Nonspecific peroxygenases and their application in the hydroxylation of steroid compounds

Abstract

This invention belongs to the field of genetic engineering technology, and particularly relates to a non-specific peroxygenase and its application in the hydroxylation of steroidal compounds. The amino acid sequence of the non-specific peroxygenase is selected from at least one of SEQ ID NO. 1-5. The multiple UPO enzymes provided by this invention exhibit good compatibility with various steroidal compounds, demonstrating excellent selectivity and / or high yield in catalyzing the 11β-, 16α-, or 6β-hydroxylation of steroids. Furthermore, the reaction system is flexible and controllable; in addition to initiating the reaction with hydrogen peroxide, the enzyme-catalyzed reaction can be activated directly using oxygen present in the environment and an external small-molecule reducing agent, which is beneficial for large-scale production. This invention opens up a novel biocatalytic technology pathway for the green synthesis and large-scale production of hydroxylated steroidal compounds, and has significant industrial application value and good potential in the bio-enzymatic hydroxylation of steroidal compounds at specific sites.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to nonspecific peroxygenases and their application in the hydroxylation of steroidal compounds. Background Technology

[0002] Steroids are a class of compounds with a basic cyclopentane-polyhydrophenanthrene skeleton. They are important active pharmaceutical ingredients and intermediates in drug synthesis, widely used in the pharmaceutical and health fields due to their rich reaction types and diverse biological activities. The physiological effects of steroids are closely related to their structural characteristics, particularly the type, position (regioselectivity), and three-dimensional orientation (stereoselectivity) of functional groups attached to the tetracyclic core. Selective hydroxylation of the inert carbon-hydrogen bonds (CH) of steroids is a key structural modification strategy. Common hydroxylation reactions include those on 6β, 9α, 11α, 11β, 16α, 17α, and 19-angular methyl groups. Hydroxylation can affect the polarity of steroids, regulating their pharmacological properties such as anti-inflammatory, anabolic, or hormonal activities. Moreover, hydroxylated steroids are often used as general intermediates in the synthesis of numerous steroid drugs. Therefore, efficient hydroxylation technology for steroids is of great significance to organic synthesis, the chemical industry, and the pharmaceutical industry.

[0003] The preparation of steroidal compounds or their intermediates mainly includes chemical and biological methods. Conventional chemical methods for steroid hydroxylation face significant challenges, including harsh reaction conditions (requiring strong oxidants, high temperature / high pressure), the use of toxic reagents / heavy metals, and difficulties in separating stereoisomers, severely limiting their industrial applications. In contrast, biocatalytic pathways, particularly those using cytochrome P450 monooxygenases (P450s), exhibit advantages such as high reaction efficiency, strong selectivity, and mild reaction conditions, providing a better synthetic strategy for steroid hydroxylation.

[0004] Currently, numerous P450s from bacteria and eukaryotes have been discovered capable of catalyzing the hydroxylation of various steroids. For example, Bacillus is one of the main sources of P450 steroid hydroxylases. Although some P450s have been used for the hydroxylation of specific steroids and have been engineered, they generally suffer from low selectivity, unsatisfactory catalytic activity, and a high dependence on reducing coenzymes and complex electron transport chains (reducing chaperone proteins). On the other hand, while there are more reported eukaryotic P450s with steroid hydroxylation capabilities than prokaryotes, most of them are membrane-bound enzymes. These characteristics greatly hinder their practical application and widespread development in the field of steroid hydroxylation.

[0005] Unspecific peroxygenases (UPOs) are a class of fungal oxidases with protein structures similar to P450s, both containing heme cofactors coordinated to cysteine ​​ligands. This makes the catalytic mechanisms of UPOs similar to those of P450s. Compared to P450s, UPOs have a broader substrate range, directly using hydrogen peroxide (H2O2) or organic hydroperoxides (R-OOH) as oxidants, eliminating the need for complex electron transport chains and expensive cofactors. Furthermore, most UPOs exhibit superior robustness to pH, organic solvents, and other physicochemical factors, and can catalyze a variety of reactions, including hydroxylation and epoxidation. Therefore, UPOs, as a new generation of biocatalysts, show great potential for enzymatic synthesis of active molecules. Some UPOs (such as Agrocybe aegerita UPO, AaeUPO) have been shown to oxidize the side chains of sterols such as vitamin D3 and phytosterols. However, hydroxylation reactions targeting the steroid nucleus structure remain a significant challenge. Currently, only a few UPOs, such as Chaetomium globosum UPO (CglUPO), exhibit detectable activity; however, 90% of their catalytic products are epoxidation byproducts, resulting in extremely low hydroxylation efficiency (selectivity <10%). Given the advantages of UPOs and their untapped potential in steroid nucleus functionalization, there is an urgent need to develop novel UPOs or their variants with high catalytic activity, especially high selective hydroxylation capabilities. Summary of the Invention

[0006] To address the problem of the lack of nonspecific peroxygenases suitable for the hydroxylation of compounds with steroidal nucleus structures in existing technologies, this invention provides a nonspecific peroxygenase, its encoding gene, an expression vector containing the encoding gene, and a recombinant strain. Furthermore, it provides the application of the aforementioned nonspecific peroxygenase and recombinant strain in the preparation of hydroxylated steroidal compounds. This invention is achieved through the following technical solutions:

[0007] A first aspect of the present invention provides a nonspecific peroxygenase having an amino acid sequence selected from at least one of SEQ ID NO. 1-5.

[0008] Furthermore, the amino acid sequence of the nonspecific peroxygenase is selected from at least one of SEQ ID NO.4-5.

[0009] A second aspect of the present invention provides a nucleic acid molecule that encodes a nonspecific peroxygenase as described above.

[0010] A third aspect of the present invention provides an expression vector comprising the nucleic acid molecule described above.

[0011] Furthermore, the expression vector also includes a gene sequence encoding a signal peptide, the amino acid sequence of which is shown in SEQ ID NO.6.

[0012] A fourth aspect of the present invention provides a recombinant strain comprising the nucleic acid molecule or expression vector described above.

[0013] The fifth aspect of the invention provides the use of the nonspecific peroxygenase or recombinant strain described above in the preparation of hydroxylated steroidal compounds; said steroidal compounds are selected from at least one of estradiol-4,9-diene-3,17-dione, testosterone, nandrolone, 1,4-androstenedione, and methyltetraene compounds.

