Steroid compound 14α-hydroxylase and its applications

Abstract

The present invention relates to steroidal compound 14α-hydroxylase and its applications. Specifically selected from enzyme P450 Y , enzyme P450 YM1 or enzyme P450 YM2 ; the amino acid sequence of enzyme P450 Y is shown as SEQ ID No.1; the amino acid sequence of enzyme P450 YM1 is shown as SEQ ID No.2; the amino acid sequence of enzyme P450 YM2 is shown as SEQ ID No.3. The present invention also constructs an engineered strain that can specifically synthesize the C14α-hydroxylated product of steroidal compounds, and under mild conditions, the C14α-hydroxylated product of 11-deoxycortisol (cortexolone, RSS) with a chiral purity exceeding 98% can be obtained within 24 hours. The reaction process is simple and easy to operate, green and pollution-free, and has good industrial application prospects.

Description

Technical Field

[0001] The invention belongs to the field of enzymology and microbial application, and in particular relates to steroid compound 14α-hydroxylase and application thereof. Background Art

[0002] Steroids are natural organic compounds found widely in animals, plants, and fungi. They possess a variety of physiological and pharmacological activities and are widely used in anti-tumor, anti-inflammatory, and antifungal drugs. In recent years, over 300 steroid molecules have been approved for drug development. In 2020, global sales of steroid products exceeded US$150 billion, making them the second-largest class of drugs in terms of market value, after antibiotics.

[0003] Steroidal compounds typically consist of a tetracyclic nucleus of a cyclopentanepolyhydrophenanthrene and three side chains forming a basic carbon skeleton. The different arrangements of functional groups, particularly the hydroxyl groups, on the sterane nucleus give steroidal molecules distinct physiological functions. For example, 11β-OH is crucial for the biological activity of glucocorticoids, 14β-OH is a core and important structural feature of all cardiac glycosides, and 14α-OH derivatives can be used as precursors for the synthesis of a range of compounds, including some chemotherapeutic agents for human breast cancer or estrogen-dependent uterine tumors, and veterinary progestogens.

[0004] How to specifically and efficiently modify hydroxyl groups at specific sites on the steroid nucleus has always been an important and core issue in the steroid field. Currently, there are two main methods for modifying hydroxyl groups on the steroid nucleus: chemical synthesis and biotransformation. Compared with chemical synthesis, biotransformation has the following advantages: (i) higher regio- and stereospecificity of the catalytic reaction; (ii) lower cost, with the substrates generally being cholesterol or diosgenin; and (iii) more environmentally friendly processes (i.e., mild reaction conditions, aqueous media, etc.). Similarly, over the past few decades, other chemical steps in steroid synthesis have been replaced by microbial biotransformation, resulting in more competitive and robust production processes. For example, the steroid hormone testosterone (TS) is chemically synthesized from the intermediate 4-androstene-3,17-dione (AD), which can be obtained from natural sterols through microbial biotransformation.

[0005] Typically, biotransformation of steroidal compounds to obtain hydroxylated products often uses bacterial or fungal strains that can produce the target product. However, direct use of the original strain for biotransformation usually has the following problems: (i) the substrate conversion rate is generally low because the activity of the enzyme on the substrate has not been optimized by natural evolution, and the electron transfer efficiency in the multi-component system is relatively low; (ii) it is difficult to find microbial strains with both complete regioselectivity and stereoselectivity. Most strains will accumulate a large amount of by-products during the production process, mainly because the original strain often has multiple enzymes that can utilize the same substrate in the cell, and the single enzyme selectivity is poor; (iii) fungi usually have slow vegetative growth (1-2 weeks), and the mycelial morphology is poorly homogeneous during liquid culture. There is a lack of genetic manipulation tools, and there is a risk of infecting humans and causing disease.

[0006] Despite this, compared to the complexity and low yield (2% w / w) of chemical synthesis, the method of obtaining steroid C14 hydroxylated products by fungal whole-cell conversion has been widely used. The main fungal species include Schizomyces, Absidia, Mucor, and Cochliobolus, and their substrate conversion rates have reached 13-32%. Currently, only one fungal steroid 14α-hydroxylase has been confirmed, but it has only about 40% 14α-hydroxylation activity towards 11-deoxycorticosterol RSS. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a steroid compound 14α hydroxylase and its application. Specifically, a P450 derived from Cochliobolus lunatus CX3 is provided. Y Its mutant P450 YM1 / P450 YM2 , and the engineered strain for biosynthesizing steroid 14α-hydroxylation products constructed on this basis can specifically and efficiently prepare the 14α-hydroxylation product of the steroid 11-deoxycorticosterol RSS.

