Curvularia lunata and application thereof in catalyzing conversion of 4-HBC to produce 9alpha, 14alpha-dihydroxy-HBC

Abstract

The invention relates to the technical field of microorganisms, in particular to curvularia lunata and application of the curvularia lunata to production of 9 alpha, 14 alpha-dihydroxy-HBC by catalyzing conversion of 4-HBC. The invention provides Curvularia lunata SYP-GBC, which is preserved in the General Microbiological Culture Collection Center of the China Committee for Culture Collection of Microorganisms, and the preservation number is CGMCC No.41964. The invention further provides a preparation method of the SYP-GBC. The strain can efficiently and specifically biologically convert 4-HBC into 9 alpha, 14 alpha-dihydroxy-HBC, has high selectivity, and can realize higher yield and yield. Based on the strain, the invention provides a method for producing 9alpha, 14alpha-dihydroxy-HBC through bioconversion, the method has the advantages of high substrate conversion rate, simplicity and convenience in operation, low cost and the like, and an efficient way is provided for green synthesis of steroid drug intermediates.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and more particularly to a Crested Spore strain and its application in the catalytic conversion of 4-HBC to 9α,14α-dihydroxy-HBC. Background Technology

[0002] 9α,14α,22-trihydroxy-23,24-bischolesterol-4-en-3-one (abbreviated as 9α,14α-dihydroxy-HBC) is an important intermediate in the synthesis of steroid hormone drugs. Its chemical synthesis faces challenges such as poor regioselectivity, numerous byproducts, and severe environmental pollution. Microbial transformation, with its advantages of high efficiency, specificity, and mild conditions, has become a key technology for the structural modification of steroid compounds. In existing technologies, the hydroxylation reaction of steroids by *Curvularia lunata* mainly focuses on sites such as C11β and C14α, but research on the 9α,14α dihydroxylation of 4-HBC is limited, and the low conversion rate restricts its industrial application. Therefore, there is an urgent need to develop strains capable of efficiently and specifically transforming 4-HBC to synthesize 9α,14α-dihydroxy-HBC. Summary of the Invention

[0003] This invention provides a Crested Cyclosporium strain and its application in the catalytic conversion of 4-HBC to 9α,14α-dihydroxy-HBC.

[0004] Specifically, the present invention provides the following technical solutions.

[0005] In a first aspect, the present invention provides *Crescentia spp.* Curvularia lunata The strain SYP-GBC was deposited on April 28, 2025, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China) and classified as *Crescentis crescentis*. Curvularia lunata The accession number is CGMCC No.41964.

[0006] This invention obtains a strain of Crescentella spp. through screening. Curvularia lunata This strain can efficiently and specifically catalyze the dihydroxylation of 21-hydroxy-20-methylpregn-4-en-3-one (4-HBC) at the 9α and 14α positions to synthesize 9α,14α-dihydroxy-HBC. It has high yield and efficiency of 9α,14α-dihydroxy-HBC and can be fermented using inexpensive culture medium raw materials, which helps to reduce production costs.

[0007] Secondly, the present invention provides a microbial preparation comprising the above-described Curvularia lunata (… Curvularia lunataSYP-GBC.

[0008] Preferably, in the microbial preparation, Curvularia crescentis (… Curvularia lunata SYP-GBC exists in the form of live bacteria.

[0009] The microbial preparation may be a solid or liquid formulation. Except for containing *Curvularia crescentis* (… Curvularia moonlit In addition to SYP-GBC, the microbial preparation may also contain excipients permitted in the field of microbial preparations, including but not limited to carriers and cryoprotectants (e.g., sugars, polyols, etc.). For example, the liquid preparation can be prepared by introducing Curvularia crescentis (… Curvularia lunata The solid formulation was prepared by mixing the seed culture of SYP-GBC with 50% glycerol at a 1:1 volume ratio. Curvularia lunata SYP-GBC was prepared by freeze-drying a mixture of SYP-GBC and a bran carrier (e.g., at a mass ratio of 1:5).

[0010] Thirdly, the present invention provides a method for preparing the above-described microbial preparation, the method comprising processing the *Curvularia lunata* (…). Curvularia lunata The steps for culturing SYP-GBC are as follows: the culture temperature is preferably 25-30℃.

[0011] Fourthly, the present invention provides the aforementioned Crested Crested Spore ( Curvularia lunata The use of SYP-GBC or the aforementioned microbial preparation in the preparation of 9α,14α-dihydroxy-HBC.