[0014] The sixth aspect of the present invention provides a method for synthesizing a hydroxylated steroid compound, comprising the following steps: mixing a nonspecific peroxygenase and a steroid compound and carrying out a catalytic reaction; wherein the steroid compound is selected from at least one of estradiol-4,9-diene-3,17-dione, testosterone, nandrolone, 1,4-androstenedione and methyltetraene compounds.

[0015] Furthermore, the catalytic reaction is carried out at a pH of 5.5-8.0 and a temperature of 25-40℃.

[0016] Furthermore, the reaction system of the catalytic reaction also includes a reducing agent. Even further, the reducing agent is selected from ascorbic acid, dehydroascorbic acid, gallic acid, or pyrogallic acid.

[0017] The advantages and positive effects of this invention are as follows: The multiple UPO enzymes provided by this invention exhibit good compatibility with various steroidal compounds (including estradiol-4,9-diene-3,17-dione, testosterone, nandrolone, 1,4-androstenedione, and methyltetraene compounds). They demonstrate excellent selectivity and / or high yield in catalyzing the 11β-, 16α-, or 6β-hydroxylation of steroids, with a hydroxylation product selectivity greater than 50%, representing a significant improvement over existing CglUPO enzyme resources. Furthermore, in addition to initiating the reaction by adding hydrogen peroxide, the enzyme catalysis process can also be initiated directly using oxygen present in the environment in conjunction with a small molecule reducing agent, which enhances the flexibility of the reaction system and enables large-scale production, greatly reducing the cost of enzyme-catalyzed preparation of hydroxylated steroidal compounds. This invention opens up a novel biocatalytic technology pathway for the green synthesis and large-scale production of 11β-, 16α-, or 6β-hydroxylated steroidal compounds, possessing significant industrial application value and good potential in the enzyme-catalyzed hydroxylation of steroidal compounds at specific sites. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is a vector map of the non-specific peroxygenase expression vector pPICZA-N1 in an embodiment of the present invention;

[0020] Figure 2 This is a chromatogram of the product of steroid substrate 1 catalyzed by the nonspecific peroxygenase N1 F66G / L215V mutant in an embodiment of the present invention;

[0021] Figure 3 This is the chromatogram of the product of steroid substrate 2 catalyzed by the nonspecific peroxygenase N1 F66G / L215V mutant in an embodiment of the present invention;

[0022] Figure 4 This is the chromatogram of the product of steroid substrate 3 catalyzed by the nonspecific peroxygenase N1 F66G / L215V mutant in this embodiment of the invention;

[0023] Figure 5 This is the chromatogram of the product of steroid substrate 4 catalyzed by the nonspecific peroxygenase N1 F66G / L215V mutant in this embodiment of the invention;

[0024] Figure 6 This is the chromatogram of the product of steroid substrate 5 catalyzed by the nonspecific peroxygenase N1 F66G / L215V mutant in this embodiment of the invention;

[0025] Figure 7 This is a chromatogram of the product of steroid substrate 1 catalyzed by the nonspecific peroxygenase N1 F66G / A162L / L215V mutant in an embodiment of the present invention;

[0026] Figure 8 This is a graph showing the substrate conversion rate of the nonspecific peroxygenase N1 F66G / A162L / L215V mutant in the scaled-up reaction system over time, as described in an embodiment of the present invention. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified, the equipment and reagents used in the embodiments and experimental examples are commercially available. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0028] Based on the information contained in this application, various changes to the precise description of the invention can be readily made by those skilled in the art without departing from the spirit and scope of the appended claims. It should be understood that the scope of the invention is not limited to the defined processes, properties, or components, as these embodiments and other descriptions are merely illustrative of specific aspects of the invention. In fact, various modifications to embodiments of the invention that will be apparent to those skilled in the art or related fields are covered within the scope of the appended claims.

[0029] To better understand the invention and not to limit its scope, all figures indicating amounts, percentages, and other numerical values ​​used in this application should, in all cases, be understood to be modified by the word "approximately." Therefore, unless specifically stated otherwise, the numerical parameters listed in the specification and appended claims are approximate values ​​and may vary depending on the desired properties being sought. Each numerical parameter should at least be considered as obtained based on reported significant figures and conventional rounding methods. Furthermore, the terms "comprising," "including," "containing," "having," and similar words are non-limiting in meaning, allowing for the addition of other steps and components that do not affect the outcome.

[0030] To make the above-mentioned objectives and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below.

[0031] Selective oxygen functionalization of inert CH bonds is a crucial reaction in organic synthesis. Unspecific peroxygenases (UPOs) are self-sufficient heme-thiol salt proteases that use hydrogen peroxide as both oxygen donor and electron acceptor, without relying on redox chaperones or any cofactors to provide reducing power. Their relatively simple electron transport chains offer advantages such as simple electron transport processes, high catalytic activity, broad substrate spectrum, and high economic efficiency, making them highly efficient biocatalysts for CH bond monooxygenation reactions and among the most promising oxidases. Currently reported UPOs include AaeUPO, MroUPO, HspUPO, and the AaeUPO mutant PADA-Ⅰ. Reactions catalyzed by these enzymes include hydroxylation, epoxidation, dealkylation, aromatization, sulfur oxidation, nitrogen oxidation, dechlorination, and halide oxidation; their substrate spectrum includes alkanes, alkenes, aromatics, and fatty acids. Although some research has been conducted, the types of UPOs reported so far remain very limited, and their catalytic selectivity and / or activity still need improvement. There are few reports on complex compounds, such as steroidal compounds with a cyclopentane-polyhydrophenanthrene tetracyclic core structure. Currently, only CglUPO has shown hydroxylation activity for steroidal compounds; however, approximately 90% of its catalytic products are epoxidation byproducts, resulting in extremely low hydroxylation efficiency (selectivity <10%). Therefore, developing novel UPOs to improve their catalytic performance, especially hydroxylation selectivity, for steroidal compounds has significant application value, which is crucial for reducing impurity content in the products and simplifying purification.

[0032] Combining database mining of novel enzyme sources with mutagenesis is a crucial approach for discovering new UPO enzyme sources and engineering enzymes. This invention reconstructs the ancestral sequence of a nonspecific peroxygenase (named N1, amino acid sequence see SEQ ID NO.1) using ancestral sequence reconstruction (ASR). The reconstructed UPON1 exhibits a broad steroid substrate spectrum and significantly improved selectivity; compared to CglUPO, the selectivity for substrate hydroxylation is increased to 63%, greatly expanding the application boundaries of this type of enzyme in oxygen functionalization reactions and providing an important UPO enzyme resource for specific hydroxylation reactions at the 11β-, 16α-, or 6β- sites of steroid compounds. However, its catalytic activity needs further improvement; the conversion rate of substrates is less than 20% at low enzyme concentrations, requiring higher enzyme concentrations to ensure product yield.

