F87I / T260G;

[0103] -F87V / T260G;

[0104] -F87S / T260G;

[0105] -F87T / T260G;

[0106] -F87A / A184I;

[0107] -F87I / A184I;

[0108] -F87V / A184I;

[0109] -F87S / A184I;

[0110] -F87T / A184I;

[0111] -F87A / S72A;

[0112] -F87I / S72A;

[0113] -F87V / S72A;

[0114] -F87S / S72A;

[0115] -F87T / S72A;

[0116] -A82M / F87A / T260G;

[0117] -A82M / F87I / T260G;

[0118] -A82M / F87V / T260G;

[0119] -A82M / F87S / T260G;

[0120] -A82M / F87T / T260G;

[0121] -A82M / F87A / A184I;

[0122] -A82M / F87I / A184I;

[0123] -A82M / F87V / A184I;

[0124] -A82M / F87S / A184I;

[0125] -A82M / F87T / A184I;

[0126] -A82M / F87A / E435I, E435M or E435T; -A82M / F87I / E435I, E435M or E435T; -A82M / F87V / E435I, E435M or E435T; -A82M / F87S / E435I, E435M or E435T; -A82M / F87T / E435I, E435M or E435T; -A82M / F87A / E435I;

[0127] -A82M / F87A / E435M;

[0128] -A82M / F87I / E435I;

[0129] -A82M / F87I / E435M;

[0130] -A82M / F87V / E435I;

[0131] -A82M / F87V / E435M;

[0132] -A82M / F87S / E435I;

[0133] -A82M / F87S / E435M;

[0134] -A82M / F87T / E435I;

[0135] -A82M / F87T / E435M;

[0136] -F87A / A184I / T260G;

[0137] -F87I / A184I / T260G;

[0138] -F87V / A184I / T260G;

[0139] -F87S / A184I / T260G;

[0140] -F87T / A184I / T260G;

[0141] -F87A / A184I / S72A;

[0142] -F87I / A184I / S72A;

[0143] -F87V / A184I / S72A;

[0144] -F87S / A184I / S72A;

[0145] -F87T / A184I / S72A;

[0146] -F87A / A184I / E435I, E435M or E435T;

[0147] -F87I / A184I / E435I, E435M or E435T;

[0148] -F87V / A184I / E435I, E435M or E435T;

[0149] -F87S / A184I / E435I, E435M or E435T;

[0150] -F87T / A184I / E435I, E435M or E435T;

[0151] -F87A / A184I / E435I;

[0152] -F87A / A184I / E435M;

[0153] -F87I / A184I / E435I;

[0154] -F87I / A184I / E435M;

[0155] -F87V / A184I / E435I;

[0156] -F87V / A184I / E435M;

[0157] -F87S / A184I / E435I;

[0158] -F87S / A184I / E435M;

[0159] -F87T / A184I / E435I;

[0160] -F87T / A184I / E435M;

[0161] -F87A / A82M / A184I / T260G;

[0162] -F87I / A82M / A184I / T260G;

[0163] -F87V / A82M / A184I / T260G;

[0164] -F87S / A82M / A184I / T260G;

[0165] -F87T / A82M / A184I / T260G;

[0166] -F87A / A82M / T260G / S72A;

[0167] -F87I / A82M / T260G / S72A;

[0168] -F87V / A82M / T260G / S72A;

[0169] -F87S / A82M / T260G / S72A;

[0170] -F87T / A82M / T260G / S72A;

[0171] -F87A / A82M / A184I / T260G / S72A;

[0172] -F87I / A82M / A184I / T260G / S72A;

[0173] -F87V / A82M / A184I / T260G / S72A;

[0174] -F87S / A82M / A184I / T260G / S72A;

[0175] -F87T / A82M / A184I / T260G / S72A;

[0176] -F87A, F87I, F87V, F87S or F87T / A82M / A184I / T260G / E435I, E435M or E435T; -F87A / A82M / A184I / T260G / E435I;

[0177] -F87A / A82M / A184I / T260G / E435M;

[0178] -F87I / A82M / A184I / T260G / E435I;

[0179] -F87I / A82M / A184I / T260G / E435M;

[0180] -F87V / A82M / A184I / T260G / E435I;

[0181] -F87V / A82M / A184I / T260G / E435M;

[0182] -F87S / A82M / A184I / T260G / E435I;

[0183] -F87S / A82M / A184I / T260G / E435M;

[0184] -F87T / A82M / A184I / T260G / E435I;

[0185] -F87T / A82M / A184I / T260G / E435M;

[0186] -F87A, F87I, F87V, F87S or F87T / A82M / A184I / T260G / S72A / E435I, E435M or E435T;

[0187] -F87A / A82M / A184I / T260G / S72A / E435I;

[0188] -F87A / A82M / A184I / T260G / S72A / E435M;

[0189] -F87I / A82M / A184I / T260G / S72A / E435I;

[0190] -F87I / A82M / A184I / T260G / S72A / E435M;

[0191] -F87V / A82M / A184I / T260G / S72A / E435I;

[0192] -F87V / A82M / A184I / T260G / S72A / E435M;

[0193] -F87S / A82M / A184I / T260G / S72A / E435I;

[0194] -F87S / A82M / A184I / T260G / S72A / E435M;

[0195] -F87T / A82M / A184I / T260G / S72A / E435I;

[0196] -F87T / A82M / A184I / T260G / S72A / E435M;

[0197] -F87A, F87I, F87V, F87S or F87T / A82M / A184I / T260G / S72A / E435I, E435M or E435T / L29M;

[0198] -F87A / A82M / A184I / T260G / S72A / E435I / L29M;

[0199] -F87I / A82M / A184I / T260G / S72A / E435I / L29M;

[0200] -F87V / A82M / A184I / T260G / S72A / E435I / L29M;

[0201] -F87S / A82M / A184I / T260G / S72A / E435I / L29M;

[0202] -F87T / A82M / A184I / T260G / S72A / E435I / L29M;

[0203] -F87A / A82M / A184I / T260G / S72A / E435M / L29M;

[0204] -F87I / A82M / A184I / T260G / S72A / E435M / L29M;

[0205] -F87V / A82M / A184I / T260G / S72A / E435M / L29M;

[0206] -F87S / A82M / A184I / T260G / S72A / E435M / L29M;

[0207] -F87T / A82M / A184I / T260G / S72A / E435M / L29M;

[0208] -F87A / A82M / A184I / T260G / S72A / E435T / L29M;

[0209] -F87I / A82M / A184I / T260G / S72A / E435T / L29M;

[0210] -F87V / A82M / A184I / T260G / S72A / E435T / L29M;

[0211] -F87S / A82M / A184I / T260G / S72A / E435T / L29M;

[0212] -F87T / A82M / A184I / T260G / S72A / E435T / L29M;

[0213] or one or more corresponding substitutions at one or more positions corresponding to the amino acid residue positions of SEQ ID NO:2 in the polypeptide chain of the wild-type CYP102A enzyme.

[0214] In addition to any combination of mutations described above, the CYP102A enzyme may also contain one or more substitutions at one or more positions corresponding to amino acid residue positions 47, 51, 74, 171, 188, 239, 259, 307, 319 and / or 353 of SEQ ID NO:2. The one or more substitutions may be selected from R47L, Y51F, A74G, H171L, N239H, I259V, L188Q, Q307H, N319Y and / or L353I in SEQ ID NO:2, or from one or more corresponding substitutions at one or more positions corresponding to amino acid residue positions 47, 51, 74, 171, 188, 239, 259, 307, 319 and / or 353 in the polypeptide chain of the wild-type CYP102A enzyme.

[0215] When describing a specific mutant of CYP102A, the letter of the amino acid residue present in the wild-type CYP102A is followed by the site and then the amino acid in the mutant. These sites may be associated with the numbering shown in SEQ ID NO:2. To represent multiple mutations in the same protein, each mutation is listed separated by a slash. Particularly preferred mutants can be described using the entry numbers provided in Tables 1 - 5 of the Examples.

[0216] Although the mutations are defined by reference to positions in CYP102A1, the present invention also encompasses equivalent substitution mutations at homologous or corresponding positions in the polypeptide chains of CYP102A homologs that have at least 39.8% amino acid identity with SEQ ID NO:2. The equivalent sites are determined by reference to amino acids 1 - 456 of SEQ ID NO:2. The homologous or corresponding sites can be readily deduced by aligning the sequence of the homolog with the amino acid sequence of positions 1 - 456 of CYP102A1 (SEQ ID NO:2) based on the homology between the sequences. The PILEUP and BLAST algorithms can be used to align these sequences. When the homologous or corresponding amino acid is an active site residue, it will generally be located in a position in the active site of the homolog similar to any particular amino acid discussed herein.

[0217] Despite having a highly conserved tertiary structure, it is well known to those skilled in the art that the P450 enzyme superfamily has a low homology primary structure among proteins and enzymes, which is not common. The amino acid identity of P450 enzymes in different families is as low as 20%. A sample alignment between CYP102A1 and structurally characterized P450 enzymes is shown in Table A. In the phylogenetic classification of the P450 superfamily, enzymes with an amino acid identity of only 40% are placed within the same family, while closely related members within the family (>55% identity) are grouped into subfamilies (e.g., see Table B). It is increasingly recognized that in some cases, the 40% cut-off for classifying enzymes into the same family may be too high, and more consideration may be needed in the future for enzyme activity and the generally higher homology observed for active site residues.