[0008] The present invention designs and optimizes P450 Y The amino acid sequence of enzyme P450 YM1 and enzyme P450 YM2 . And verified that compared with the wild-type enzyme P450 Y , enzyme P450 YM1 and enzyme P450 YM2 The enzyme activity was maintained or enhanced, and its regioselectivity was significantly improved, with the ratio of C14α hydroxylation product of catalyzing the synthesis of steroid 11-deoxycorticosterol RSS approaching 98%.

[0009] The purpose of the present invention can be achieved by the following technical solutions:

[0010] The present invention provides a steroid compound 14α hydroxylase selected from enzyme P450 Y , enzyme P450 YM1 or enzyme P450 YM2 ;

[0011] Among them, enzyme P450 Y The amino acid sequence is shown in SEQ ID No. 1;

[0012] enzyme P450 YM1 The amino acid sequence is shown in SEQ ID No. 2;

[0013] P450 enzymes YM2 The amino acid sequence is shown in SEQ ID No.3.

[0014] In the present invention, enzyme P450 Y It is a wild-type enzyme from Cochliobolus lunatus CX3 (genome NCBI ID: ASM74333v1). YM1 P450 Y The L209R mutant of P450 YM2 P450 Y The T302Y mutant.

[0015] The present invention also provides a method for preparing an engineered strain capable of synthesizing steroid 14α-hydroxylation products, comprising the following steps:

[0016] Step 1: PCR amplification to obtain enzyme P450 Y , enzyme P450 YM1 or enzyme P450 YM2 Genes, get P450 Y Genes, P450 YM1 Genes, P450 YM2 Gene; a P450 reductase gene, namely, a CPR gene, was amplified from a cDNA library of the fungus Cochliobolus lunatus CX3, and its amino acid sequence is shown in SEQ ID No.4;

[0017] Step 2: Take the P450 obtained in step 1 Y Genes, P450 YM1 Gene or P450 YM2 One of the genes and the CPR gene fragment were simultaneously cloned into an expression vector, and positive plasmids were screened;

[0018] Step 3: The positive plasmid obtained in step 2 is transferred into the host bacteria to construct an engineered strain that can synthesize steroid 14α-hydroxylation products.

[0019] In one embodiment of the present invention, in step 2, the expression vector is pXW02 (LEU2 marker).

[0020] In one embodiment of the present invention, the host bacteria in step three is Saccharomyces cerevisiae BY4741.

[0021] The present invention further provides an engineered strain capable of synthesizing steroid 14α hydroxylation products.

[0022] The present invention further provides a method for preparing an engineered bacterial strain culture capable of synthesizing steroid 14α-hydroxylation products, comprising the following steps:

[0023] Step A: Cultivate the engineered strain capable of synthesizing steroid 14α-hydroxylation products in YPD medium at 28-30°C, 250 rpm for 2 days;

[0024] Step B: Collect the bacterial cells by centrifugation and resuspend them in potassium phosphate buffer to obtain the engineered strain culture capable of synthesizing steroid 14α-hydroxylation products.

[0025] In one embodiment of the present invention, in step A, the YPD medium contains 1% yeast powder, 2% peptone and 2% glucose.

[0026] In one embodiment of the present invention, in step B, the centrifugation condition is: centrifugation is performed at 6,000 rpm.

[0027] In one embodiment of the present invention, in step B, the concentration of potassium phosphate buffer is 50 mM, pH 7.2.

[0028] The present invention further provides the use of steroid compound 14α hydroxylase or an engineered strain capable of synthesizing steroid 14α hydroxylation products or an engineered strain culture capable of synthesizing steroid 14α hydroxylation products in synthesizing steroid 14α hydroxylation products.

[0029] The present invention further provides a method for preparing a steroid 14α-hydroxylated product, comprising the following steps:

[0030] Step (1): dissolving a steroid compound as a reaction substrate in N,N-dimethylformamide;

[0031] Step (2): adding the reaction substrate and inducer in step (1) to a culture of steroid compound 14α-hydroxylase or an engineered strain capable of synthesizing steroid 14α-hydroxylation products;

[0032] Step (3): The mixed solution of step (2) is biotransformed on a shaker to obtain a steroid 14α-hydroxylated product.