[0033] To reduce enzyme usage and further lower production costs, this invention uses the UPO N1 amino acid sequence shown in SEQ ID NO.1 as a base, mutating key active sites to construct a series of mutant libraries. The mutant genes were then inserted into the pPICZA expression vector and transformed into Pichia pastoris for extracellular secretory expression. By comparing the catalytic activity and selectivity of the fermentation supernatant (i.e., crude UPO enzyme solution) of various recombinant strains in the hydroxylation of steroid compounds, several novel UPO mutants with significantly enhanced catalytic activity and / or selectivity were screened: F66G, L215V, F66G+L215V, and F66G+L215V+A162L. These mutants are compatible with multiple steroid substrates and exhibit excellent substrate conversion rates and product selectivity.

[0034] Based on this, one embodiment of the present invention provides a nonspecific peroxygenase, wherein the nonspecific peroxygenase is selected from any one of (a)-(e) below:

[0035] (a) The amino acid sequence is shown in SEQ ID NO.1;

[0036] (b) Mutate the 66th position of the amino acid sequence shown in SEQ ID NO.1 from F to G (F66G mutant);

[0037] (c) Mutate the 215th position of the amino acid sequence shown in SEQ ID NO.1 from L to V (L215V mutant);

[0038] (d) Mutate the 66th position of the amino acid sequence shown in SEQ ID NO.1 from F to G and the 215th position from L to V, i.e., the mutant containing the mutation sites in (b) and (c) (F66G / L215V mutant).

[0039] (e) The amino acid sequence shown in SEQ ID NO.1 is mutated from F to G at position 66, from A to L at position 162, and from L to V at position 215, i.e., the mutant containing (d) and with the addition of the A162L mutation site (F66G / A162L / L215V mutant).

[0040] The amino acid sequences of the nonspecific peroxygenases (b)-(e) are shown in SEQ ID NO.2-5, respectively.

[0041] This invention, through ancestral sequence reconstruction and mutational modification, combined with catalytic activity and selective screening for steroid compound hydroxylation reactions, yielded a novel ancestral sequence of UPO and different mutants of this ancestral sequence: F66G, L215V, F66G / L215V, and F66G / A162L / L215V. These enzymes can catalyze the 11β-, 16α-, or 6β-hydroxylation reactions of various steroid compounds, effectively solving the predicament of enzyme scarcity in the efficient hydroxylation reactions of steroid compounds in existing technologies. Using Pichia pastoris as the host strain for UPO expression, the obtained ancestral enzymes and various mutants were heterologously expressed extracellularly. The resulting fermentation supernatant exhibited high enzyme activity, demonstrating good compatibility with multiple steroid substrates (including estradiol-4,9-diene-3,17-dione, testosterone, nandrolone, 1,4-androstenedione, and methyltetraene compounds), along with excellent selectivity and / or high yields. The selectivity of the hydroxylation products was greater than 50%, significantly outperforming existing CglUPO enzyme resources. Furthermore, optimizing the enzyme concentration could effectively improve product conversion rates. Moreover, these enzymes of the present invention, in addition to being activated or initiating the reaction process via hydrogen peroxide, can also directly utilize oxygen present in the environment and synergistically initiate the hydroxylation reaction with small molecule reducing agents such as ascorbic acid. This allows for more flexible and controllable reaction conditions, facilitating large-scale production and significantly reducing the cost of enzyme-catalyzed preparation of hydroxylated steroid compounds. The implementation of this invention opens up a completely new biocatalytic technology route for the green synthesis and large-scale production of 11β-, 16α-, or 6β-hydroxylated steroidal compounds, and has important industrial application value and good potential in the hydroxylation of steroidal compounds at specific sites by bioenzymes.

[0042] Based on a comprehensive comparison of catalytic efficiency, substrate conversion, product selectivity, and yield, the non-specific peroxygenase is preferably at least one of the F66G / L215V and F66G / A162L / L215V mutants with ancestral sequences, the amino acid sequences of which are shown in SEQ ID NO.4-5, respectively.

[0043] Another embodiment of the present invention provides a nucleic acid molecule encoding a nonspecific peroxygenase as described above.

[0044] The advantages of the nucleic acid molecule over the prior art are the same as those of the nonspecific peroxygenase over the prior art as described above, and will not be repeated here.

[0045] Nucleic acid molecules include DNA molecules (such as genomic DNA or cDNA) and / or RNA molecules (such as mRNA), and can be single-stranded or double-stranded. The sequence of a nucleic acid molecule can be derived from the UPO amino acid (AA) sequence using conventional methods such as codon coding rules. The full-length sequence of a nucleic acid molecule or a fragment thereof can usually be obtained using PCR amplification, recombination, or artificial synthesis.

[0046] Another embodiment of the present invention provides an expression vector comprising the nucleic acid molecules described above.

[0047] The advantages of the expression vector over the prior art are the same as those of the nonspecific peroxygenase over the prior art as described above, and will not be repeated here.

[0048] Optionally, the expression vector further includes a gene sequence encoding a promoter, wherein the nonspecific peroxygenase is located downstream of the promoter. The promoter is used to initiate the transcription and translation process of UPO and can be adaptively selected based on the expression of the recipient cell and the nonspecific peroxygenase.

[0049] Optionally, the expression vector further includes a gene sequence encoding a signal peptide, with the nonspecific peroxygenase located downstream of the signal peptide. The signal peptide guides the secretory expression of UPO in the host bacteria, facilitating isolation and purification.

[0050] The signal peptide undergoes adaptive selection based on the recipient cell. The ancestral sequence of this invention contains the signal peptide MKASLLLILTVVAAAAA, which can be secreted and expressed in Pichia pastoris. Of course, those skilled in the art can replace it with different signal peptides according to actual needs, such as using the commonly used yeast signal peptide α-factor, and this invention does not impose any special limitations on this.

[0051] Optionally, the expression vector includes a prokaryotic expression vector, a eukaryotic expression vector, or a viral expression vector (such as lentivirus or adenovirus). Correspondingly, the recipient cells transformed or transfected by the expression vector can be prokaryotic cells or eukaryotic cells, selected according to the type of expression vector. For example, when using a prokaryotic expression vector, prokaryotic cells are selected as recipient cells. Common examples of prokaryotic cells include Escherichia coli, Bacillus, Corynebacterium, Fermentosum motilityis, and Streptomyces. When using a eukaryotic expression vector, eukaryotic cells are selected as recipient cells. Common examples of eukaryotic cells include Saccharomyces cerevisiae, Pichia pastoris, Saccharomyces cerevisiae, and filamentous fungi.