[0012] Preferably, the preparation of 9α,14α-dihydroxy-HBC uses 21-hydroxy-20-methylpregn-4-en-3-one as a substrate.

[0013] Preferably, the application includes: using 21-hydroxy-20-methylpregn-4-en-3-one as a substrate, utilizing the crescent-shaped spore ( Curvularia lunata SYP-GBC or the aforementioned microbial preparation can be used to convert 21-hydroxy-20-methylpregn-4-en-3-one into 9α,14α-dihydroxy-HBC.

[0014] Fifthly, the present invention provides the aforementioned Crested Sporula ( Curvularia lunata The use of SYP-GBC or the aforementioned microbial preparation in the preparation of fermentation agents for the synthesis of 9α,14α-dihydroxy-HBC or in the construction of production strains of 9α,14α-dihydroxy-HBC.

[0015] Preferably, the fermentation agent contains the crescent-shaped spores ( Curvularia lunata SYP-GBC.

[0016] Preferably, the production strain for constructing 9α,14α-dihydroxy-HBC is the *Crescentis* species (…). Curvularia moonlit SYP-GBC is the starting strain. A high-yield strain of 9α,14α-dihydroxy-HBC is constructed by gene editing (e.g., CRISPR-Cas9 gene editing) of the starting strain.

[0017] As an example, with the aforementioned Crested Crested Spore ( Curvularia lunata SYP-GBC is the starting strain, overexpressing the P450 enzyme or its variant gene, and / or overexpressing the hydroxylase or its variant gene to further improve transformation efficiency.

[0018] Sixthly, the present invention provides the aforementioned Crested Crested Spore ( Curvularia lunata The application of SYP-GBC or the aforementioned microbial preparation in catalyzing the 9α and 14α-hydroxylation of steroidal compounds.

[0019] Preferably, the steroidal compound is 21-hydroxy-20-methylpregn-4-en-3-one.

[0020] In a seventh aspect, the present invention provides a method for microbial synthesis of 9α,14α-dihydroxy-HBC, the method comprising: using 21-hydroxy-20-methylpregn-4-en-3-one as a substrate, and utilizing the *Crescentis* ( Curvularia lunata SYP-GBC or the aforementioned microbial preparation bioconverts 21-hydroxy-20-methylpregn-4-en-3-one into 9α,14α-dihydroxy-HBC.

[0021] Preferably, the method includes: placing the crescent-shaped spores ( Curvularia lunata SYP-GBC or the microbial preparation described herein may be inoculated into a fermentation medium containing 21-hydroxy-20-methylpregn-4-en-3-one for biotransformation.

[0022] Preferably, the fermentation medium used for the biotransformation includes 21-hydroxy-20-methylpregn-4-en-3-one, as well as a carbon source, a nitrogen source, and inorganic salts.

[0023] The carbon source is preferably galactose. The nitrogen source is preferably ammonium sulfate.

[0024] This invention targets Crescentella lunata ( Curvularia lunata SYP-GBC underwent fermentation medium optimization and found that using galactose as a carbon source and ammonium sulfate as a nitrogen source was more conducive to improving conversion efficiency, thereby increasing the yield of 9α,14α-dihydroxy-HBC.

[0025] Preferably, the 21-hydroxy-20-methylpregn-4-en-3-one is added to the biotransformation system after being solubilized with methanol. The final concentration of 21-hydroxy-20-methylpregn-4-en-3-one in the transformation system is preferably 1-50 g / L, more preferably 1-30 g / L.

[0026] Preferably, the fermentation medium comprises the following components: galactose 25-35 g / L, ammonium sulfate 15-25 g / L, KH₂PO₄ 1-2 g / L, K₂HPO₄ 1-3 g / L, MgSO₄·7H₂O 0.2-0.8 g / L, and 21-hydroxy-20-methylpregn-4-en-3-one 1-50 g / L. The pH of the fermentation medium is preferably 5-6.

[0027] Preferably, the temperature for the biotransformation is 25-30°C. The pH for the biotransformation is 5-6. The rotation speed for the biotransformation is preferably 200-230 rpm. The biotransformation time is 80-150 h.

[0028] Preferably, the amount of biotransformation is 5%-15%. More preferably, it is 8%-12%.