[0033] In one embodiment of the present invention, the steroid compound is the steroid 11-deoxycorticosterol RSS, the structure of which is shown below:

[0034]

[0035] In one embodiment of the present invention, in step (1), the steroid compound is dissolved in N,N-dimethylformamide to a final concentration of 150 mg / L.

[0036] In one embodiment of the present invention, in step (2), the inducer is ethanol.

[0037] In one embodiment of the present invention, in step (3), the mixed solution of step (2) is biotransformed on a shaker for 24 hours.

[0038] In one embodiment of the present invention, in step (3), the shaking conditions are 30° C. and 250 rpm.

[0039] In one embodiment of the present invention, the steroid 14α-hydroxylation product (abbreviated as 14αOH-RSS) has the following structural formula:

[0040]

[0041] In one embodiment of the present invention, the system for preparing steroid 14α-hydroxylated products is: Y / P450 YM1 / P450 YM2 and a Saccharomyces cerevisiae strain co-expressing a plasmid for its CPR gene, a steroid substrate RSS, and a potassium phosphate buffer. Specifically, the yeast strain is BY4741, the growth OD reaches 7.0, the substrate RSS is dissolved in N,N-dimethylformamide and added to the reaction system at a final concentration of 150 mg / L, the buffer is a potassium phosphate buffer with a concentration of 50 mM, pH 7.2, and the conversion time is 24 hours. After the reaction product is extracted with ethyl acetate, it is verified by ultra-performance liquid chromatography (UPLC), and the generation of 14αOH-RSS is monitored, and in the enzyme P450 YM1 and enzyme P450 YM2 The catalytic product of 14αOH-RSS accounts for 98%. YM1 and enzyme P450 YM2 The engineered strain and biocatalytic method for synthesizing steroid C14α hydroxylation products can specifically and efficiently achieve the synthesis of C14α hydroxylation products of steroid compounds.

[0042] The P450 obtained by the present invention Y / P450 YM1 / P450 YM2, which can efficiently catalyze steroid substrates such as RSS to produce 14α-hydroxylated products. YM1 and enzyme P450 YM2 While maintaining enzyme activity, its regional selectivity is increased to nearly 100%, which can specifically achieve the full loading process of the C14α hydroxyl group of steroid compounds.

[0043] The engineered strain and biocatalytic method provided by this invention can efficiently and specifically convert steroidal substrates, such as RSS, into C14α-hydroxylated products under mild conditions. Regioselectivity, i.e., product chiral purity, exceeds 98%, and the bioconversion time is within 24 hours, demonstrating promising industrial applications. This bioenzymatic alternative to chemical methods for the specific 14α-hydroxylation of steroidal compounds is simple, efficient, easy to operate, low-cost, and environmentally friendly, offering promising industrial applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 To utilize wild-type P450 Y Ultra-high performance liquid chromatography (HPLC) results of the engineered strain and biocatalytic method for converting the steroid substrate RSS into 14αOH-RSS.

[0045] Figure 2 To utilize mutant P450 YM1 Ultra-high performance liquid chromatography (HPLC) results of the engineered strain and biocatalytic method for converting the steroid substrate RSS into 14αOH-RSS.

[0046] Figure 3 To utilize mutant P450 YM2 Ultra-high performance liquid chromatography results of the engineered strain and biocatalytic method for converting the steroid substrate RSS into 14αOH-RSS.

[0047] Figure 4 P450 Y / P450 YM1 / P450 YM2 The reaction process and results of efficiently catalyzing the steroid substrate RSS to produce 14α-hydroxylation products. DETAILED DESCRIPTION

[0048] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0049] Example

[0050] 1. Enzyme P450 Y Acquisition of CPR genes

[0051] The P450 of Cochliobolus lunatus CX3 strain was obtained by using the NCBI database and Pfam comparison.Y Enzyme and CPR gene sequences. RNA was extracted from Cochliobolus lunatus CX3 strain and reverse transcribed to obtain cDNA.

[0052] 2. Enzyme P450 Y Construction of co-expression vector with CPR

[0053] The linearized plasmid vector pXW02 (LEU2 marker) was obtained by inverse PCR. Y Perform homologous recombination with the CPR and linearized plasmid vector. Transform the homologous recombination product into competent E. coli DH5α cells, and select transformants on LB plates containing ampicillin. Identify positive clones by sequencing.