[0218] Therefore, enzymes containing a sequence with at least 40% amino acid identity to CYP102A1 (SEQ ID NO:2) can also be easily identified based on the "P450 fold"; and by understanding the conservation of the α-helix and β-strand arrangements of the P450 fold shared throughout the enzyme family, it can assist in aligning homologous sequences and introducing equivalent mutations at corresponding or homologous positions.

[0219] Table A. Sequence similarity between the heme domain of CYP102A1 (amino acid residues 1 - 470) and various structurally characterized cytochrome P450 enzymes.

[0220]

[0221]

[0223] CYP165C (P450oxyC) 48 / 204 (23%) 90 / 204 (44%) 41 / 204 (20%)

[0224] CYP119A2 44 / 166 (26%) 70 / 166 (42%) 35 / 166 (21%)

[0225] CYP152A1 (P450BS) 41 / 148 (27%) 62 / 148 (41%) 20 / 148 (13%)

[0226] CYP121 44 / 184 (23%) 72 / 184 (39%) 35 / 184 (19%) Table B. Sequence similarity between the heme domain of CYP102A1 (amino acid residues 1 - 470 of SEQ ID NO:2) and other members of the CYP102A subfamily. Note that although belonging to the same subfamily, the homology between CYP102A25 and CYP102A1 is only 39.8%, which is almost the same as the homology between CYP102A1 and another member of the CYP102 subfamily, CYP102B1.

[0227]

[0228]

[0229] It should be understood that members of the CYP102A family are fusions of an electron transfer reductase domain and a heme monooxygenase domain. These domains can be cleaved by proteolysis or by truncating the full - length gene. The active site (substrate - binding pocket) is located in the heme domain. Some members of the CYP102 family are not fusion proteins, but their sequence homology with the CYP102A heme domain is 40%. Thus, in these cases, sequence homology can be determined based solely on the heme domain. Residues equivalent to those in CYP102A described herein in these enzymes can be identified by sequence homology analysis and structural analysis known to those skilled in the art.

[0230] Amino acids in the active site refer to those that line or define the substrate - binding site during catalysis, or those that line or define the site through which the substrate must pass before reaching the catalytic site. Thus, such amino acids usually interact with the substrate during entry into the catalytic site or during catalysis. This interaction usually occurs through electrostatic interactions (between charged or polar groups), hydrophobic interactions, hydrogen bonds, or van der Waals forces. Active - site amino acids can be identified by sequence alignment and by reference to the known crystal structure of the wild - type CYP102A heme domain or the crystal structure of its homologs.

[0231] When the mutated residue is not an active - site residue, homologous or corresponding positions can be inferred by computer or manual alignment of the sequences of homologs with CYP102A1, which can be assisted by knowing the residues flanking the mutated position in CYP102A1 listed in SEQ ID NO:2. Thus, for example, the 10 N - terminal and C - terminal flanking residues at the following positions in CYP102A1 are:

[0232] CDESRFDKNL(S72)QALKFVRDFA

[0233] DKNLSQALKF(V78)RDFAGDGLFT

[0234] SQALKFVRDF(A82)GDGLFTSWTH

[0235] FVRDFAGDGL(F87)TSWTHEKNWK

[0236] DQPHPFITSM(V178)RALDEAMNKL

[0237] ITSMVRALDE(A184)MNKLQRANPD

[0238] DDENIRYQII(T260)FLIAGHETTS

[0239] NIRYQIITFL(I263)AGHETTSGLL

[0240] IRYQIITFLI(A264)GHETTSGLLS

[0241] IITFLIAGHE(T268)TSGLLSFALY

[0242] LNEALRLWPT(A328)PAFSLYAKED

[0243] EYPLEKGDEL(M354)VLIPQLHRDK

[0244] DHTNYELDIK(E435)TLTLKPEGFV

[0245] Retention of two, three or more N-terminal and / or C-terminal flanking residues may permit inference of homologous or corresponding positions for mutations to be introduced.

[0246] Similar analyses can be performed for any other positions in CYP102A1 mentioned in the specification to identify homologous or corresponding sites in naturally occurring homologues.

[0247] The properties of the amino acids substituted at the CYP102A positions described herein (or equivalent positions as defined above) mainly depend on the requirements for the mutants to exhibit enhanced monooxygenase activity and / or altered product specificity. Thus, the introduced amino acids will generally enhance monooxygenase activity and / or alter product specificity. When referring to a specific substitution in CYP102A, it should be understood that, according to the present invention, any substitution of other amino acid residues at the same position, the effect of which on the oxidative activity and / or product specificity of the CYP102A enzyme is redundant or similar to the effect of the said specific substitution, is included. Similarly, when a specific substitution also affects other parameters of the CYP102A enzyme (such as substrate specificity), it should be understood that, according to the present invention, the substitution of other amino acid residues that cause redundant or similar effects can also be considered.

[0248] In some embodiments, the substitution introduces a conservative change, i.e., an amino acid is replaced with another amino acid having a similar chemical structure, similar chemical properties or similar side-chain volume. The introduced amino acids may have similar polarity, hydrophilicity or hydrophobicity compared to the amino acids they replace. Conservative amino acid changes are well known in the art and can be selected according to the changes defined in Table C. When amino acids have similar polarity, this can also be determined by referring to the hydrophilicity scale of the amino acid side chains (Table D). The side-chain volumes of the twenty naturally occurring amino acids can be grouped (from smallest to largest) as follows: Gly, Ala, Ser, Cys, Thr≈Asp≈Pro≈Asn, Val≈Glu≈Gln≈His, Ile≈Leu≈Met≈Lys, Phe≈Arg≈Tyr≈Trp.

[0249] Table C. Physical properties of amino acids

[0250]

[0251]

[0252] Table D. Hydrophilicity scale

[0253]

[0254] Conservative amino acid changes can also be determined by referring to the "Point Accepted Mutation" (PAM) or "BLOcks Substitution Matrix" (BLOSUM) series of scoring matrices for amino acid sequence conservation. Thus, conservative amino acid changes can be members of an equivalent group, i.e., a group of amino acids that have positive scores for each other in the similarity representation of the scoring matrix selected for aligning the reference polypeptide chain and the mutant polypeptide chain.

[0255] It should be understood that the physical property definitions provided in Table C are not considered as limitations to the present invention. For example, the amino acid proline is classified as non-polar, but it also has a rigid property and can cause changes in secondary structure. For example, proline often appears at the end of a helical structure. In addition, depending on the specific environment of the side chain of a given amino acid residue, for example, the amino acid tyrosine, which is usually classified as hydrophobic due to its aromatic ring, may have a similar functional effect with a polar amino acid residue (such as threonine) through its hydroxyl group. Therefore, for the purposes of the present invention, tyrosine can be considered as an amino acid with both hydrophobic and polar properties. In addition, the amino acids described as polar or hydrophilic can be uncharged or charged, and can also be basic or acidic. It is well known that the dissociation constant (pKa) value of the amino acid histidine is close to 7. Therefore, under neutral pH conditions, depending on the protein environment, its side chain may be protonated or not protonated, and thus may be charged or uncharged. Therefore, for the purposes of the present invention, histidine can be considered as an amino acid residue with both polar charged or polar uncharged properties.

[0256] In the polypeptide chain, the substitution at the site corresponding to amino acid residue 435 of SEQ ID NO:2 can be a substitution with a neutral amino acid. Preferably, the substitution at the site corresponding to amino acid residue 435 of SEQ ID NO:2 in the polypeptide chain can be a substitution with a hydrophobic neutral amino acid. The substitution at the site corresponding to amino acid residue 435 of SEQ ID NO:2 in the polypeptide chain can be a substitution selected from E435I, E435M, and E435T, preferably E435I and E435M, and more preferably E435I.

[0257] In the polypeptide chain, the substitution at the site corresponding to amino acid residue 82 of SEQ ID NO:2 can be a substitution with a neutral and / or hydrophobic amino acid. Residue 82 of SEQ ID NO:2 is located distally to the heme in the substrate binding pocket. Preferably, the substitution at the site corresponding to amino acid residue 82 of SEQ ID NO:2 in the polypeptide chain can be a substitution with a residue that can block the residue at the distal end of the substrate binding pocket from the heme, such as a substitution that increases the side chain volume of the residue at position 82. The substitution at the site corresponding to amino acid residue 82 of SEQ ID NO:2 in the polypeptide chain can be a substitution selected from A82M, A82L, A82I, A82F, and A82W.

[0258] In the polypeptide chain, the substitution at the site corresponding to amino acid residue 184 of SEQ ID NO:2 can be a substitution with an aliphatic, hydrophobic, and / or neutral amino acid. The substitution at the site corresponding to amino acid residue 184 of SEQ ID NO:2 in the polypeptide chain can be A184I.