[0029] The seed culture medium used for preparing the seed liquid for transplantation comprises the following components: potato starch 40-50 g / L, yeast extract 1-5 g / L, corn steep liquor 5-15 g / L, CaCO3 1-5 g / L, MgSO4·7H2O 0.2-0.8 g / L, and FeSO4·7H2O 0.03-0.07 g / L. The preferred pH is 6.0-6.8.

[0030] Preferably, the temperature for seed culture is 25-30℃.

[0031] The above method further includes, after the biotransformation is completed, the step of purifying 9α,14α-dihydroxy-HBC by ethyl acetate extraction and semi-preparative HPLC separation of the obtained transformation solution.

[0032] In some embodiments of the present invention, the method for microbial synthesis of 9α,14α-dihydroxy-HBC includes the following steps: (1) Seed culture: The activated Crested Spores ( Curvularia lunata SYP-GBC was inoculated into seed culture medium and cultured at 28±1℃ and 200±20 rpm for 48±2 h to obtain seed liquid; (2) Fermentation transformation: The seed liquid was inoculated into the fermentation medium at a transformation rate of 8%-12% (v / v), and 4-HBC was added to a final concentration of 1-3 g / L. The mixture was transformed at 28±1℃ and 220±20 rpm for 120±2 h, and the transformation liquid was collected. (3) Product purification: The conversion solution was extracted with ethyl acetate and purified by semi-preparative HPLC to obtain pure 9α,14α-dihydroxy-HBC.

[0033] In step (2) above, the product generation was monitored by HPLC during the conversion process. The detection conditions were: C18 column (250 mm × 4.6 mm, 5 μm), mobile phase acetonitrile-water (60:40, v / v), flow rate 0.8 mL / min, and detection wavelength 254 nm.

[0034] In step (3) above, the separation and purification conditions for semi-preparative liquid chromatography were as follows: SinoChrom ODS-BP column (250 mm × 10 mm, 5 μm), mobile phase acetonitrile-water (26:74, v / v), flow rate 3.5 mL / min, and collection of the elution peak with a retention time of 34.1 min. After purification using the above method, the purity of 9α,14α-dihydroxy-HBC was ≥95%.

[0035] Eighthly, the present invention provides the use of 9α,14α-dihydroxy-HBC in the preparation of anti-inflammatory drugs.

[0036] The beneficial effects of this invention include at least the following: This invention provides Crescentella spp. ( Curvularia lunata The strain SYP-GBC efficiently and specifically converts 21-hydroxy-20-methylpregn-4-en-3-one (4-HBC) to 9α,14α-dihydroxy-HBC with high selectivity, specifically catalyzing the dihydroxylation of 4-HBC at the 9α,14α position with few byproducts, achieving high yield and production. Based on this strain, this invention provides a method for the biotransformation production of 9α,14α-dihydroxy-HBC, using 4-HBC as a substrate and utilizing *Curvularia crescentis* (…). Curvularia lunata The SYP-GBC method for converting 4-HBC to 9α,14α-dihydroxy-HBC achieves a conversion yield of 42.56%, and utilizes inexpensive culture medium raw materials such as galactose and ammonium sulfate, reducing culture medium costs. It boasts advantages such as high substrate conversion rate, simple operation, and low cost, making it easy to scale up production. It can be used for the industrial production of 9α,14α-dihydroxy-HBC, providing an efficient route for the green synthesis of steroidal drug intermediates and laying the foundation for the large-scale production of 9α,14α-dihydroxy-HBC. Attached Figure Description

[0037] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0038] Figure 1 The image shows the colony morphology of Curvularia crescentis SYP-GBC in Example 1 of this invention (PDA medium, cultured at 28°C for 7 days). The left image shows the front of the colony, and the right image shows the back of the colony.

[0039] Figure 2 This is the HPLC chromatogram (retention time 34.1 min) of the biotransformation of *Curvularia crescentis* SYP-GBC into 9α,14α-dihydroxy-HBC in Example 3 of this invention. In this chromatogram, A represents the HPLC detection result of the transformation solution of *Curvularia crescentis* SYP-GBC into 4-HBC (A: *Curvularia crescentis* SYP-GBC strain untransformed control group; B: ...). Cosolvent control group; C: Crescentella syringae SYP-GBC conversion solution), the upper figure of B is the semi-preparative HPLC result of product 1, and the lower figure is the semi-preparative HPLC result of product 2. Both product 1 and product 2 are 9α,14α-dihydroxy HBC.