[0052] Typical vectors include plasmids (such as the pUC series, pET series, pWB series, pGEX series, pDXW series, pBR322, pEZ15a, pTZ28a, pMA5, pPICZα, PIC9K, pSET152), viral vectors, bacteriophages (such as λgt4λB, λ-Charon, λΔz1, and M13), entrapments, and mini-chromosomes. Plasmids are the most commonly used vectors, and in the context of this invention, unless otherwise specified, plasmids and vectors are used interchangeably.

[0053] Pichia pastoris, as an important protein expression host, possesses advantages such as mature culture conditions, convenient genetic manipulation, strong metabolic activity, and high protein secretion efficiency, and has wide applications in the field of bio-fermentation. In a typical embodiment of this invention, a Pichia pastoris expression system is used, employing the Pichia pastoris expression vector pPICZA, with Pichia pastoris X33 as the host (or recipient) cell, and the ancestral sequence of the original signal peptide. The ancestral sequence containing the signal peptide or its mutant gene sequence is placed downstream of the pPICZA promoter to construct a recombinant expression vector. After obtaining recombinant Pichia pastoris transformed with the recombinant expression vector, secretory expression of UPO can be achieved through conventional fermentation culture. The fermentation supernatant of the recombinant strain can serve as a high-quality enzyme source for the hydroxylation catalysis of steroids, which is beneficial for improving UPO production efficiency and reducing production costs, as well as lowering the conversion cost of hydroxylated steroids.

[0054] In optional embodiments, the expression vector may also be pPIC9, pPIC9k, pHIL-S1, pPICZa, pHIL-D2, pA0815, pPIC3K, pPIC3.5K, pPICZB, pGAPZ, pGAPZa, etc.; the Pichia pastoris host strain may also be GS115, KM71H, SMD116, etc.

[0055] In a preferred embodiment of the present invention, the method for inserting a nucleic acid molecule into an expression vector includes: synthesizing the above-mentioned nucleic acid molecule through whole-genome synthesis, designing first and second restriction enzyme sites at both ends of the nucleic acid molecule, treating the expression vector with the restriction enzymes corresponding to the first and second restriction enzyme sites to obtain a linearized vector, and linking the nucleic acid molecule and the linearized vector to obtain a recombinant expression vector.

[0056] Optionally, the first and second restriction sites are EcoRI and NotI, respectively.

[0057] In another embodiment of the present invention, a recombinant strain is provided, the recombinant strain containing the nucleic acid molecule as described above or the expression vector as described above.

[0058] Optionally, the starting strain of the recombinant strain is Pichia pastoris, specifically Pichia pastoris X33.

[0059] Nucleic acid molecules or expression vectors can be transformed or transfected into host cells by various methods known in the art, including: CaCl2 transformation, lithium acetate transformation, calcium phosphate-DNA coprecipitation, electroporation, gene gun bombardment, microinjection, conjugation transfer, liposome-mediated transfection, liposome fusion, lipid transfection, and protoplast fusion, etc.

[0060] This invention also provides a method for preparing nonspecific peroxygenase, comprising the following steps: culturing the recombinant strain as described above, centrifuging the fermentation culture, filtering the supernatant, and collecting the filtrate to obtain a crude enzyme solution containing nonspecific peroxygenase.

[0061] The embodiments of the present invention further provide the application of the nonspecific peroxygenase, nucleic acid molecule, expression vector or recombinant strain as described above in the preparation of hydroxylated steroidal compounds.

[0062] Optionally, the steroid compound is selected from at least one of estradiol-4,9-diene-3,17-dione, testosterone, nandrolone, 1,4-androstenedione, and methyltetraene compounds; its structural formula is shown in Formulas 1-5 below:

[0063] .

[0064] The present invention relates to a nonspecific peroxygenase that 11β-hydroxylates estradiol-4,9-diene-3,17-dione (Formula 1), 16α-hydroxylates testosterone (Formula 2), 16α-hydroxylates nandrolone (Formula 3), 6β-hydroxylates 1,4-androstenedione (Formula 4), and 6β-hydroxylates methyltetraene (Formula 5). The structural formulas of the hydroxylated steroid products are shown in Formulas 1a-5a below:

[0065] .

[0066] The present invention further provides a method for synthesizing hydroxylated steroidal compounds, comprising the following steps: mixing a nonspecific peroxygenase and a steroidal compound, and carrying out a catalytic reaction.

[0067] In the catalytic reaction of nonspecific peroxygenase, the reaction system has a pH of 5.5-8.0 and a temperature of 25-40°C. Preferably, the pH is 7.0 and the temperature is 30°C.

[0068] This invention allows for the initiation of the hydroxylation reaction of UPO via conventional external hydrogen peroxide. It is important to note that under these reaction conditions, the rate of hydrogen peroxide addition must be controlled to avoid localized high concentrations that could inactivate the enzyme.

[0069] Because high concentrations of hydrogen peroxide can easily inactivate enzymes, the addition rate and concentration must be strictly controlled. In a preferred embodiment, this invention employs a synergistic approach of "oxygen + reducing agent" to initiate the hydroxylation reaction. Under these reaction conditions, oxygen can be directly obtained from the air without additional supplementation; the reducing agent is added to the reaction solution simultaneously with the enzyme. Optionally, the reducing agent is selected from ascorbic acid, dehydroascorbic acid, gallic acid, and pyrogallic acid. The concentration of the reducing agent is adaptively adjusted according to the concentration of UPO enzyme in the reaction system and the consumption rate of the reducing agent; generally, the concentration of the reducing agent is 100 mM.

[0070] Optionally, the concentration of UPO enzyme in the reaction system is 1-20 μM, and the concentration of steroid compound is 1-40 mM.

[0071] The present invention will be further illustrated below with reference to specific embodiments. Experimental methods in the following embodiments, unless otherwise specified, were generally performed under conventional conditions, such as those described in *Molecular Cloning: A Laboratory Manual (4th Edition)* published by Cold Spring Harbor Laboratory, or as recommended by the manufacturer. The sequencing work during the whole-gene synthesis, primer synthesis, and expression vector construction processes in the following embodiments was outsourced to Sangon Biotech Co., Ltd. The protein, gene, and primer sequences involved are shown in Table 1.