[0259] In the polypeptide chain, the substitution at the site corresponding to amino acid residue 260 of SEQ ID NO:2 can be a substitution with an aliphatic, hydrophobic, and / or neutral amino acid. Preferably, the substitution at the site corresponding to amino acid residue 260 of SEQ ID NO:2 in the polypeptide chain can be a substitution that reduces the side-chain volume of the residue at site 260, such as a substitution with S, C, A, or G. The substitution at the site corresponding to amino acid residue 260 of SEQ ID NO:2 in the polypeptide chain can be T260A or T260G.

[0260] In the polypeptide chain, the substitution at the site corresponding to amino acid residue 72 of SEQ ID NO:2 can be a substitution with an aliphatic, hydrophobic, and / or neutral amino acid. Preferably, the substitution at the site corresponding to amino acid residue 72 of SEQ ID NO:2 in the polypeptide chain can be a substitution that reduces the side-chain volume of the residue at site 72. The substitution at the site corresponding to amino acid residue 72 of SEQ ID NO:2 in the polypeptide chain can be S72A or S72G.

[0261] In the polypeptide chain, the substitution at the site corresponding to amino acid residue 78 of SEQ ID NO:2 can be a substitution with an aromatic, hydrophobic, and / or neutral amino acid. Preferably, the substitution at the site corresponding to amino acid residue 78 of SEQ ID NO:2 in the polypeptide chain can be a substitution that increases the side-chain volume of the residue at site 78. The substitution at the site corresponding to amino acid residue 78 of SEQ ID NO:2 in the polypeptide chain can be V78F.

[0262] In the polypeptide chain, the substitution at the site corresponding to amino acid residue 87 of SEQ ID NO:2 can be a substitution with an aliphatic, hydrophobic, and / or neutral amino acid. Preferably, the substitution at the site corresponding to amino acid residue 87 of SEQ ID NO:2 in the polypeptide chain is any substitution that can achieve the oxidation of the steroid substrate, such as A / V / I / S / T. The substitution at the site corresponding to amino acid residue 87 of SEQ ID NO:2 in the polypeptide chain can be selected from F87A, F87V, and F87I. Preferably, the mutant CYP102A enzyme comprises one or more substitutions disclosed herein and incorporates a substitution with an aliphatic and neutral amino acid at the site corresponding to amino acid residue 87 of SEQ ID NO:2 in the polypeptide chain, such as a substitution selected from F87A, F87V, F87I, F87S, and F87T.

[0263] In the polypeptide chain, the substitution at the site corresponding to amino acid residue 178 of SEQ ID NO:2 can be a substitution with a hydrophobic and / or neutral amino acid. Preferably, the substitution at the site corresponding to amino acid residue 178 of SEQ ID NO:2 in the polypeptide chain can be a substitution that increases the side-chain volume of the residue at position 178. The substitution at the site corresponding to amino acid residue 178 of SEQ ID NO:2 in the polypeptide chain is selected from V178F, V178W, V178L, and V178I.

[0264] In the polypeptide chain, the substitution at the site corresponding to amino acid residue 263 of SEQ ID NO:2 can be a substitution with an aliphatic and / or neutral amino acid. Preferably, the substitution at the site corresponding to amino acid residue 263 of SEQ ID NO:2 in the polypeptide chain can be a substitution that reduces the side-chain volume of the residue at position 263. The substitution at the site corresponding to amino acid residue 263 of SEQ ID NO:2 in the polypeptide chain can be I263G.

[0265] In the polypeptide chain, the substitution at the site corresponding to amino acid residue 264 of SEQ ID NO:2 can be a substitution with an aliphatic and / or neutral amino acid. Preferably, the substitution at the site corresponding to amino acid residue 263 of SEQ ID NO:2 in the polypeptide chain can be a substitution that reduces the side-chain volume of the residue at position 263. The substitution at the site corresponding to amino acid residue 264 of SEQ ID NO:2 in the polypeptide chain can be A264G.

[0266] In the polypeptide chain, the substitution at the site corresponding to amino acid residue 268 of SEQ ID NO:2 can be a substitution with a polar, hydrophilic, and / or neutral amino acid. Preferably, the substitution at the site corresponding to amino acid residue 268 of SEQ ID NO:2 in the polypeptide chain can be a substitution that reduces the side-chain volume of the residue at position 268. The hydroxyl group in the side chain of residue 268 is crucial for catalysis. Therefore, the substitution at the site corresponding to amino acid residue 268 of SEQ ID NO:2 in the polypeptide chain can be T268S.

[0267] In the polypeptide chain, the substitution at the site corresponding to amino acid residue 328 of SEQ ID NO:2 can be a substitution with an aliphatic and / or neutral amino acid. Preferably, the substitution at the site corresponding to amino acid residue 328 of SEQ ID NO:2 in the polypeptide chain can be a substitution that reduces the side-chain volume of the residue at position 328. The substitution at the site corresponding to amino acid residue 328 of SEQ ID NO:2 in the polypeptide chain can be A328G.

[0268] In the polypeptide chain, the substitution at the site corresponding to amino acid residue 354 of SEQ ID NO:2 can be a substitution with an aromatic, hydrophobic, and / or neutral amino acid. Preferably, the substitution at the site corresponding to amino acid residue 354 of SEQ ID NO:2 in the polypeptide chain can be a substitution that increases the side-chain volume of the residue at position 354. The substitution at the site corresponding to amino acid residue 354 of SEQ ID NO:2 in the polypeptide chain can be M354F.

[0269] Mutant CYP102A enzyme

[0270] The present invention also relates to a mutant CYP102A enzyme, which comprises a heme monooxygenase domain containing a P450 fold structure, and the mutant CYP102A enzyme comprises substitutions at one or more sites corresponding to amino acid residue sites 435, 82, 184, 260, and / or 72 of SEQ ID NO:2 in the polypeptide chain of the wild-type CYP102A enzyme, so as to enhance the monooxygenase activity of the mutant enzyme and / or alter its product selectivity. The enhanced monooxygenase activity can be enhanced monooxygenation of ring-opened steroids, preferably enhanced monooxygenation of vitamin D, more preferably enhanced monooxygenation of vitamin D 3 or vitamin D 2 The altered product selectivity can be the generation of a ring-opened steroid product oxidized at C25, preferably vitamin D oxidized at C25, more preferably vitamin D oxidized at C25 3 or vitamin D 2 . The wild-type CYP102A enzyme does not act as a monooxygenase for ring-opened steroid products.

[0271] The conversion rate of the mutant CYP102A enzyme (for example, when used in the method of the present invention) can be at least 10%, such as at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80% or higher. The turnover number of the mutant CYP102A enzyme can be at least 50, such as at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, or at least 800 or higher. The mutant CYP102A enzyme can have a conversion rate of at least 10% and a TON of at least 50.

[0272] Substitutions at one or more sites corresponding to amino acid positions 435, 82, 184, 260, and / or 72 of SEQ ID NO:2 can be any of the substitutions discussed above with respect to the methods of the present invention. The mutant CYP102A enzyme can comprise one or more additional substitutions discussed above with respect to the methods of the present invention.

[0273] In addition, a method for oxidizing an organic compound substrate is provided, which comprises the step of contacting the organic compound substrate with the mutant CYP102A enzyme of the present invention. The organic compound is generally any organic compound capable of being oxidized by a monooxygenase. The suitability of any organic compound for oxidation by a monooxygenase can be routinely determined by the methods described herein. The organic compound can be a steroid, preferably an open-ring steroid.

[0274] The oxidation process results in the formation of a carbon-oxygen bond (C–O) in the compound, typically by oxidation of a carbon-hydrogen bond to form an alcohol, but may also result in the formation of an epoxide by oxidation of a carbon-carbon bond (C=C). Thus, oxidation can introduce an alcohol group, an aldehyde group, a ketone group, or an epoxy group. Alternatively, oxidation may result in further oxidation of an oxygen-containing group, such as conversion of an alcohol group to an aldehyde or a ketone. One, two, or more carbon atoms may be attached in the same substrate molecule. Oxidation may also result in N-dealkylation and O-dealkylation of the substrate molecule.

[0275] The substrate can be a natural substrate of the wild-type CYP102A enzyme or a substrate that is not normally a substrate of the wild-type enzyme but can be utilized as a substrate in the mutant enzyme. Examples of natural substrates of the CYP102A enzyme include branched and straight-chain fatty acids, which are hydroxylated by wild-type CYP102A1 at the sub-terminal positions (ω-1 to ω-3). Preferred examples are lauric acid, undecanoic acid, capric acid, pelargonic acid, and caprylic acid. Examples of non-natural substrates of the wild-type CYP102A enzyme that can be used with the mutant CYP102A enzyme include steroids, such as open-ring steroids.

[0276] Other products of the present invention

[0277] The present invention also relates to a polynucleotide comprising a sequence encoding the mutant CYP102A enzyme of the present invention.

[0278] The polynucleotide can be in the form of a vector. The vector is generally a transposon, plasmid, viral, or phage vector. It generally contains an origin of replication. It generally contains one or more selectable marker genes, such as an ampicillin resistance gene in the case of a bacterial plasmid. The vector is generally introduced into a host cell using conventional techniques, including calcium phosphate precipitation, DEAE-dextran transfection, or electroporation.

