Genetic engineering strain for producing steroid drug intermediate by converting diosgenin as well as construction method and application of genetic engineering strain

Abstract

The invention provides a genetic engineering strain for producing a steroid drug intermediate by converting diosgenin as well as a construction method and application of the genetic engineering strain, and belongs to the technical field of biochemical engineering. The method comprises the following steps: carrying out single gene knockout inactivation or combined knockout inactivation on a key gene kstD and / or kshA for degrading a mother nucleus of a steroid compound in a microorganism, a key gene BV-2539 for catalyzing conversion of the steroid compound from C-21 to C-19, and a key negative transcriptional regulation factor IclR-2535; and carrying out single gene overexpression or combined overexpression on key genes kstD2, kshA3, kshA4 and BV-2539 for catalyzing synthesis of a C-19 steroid drug intermediate in a microbial diosgenin pathway, so as to construct a genetic engineering strain. According to the method, the green manufacturing of synthesizing the steroid drug intermediate by converting the diosgenin through the genetic engineering strain is realized for the first time, the production steps of the steroid drug are simplified, the conversion rate of the steroid drug intermediate and the production efficiency of the steroid drug are greatly improved, and the method has good social value and economic value and is beneficial to industrial production.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological chemical industry, in particular to a genetically engineered strain for converting diosgenin to produce a steroid drug intermediate and a construction method and application thereof. BACKGROUND

[0002] Steroid drugs are the second largest class of drugs next to antibiotics, with a series of therapeutic effects such as anti-inflammatory, anti-viral, anti-cancer, and anti-allergic. At present, the preparation of steroid drugs usually follows the semi-synthetic route of "natural steroid compound-steroid drug intermediate-steroid drug", that is, taking natural steroid compound as raw material, first obtaining steroid drug intermediate through chemical reaction or biological transformation combined method, and then synthesizing steroid drug after modification. Among them, the raw materials of natural steroids are mainly diosgenin (about 55%) from Dioscorea deltoidea (Huangjian), phytosterol (about 15%) from soybean, cholesterol (about 10%) from animal sources, and hecogenin (about 10%) from Gynostemma pentaphyllum, etc. Although the raw materials for synthesizing steroid drugs are still being explored and innovated in the processes of extraction, preparation and post-processing, it can be clearly seen that the steroid drug intermediate is an indispensable key precursor in the synthesis of steroid drugs, and its supply determines the prospects of the steroid drug market to some extent.

[0003] The strict requirements of pharmaceutical industry standards for raw materials make diosgenin, as a monomer, favored by steroid drug factories, which leads to diosgenin being one of the most important starting materials in the steroid pharmaceutical industry, and being called "mother of hormones" and "pharmaceutical gold", which has special significance to the modern pharmaceutical system. Through the traditional "Marker degradation" method, diosgenin is converted into steroid drug intermediate 16-dienolone acetate (16-DPA) by chemical degradation, and more than 400 kinds of steroid drugs can be obtained by drug modification. The process route is mature and has a long history, and has obtained the recognition of the US FDA and foreign pharmaceutical companies. However, the planting of key raw materials Huangjian is greatly affected by market fluctuations, the supply of raw materials is limited, and the production of diosgenin produces a large amount of acid wastewater and 16-DPA production involves high temperature, strong acid and strong base, toxic reagents and heavy metal chromium pollution, etc. Environmental problems, and the yield of synthetic C-21 steroid drug intermediates in the prior art is less than 50%, and there are many problems such as reaction by-products, which restrict the development of diosgenin industry.

[0004] With the development of enzyme engineering, metabolic engineering and synthetic biology, the clean production and raw material supply of diosgenin have been gradually solved; but in addition to clean production, the research on green conversion of diosgenin to steroid drug is another important field of concern. Developing a technology based on green synthesis of steroid drug intermediates from diosgenin has become a key problem to be broken through in the steroid pharmaceutical industry. It is worth noting that the multi-step chemical synthesis of steroid drug intermediates is replaced by bioconversion, which provides a solution to this problem. However, the lack of microbial strains hinders the bioconversion of diosgenin to steroid drug intermediates; and the genetically engineered strain for converting diosgenin to steroid drug intermediates has not been reported. At present, the steroid pharmaceutical industry urgently needs a strain that can convert diosgenin to steroid drug intermediates.