[0072] Table 1. Enzyme and gene sequences involved in the embodiments of the present invention.

[0073]

[0074] Note: Shaded and underlined areas indicate sites to be mutated; bolded shaded areas indicate amino acid mutation sites; and bolded underlines indicate codon mutation sites. Table 1 provides an example of the UPO N1 gene sequence. The gene sequences of each mutant are obtained by replacing the amino acid codons at the N1 gene mutation sites with the codons of the mutated amino acids. For example, when constructing the F66G mutant, the TCT codon at amino acid position 66 of the gene sequence is replaced with GGT, thus obtaining the coding gene of the N1 F66G mutant. Additionally, in the primers, F represents the upstream primer and R represents the downstream primer.

[0075] In the following examples, the YPD solid medium consisted of 20 g / L tryptone, 10 g / L yeast extract, 20 g / L glucose, and 20 g / L agarose. The BMGY liquid medium (pH 6.0) consisted of 20 g / L tryptone, 10 g / L yeast extract, 10 mL / L glycerol, 13.4 g / L amino-free yeast nitrogen source, and 0.1 mol / L phosphate. The BMMY liquid medium (pH 6.0) consisted of 20 g / L tryptone, 10 g / L yeast extract, 13.4 g / L amino-free yeast nitrogen source, 0.1 mol / L phosphate, and 10 mL / L methanol.

[0076] I. Synthesis and Expression of Nonspecific Peroxygenase N1

[0077] The evolutionary relationships of proteins in evolutionary history were inferred using ancestral sequence reconstruction technology. Multiple sequence alignment was performed using MUSCLE, followed by phylogenetic tree construction using IQ-TREE. Finally, the ancestral sequence was predicted using FastML, and the non-specific peroxygenase (UPO) was reconstructed to obtain the ancestral enzyme. Based on the ancestral sequence, the amino acid sequence shown in SEQ ID NO.1 was reconstructed and named UPON1. Its predicted signal peptide sequence is MKASLLLILTVVAAAAA. Using the Pichia pastoris expression system, the gene sequence of UPO N1 was optimized using Pichia pastoris-preferred codons. The optimized gene sequence is shown in SEQ ID NO.7, and the gene sequence of the signal peptide is shown in SEQ ID NO.8.

[0078] The UPO N1 gene sequence containing the signal peptide was synthesized from the whole genome. EcoRI and NotI restriction enzyme sites were designed at both ends during synthesis. The expression vector pPICZA was double-digested with EcoRI and NotI. The N1 gene was then ligated to the linearized pPICZA, and the N1 gene expression was placed under the AOX promoter of the vector to obtain the recombinant plasmid pPICZA-N1. Its vector map is shown below. Figure 1 In this context, signal represents the signal peptide, N1 represents the N1 gene, AOX1 promoter represents the AOX1 promoter, AOX1 terminator represents the AOX1 terminator, EM7 promoter represents the EM7 promoter, CYC1 terminator represents the CYC1 terminator, ori represents the replication initiator, and BleoR represents bleomycin resistance.

[0079] The recombinant plasmid pPICZA-N1 was linearized using PmeI restriction enzyme. The linearized product was recovered after detection by 1% agarose gel electrophoresis. The recovered product was electroporated into Pichia pastoris X33 competent cells. The transformation products were plated on YPD solid medium (YPD+Z) containing 100 μg / mL bleomycin for screening positive transformants. Single clones of different transformants were picked and added to 50 μL of sterile water. The cell walls were disrupted by a heat-cooling cycle (i.e., microwave heating for 5 min, followed by incubation at -20℃ for 5 min, repeated 3 times) to obtain the single-clone lysate. Positive transformants were verified by Pichia pastoris colony PCR. The detection primers were N1-F (sequence shown in SEQ ID NO. 9) and 3AOX-R (sequence shown in SEQ ID NO. 10). The PCR amplification system was 20 μL, including: 1 μL of monoclonal bacterial lysate, 1 μL each of primers N1-F and 3AOX, 0.3 μL of KOD FX polymerase (purchased from TOYOBO, catalog number 5305002), 10 μL of 2× PCR buffer for KOD FX, 4 μL of 2 mMdNTPs, and finally, sterile distilled water was added to bring the volume to 10 μL. The PCR reaction program included: 94℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 56℃ annealing for 30 s, 68℃ extension for 1 min, and 30 cycles of denaturation-annealing-extension; and a final extension at 72℃ for 5 min. The PCR products were detected by 1% agarose gel electrophoresis, and positive transformants were sent for sequencing.

[0080] Selected single colonies with correct sequencing were cultured in 100 mL of BMGY liquid culture medium at 30°C and 250 rpm for 48 hours. The bacterial cells were then collected, and the precipitate was resuspended in 50 mL of BMMY liquid culture medium with the addition of 1% (v / v) methanol. The culture was then inducible to express nonspecific peroxygenase at 30°C and 250 rpm.

[0081] II. Construction of Nonspecific Peroxygenase N1 Mutant

[0082] Using recombinant plasmid pPICZA-N1 as a template, mutation primers were designed, with the bases to be mutated placed in the middle of the primers. For the F66G mutation site, pPICZA-N1 was amplified using primer pairs N1-66G-F (sequence shown in SEQ ID NO.11) and N1-110-R (sequence shown in SEQ ID NO.12) to obtain the recombinant plasmid pPICZA-N1 F66G. For the L215V mutation site, pPICZA-N1 was amplified using primer pairs N1-215V-F (sequence shown in SEQ ID NO.13) and pPICZ-R (sequence shown in SEQ ID NO.14) to obtain the recombinant plasmid pPICZA-N1 L215V. In the combined mutation, for the F66G / L215V mutation site, the recombinant plasmid pPICZA-N1 F66G was used as a template and amplified using primer pairs N1-215V-F and pPICZ-R, or the recombinant plasmid pPICZA-N1 L215V was used as a template and amplified using primer pairs N1-66G-F and N1-110-R, to obtain the recombinant plasmid pPICZA-N1 F66G / L215V; for the F66G / A162L / L215V mutation site, the recombinant plasmid pPICZA-N1 F66G / L215V was used as a template and amplified using primer pairs N1-162L-F (sequence shown in SEQ ID NO.15) and N1-210-R (sequence shown in SEQ ID NO.16), to obtain the recombinant plasmid pPICZA-N1 F66G / A162L / L215V.