[0279] The present invention also relates to a cell expressing the mutant CYP102A enzyme of the present invention. The cell can be a prokaryotic cell. The cell can be a eukaryotic cell. The cell can be a bacterial strain, such as Escherichia coli, Pseudomonas sp., Rhodococcus sp. or Bacillus sp. The cell can also be a yeast strain, such as Pichia sp.

[0280] The present invention also relates to a transgenic animal or plant comprising a cell expressing the mutant CYP102A enzyme of the present invention. The transgenic animal can be a non-mammal, such as a non-human animal. The animal or plant is transgenic for one or more polynucleotides encoding the mutant CYP102A enzyme. They can be homozygous or heterozygous for these polynucleotides, which are typically transiently introduced into the cell or stably integrated into the cell. The plant or animal can be obtained by transforming a suitable cell (such as an embryonic stem cell, callus or germ cell), fertilizing the cell if necessary, allowing the cell to develop into an animal or plant and, if necessary, breeding the animal or plant. The animal or plant can be obtained by sexual or asexual reproduction (such as cloning), breeding the animal or plant of the present invention or an F1 generation organism (or any generation derived from the F1 generation, or a chimera developed from a transformed cell).

[0281] Cells are typically generated by introducing a vector comprising a polynucleotide encoding the mutant CYP102A enzyme of the present invention into the cell (i.e., transforming the cell). It should be understood that due to the degeneracy of nucleotide codons, more than one polynucleotide can encode each mutant CYP102A enzyme. It should also be understood that the nucleotide sequence can be engineered to exhibit codon preference suitable for a particular cell or organism. The vector can be integrated into the genome of the cell or remain episomal. The cell can develop into an animal or plant. Typically, the coding sequence of the polynucleotide is operably linked to a control sequence capable of providing expression of the coding sequence in the host cell. The control sequence is typically a promoter, typically a promoter in a cell expressing a monooxygenase.

[0282] The term "operably linked" means juxtaposition wherein the components described are in a relationship that permits them to function in their intended manner. A control sequence "operably linked" to a coding sequence is linked in such a way that expression of the coding sequence can be achieved under conditions compatible with the control sequence.

[0283] The method disclosed herein can be implemented in cells expressing the mutant CYP102A enzyme. The method can be implemented in vitro, such as under culture conditions, or in vivo or in planta.

[0284] Organic compound

[0285] Using the method of the present invention, the inventors also obtained a method for obtaining two novel compounds, namely 25,26-epoxyvitamin D 3 [IUPAC name: (1S,Z)-3-(2-((1R,3aS,7aR,E)-7a-methyl-1-((2R)-5-(2-methyloxirane-2-yl)pentan-2-yl)octahydro-4H-inden-4-ylidene)ethylidene)-4-methylenecyclohexan-1-ol] and 25,28-dihydroxyvitamin D 2 [IUPAC name: (3S,6R,E)-6-((1R,3aS,7aR,E)-4-((Z)-2-((S)-5-hydroxy-2-methylenecyclohexyl)ethylidene)-7a-methyloctahydro-1H-inden-1-yl)-2-methylhept-4-ene-2,3-diol]. To the inventors' knowledge, these compounds have not been generated previously. The inventors obtained these compounds using the mutant CYP102A enzyme of the present invention, as described in Examples 2 and 4 respectively.

[0286] 25,26-epoxyvitamin D 3 and 25,28-dihydroxyvitamin D 2 are structurally similar to 25-hydroxyvitamin D 3 and 25-hydroxyvitamin D 2 respectively, and may thus be substrates for CYP27B1 oxidation to 1-hydroxy-25,26-epoxyvitamin D 3 and 1,25,28-trihydroxyvitamin D 2 . Due to the high structural similarity to the natural ligand of the vitamin D receptor, 1-hydroxy-25,26-epoxyvitamin D 3 and 1,25,28-trihydroxyvitamin D 2 are likely to be ligands of the vitamin D receptor as well.

[0287] 25,26-epoxyvitamin D 3 and 25,28-dihydroxyvitamin D 2 can be used as substitutes for 25-hydroxyvitamin D 3 and 25-hydroxyvitamin D 2 in pharmaceutical compositions, health supplements or animal feeds.

[0288] Composition

[0289] The method of the present invention may include oxidizing vitamin D 3 to 25-hydroxyvitamin D 3 or 1,25-dihydroxyvitamin D 3 , and formulating the 25-hydroxyvitamin D 3 or 1,25-dihydroxyvitamin D 3 into a pharmaceutical composition, a health supplement or an animal feed. The method of the present invention may also include oxidizing vitamin D 2 to 25-hydroxyvitamin D 2 , and formulating the 25-hydroxyvitamin D 2 into a pharmaceutical composition, a health supplement or an animal feed.

[0290] Also disclosed herein is a pharmaceutical composition, a health supplement or an animal feed, which comprises 25-hydroxyvitamin D 3 , 1,25-dihydroxyvitamin D 3 or 25-hydroxyvitamin D 2 produced by the method of the present invention.

[0291] The pharmaceutical composition, the health supplement or the animal feed may comprise one or more additional reagents. For example, the pharmaceutical composition may comprise one or more pharmaceutically acceptable excipients, carriers, diluents, buffers, stabilizers or other materials well known to those skilled in the art. Such materials should be non-toxic and not interfere with the efficacy of the active ingredient. The pharmaceutical carrier or diluent may be, for example, an isotonic solution.

[0292] The exact nature of the carrier or other materials may depend on the route of administration, such as oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular and intraperitoneal routes. For example, solid oral dosage forms may contain the active substance and the following components: diluents, such as lactose, glucose, sucrose, cellulose, corn starch or potato starch; lubricants, such as silica, talc, stearic acid, magnesium stearate or calcium stearate and / or polyethylene glycol; binders, such as starch, gum arabic, gelatin, methylcellulose, carboxymethylcellulose or polyvinylpyrrolidone; disintegrants, such as starch, alginic acid, alginates or sodium carboxymethyl starch; effervescent mixtures; colorants; sweeteners; wetting agents, such as lecithin, polysorbates, lauryl sulfates; and non-toxic and pharmacologically inactive substances commonly used in pharmaceutical formulations. Such pharmaceutical preparations can be prepared by known methods, such as by mixing, granulating, tableting, sugar coating or film coating processes.

[0293] Oral formulations contain commonly used excipients such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. These compositions are in the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders, and contain 10% to 95% of the active ingredient, preferably 25% to 70%. If the pharmaceutical composition is lyophilized, the lyophilized material can be reconstituted before administration, for example, as a suspension. The reconstitution is preferably carried out in a buffer solution.

[0294] Capsules, tablets and pills for oral administration to an individual may be provided with an enteric coating, which contains, for example, Eudragit “S”, Eudragit “L”, cellulose acetate, cellulose acetate phthalate or hydroxypropyl methylcellulose.

[0295] Liquid dispersions for oral administration can be syrups, emulsions or suspensions. The syrup may contain, for example, sucrose or sucrose containing glycerol and / or mannitol and / or sorbitol as a carrier.

[0296] Suspensions and emulsions may contain, for example, natural gums, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose or polyvinyl alcohol as a carrier. Suspensions or solutions for intramuscular injection may contain the active substance and a pharmaceutically acceptable carrier such as sterile water, olive oil, ethyl oleate, glycols (such as propylene glycol), and, if necessary, an appropriate amount of lidocaine hydrochloride.

[0297] Solutions for intravenous administration or infusion may contain, for example, sterile water as a carrier, or preferably they may be in the form of sterile, aqueous, isotonic salt solutions.

[0298] For suppositories, conventional binders and carriers may include, for example, polyethylene glycol or triglycerides; such suppositories may be made from a mixture containing 0.5% to 10% (preferably 1% to 2%) of the active ingredient.

[0299] Administration can be in a “prophylactically effective amount” or “therapeutically effective amount” (depending on the situation, although prophylaxis may also be considered a therapy), which is sufficient to confer a benefit on the individual, such as an effective amount for preventing or delaying the onset of a disease or disorder, alleviating one or more symptoms, inducing or prolonging a remission period or delaying recurrence or relapse.

[0300] Administration may not need to reach a therapeutically effective amount. Administration can be in an amount that ensures the recommended daily intake of vitamin D. The individual may already have a source of vitamin D and may therefore only need to reach the recommended daily intake amount.

[0301] The dosage can be determined according to various parameters, especially according to the substance used, the species, the age, weight and condition of the individual to be treated, the route of administration, and the required regimen. A physician can determine the required route of administration and dosage for any specific individual. The typical daily dose for humans is about 20 micrograms of vitamin D per day. For example, the Endocrine Society has stated that to maintain a serum 25(OH)D level above 75 nmol / L (30 ng / mL), adults may require at least 37.5 to 50 micrograms (1,500 - 2,000 IU) / day of vitamin D supplements, while children and adolescents may require at least 25 micrograms (1,000 IU) / day. The recommended daily allowance (RDA) of vitamin D is 600 IU (15 micrograms) per day for adults. However, some experts recommend even higher doses for adults - up to 1,000 - 2,000 IU (25 - 50 micrograms) per day. The vitamin D content in breast milk is related to the mother's vitamin D status; studies have shown that the nutrient level is higher in the breast milk of mothers who take supplements containing at least 50 micrograms (2,000 IU) of vitamin D 3 per day. In terms of raising the serum 25-hydroxyvitamin D 3 level, 20 micrograms per day of 25-hydroxyvitamin D 3 has been proven to be a more effective human supplement than the same dose of vitamin D 3 . The dosage of the vitamin D supplement can be set as a single dose or can be set as multiple doses, for example, taken at fixed intervals, such as administering 2, 3, or 4 doses per hour.