[0054] 3. Contains P450 Y Engineering strain construction

[0055] The recombinant plasmid was extracted and transformed into competent cells of Saccharomyces cerevisiae BY4741. Yeast transformants were screened using leucine-deficient plates and positive clones were selected by sequencing.

[0056] 4. Enzyme P450 Y Site-directed mutagenesis

[0057] Overlap PCR was used to identify P450 Y The gene was subjected to site-directed mutagenesis, with Leu209 and Thr302 mutated to Arg and Trp, respectively. The PCR product was treated with the restriction endonuclease Dpn I to remove the template and then transformed into competent Saccharomyces cerevisiae BY4741 cells. Positive clones were screened using leucine-deficient plates and sequenced. This was named P450. YM1 P450 Y The L209R mutant of P450 YM2 P450 Y The T302Y mutant.

[0058] 5. Contains P450 Y / P450 YM1 / P450 YM2 Cultivation of engineered strains and RSS transformation

[0059] The expression strain P450 constructed above Y / P450 YM1 / P450 YM2-CPR-pXW02-BY4741 was cultured in YPD medium (1% yeast extract, 2% peptone, 2% glucose) at 30°C with shaking at 250 rpm. After two days of culture, cells were harvested by centrifugation at 6,000 rpm and resuspended in potassium phosphate buffer (50 mM, pH 7.2). The steroid substrate RSS dissolved in N,N-dimethylformamide was added to a final concentration of 150 mg / L. Transformation was incubated at 30°C with shaking at 250 rpm for 24 hours.

[0060] 6. Ultra-high performance liquid chromatography (UPLC) detection of products

[0061] The conversion system solution was extracted twice with twice the volume of ethyl acetate and centrifuged at 15,000 rpm for 15 minutes. The ethyl acetate layer was concentrated on a rotary concentrator. Methanol was added for reconstitution and the solution was centrifuged at 15,000 rpm for 15 minutes. The supernatant was filtered through a 0.22 μm filter and analyzed by ultra-high performance liquid chromatography (UPLC). UPLC detection conditions were: Agilent 1290 Infinity II LC system, C18 reversed-phase column (InfinityLab Poroshell 120); mobile phase A was 0.1% formic acid in water, mobile phase B was acetonitrile, gradient elution; column temperature was 25°C, elution rate was 0.6 ml / min, and DAD UV detection wavelength was 242 nm.

[0062] After testing, it was found to contain wild-type P450 Y The biotransformation system can catalyze the steroid substrate RSS to produce a variety of hydroxylation products, including 14α hydroxylation, 11β hydroxylation, 7α hydroxylation, etc. Among them, the 14α hydroxylation product accounts for 31%, and the chromatographic results are shown in the figure. Figure 1 According to the peak area calculation, the production rate of 14α-hydroxylation product of RSS is 23.7 mg -1 ·L -1 ·d -1 .

[0063] Contains P450 YM1 The biotransformation system can convert almost all of the steroidal substrate RSS into 14α-hydroxylated products, accounting for nearly 98%. The chromatographic results are shown in the figure. Figure 2 According to the peak area calculation, the production rate of C14α hydroxylation product of RSS is 66.8 mg -1 ·L -1 ·d -1 .

[0064] Contains P450 YM2 The biotransformation system can convert almost all of the steroidal substrate RSS into 14α-hydroxylated products, accounting for nearly 98%. The chromatographic results are shown in the figure. Figure 3 According to the peak area calculation, the production rate of C14α hydroxylation product of RSS is 107.8 mg-1 ·L -1 ·d -1 .

[0065] P450 Y / P450 YM1 / P450 YM2 The reaction process and reaction results of the steroid substrate RSS to produce 14α-hydroxylated products are as follows: Figure 4 shown.