[0083] The PCR amplification system, in 20 μL volumes, included: 1 μL pPICZA-N1 (10 ng), 1 μL each of the corresponding 10 μM forward and reverse primers, 10 μL Primer STAR Max high-fidelity DNA polymerase (purchased from Takara), and finally, sterile distilled water to bring the volume to 25 μL. The PCR reaction program was as follows: 98℃ pre-denaturation for 5 min; 98℃ denaturation for 10 s, 56℃ annealing for 15 s, 72℃ extension for 90 s, and 30 cycles of denaturation-annealing-extension, followed by a final extension at 72℃ for 5 min. The PCR products were then stored at 12℃.

[0084] The PCR products obtained above were digested with DpnI restriction enzyme at 37°C for 2 h, and the digested products were completely transformed into *E. coli* DH5α. The transformed products were then evenly spread on low-salt LB agar plates containing 25 μg / mL bleomycin and incubated overnight at 37°C for 12 h. Positive transformants were then screened from the plates. Single colonies were picked and cultured in 5 mL of low-salt LB liquid medium containing 25 μg / mL bleomycin, followed by sequencing. The sequencing results were compared with the gene sequence of the UPO mutant using SnapGene software, and plasmids were extracted from the correctly sequenced strains.

[0085] The above recombinant plasmids were electroporated into Pichia pastoris X33. The successfully transformed recombinant strains were added to BMMY liquid medium for culture, and 1% (v / v) methanol was added. The culture was induced to express the recombinant strains at 30℃ and 250 rpm.

[0086] III. Catalytic Activity Analysis of Nonspecific Peroxygenase N1 and its Mutants

[0087] 3.1 Substrate Spectrum Investigation of UPO N1

[0088] After inducing expression of the recombinant strain transformed with the recombinant plasmid pPICZA-N1 for 5 days, the bacterial culture was collected and centrifuged at 4000 rpm for 10 min. The precipitate was removed, and the supernatant was collected. A steroid substrate with a final concentration of 1 mM was added to 200 mL of the supernatant, followed by the addition of 100 mM sodium ascorbate to initiate the reaction. The reaction was carried out at 30 °C and 220 rpm for 48 hours. After the reaction, the product was extracted with 600 mL of ethyl acetate, and the organic phases were combined and dried over Na2SO4 for 6 hours. The organic solvent was then evaporated, and the residue was purified by silica gel column chromatography to obtain the purified product.

[0089] In this embodiment, estradiol-4,9-diene-3,17-dione, testosterone, nandrolone, 1,4-androstenedione, and methyltetraene compounds were selected as substrates, hereinafter referred to as steroidal substrates 1-5. UPO N1 catalyzed the formation of products 1a-5a from substrates 1-5. The reaction equations for the hydroxylation of steroidal substrates 1-5 to products 1a-5a catalyzed by UPO N1 are as follows, with hydroxylation sites at 11β, 16α, or 6β:

[0090]

[0091] The product structure was identified using nuclear magnetic resonance (NMR). NMR analysis: Spectra were recorded on a Bruker AV-400 spectrometer. For 1 1H NMR spectra, chemical shifts are reported in ppm, with the internal standard tetramethylsilane (TMS) chemical shift value set at 0 ppm as the standard. For 13 C10 NMR spectra, chemical shifts reported in ppm, with the internal standard chloroform signal at 77.0 ppm as the standard. Data report: s = singlet, d = doublet, t = triplet, q = quartet, m = multipeak or unresolved, coupling constant in Hz, integral.

[0092] The reaction product 1a of steroid substrate 1 catalyzed by UPO N1, namely 11β-hydroxyestradiol-4,9-diene-3,17-dione, has the following 1H NMR data: 1¹H NMR (CD3OD, 400 MHz) δ 5.78 (¹H, s, H⁻⁴), 5.07 (¹H, dd, 4.4, 1.9 Hz, H⁻¹¹), 1.21 (³H, s, H⁻¹⁸); Carbon NMR data are as follows: 13 C NMR (CD3OD, 100 MHz) δ221.8 (C-17), 202.2 (C-3), 160.2 (C-5), 147.0 (C-9), 130.5 (C-10), 124.4 (C-4), 66.5 (C-11), 51.9 (C-14), 48.3 (C-13), 39.5 (C-12), 37.9 (C-2), 36.5 (C-8), 36.3 (C-16), 31.5 (C-6), 27.5 (C-7), 26.3 (C-1), 22.7 (C-15), 15.8 (C-18).

[0093] The 1H NMR spectrum data of 2a, 16α-hydroxytestosterone, the product of the reaction of steroid substrate 2 catalyzed by UPO N1, are as follows: 1 ¹H NMR (DMSO-d6, 400 MHz) δ 5.62 (1H, s, H-4), 4.65 (1H, brs, 17-OH), 4.59 (1H, brs, 16-OH), 3.81 (1H, t, 7.3 Hz, H-16), 3.20 (1H, d, 5.5 Hz, H-17), 1.13 (3H, s, H-19), 0.67 (3H, s, H-18); Carbon NMR data are as follows: 13 C NMR (DMSO-d6, 100 MHz) δ198.1 (C-3), 171.0 (C-5), 123.2 (C-4), 88.6 (C-17), 76.6 (C-16), 53.4 (C-9), 47.8 (C-14), 42.9 (C-13), 38.3 (C-10), 36.4 (C-12), 35.1 (C-1), 34.7 (C-8), 34.3 (C-15), 33.7 (C-2), 32.1 (C-6), 31.4 (C-7), 19.9 (C-11), 16.9 (C-19),12.4 (C-18).

[0094] The 1H NMR spectrum of 16α-hydroxynandrolone, the product of the reaction of steroid substrate 3 catalyzed by UPO N1, is as follows: 1¹H NMR (DMSO-d6, 400 MHz) δ 5.72 (1H, s, H-4), 4.67 (1H, brs, 17-OH), 4.60 (1H, brs, 16-OH), 3.81 (1H, t, 7.2 Hz, H-16), 3.22 (1H, d, 5.5 Hz, H-17), 0.69 (3H, s, H-18); Carbon NMR data are as follows: 13 C NMR (DMSO-d6, 100 MHz) δ 198.5 (C-3), 166.9 (C-5), 123.8 (C-4), 88.6 (C-17), 76.6 (C-16), 49.1 (C-9), 47.1 (C-14), 43.1 (C-13), 41.8 (C-10), 39.4 (C-8), 36.4 (C-12), 36.2 (C-2), 34.7 (C-6), 34.2 (C-15), 30.4 (C-7), 26.1 (C-1), 25.3 (C-11), 12.4 (C-18).