[0040] Figure 3 This is the ESI-MS spectrum of the conversion product in Example 3 of the present invention.

[0041] Figure 4 The conversion product in Example 3 of this invention 1 H-NMR spectrum (CDCl3, 400 MHz).

[0042] Figure 5 The conversion product in Example 3 of this invention 13 C-NMR spectrum (100 MHz, CDCl3).

[0043] Figure 6 This is the HMBC spectrum of the conversion product in Example 3 of the present invention.

[0044] Figure 7 This refers to the key HMBC correlation of the conversion product in Example 3 of the present invention. Detailed Implementation

[0045] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0046] In the following examples, the formula for calculating the conversion yield of 9α,14α-dihydroxy-HBC is as follows: .

[0047] Example 1: Obtaining and culturing Curvularia crescentis SYP-GBC This invention obtained a strain of Crescentella spp. through screening. Curvularia lunata This strain was verified to be capable of dihydroxylation at the 9α,14α position of 4-HBC, thereby synthesizing 9α,14α-dihydroxy-HBC. The strain was named SYP-GBC. The colony morphology of *Crescentis crescentis* SYP-GBC cultured on PDA medium (potato starch 200 g / L, glucose 20 g / L, agar 20 g / L) at 28°C for 7 days is as follows. Figure 1 As shown.

[0048] Crescentoides ( Curvularia lunata SYP-GBC was deposited on April 28, 2025, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China), and classified as *Crescentis crescentis*. Curvularia lunata The accession number is CGMCC No.41964.

[0049] The activation and seed culture methods for Curvularia crescentis SYP-GBC are as follows: Activation: Streak SYP-GBC of Curvularia crescentis onto PDA medium (potato 200 g / L, glucose 20 g / L, agar 20 g / L), and incubate at 28°C for 6 days until the colony diameter is 5-6 cm and the surface is covered with black spores.

[0050] Seed culture: Spores were scraped and inoculated into 50 mL seed culture medium (250 mL Erlenmeyer flasks) and cultured at 28℃ and 200 rpm for 48 h until the dry weight of the cells reached 8.5 g / L. The seed culture medium consisted of the following: potato starch 45 g / L, yeast extract 3 g / L, corn steep liquor 10 g / L, CaCO3 3 g / L, MgSO4·7H2O 0.5 g / L, FeSO4·7H2O 0.05 g / L, pH 6.5.

[0051] Example 2: Optimization of Fermentation Conversion Process This embodiment optimizes the fermentation conversion process for the synthesis of 9α,14α-dihydroxy-HBC from Crescentella syringae SYP-GBC using 4-HBC as a substrate, including optimization of the culture medium and conversion conditions, as detailed below.

[0052] Carbon source screening: Compared with other carbon sources such as glucose, sucrose, and galactose, galactose (30 g / L) was found to be the optimal carbon source, with a yield of 27.6% for 9α,14α-dihydroxy-HBC.

[0053] Nitrogen source screening: Comparison of different nitrogen sources revealed that ammonium sulfate (20 g / L) had a better promoting effect on the synthesis of 9α,14α-dihydroxy-HBC than yeast extract and sodium nitrate, increasing the yield of 9α,14α-dihydroxy-HBC to 32.4%.

[0054] Orthogonal experiment optimization: using L9(3) 4 The optimal conditions were determined by orthogonal experiments: galactose 30 g / L, ammonium sulfate 20 g / L, pH 5.5, and transfection amount 10%. Under these conditions, the yield of 9α,14α-dihydroxy-HBC reached 42.56%.

[0055] After the above optimization, the final fermentation medium formula is as follows: galactose 30 g / L (carbon source), ammonium sulfate 20 g / L (nitrogen source), KH2PO4 1.6 g / L, K2HPO4 2.0 g / L, MgSO4·7H2O 0.5 g / L, initial pH 5.5; the substrate 4-HBC is added to the fermentation medium to a final concentration of 2.0 g / L, wherein 4-HBC is dissolved in methanol at a concentration of 6%.

[0056] After optimization, the final conversion process parameters are as follows: conversion amount: 10% (v / v); culture conditions: 28℃, 220rpm, conversion time 120 h.