[0066] The enzyme P450 involved in the scheme of the present invention Y The amino acid sequence is as follows:

[0067] MDPQTVELVLRALQTTAIAAVLFAAWTYIPKLQYKVHVRKLPSLTSEGSTKARDSFMASAKKLYQDGYEKFKDSAYTLINENGKENVVVPTRFLPELRKLSDSILSFPEAITEDMEVRYTKLIVEH PTSVDTIRTKLTPALPRLNPAICRDVDNAVKSYLPPCDDWTEVNINERLVRIVAKVSGTIFVGPELSGDPDYLDAGCFYTVDLMNAVTAMKKLSPWLKPFLASRTPEIIALRAREKQAERVLRPIVE ERITAKANDPNWQEPDDVLQWMINKSDGKESVASLAKAQLGLIFAAIHTTSTTVTNIMYTLAVMPEYLQPLREEIPNGMAHKGGVITFRALQKMEKLDSYMKEVLRFYGPVMTSFTRRRTLKGITLS NGQYIPAGVLIEVPAAAAVYKDDAFYPSSDTFDGFRAFKARSTGKATDIARNQFVTSNEENLTFGYGRHACPGRFFAANEIKMIISRLILDYDIKMPNGETERYPQIEIGKVSMPNPFKTLAFKRVVV

[0068] P450 enzymes YM1 The amino acid sequence is as follows:

[0069] MDPQTVELVLRALQTTAIAAVLFAAWTYIPKLQYKVHVRKLPSLTSEGSTKARDSFMASAKKLYQDGYEKFKDSAYTLINENGKENVVVPTRFLPELRKLSDSILSFPEAITEDMEVRYTKLIVEHPTSVDTIRTKLTPALPRLNPAICRDVDNAVKSYLPPCDDWTEVNINERLVRIVAKVSGTIFVGPELSGDPDYLDAGCFYTVDRMNAVTAMKKLSPWLKPFLASRTPEIALRAREKQAERVLRPIVE ERITAKANDPNWQEPDDVLQWMINKSDGKESVASLAKAQLGLIFAAIHTTSTTVTNIMYTLAVMPEYLQPLREEIPNGMAHKGGVITFRALQKMEKLDSYMKEVLRFYGPVMTSFTRRTLKGITLSNGQYIPAGVLIEVPAAAVYKDDAFYPSSDTFDGFRAFKARSTGKATDIARNQFVTSNEENLTFGYGRHACPGRFFAANEIKMIISRLILDYDIKMPNGETERYPQIEIGKVSMPNPFKTLAFKRVVV

[0070] P450 YM2 The amino acid sequence is as follows:

[0071] MDPQTVELVLRALQTTAIAAVLFAAWTYIPKLQYKVHVRKLPSLTSEGSTKARDSFMASAKKLYQDGYEKFKDSAYTLINENGKENVVVPTRFLPELRKLSDSILSFPEAITEDMEVRYTKLIVEHPTSVDTIRTKLTPALPRLNPAICRDVDNAVKSYLPPCDDWTEVNINERLVRIVAKVSGTIFVGPELSGDPDYLDAGCFYTVDLMNAVTAMKKLSPWLKPFLASRTPEIIALRAREKQAERVLRPIVEERITAKANDPNWQEPDDVLQWMINKSDGKESVASLAKAQLGLIFAAIHYTSTTVTNIMYTLAVMPEYLQPLREEIPNGMAHKGGVITFRALQKMEKLDSYMKEVLRFYGPVMTSFTRRTLKGITLSNGQYIPAGVLIEVPAAAVYKDDAFYPSSDTFDGFRAFKARSTGKATDIARNQFVTSNEENLTFGYGRHACPGRFFAANEIKMIISRLILDYDIKMPNGETERYPQIEIGKVSMPNPFKTLAFKRVVV

[0072] The amino acid sequence of P450 reductase CPR is specifically as follows:

[0073] MAQLDTLDIIVLAVLLAGTVAYFTKGTYWAVSADPYGSSLATANGAAKAGKSRNIIEKMDETDKNCVVFYGSQTGTAEDYASRISKEGHSRFGLKTMVADLEEYDYDNLDTFPEDKLAVFVLATYGEGEPTDNAVEFYEFLGSEDIAFSQGGGIEDKPLSNLHYVAFGLGNNTY EHYNSMVRNVDKYLTRLGAKRLGTAGEGDDGAGTMEEDFLAWKEPMWAAVAEKMGLEEREAMYEPVFEVTEKPDLSPEDDTVYLGEPNKNHLEGTQKGPFNANNPFIAPIVESAELFKDSDRNCLHMEISIAGSNLSYTTGDHIAIWPTNAGKEVDRLFKVLGKEDKRHTVISV RGLDPTAKVPFPSPTTYDAAIRYHIEINAAVSRQLVSVIAQFAPNDDIKAEIVKLGGDKDYFKEQVTDRNLNLAQLLEITGKGATWDKIPFSFLFETMVKIQPRYYSISSSSLVQKDKISITAVVESIEKPGAPYALKGVTTNYLLALKQKQHGDPNPDPHGLNYSITGPRNKY DGIHVPVHVRHSNFKLPSDPSKPIIMVGPGTGVAPFRGFVQERAAQAKAGQNVGKTILFFGCRRQSEDFMYADEWKQYQQDLGDKFEMHTAFSRDGPQKVYVQHKLEENGEEVNRLLEQKAYFYVCGDAAHMAREVNTLLGKIIAKYRNVSETKGEEIVKAMRASNQYQEDVWS