[0095] The 1H NMR spectrum of 4a, the product of the reaction of steroid substrate 4 catalyzed by UPO N1, namely 6β-hydroxy-1,4-androstenedione, is as follows: 1 ¹H NMR (DMSO-d6, 400 MHz) δ 7.16 (1H, d, 10.1 Hz, H⁻¹), 6.09 (1H, dd, 10.1, 1.9 Hz, H⁻²), 6.05 (1H, d, 1.9 Hz, H⁻⁴), 5.33 (1H, s, 6-OH), 4.40 (1H, m, H⁻⁶), 1.37 (3H, s, H⁻¹⁹), 0.87 (3H, s, H⁻¹⁸); Carbon NMR data are as follows: 13 C NMR (DMSO-d6,100 MHz) δ 219.3 (C-17), 185.6 (C-3), 167.2 (C-5), 157.5 (C-1), 126.1 (C-2), 124.5 (C-4), 71.8 (C-6), 51.6 (C-9), 49.5 (C-14), 47.1 (C-13), 43.2 (C-10),38.9 (C-7), 35.3 (C-16), 31.0 (C-12), 29.4 (C-8), 21.6 (C-15), 21.4 (C-11),20.1 (C-19), 13.5 (C-18).

[0096] The 1H NMR spectrum of the reaction product 5a, namely 6β-hydroxymethyltetraene, catalyzed by UPO N1 is as follows: 1 H NMR (DMSO-d6, 400 MHz) δ 7.38 (1H, d, 10.2 Hz, H-1), 6.92 (1H, dd, 3.4,1.9 Hz, H-16), 6.14 (1H, dd, 10.2, 1.9 Hz, H-2), 6.05 (1H, d, 1.9 Hz, H-4), 5.56 (1H, dt, 6.1, 1.9 Hz, H-11), 5.46 (1H, d, 2.7 Hz, 6-OH), 4.43 (1H, m, H-6), 2.23 (3H, s, H-21), 1.58 (3H, s, H-19), 0.85 (3H, s, H-18); Carbon spectrum data are: 13 C NMR (DMSO-d6, 100 MHz) δ 196.0 (C-20), 185.6 (C-3), 165.4 (C-5), 156.4 (C-1), 152.5 (C-17), 145.0 (C-16), 143.8 (C-9), 125.6 (C-2), 124.4 (C-4), 119.9(C-11), 71.2 (C-6), 52.3 (C-14), 45.6 (C-10), 44.0 (C-13), 40.7 (C-7), 37.4(C-12), 32.5 (C-15), 29.2 (C-8), 28.6 (C-19), 26.9 (C-21), 15.3 (C-18).

[0097] 3.2 Activity and selectivity of UPO N1 mutant catalyzing substrate 1

[0098] Recombinant strains transformed with plasmids pPICZA-N1, pPICZA-N1 F66G, pPICZA-N1 L215V, and pPICZA-N1F66G / L215V were induced to express for 5 days. After centrifugation at 4000 rpm for 15 min at 4℃, the supernatant was collected, filtered through a 0.45 μm PVDF membrane, and concentrated 10-fold using a 10 kDa ultrafiltration tube (Amicon Ultra-15). The concentrated supernatant was diluted 20-fold, and the UPO concentration was determined using carbon monoxide (CO) differential spectroscopy on a spectrophotometer. A catalytic reaction system was established in a 25 mL ground glass stoppered reaction flask. The enzyme-catalyzed reaction system consisted of: 100 mM potassium phosphate buffer (pH 7.0), 1 mM steroid substrate 1, and a final concentration of 2 μM UPO enzyme (N1 or its mutant). After mixing, 100 mM sodium ascorbate was added to initiate the reaction, and the reaction flask was placed at 30°C and 250 rpm for 12 hours. After the reaction was completed, an equal volume of ethyl acetate was added to terminate the reaction. After vigorous shaking and mixing, the mixture was centrifuged at 12000 rpm for 1 minute to achieve phase separation. The upper organic phase was collected and analyzed by nuclear magnetic resonance (NMR). NMR of the reaction products of N1 F66G, N1 L215V, and N1 F66G / L215V mutants were analyzed. 1 HNMR and 13 The C NMR spectral data are the same as those of UPO N1, and will not be shown again here.

[0099] In addition, 200 μL of the upper organic phase was collected, evaporated to dryness, and then redissolved in 200 μL of chromatographic grade methanol. The solution was filtered through a 0.22 μm microporous membrane and analyzed by high-performance liquid chromatography (HPLC) to determine the substrate conversion and selectivity. The HPLC analysis procedure was as follows: an automated Shimadzu LC-2010 HPLC system (Japan) equipped with four MTP racks was used. The chromatographic column was a reversed-phase Zorbax Eclipse XDB-C18 column (250 × 4.6 mm, 5 μm), the temperature was 40 °C, the mobile phase was an acetonitrile-water aqueous solution mixed with ultrapure water at a volume ratio of 45:55, the flow rate was 1.2 mL / min, and the UV detection wavelength was 310 nm.

[0100] The substrate and product concentrations were determined by HPLC. The substrate conversion rate was calculated based on the initial substrate concentration and the final substrate concentration. The product selectivity was calculated based on the amount of hydroxylated product generated. The yield (or productivity) of the purified product from the reaction solution was then calculated. The relevant formulas are as follows: Conversion rate = (Initial substrate concentration - Residual substrate concentration) / Initial substrate concentration × 100%; Selectivity = Concentration of hydroxylated product at a specific position / (Initial substrate concentration - Residual substrate concentration) × 100%; Product productivity = Purified concentration of hydroxylated product at a specific position / Concentration in the reaction solution.

[0101] The relevant results are shown in Table 2. It can be seen that the mutants have significantly improved catalytic activity for the hydroxylation of steroidal compounds compared with the ancestral enzyme UPO N1. Except for the F66G mutant, which has the same product selectivity as UPO N1, the substrate conversion rate and product selectivity of the other mutants are higher than those of UPO N1. In particular, the enzyme activity of the combined mutant N1 F66G / L215V is significantly improved. Figure 2-6 The HPLC chromatograms of the products of steroid substrates 1-5 catalyzed by the N1 F66G / L215V mutant are shown as examples. From top to bottom, they are substrate standards (1-5), reaction mixtures (1A-5A), and purified products (1a-5a), represented by black lines, pink lines, and blue lines, respectively.