[0057] This embodiment provides a method for preparing 9α,14α-dihydroxy-HBC using Curvularia crescentis SYP-GBC as a substrate with 4-HBC as the substrate. The steps are as follows: (1) Seed culture: Spores of activated Crested spores SYP-GBC were scraped and inoculated into 50 mL seed culture medium (250 mL Erlenmeyer flask), and cultured at 28℃ and 200 rpm for 48 h to obtain seed liquid with a cell dry weight of 8.5 g / L; The composition of the seed culture medium is as follows: potato starch 45 g / L, yeast extract 3 g / L, corn steep liquor 10 g / L, CaCO3 3 g / L, MgSO4·7H2O 0.5 g / L, FeSO4·7H2O 0.05 g / L, pH 6.5.

[0058] (2) Fermentation transformation: The seed culture was inoculated into the fermentation medium (50 mL / 250 mL Erlenmeyer flask) at a transformation rate of 10% (v / v), and 4-HBC (6% dissolved in methanol) was added to a final concentration of 2.0 g / L. The transformation was carried out at 28℃ and 220 rpm for 120 h, and the transformation liquid was collected. The fermentation medium formula was as follows: galactose 30 g / L (carbon source), ammonium sulfate 20 g / L (nitrogen source), KH2PO4 1.6 g / L, K2HPO4 2.0 g / L, MgSO4·7H2O 0.5 g / L, initial pH 5.5. During the transformation process, the product formation was monitored by HPLC. The detection conditions were: C18 column (250 mm × 4.6 mm, 5 μm), mobile phase acetonitrile-water (60:40, v / v), flow rate 0.8 mL / min, and detection wavelength 254 nm.

[0059] (3) Product purification: The conversion solution was extracted with ethyl acetate and purified by semi-preparative HPLC to obtain pure 9α,14α-dihydroxy-HBC; the purification conditions of semi-preparative HPLC were as follows: SinoChrom ODS-BP column (250 mm × 10 mm, 5 μm), mobile phase was acetonitrile-water (26:74, v / v), flow rate was 3.5 mL / min, and the elution peak with a retention time of 34.1 min was collected.

[0060] Using the above method, the yield of 9α,14α-dihydroxy-HBC in the fermentation broth of Curvularia syP-GBC was significantly increased, with a conversion yield of 42.56%, which is 35.28% higher than the yield of the same strain in the unoptimized fermentation medium (7.28%), and the product purity is ≥95%. The unoptimized fermentation medium formulation was as follows: sucrose: 30.00 g / L, yeast extract: 10.00 g / L, corn steep liquor: 10.00 g / L, potassium dihydrogen phosphate (K2HPO4): 2.00 g / L, potassium monohydrogen phosphate (KH2PO4): 1.60 g / L, magnesium sulfate (MgSO4·7H2O): 0.50 g / L, ferrous sulfate (FeSO4·7H2O): 0.05 g / L, pH adjustment: 6.5 (corrected using 1 mol / L HCl or NaOH solution).

[0061] Example 3: Identification of biotransformation products of Curvularia crescentis SYP-GBC The biotransformation products prepared using the method of Example 2 were identified as follows.

[0062] 1. HPLC analysis: Results are as follows Figure 2 As shown, the retention time of the target product was 34.1 min. The HPLC analysis included high-performance liquid chromatography (HPLC) detection of the conversion solution and semi-preparative HPLC purification detection of the target product, with the parameters shown in Tables 1 and 2, respectively.

[0063] Table 1 High Performance Liquid Chromatography Conditions

[0064] Table 2 Semi-preparative high performance liquid chromatography conditions

[0065] 2. Structural Confirmation: The structure of the transformation product was analyzed using mass spectrometry (MS) and nuclear magnetic resonance (NMR). The mass spectrometry results showed that ( Figure 3 ), ESI-MS m / z [M+Na + =385.2 (molecular weight 362.28); 1 H-NMR and 13 C-NMR data (Table 3, Figure 4 and Figure 5 The HMBC spectra of the transformed product matched those of the target product 9α,14α-dihydroxy-HBC (δH 0.89 (s, H-18), 1.31 (s, H-19), δC 77.3 (C-9), 87.1 (C-14)). The HMBC spectra and key HMBC correlations of the transformed product are shown below. Figure 6 and Figure 7 As shown. The transformation product was identified as 9α,14α-dihydroxy-HBC.