[0005] Therefore, the present application takes microorganisms capable of decomposing and metabolizing diosgenin as the starting strain, and constructs a genetically engineered strain based on a rational metabolic engineering strategy to efficiently convert diosgenin to steroid drug intermediates. SUMMARY

[0006] In view of the above problems of the prior art, the present application provides a genetically engineered strain for converting diosgenin to steroid drug intermediates, and a construction method and application thereof. The genetically engineered strain is obtained by metabolic engineering blocking or strengthening pathway modification of microorganisms capable of decomposing and metabolizing diosgenin, and is applied to the preparation of steroid drug intermediates such as 9-hydroxyandrostenedione, androstadienedione, androstenedione, 9,16-dihydroxyprogesterone, 16-dehydroprogesterone, 9-hydroxy-16-dehydroprogesterone, and 1,16-bishydrodehydroprogesterone. A new bioconversion method is used to replace the multi-step chemical degradation of diosgenin, thereby solving the problems of low yield and environmental unfriendliness in the synthesis of steroid drug intermediates in the prior art.

[0007] To achieve the above-mentioned object, the specific technical solutions of the present application are as follows:

[0008] In a first aspect, the present application provides a construction method of a genetically engineered strain for converting diosgenin to steroid drug intermediates, comprising the following steps:

[0009] 1) selecting microorganisms capable of decomposing and metabolizing diosgenin;

[0010] 2) single gene knockout inactivation or combined knockout inactivation of key genes for degradation of steroid compound nucleus, key genes for catalyzing conversion of C-21 to C-19 steroid compounds, and key negative transcriptional regulatory factors in the microorganism; wherein the key genes for degradation of steroid compound nucleus include kstD and / or kshA, the key genes for catalyzing conversion of C-21 to C-19 steroid compounds include BV-2539, and the key negative transcriptional regulatory factors include IclR-2535.

[0011] Further, the microorganism capable of degrading dioscin in step 1) comprises a non-pathogenic microorganism in the genus Mycobacterium, Mycolicibacterium, Mycolicibacillus, Mycolicibacter or Mycobacteroides. For example, the microorganism is Mycolicibacterium sp. HK-90 in the genus Mycobacterium, and the accession number of the Mycolicibacterium sp. HK-90 is CCTCC M2024221.

[0012] Further, the kstD in step 2) comprises kstD1, kstD2 and kstD3, and the kshA comprises kshA1, kshA2, kshA3 and kshA4.

[0013] Further, the nucleotide sequences of kstD1, kstD2 and kstD3 are respectively the sequences shown in SEQ ID No: 1-3, or the nucleotide sequences of kstD1, kstD2 and kstD3 are respectively the orthologous gene sequences of the sequences shown in SEQ ID No: 1-3; and the nucleotide sequences of kshA1, kshA2, kshA3 and kshA4 are respectively the sequences shown in SEQ ID No: 4-7, or the nucleotide sequences of kshA1, kshA2, kshA3 and kshA4 are respectively the orthologous gene sequences of the sequences shown in SEQ ID No: 4-7.

[0014] Further, step 2) comprises combined knockout inactivation of kstD1, kstD2 and kstD3;

[0015] or combined knockout inactivation of kshA1, kshA2, kshA3 and kshA4;

[0016] or combined knockout inactivation of kstD1, kstD2, kstD3, kshA1, kshA2, kshA3 and kshA4;

[0017] or combined knockout inactivation of kstD1, kstD2, kstD3 and IclR-2535;

[0018] or combined knockout inactivation of kshA1, kshA2, kshA3, kshA4 and IclR-2535;

[0019] or combined knockout inactivation of kstD1, kstD2, kstD3, kshA1, kshA2, kshA3, kshA4 and IclR-2535;

[0020] or kstD1, kstD2, kstD3, BV-2539 and IclR-2535 are subjected to combined knockout inactivation;

[0021] or kshA1, kshA2, kshA3, kshA4, BV-2539 and IclR-2535 are subjected to combined knockout inactivation;

[0022] or kstD1, kstD2, kstD3, kshA1, kshA2, kshA3, kshA4, BV-2539 and IclR-2535 are subjected to combined knockout inactivation.

[0023] Further, the nucleotide sequence of BV-2539 is the sequence shown in SEQ ID No: 8, or is the orthologous gene sequence of the sequence shown in SEQ ID No: 8; the nucleotide sequence of IclR-2535 is the sequence shown in SEQ ID No: 9, or is the orthologous gene sequence of the sequence shown in SEQ ID No: 9.