[0302] Vitamin D 3 is a key component in agricultural feed. It is present in fish feed at about 25 micrograms / kg, in poultry feed at about 125 micrograms / kg, for cattle at about 250 micrograms / kg, and in dairy cattle feed up to about 750 micrograms / kg. 25-Hydroxyvitamin D 3 has been used as a more beneficial poultry feed additive, about 70 micrograms / kg of feed.

[0303] Examples of the above techniques and protocols can be found in Remington’s Pharmaceutical Sciences, 20th Edition (published in 2000 by Lippincott, Williams & Wilkins).

[0304] Examples

[0305] Example 1. Design and engineering of CYP102A1 for vitamin D 3 oxidation

[0306] The wild-type CYP102A1 enzyme for vitamin D 3Oxidation is inactive. The inventors investigated whether the enzyme could be engineered to provide such activity.

[0307] Since vitamin D 3 is formed by the opening of ring B of the steroid core, a CYP102A1 variant with steroid oxidation activity was selected as the starting point for exploring vitamin D 3 oxidation activity. Fifteen years ago, the steroid oxidation of engineered CYP102A1 was first reported when the oxidation of testosterone was studied. Subsequent reports described the engineering of CYP102A1 for testosterone and progesterone oxidation by directed evolution, combinatorial active-site saturation testing (CAST), and iterative saturation mutagenesis (ISM). In addition, the rational engineering of CYP102A1 using an enzyme variant library of CYP102A1 has been reported to oxidize steroids including androstenedione, testosterone, and dehydroepiandrosterone. However, there has been no report on the oxidation of steroids with a long side chain at C17, such as cholesterol, by CYP102A1, which is more closely related to vitamin D 3 than others.

[0308] Regarding the oxidation activity of vitamin D 3 a library of approximately 100 CYP102A1 variants that showed high activity and different product profiles for steroid oxidation were screened (see Chen et al., ACS Catal. 2020, 10, 8334 - 8343). These variants were designed by glycine mutagenesis of residues near the heme; mutation combinations such as F87A, I263G, A264G, and A328G were found to promote steroid oxidation. In the study by Chen et al., the I263G mutation was found to play a key role and was the base mutation for adding other mutations. Molecular dynamics simulations showed that this mutation changed the conformation of the I helix, resulting in local unwinding of the helix near the mutation, thus creating space for accommodating the enlarged steroid. However, this mutation was ineffective in promoting vitamin D 3 oxidation, and none of the variants showed significant vitamin D 3 oxidation activity.

[0309] Therefore, a different approach was needed. When designing new variants that might show C25 oxidation of vitamin D 3 the inventors noted that the known mutation F87A was required for steroid oxidation activity; this was selected as the base mutation in the new variants. For C25 oxidation, vitamin D 3 needs to bind to the isopropyl terminal of the C17 side chain within the active-site pocket, which is closest to the oxygen atom of the ferryl intermediate. This was confirmed to be achievable by placing the A ring deep within the pocket (e.g., in the region near the A82 side chain at the distal end of the substrate pocket). Alternatively, this end of the pocket can be closed to force vitamin D3 The substrate binds to the C17 side chain near the heme iron. The inventors determined that mutations including A82M are suitable. The side chain volume of the residues near the heme can be reduced, thus creating space for vitamin D 3 binding. The screening results showed that the I263G mutation from Chen et al. was ineffective in promoting vitamin D 3 oxidation. This is because the I263 side chain is located at a high position in the substrate pocket, while the other I-helix residues T260 and A264 are close to the heme. The inventors replaced T260 and A264 with glycine, and also replaced another residue A328 adjacent to the heme with glycine. The side chain alcohol group of Thr268 is crucial for oxygen binding and oxygen-oxygen (O–O) bond cleavage to form a high-valent iron (compound I species [(P ·+ )Fe IV (=O)]), which is the active intermediate for carbon-hydrogen (C–H) bond oxidation. Replacing T268 with Ser retains the hydroxyl group on the side chain to achieve oxygen binding and activation, but removes the side chain methyl group, creating space for accommodating the substrate. The inventors identified the substrate channel residues S72, A330, and S332 for substitution with residues having larger side chains to facilitate the approach of the substrate to the heme binding. The inventors used many variants such as the I263G mutation as controls. As shown in Example 2, a new CYP102A1 variant library was generated and screened for vitamin D 3 oxidation.

[0310] Example 2. Initial enzyme library screening and characterization of vitamin D 3 oxidation products

[0311] HPLC analysis of the organic extracts from the in vitro screening scale (0.5 mL) reactions showed that 11 out of 48 new variants had more than 10% vitamin D 3 conversion activity (total turnover number about 50, Table 1). The active variants were selected for preparative-scale reactions, from which three oxidation products were isolated and characterized by NMR and MS data as the target products 25-hydroxyvitamin D 3 (1), diol 23,25-dihydroxyvitamin D 3 (2), and epoxide 25,26-epoxyvitamin D 3 (3). Diol 2 was likely formed by further oxidation of 1 at C23. Epoxide 3 may originate from the initial desaturation of the C25–C26 bond to form a terminal olefin, followed by epoxidation.

[0312] The product distribution (Table 1) shows that the combination of mutations A82M, F87A, A184I, T260G, and T268S promotes the 25-hydroxylation of vitamin D3 to produce 1. Extremely high (>99%) selectivity is possible; variants R19 / F87A / T268S and R19 / F87A / A82M / T260G / A328G / A184I / A330V (Entries 6 and 10 in Table 1) produce >99% of 1, although with lower activity. The most active variants (such as R19 / F87A / A82M / T260G / A184I / A328G, with a total turnover number TON = 285 for the production of 1) have a lower but still significant selectivity for 1 (>80%) (Entry 5 in Table 1). Variant R19 / F87A / A82M / T260G / A184I produces 90% of 1 but with lower activity (TON = 180, Entry 4 in Table 1).

[0313] Table 1 (First-generation mutants)

[0314]

[0315] Table 1. Selected cytochrome P450 BM3 Activity and product selectivity (HPLC analysis) of the variants-catalyzed hydroxylation of cholecalciferol (vitamin D 3 ). The substrate-to-enzyme concentration ratio was 500:1 (1 mM vitamin D 3 and 2 μM CYP102A1 enzyme). Conv. refers to the percentage of vitamin D 3 substrate converted to the product. TON refers to the turnover number of the variant for the formation of 25-hydroxyvitamin D 3 (1). K19 = H171L / Q307H / N319Y; R19 = R47L / Y51F / K19.

[0316] The results show that the mutation combination of F87A / A82M promotes the CYP102A1-mediated hydroxylation of vitamin D 3Oxidation; the only active variants without this combination are the variants containing the T268S mutation (Entries 10 and 11 in Table 1). As shown by the preliminary screening, the I263G mutation is ineffective, and the I263G / A264G combination is also ineffective. For example, the K19 / F87A / A82M / I263G / A264G variant in the new library is inactive (data not shown). When the A330 mutation was introduced to a residue with a larger side chain in the most active variant of this new library, the activity decreased by six-fold (Entries 6 - 8). In the context of the first-generation mutants, the S72 mutation to a residue with a larger side chain also decreased the activity (Entry 9). The difference in the success rate of combined mutations to promote the formation of the desired product is further illustrated by the lower activity of the K19 / F87A / A82M / T260G / T268S variant, which contains the activity-promoting mutations A82M and T268S. Similarly, although the R19 / F87A / A82M / T260G / A184I variant oxidizes vitamin D 3 (Entry 4), the K19 / F87A / A82M / T260G / A184I variant is inactive. The difference between these variants lies in that compared with K19, the R47L / Y51F combination is added in the R19 base variant.

[0317] It is concluded that the base mutations K19 and R19 interfere with the effects of the activity-enhancing mutations at A82, F87, T260G, etc. When designing a new generation of variants for vitamin D 3 oxidation, the K19 and R19 base mutations were removed. The most effective mutation combination F87A / A82M / T260G was retained as the starting point, and the same was true for the A184I mutation that enhances both activity and selectivity.

[0318] Example 3. Second-generation variants

[0319] The F87A / A82M / A184I / T260G variant showed a higher conversion rate than the analogues with the added R19 mutation combination (Entry 22 in Table 2 and Entry 4 in Table 1). Therefore, the screening reaction was carried out at a higher substrate-to-enzyme concentration ratio (1000:1). Under these conditions, the F87A / A82M / A184I / T260G variant showed a 40% conversion rate, 75% selectivity, and a turnover number of 300 for the formation of 25-(OH)-D 3 . No activity was detected for the wild-type enzyme and the F87A single mutant variant. Interestingly, the F87A / A82M variant for 25-(OH)-D 3showed a 28% conversion rate and 72% selectivity, but when the A184I and T260G mutations were added to the F87A / A82M double mutant variant, both decreased the activity and selectivity (Table 2, entries 14 and 16 - 17), while combining all four mutations together increased both the activity and selectivity (entry 22). The results highlight the important role of mutation combinations in enhancing activity and / or selectivity.