[0102] Table 2. Conversion and selectivity of UPO N1 and its mutants for steroid substrate 1

[0103]

[0104] 3.3 Substrate spectral analysis of UPO N1 F66G / L215V

[0105] Recombinant strains transformed with recombinant plasmid pPICZA-N1 F66G / L215V were induced to express for 5 days. The supernatant was collected by centrifugation, filtered, and concentrated to obtain the enzyme solution, which was used to catalyze the hydroxylation of steroid substrates 1-5. The enzyme catalytic reaction system consisted of: 100 mM potassium phosphate buffer (pH 7.0), 1 mM steroid substrate, and a final concentration of 4 μM UPO enzyme (N1 or its mutant). After mixing, 100 mM sodium ascorbate was added to initiate the reaction, and the reaction flask was placed at 30℃ and 250 rpm for 12 hours. After the reaction, the reaction solution was collected, extracted with ethyl acetate, reconstituted with methanol, and filtered through a 0.22 μm microporous membrane. HPLC analysis was performed to determine the substrate conversion rate and selectivity. The UV detection wavelength for steroid substrate 1 was 310 nm, and the UV detection wavelength for steroid substrates 2-5 was 254 nm.

[0106] Table 3 shows the enzyme activity data of N1F66G / L215V catalyzing steroid substrates 1-5. It can be seen that the N1F66G / L215V mutant of this invention exhibits good compatibility with steroid substrates 1-5, and shows higher specific hydroxylation activity for steroid substrate 1.

[0107] Table 3. Conversion and selectivity of substrates 1-5 catalyzed by the N1 F66G / L215V mutant.

[0108]

[0109] 3.4 Enzyme activity analysis of UPO N1 F66G / A162L / L215V

[0110] To further improve enzyme activity, the present invention introduces the A162L mutation site into the F66G / L215V mutant, and compares the catalytic activity of substrate 1 by the N1F66G / L215V and N1F66G / A162L / L215V mutants. The enzyme concentration is reduced to 0.5 μM and the substrate concentration is increased to 2 mM, while other conditions are the same as above.

[0111] Results (see Table 4 and) Figure 7 The results showed that mutating amino acid residue A to L at position 162 significantly improved the enzyme activity of the F66G / L215V mutant, with a substantial increase in substrate conversion and product selectivity.

[0112] Table 4. Conversion and selectivity of substrate 1 catalyzed by the N1 F66G / L215V and F66G / A162L / L215V mutants.

[0113]

[0114] 3.5, UPO N1 F66G / A162L / L215V industrial scale-up reaction system

[0115] The recombinant strain pPICZA-N1 F66G / A162L / L215V was cultured and transformed in a 5 L fermenter. After fermentation, the supernatant was collected and centrifuged at 4℃ and 4000 rpm for 30 min to remove bacterial cells. 1 L of the clarified supernatant (approximately 20 μM enzyme concentration) was transferred to a new 5 L fermenter, and 10 g of steroidal substrate 1, pre-dissolved in 200 mL of 30% (w / v) hydroxypropyl-β-cyclodextrin solution, was added to a final substrate concentration of 10 g / L. Sodium ascorbate was added to a final concentration of 100 mM to initiate the reaction. The reaction was carried out under strictly controlled conditions: aeration rate of 1 L / min, constant temperature of 30℃ (±0.5℃), stirring rate of 500 rpm, and 20 g of sodium ascorbate added every 24 hours. Samples were taken every few hours during the reaction, and the product formation was monitored using high-performance liquid chromatography (HPLC). The reaction was terminated when the substrate conversion rate remained constant. After 48 hours of reaction, the final conversion rate reached 94.8% (see...). Figure 8 ), generating 9.2 g of the target product 1a.

[0116] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A non-specific peroxygenase, characterized in that, The amino acid sequence of the non-specific peroxygenase is selected from at least one of SEQ ID NO. 1-5.

2. The non-specific peroxygenase according to claim 1, characterized in that, The amino acid sequence of the non-specific peroxygenase is selected from at least one of SEQ ID NO. 4-5.

3. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the non-specific peroxygenase according to claim 1.

4. An expression vector, characterized by, The expression vector comprises the nucleic acid molecule according to claim 3.

5. The expression vector of claim 4, wherein, The expression vector further comprises a gene sequence encoding a signal peptide, and an amino acid sequence of the signal peptide is shown as SEQ ID NO.

6.

6. A recombinant bacterial strain, characterized in that, The recombinant strain comprises the nucleic acid molecule according to claim 3 or the expression vector according to any one of claims 4-5.

7. Use of a non-specific peroxygenase according to any one of claims 1 to 2, or a recombinant strain according to claim 6, for the preparation of a hydroxylated steroid compound, characterized in that, The steroid compound is selected from at least one of estr-4,9-dien-3,17-dione, testosterone, nandrolone, 1,4-androstenedione and methyltetraene; The non-specific peroxygenase or the recombinant strain respectively uses estr-4,9-dien-3,17-dione, testosterone, nandrolone, 1,4-androstenedione and methyltetraene as substrates to prepare 11beta-hydroxyestr-4,9-dien-3,17-dione, 16alpha-hydroxytestosterone, 16alpha-hydroxynandrolone, 6beta-hydroxy-1,4-androstenedione and 6beta-hydroxymethyltetraene.

8. A method of synthesizing a hydroxylated steroid compound, characterized by, The method comprises the following steps: The non-specific peroxygenase and the steroid compound are mixed to perform a catalytic reaction, wherein the amino acid sequence of the non-specific peroxygenase is selected from at least one of SEQ ID NO. 1-5, and the steroid compound is selected from at least one of estr-4,9-dien-3,17-dione, testosterone, nandrolone, 1,4-androstenedione and methyltetraene.

9. The method of synthesis of hydroxylated steroidal compounds according to claim 8, wherein, The pH value of the catalytic reaction is 5.5-8.0, and the temperature is 25-40℃.

10. The method of synthesis of hydroxylated steroidal compounds as claimed in claim 8, wherein, The reaction system of the catalytic reaction further comprises a reducing agent, and the reducing agent is selected from ascorbic acid, dehydroascorbic acid, gallic acid or pyrogallic acid. The pH value of the catalytic reaction is 5.5-8.0, and the temperature is 25-40℃. The reaction system of the catalytic reaction further comprises a reducing agent, and the reducing agent is selected from ascorbic acid, dehydroascorbic acid, gallic acid or pyrogallic acid.

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