[0074] The above description of the embodiments is intended to facilitate understanding and use of the invention by those skilled in the art. It will be apparent that those skilled in the art can readily make various modifications to these embodiments and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, the present invention is not limited to the above-described embodiments. Improvements and modifications made by those skilled in the art based on the disclosure of the present invention, without departing from the scope of the present invention, should be within the scope of protection of the present invention.

Claims

1. Steroid compound 14α-hydroxylase, characterized in that, Selected from enzyme P450 YM1 P450 YM2 ; wherein, Enzyme P450 YM1 The amino acid sequence is shown in SEQ ID No. 2; Enzyme P450 YM2 The amino acid sequence of which is shown in SEQ ID No.

3.

2. Preparation method of an engineered strain capable of synthesizing steroid 14α-hydroxylated product, characterized in that, comprising the following steps: Step 1: Obtain enzyme P450 by PCR amplification YM1 or the gene of enzyme P450 YM2 to obtain the P450 YM1 gene or P450 YM2 gene; Amplify and obtain the P450 reductase gene, i.e., CPR gene, from the cDNA library of fungus Cochliobolus lunatus CX3. The amino acid sequence thereof is as shown in SEQ ID No.4; Step 2: Clone one of the P450 YM1 genes or P450 YM2 genes and the CPR gene fragment into an expression vector simultaneously, and screen for positive plasmids; Step 3: Transfer the positive plasmid obtained in Step 2 into a host bacterium to construct an engineered strain capable of synthesizing steroid 14α-hydroxylated product; Among them, the amino acid sequence of enzyme P450 YM1 is shown in SEQ ID No. 2; Enzyme P450 YM2 The amino acid sequence thereof is as shown in SEQ ID No.

3.

3. The preparation method of an engineered strain capable of synthesizing steroid 14α-hydroxylated product according to claim 2, characterized in that, the host bacterium in Step 3 is Saccharomyces cerevisiae BY4741.

4. Engineered strain capable of synthesizing steroid 14α-hydroxylated product, characterized in that, prepared by the method described in any one of claims 2-3.

5. Preparation method of a culture of an engineered strain capable of synthesizing steroid 14α-hydroxylated product, characterized in that, comprising the following steps: Step A: Culture the engineered strain capable of synthesizing steroid 14α-hydroxylated product described in claim 4 in YPD medium; Step B: Centrifuge to collect the bacterial cells and resuspend them in potassium phosphate buffer to obtain a culture of the engineered strain capable of synthesizing steroid 14α-hydroxylated product.

6. Application of the steroid compound 14α-hydroxylase described in claim 1 or the engineered strain capable of synthesizing steroid 14α-hydroxylated product described in claim 4 or the culture of the engineered strain capable of synthesizing steroid 14α-hydroxylated product obtained by the method of claim 5 in synthesizing steroid 14α-hydroxylated product using steroid compounds, characterized in that, the steroid compound is steroid 11-deoxypiosterol RSS, and its structure is as follows: ； The structural formula of the steroid 14α-hydroxylated product is as follows: 。 7. Method for preparing steroid 14α-hydroxylated product, characterized in that, comprising the following steps: Step (1): Dissolve the steroid compound used as a reaction substrate in N,N-dimethylformamide; Step (2): Add the reaction substrate and inducer in Step (1) to the steroid compound 14α-hydroxylase described in claim 1 or the culture of the engineered strain capable of synthesizing steroid 14α-hydroxylated product obtained by the method of claim 5; Step (3): Biotransform the mixture in Step (2) on a shaker to obtain the steroid 14α-hydroxylated product; the steroid compound is steroid 11-deoxypiosterol RSS, and its structure is as follows: ； The structural formula of the steroid 14α-hydroxylated product is as follows: 。 8. The method for preparing steroid 14α-hydroxylated product according to claim 7, characterized in that, in Step (2), the inducer is ethanol.

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