[0320]

[0321]

[0322] Table 2. Activity and product selectivity (HPLC analysis) of cholecalciferol (vitamin D 3 ) hydroxylation catalyzed by second - generation CYP102A1 variants. The substrate - to - enzyme concentration ratio was 1000:1 (2 mM vitamin D 3 , 2 μM CYP102A1 enzyme). Conv. refers to the percentage of substrate converted to product, and TON refers to the turnover number of the variant that generates 25 - hydroxyvitamin D 3 (1). –: Activity not detected.

[0323] Subsequently, vitamin D 3 was computationally docked into the active site of the molecular dynamics simulation structure of the F87A / A82M / A184I / T260G variant. Based on the substrate - binding model, active - site residues within the range of the bound substrate were selected for substitution with amino acid residues having smaller and larger side chains to explore their effect on vitamin D 3 oxidation. Mutations were introduced at P6, L19, L20, P25, V26, L29, K69, S72, A74, L75, V78, V178, L181, M185, L188, I259, A330, S332, M354, Q403, L437, and T438. We also targeted salt - bridge and hydrogen - bond interactions connecting secondary - structure elements, aiming to relax the structure and introduce greater flexibility to allow non - native substrates such as vitamin D 3 to enter and bind. For this purpose, hydrophobic substitutions were introduced at residues R79, N239, R255, E435, and K440.

[0324] Except for the S72A and E435M mutations, most of the mutations introduced at these target residues had little effect on the activity of the F87A / A82M / A184I / T260G parental variant or completely abolished the activity (see Tables 2 and 3). The S72A mutation, when added to various precursor variants, increased the vitamin D 3The oxidation activity is increased by 50% to twice (see, for example, Table 2, entries 14 and 15, 17 and 19, 16 and 21). When the E435M mutation is added to the F87A / A82M / A184I / T260G / S72A variant, the conversion rate of vitamin D 3 is significantly increased from 37% to 79%, and the selectivity for C25 hydroxylation is increased from 63% to 73%, resulting in the F87A / A82M / A184I / T260G / S72A / E435M variant having a TON of 570 for the formation of 25-hydroxyvitamin D 3 (entry 29). This activity enhancement effect of the E435M mutation is observed in other precursors (see, for example, Table 2, entries 14 and 18, 17 and 20, 16 and 23, etc.). The carboxylate group of the E435 side chain forms a hydrogen bond with the backbone carbonyl oxygen of V26. The disruption of this bonding interaction provides greater flexibility for this part of the P450 BM3 substrate channel, which can promote the oxidation of vitamin D 3 . Replacing E435 with other hydrophobic residues (Ala, Val, Ile, Leu, Phe) shows that the E435I mutation is even more effective than E435M. Among them, the F87A / A82M / A184I / T260G / S72A / E435I variant has a TON of 620 for the formation of 25-OH-D 3 through increased conversion rate and C25 selectivity (entry 31). Li et al. (Appl. Biochem. Biotechnol. 2008, 144, 27–36) reported that the E435T mutation increased the indigo formation activity of the A74G / F87V / L188Q variant of CYP102A1 through indole oxidation, but the hydrophobic substitution effect was poor. For example, the activity of the best variant containing the E435F mutation was about 60% of the activity of the E435T variant. The E435T mutation was found to be less effective than the E435I and E435M mutations in promoting the oxidation of vitamin D 3 (entry 35).

[0325] Subsequently, different hydrophobic substitutions were introduced at F87. The F87I mutation increased the activity of the E435I and E435M variants by about 10%, with a TON of about 700 (Table 2 entries 32 and 36), while the F87V mutation decreased the selectivity for C25 (entries 33 and 34).

[0326]

[0327]

[0328]

[0329]

[0330] Table 3. Activity and product selectivity (HPLC analysis) of cholecalciferol (vitamin D 3 ) oxidation catalyzed by CYP102A1 variants. The substrate-to-enzyme concentration ratio was 1000:1 (2 mM vitamin D 3 , 2 μM CYP102A1 enzyme). Conv. refers to the percentage of the substrate converted to product, and TON refers to the turnover number of the variant that generates 25-hydroxyvitamin D 3, 1. –: No activity detected.

[0331] Example 4. Oxidation of vitamin D2

[0332] Vitamin D 2 differs from D 3 in that vitamin D 2 has a C22–C23 double bond and a C24 methyl group. The rigidity of the double bond and the altered steric requirements introduced by the methyl group can affect P450 substrate recognition, as demonstrated by the oxidation of vitamin D 2 to its circulating form 25-hydroxyvitamin D 3 by a different hepatic P450 enzyme (CYP2R1) than vitamin D 2 (CYP27A1). Similarly, CYP2R1 does not oxidize vitamin D 3 . Therefore, this study investigated whether CYP102A1 variants capable of C25 oxidation of vitamin D 3 also have vitamin D 2 oxidation activity.

[0333]

[0334]

[0335] Table 4. Activity and product selectivity (HPLC analysis) of hydroxylation of calciferol (vitamin D 2 ) by second-generation CYP102A1 variants. The substrate-to-enzyme concentration ratio was 1000:1 (2 mM vitamin D 2 , 2 μM CYP102A1 enzyme). Conv. refers to the percentage of the substrate converted to product, and TON refers to the turnover number of the variant that generates 25-hydroxyvitamin D 2 (4). –: No activity detected.

[0336] Wild-type CYP102A1, F87A variant, and second-generation variants were tested for vitamin D 2 oxidation. Neither the wild-type nor the F87A variant showed vitamin D 2 oxidation activity. The second-generation variants had activity, but with vitamin D 3has a lower activity level compared thereto (see Table 4). The major product purified from the preparative-scale reaction with the F87A / A82M / A184I / T260G / E435M variant was identified as 25-hydroxyvitamin D by NMR and MS data 2 (4) (selectivity 94%, TON = 330, see Table 4 entry 55). Some variants also gave 25,28-dihydroxyvitamin D 2 . The activity and selectivity trends are similar to those of vitamin D 3 (compare Table 4 with Table 5); for example, the F87A / A82M variant showed relatively low activity; introduction of the A184I / T260G double mutation enhanced the C25 oxidation activity and selectivity, and with the addition of the S72A, E435M, and E435I mutations, the activity and selectivity further increased. The most active and selective variant for vitamin D 3 oxidation, F87I / A82M / A184I / T260G / S72A / E435I, was also the most active and selective for 25-hydroxylation of vitamin D 2 (conversion 88%, selectivity 92%, TON = 810, Table 4 entry 61).

[0337]

[0338]

[0339]

[0340]

[0341] Table 5. Activity and product selectivity (HPLC analysis) of calciferol (vitamin D 2 ) oxidation catalyzed by CYP102A1 variants. The substrate to enzyme concentration ratio was 1000:1 (2 mM vitamin D 2 , 2 μM CYP102A1 enzyme). Conv. refers to the percentage of substrate converted to product, and TON refers to the turnover number of the variant that generates 25-hydroxyvitamin D 2, 4. –: No activity detected.

[0342] Example 5. Materials and Methods

[0343] Screening of vitamin D 3 and D 2 for oxidation by CYP102A1 variants

[0344] Vitamin D 3 and D 2Dissolved in EtOH to serve as a 100 mM stock solution. The screening-scale reaction was carried out in a 24-well plate in 0.5 mL volume of 200 mM phosphate buffer (pH 7.9). The final concentration of CYP102A1 variant was 2 μM; vitamin D 3 was 1 mM (500:1) or 2 mM (1000:1) and vitamin D 2 was 2 mM (1000:1). Methyl-β-cyclodextrin (10 mM) was added to improve the solubility of vitamin D 3 in the aqueous buffer. GDH (20 U / mL) and glucose (100 mM) were used to regenerate the NADPH cofactor. NADP+ (40 μM) was added to initiate the reaction. The screening plate was shaken at 120 rpm at 20 °C in the dark for 72 h, followed by extraction with 300 μL of ethyl acetate. After phase separation by centrifugation at 14300 g, the organic extract was analyzed by reversed-phase HPLC on a C18 column (4.6 mm × 10 cm, 5 μm), eluted with an aqueous solution of 95% acetonitrile at a flow rate of 1 mL / min for 20 min. The retention times were: 23,25-dihydroxyvitamin D 3 (2), 1.50 min; 25-hydroxyvitamin D 3 (1), 2.27 min; 25,26-epoxyvitamin D 3 (3), 3.10 min; vitamin D 3 , 8.35 min; 25-hydroxyvitamin D 2 (5), 2.51 min; vitamin D 2 , 8.37 min.