[0066] Table 3. Biotransformation products 1 H (400 MHz) and 13 C NMR (100 MHz) data (CDCl3)

[0067] Example 4: Scale-up fermentation verification in a fermenter The method for preparing 9α,14α-dihydroxy-HBC using 4-HBC as a substrate with Crested spore SYP-GBC as provided in Example 2 (the optimized and finalized method) was used for fermentation and transformation in a 5 L fermenter. The final concentration of the substrate 4-HBC was adjusted to 20 g / L, the stirring speed was 300 rpm, and the aeration rate was 1.0 vvm.

[0068] The yield of 9α,14α-dihydroxy-HBC was 8.5 g / L, with a yield of 42.1%.

[0069] In summary, the Crested Spore SYP-GBC provided by this invention can convert 4-HBC to 9α,14α-dihydroxy-HBC. By optimizing the culture medium and transformation conditions, Crested Spore SYP-GBC can efficiently catalyze the conversion of 4-HBC to 9α,14α-dihydroxy-HBC with high yield and production rate. Moreover, the process is stable and reliable, suitable for industrial production, and provides a new route for the green synthesis of steroidal drug intermediates.

[0070] Example 5: Pharmacological effects of 9α,14α-dihydroxy-HBC The in vitro anti-inflammatory activity of 9α,14α-dihydroxy-4-HBC prepared by the methods in Examples 2 and 4 was evaluated using a conventional LPS-induced RAW264.7 cell model. A blank control group, an LPS model group (LPS treatment), a positive control group (LPS + 50 µM L-arginine treatment), and an experimental group (LPS + 50 µM 9α,14α-dihydroxy-4-HBC) were set up. Cell viability and NO production were measured in each group. The results showed that, compared with the blank control group, the cell viability was 95.72% at a concentration of 50 µM 9α,14α-dihydroxy-4-HBC. The results of NO production detection showed that, compared with the LPS model group, the NO production inhibition rate of the positive control group was 58.21%, while the NO production inhibition rate of the experimental group reached 51.22%. This indicates that 9α,14α-dihydroxy-4-HBC has a strong inhibitory effect on NO production in the LPS-induced RAW264.7 cell inflammation model and can be used as a candidate compound for treating inflammation.

[0071] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Crescentella ( Curvularia lunata SYP-GBC, characterized in that, It is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 41964.

2. A microbial preparation, characterized in that, The microbial preparation comprises Curvularia lunata as described in claim 1 (… Curvularia lunata SYP-GBC.

3. The Crested Crested Spore (as described in claim 1) Curvularia lunata The use of SYP-GBC or the microbial preparation of claim 2 in the preparation of 9α,14α-dihydroxy-HBC.

4. The application according to claim 3, characterized in that, The preparation of 9α,14α-dihydroxy-HBC uses 21-hydroxy-20-methylpregn-4-en-3-one as a substrate.

5. The Crested Spore as described in claim 1 ( Curvularia lunata The use of SYP-GBC or the microbial preparation of claim 2 in the preparation of fermentation agents for the synthesis of 9α,14α-dihydroxy-HBC or in the construction of production strains of 9α,14α-dihydroxy-HBC.

6. The Crested Spore as described in claim 1 ( Curvularia lunata The application of the microbial preparation described in SYP-GBC or claim 2 in catalyzing the 9α- and 14α-hydroxylation reactions of steroidal compounds.

7. A method for microbial synthesis of 9α,14α-dihydroxy-HBC, characterized in that, The method includes: using 21-hydroxy-20-methylpregn-4-en-3-one as a substrate, and utilizing the Crested Sporula as described in claim 1 (… Curvularia lunata The microbial preparation of claim SYP-GBC or claim 2 bioconverts 21-hydroxy-20-methylpregn-4-en-3-one into 9α,14α-dihydroxy-HBC.

8. The method according to claim 7, characterized in that, The fermentation medium used for the biotransformation includes 21-hydroxy-20-methylpregn-4-en-3-one, as well as a carbon source, a nitrogen source, and inorganic salts. Preferably, the carbon source is galactose, and / or the nitrogen source is ammonium sulfate.

9. The method according to claim 8, characterized in that, The fermentation medium comprises the following components: galactose 25-35 g / L, ammonium sulfate 15-25 g / L, KH2PO4 1-2 g / L, K2HPO4 1-3 g / L, MgSO4·7H2O 0.2-0.8 g / L, and 21-hydroxy-20-methylpregn-4-en-3-one 1-50 g / L.

10. The method according to any one of claims 7 to 9, characterized in that, The biotransformation is performed at a temperature of 25-30°C; and / or at a pH of 5-6; and / or at a conversion rate of 5%-15%.

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