[0345] Preparation-scale oxidation of vitamin D 3 The reaction catalyzed by the R19 / F87A / A82M / A184I / T260G variant demonstrated the scalability of engineered CYP102A1 for the selective 25-hydroxylation of vitamin D

[0346] in vitro. The reaction was scaled up to 500 mL, and the concentrations of the other components were the same as those in the screening-scale reaction except that the substrate concentration was increased to 3 mM (600 mg, 1.2 g / L) and 6 μM enzyme was used. The reaction mixture was stirred at ambient temperature in the dark for 72 h. Aliquots were taken periodically to monitor the reaction progress by GC. After 36 h, a second aliquot (6 μM) of the enzyme was added. After the conversion reached >90% after 72 h, the reaction mixture was extracted three times with an equal volume of ethyl acetate. The combined extracts were washed with brine, dried over Na 3 29 2 (SO 4 4), and the solvent was removed by rotary evaporation. The crude mixture was purified by silica gel column chromatography; the remaining vitamin D 3 ​The substrate was eluted with a 5:1 mixture of petroleum ether (b.p. 30 - 40 °C) and ethyl acetate (petroleum / EtOAc); the epoxide (3) was eluted with a 3:1 mixture of petroleum / EtOAc; 25-hydroxyvitamin D 3 (1) was eluted with a 1:1 petroleum / EtOAc; the diol (2) was eluted with pure EtOAc. Based on the amount of converted vitamin D 3 the isolation yield of 25-(OH)-D 3 was 72% (400 mg).

[0347] Further embodiments

[0348] Further embodiments of the present invention are described below:

[0349] 1. A method for oxidatively opening a steroid ring, comprising the step of contacting the ring-opened steroid with a mutant CYP102A (member of cytochrome P450 family 102A subfamily) enzyme, wherein the CYP102A enzyme comprises a heme monooxygenase domain containing a P450 fold, and the mutant CYP102A enzyme comprises substitutions at one or more positions in the polypeptide chain of the wild-type CYP102A enzyme, thereby enhancing the monooxygenase activity of the mutant enzyme and / or altering its product selectivity.

[0350] 2. The method according to embodiment 1, wherein the mutant CYP102A enzyme comprises substitutions at one or more positions in the polypeptide chain of the wild-type CYP102A enzyme corresponding to amino acid residue positions 435, 82, 184, 260 and / or 72 of SEQ ID NO:2.

[0351] 3. The method according to embodiment 2, wherein the mutant CYP102A enzyme comprises one or more substitutions selected from E435I, E435M, E435T, A82M, A82L, A82I, A82F, A82W, A184I, T260G, T260A, S72A and / or S72G in SEQ ID NO:2, or corresponding substitutions at one or more positions in the polypeptide chain of the wild-type CYP102A enzyme corresponding to amino acid residues 435, 82, 184, 260 and / or 72 of SEQ ID NO:2.

[0352] 4. The method according to embodiment 2 or 3, wherein the mutant CYP102A enzyme further comprises one or more substitutions at one or more positions corresponding to amino acid residue positions 29, 78, 87, 178, 263, 264, 268, 328 and / or 354 of SEQ ID NO:2.

[0353] 5. The method according to embodiment 4, wherein the mutant CYP102A enzyme comprises one or more substitutions selected from L29M, V78F, F87A, F87V, F87I, F87S, F87T, V178F, V178W, V178L, V178I, I263G, A264G, T268S, A328G, and / or M354F, or corresponding substitutions at one or more positions corresponding to amino acid residue positions 29, 78, 87, 178, 263, 264, 268, 328, and / or 354 of SEQ ID NO:2.

[0354] 6. The method according to embodiment 4 or 5, wherein the mutant CYP102A enzyme comprises the substitution at position 82 of SEQ ID NO:2 or a corresponding position thereto, and the substitution at position 87 of SEQ ID NO:2 or a corresponding position thereto.

[0355] 7. The method according to embodiment 6, wherein the mutant CYP102A further comprises: the substitution at position 184 of SEQ ID NO:2 or a corresponding position thereto, the substitution at position 260 of SEQ ID NO:2 or a corresponding position thereto, the substitution at position 72 of SEQ ID NO:2 or a corresponding position thereto, and / or the substitution at position 435 of SEQ ID NO:2 or a corresponding position thereto.

[0356] 8. The method according to embodiment 7, wherein the mutant CYP102A enzyme comprises: the substitution at position 82 of SEQ ID NO:2 or a corresponding position thereto, the substitution at position 87 of SEQ ID NO:2 or a corresponding position thereto, and the substitution at position 435 of SEQ ID NO:2 or a corresponding position thereto.

[0357] 9. The method according to any one of the foregoing embodiments, wherein the mutant CYP102A enzyme comprises one of the following substitution groups in SEQ ID NO:2:

[0358] a. A82M / F87A;

[0359] b. A82M / T260G;

[0360] c. A82M / A184I;

[0361] d. A82M / S72A;

[0362] e. A82M / E435I, E435M or E435T;

[0363] f. F87A / T260G;

[0364] g. F87A / A184I;

[0365] h. F87A / S72A;

[0366] i. A82M / F87A / T260G;

[0367] j. A82M / F87A / A184I;

[0368] k. A82M / F87A / E435I, E435M or E435T;

[0369] l. F87A / A184I / T260G;

[0370] m. F87A / A184I / S72A;

[0371] n. F87A / A184I / E435I, E435M or E435T;

[0372] o. F87A / A82M / A184I / T260G;

[0373] p. F87A / A82M / T260G / S72A;

[0374] q. F87A / A82M / A184I / T260G / S72A;

[0375] r. F87A, F87I or F87V / A82M / A184I / T260G / E435I, E435M or E435T;

[0376] s. F87A, F87I, F87V, F87S or F87T / A82M / A184I / T260G / S72A / E435I, E435M or E435T; or

[0377] t. F87I / A82M / A184I / T260G / S72A / E435M / L29M;

[0378] or, corresponding substitutions at one or more positions in the polypeptide chain of the wild-type CYP102A enzyme at positions corresponding to the amino acid residue positions enumerated in a.-t. of SEQ ID NO:2.

[0379] 10. The method according to any one of the foregoing embodiments, wherein the mutant CYP102A enzyme further comprises one or more substitutions at one or more positions corresponding to amino acid residues 47, 51, 74, 171, 188, 239, 259, 307, 319, 330, and / or 353 of SEQ ID NO:2, and the one or more substitutions are optionally selected from R47L, Y51F, A74G, H171L, N239H, I259V, L188Q, Q307H, N319Y, A330V, A330I, A330L, A330W, and / or L353I in SEQ ID NO:2; or corresponding substitutions at one or more positions corresponding to amino acid residues 47, 51, 74, 171, 188, 239, 259, 307, 319, 330, and / or 353 of SEQ ID NO:2 in the polypeptide chain of the wild-type CYP102A enzyme.

[0380] 11. The method according to any one of the foregoing embodiments, wherein the mutant CYP102A enzyme comprises a fusion of a heme oxygenase domain and a reductase domain.

[0381] 12. The method according to any one of the foregoing embodiments, which is used for oxidizing vitamin D 3 .

[0382] 13. The method according to embodiment 12, which is used for preparing 25-hydroxyvitamin D 3 .

[0383] 14. The method according to embodiment 13, which further comprises oxidizing 25-hydroxyvitamin D 3 to 1,25-dihydroxyvitamin D 3 .

[0384] 15. The method according to any one of embodiments 1-11, which is used for oxidizing vitamin D 2 .

[0385] 16. The method according to embodiment 15, which is used for preparing 25-hydroxyvitamin D 2 .

[0386] 17. The method according to any one of the foregoing embodiments, wherein the mutant CYP102A enzyme is a mutant CYP102A1 enzyme.

[0387] 18. A mutant CYP102A (member of cytochrome P450 family 102A subfamily) enzyme, wherein the CYP102A enzyme comprises a heme monooxygenase domain containing a P450 fold, and the mutant CYP102A enzyme comprises substitutions at one or more positions corresponding to amino acid residues 435, 82, 184, 260, and / or 72 of SEQ ID NO:2 in the polypeptide chain of the wild-type CYP102A enzyme, thereby enhancing the monooxygenase activity of the mutant enzyme and / or altering its product selectivity.

[0388] 19. The mutant CYP102A enzyme according to embodiment 18, as defined in any one of embodiments 3 - 11 and 17.

[0389] 20. A polynucleotide comprising a sequence encoding the enzyme as defined in embodiment 18 or 19, optionally in the form of a vector.

[0390] 21. A cell expressing the enzyme as defined in embodiment 18 or 19.

[0391] 22. The cell according to embodiment 21, which is a prokaryotic cell or a eukaryotic cell.

[0392] 23. The cell according to embodiment 22, which is a strain of Escherichia coli, Pseudomonas sp., yeast, Pichia sp., Rhodococcus sp., or Bacillus sp.

[0393] 24. A transgenic animal or plant, the cells of which are as defined in any one of embodiments 21 to 23.

[0394] 25. The method according to any one of embodiments 1 to 17, wherein the ring - opened steroid is oxidized in the cell as defined in any one of embodiments 21 to 23.

[0395] 26. The method according to any one of embodiments 1 to 14, 17, and 25, comprising oxidizing vitamin D 3 to 25 - hydroxyvitamin D 3 or 1,25 - dihydroxyvitamin D 3 , and formulating the 25 - hydroxyvitamin D 3 or 1,25 - dihydroxyvitamin D 3 into a pharmaceutical composition, a health supplement, or animal feed.

[0396] 27. The method according to any one of embodiments 1 to 12, 15 to 17 and 25, comprising oxidizing vitamin D 2 to 25-hydroxyvitamin D 2 , and formulating said 25-hydroxyvitamin D 2 into a pharmaceutical composition, a health supplement or an animal feed.

[0397] 28. The compound (1S,Z)-3-(2-((1R,3aS,7aR,E)-7a-methyl-1-((2R)-5-(2-methyloxirane-2-yl)pentan-2-yl)octahydro-4H-inden-4-ylidene)ethylidene)-4-methylenecyclohexan-1-ol (i.e., 25,26-epoxyvitamin D 3 ).

[0398] 29. The compound (3S,6R,E)-6-((1R,3aS,7aR,E)-4-((Z)-2-((S)-5-hydroxy-2-methylenecyclohexylidene)ethylidene)-7a-methyloctahydro-1H-inden-1-yl)-2-methylhept-4-ene-2,3-diol (i.e., 25,28-dihydroxyvitamin D 2 ).

Claims

1. A method for oxidatively opening a steroid ring, comprising the step of contacting the ring-opened steroid with a mutant CYP102A (member of cytochrome P450 family 102A subfamily) enzyme, wherein the CYP102A enzyme comprises a heme monooxygenase domain containing a P450 fold, and the mutant CYP102A enzyme comprises substitutions at one or more positions in the polypeptide chain of the wild-type CYP102A enzyme, thereby enhancing the monooxygenase activity of the mutant enzyme and / or altering its product selectivity.

2. The method according to claim 1, wherein the mutant CYP102A enzyme comprises substitutions at one or more positions in the polypeptide chain of the wild-type CYP102A enzyme corresponding to amino acid residue positions 435, 82, 184, 260, and / or 72 of SEQ ID NO:

2.

3. The method according to claim 2, wherein the mutant CYP102A enzyme comprises one or more substitutions selected from E435I, E435M, E435T, A82M, A82L, A82I, A82F, A82W, A184I, T260G, T260A, S72A, and / or S72G in SEQ ID NO:2, or corresponding substitutions at one or more positions in the polypeptide chain of the wild-type CYP102A enzyme corresponding to amino acid residues 435, 82, 184, 260, and / or 72 of SEQ ID NO:

2.

4. The method according to claim 2 or 3, wherein the mutant CYP102A enzyme further comprises one or more substitutions at one or more positions corresponding to amino acid residue positions 29, 78, 87, 178, 263, 264, 268, 328, and / or 354 of SEQ ID NO:

2.

5. The method according to claim 4, wherein the mutant CYP102A enzyme comprises one or more substitutions selected from L29M, V78F, F87A, F87V, F87I, F87S, F87T, V178F, V178W, V178L, V178I, I263G, A264G, T268S, A328G, and / or M354F, or corresponding substitutions at one or more positions corresponding to amino acid residue positions 29, 78, 87, 178, 263, 264, 268, 328, and / or 354 of SEQ ID NO:

2.

6. The method according to claim 4 or 5, wherein the mutant CYP102A enzyme comprises the substitution at position 82 of SEQ ID NO:2 or a corresponding position thereto, and the substitution at position 87 of SEQ ID NO:2 or a corresponding position thereto.

7. The method according to claim 6, wherein the mutant CYP102A further comprises: the substitution at position 184 of SEQ ID NO:2 or the corresponding position thereto, the substitution at position 260 of SEQ ID NO:2 or the corresponding position thereto, the substitution at position 72 of SEQ ID NO:2 or the corresponding position thereto, and / or the substitution at position 435 of SEQ ID NO:2 or the corresponding position thereto.

8. The method according to claim 7, wherein the mutant CYP102A enzyme comprises: the substitution at position 82 of SEQ ID NO:2 or the corresponding position thereto, the substitution at position 87 of SEQ ID NO:2 or the corresponding position thereto, and the substitution at position 435 of SEQ ID NO:2 or the corresponding position thereto.

9. The method according to any one of the preceding claims, wherein the mutant CYP102A enzyme comprises one of the following substitution groups in SEQ ID NO:2: a. A82M / F87A; b. A82M / T260G; c. A82M / A184I; d. A82M / S72A; e. A82M / E435I, E435M or E435T; f. F87A / T260G; g. F87A / A184I; h. F87A / S72A; i. A82M / F87A / T260G; j. A82M / F87A / A184I; k. A82M / F87A / E435I, E435M or E435T; l. F87A / A184I / T260G; m. F87A / A184I / S72A; n. F87A / A184I / E435I, E435M or E435T; o. F87A / A82M / A184I / T260G; p. F87A / A82M / T260G / S72A; q. F87A / A82M / A184I / T260G / S72A; r. F87A, F87I or F87V / A82M / A184I / T260G / E435I, E435M or E435T; s. F87A, F87I, F87V, F87S or F87T / A82M / A184I / T260G / S72A / E435I, E435M or E435T; or t. F87I / A82M / A184I / T260G / S72A / E435M / L29M; or, the corresponding substitutions at one or more positions in the polypeptide chain of the wild-type CYP102A enzyme at positions corresponding to the amino acid residue positions listed in a.-t. of SEQ ID NO:

2.

10. The method according to any one of the preceding claims, wherein the mutant CYP102A enzyme further comprises one or more substitutions at one or more positions corresponding to amino acid residue positions 47, 51, 74, 171, 188, 239, 259, 307, 319, 330 and / or 353 of SEQ ID NO:2, and the one or more substitutions are optionally selected from R47L, Y51F, A74G, H171L, N239H, I259V, L188Q, Q307H, N319Y, A330V, A330I, A330L, A330W and / or L353I in SEQ ID NO:2; or corresponding substitutions at one or more positions in the polypeptide chain of the wild-type CYP102A enzyme corresponding to amino acid residue positions 47, 51, 74, 171, 188, 239, 259, 307, 319, 330 and / or 353 of SEQ ID NO:

2.

11. The method according to any one of the preceding claims, wherein the mutant CYP102A enzyme comprises a fusion of a heme monooxygenase domain and a reductase domain.

12. The method according to any one of the preceding claims, the method being for oxidizing vitamin D 3 , optionally for preparing 25-hydroxyvitamin D 3 , optionally further comprising oxidizing 25-hydroxyvitamin D 3 to 1,25-dihydroxyvitamin D 3 .

13. The method according to any one of claims 1-11, said method being for oxidizing vitamin D 2 , optionally for preparing 25-hydroxyvitamin D 2 .

14. The method according to any one of the preceding claims, wherein the mutant CYP102A enzyme is a mutant CYP102A1 enzyme.

15. A mutant CYP102A (member of the cytochrome P450 family 102A subfamily) enzyme, wherein the CYP102A enzyme comprises a heme monooxygenase domain containing a P450 fold, and the mutant CYP102A enzyme comprises substitutions at one or more positions in the polypeptide chain of the wild-type CYP102A enzyme corresponding to amino acid residue positions 435, 82, 184, 260 and / or 72 of SEQ ID NO:2, thereby enhancing the monooxygenase activity of the mutant enzyme and / or altering its product selectivity.

16. The mutant CYP102A enzyme according to claim 15, as defined in any one of claims 3 to 11 and 14.

17. A polynucleotide comprising a sequence encoding the enzyme as defined in claim 15 or 16, and the polynucleotide is optionally in the form of a vector.

18. A cell expressing the enzyme as defined in claim 15 or 16.

19. The cell according to claim 18, which is a prokaryotic cell or a eukaryotic cell, and optionally the cell is a strain of Escherichia coli, a species of Pseudomonas, yeast, a species of Pichia, a species of Rhodococcus, a species of Bacillus.

20. A transgenic animal or plant, the cells of which are as defined in claim 18 or 19.

21. The method according to any one of claims 1 to 14, wherein the ring-opened steroid is oxidized in the cell according to claim 18 or 19.

22. The method according to any one of claims 1 to 12, 14 and 21, comprising oxidizing vitamin D 3 to 25-hydroxyvitamin D 3 or 1,25-dihydroxyvitamin D 3 , and formulating the 25-hydroxyvitamin D 3 or 1,25-dihydroxyvitamin D 3 into a pharmaceutical composition, a health supplement or an animal feed.

23. The method according to any one of claims 1 to 11, 13, 14 and 21, comprising oxidizing vitamin D 2 to 25-hydroxyvitamin D 2 , and formulating said 25-hydroxyvitamin D 2 into a pharmaceutical composition, a health supplement or an animal feed.

24. A compound (1S,Z)-3-(2-((1R,3aS,7aR,E)-7a-methyl-1-((2R)-5-(2-methyloxirane-2-yl)pentan-2-yl)octahydro-4H-inden-4-ylidene)ethylidene)-4-methylenecyclohexan-1-ol (i.e., 25,26-epoxyvitamin D 3 ).

25. A compound (3S,6R,E)-6-((1R,3aS,7aR,E)-4-((Z)-2-((S)-5-hydroxy-2-methylenecyclohexylidene)ethylidene)-7a-methyloctahydro-1H-inden-1-yl)-2-methylhept-4-ene-2,3-diol (i.e., 25,28-dihydroxyvitamin D 2 ).

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