[0024] Further, the method further comprises the step of: 3) single gene overexpression or combined overexpression of key genes catalyzing the synthesis of C-19 steroid drug intermediates in the microorganism diosgenin pathway; wherein the key genes catalyzing the synthesis of C-19 steroid drug intermediates include kstD2, kshA3, kshA4, BV-2539.

[0025] Further, step 3) includes single gene overexpression of BV-2539;

[0026] or kshD2 and BV-2539 are subjected to combined overexpression;

[0027] or kshA3, kshA4 and BV-2539 are subjected to combined overexpression.

[0028] The present application obtains a genetically engineered strain by blocking or strengthening the pathway of the microorganism capable of decomposing diosgenin through metabolic engineering. The genetically engineered strain after metabolic engineering modification can be used to produce C-19 steroid drug intermediates 9-hydroxyandrostenedione, androstenedione, androstene-dione and C-21 steroid drug intermediates 9, 16-dihydroxyprogesterone, 16-dehydroprogesterone, 9-hydroxy-16-dehydroprogesterone, 1, 16-bishydroxyprogesterone by converting diosgenin. It can also be used to prepare 9-hydroxyandrostenedione, androstenedione, androstenedione by converting phytosterol.

[0029] Specifically, the construction method of the genetically engineered strain comprises the following steps:

[0030] S1, the gene knockout plasmid is electroporated into mycobacterium HK-90 competent, coated with kanamycin and hygromycin double resistance plate, and the grown clones are selected by PCR amplification screening with corresponding gene specific upstream and downstream primers.

[0031] For example, the gene knockout plasmid includes plasmid pNHEJ-Cas12a expressing Cas12a protein, and plasmids PCR-hyg-crkstd1, PCR-hyg-crkstd2, PCR-hyg-crkstd3, PCR-hyg-crksha1, PCR-hyg-crksha2, PCR-hyg-crksha3, PCR-hyg-crksha4, PCR-hyg-cr2539, PCR-hyg-cr2535 required for expressing matching target gene crRNA.

[0032] The gene knockout plasmid includes: mHust-ΔkstD (knocking out gene kstD), mHust-ΔkshA (knocking out gene kshA), mHust-ΔkstD-ΔkshA (knocking out genes kstD and kshA), mHust-ΔkstD-ΔIclR2535 (knocking out genes kstD and IclR-2535), mHust-ΔkshA-ΔIclR2535 (knocking out genes kshA and IclR-2535), mHust-ΔkstD-ΔkshA-ΔIclR2535 (knocking out genes kstD, kshA and IclR-2535), mHust-ΔkstD-ΔIclR2535-ΔBV2539 (knocking out genes kstD, IclR-2535 and BV-2539), mHust-ΔkshA-ΔIclR2535-ΔBV2539 (knocking out genes kshA, IclR-2535 and BV-2539), mHust-ΔkstD-ΔkshA-ΔIclR2535-ΔBV2539 (referred to as mHust-C21, knocking out genes kstD, kshA, IclR-2535 and BV-2539).

[0033] The construction of the plasmid PCR-hyg-crkstd1, PCR-hyg-crkstd2, PCR-hyg-crkstd3, PCR-hyg-crksha1, PCR-hyg-crksha2, PCR-hyg-crksha3, PCR-hyg-crksha4, PCR-hyg-cr2539 or PCR-hyg-cr2535 required for expressing matching target gene crRNA in the gene knockout plasmid includes the following steps:

[0034] A1: First, construct a plasmid PCR-hyg-crRNA that can synthesize crRNA in the strain. According to the PAM sequence (5'-TTN-3', N represents any one of A, T, C, G bases) inside the target gene, select a 25 bp fragment at the end of the 3' end of the PAM region as the target sequence for synthesis, and anneal the synthesized two single strands on the PCR instrument; ligate the annealed product to the plasmid PCR-hyg, that is, construct the recombinant plasmid by enzyme digestion and enzyme ligation; wherein, in addition to the 25 bp sequence fragment paired with the target sequence, the annealed product of the crRNA also contains the corresponding complementary sticky ends at both ends after enzyme digestion, so that the annealed crRNA can be directly ligated to the enzyme digestion product of PCR-hyg.

[0035] A2: The crRNA fragment with the corresponding sticky end after enzyme digestion and the PCR-hyg enzyme digestion fragment are ligated under the action of T4 DNA ligase, the ligation system is 10 μL, the vector is 50 ng, and the molar ratio of the vector to the fragment is 1:3-1:10, and the enzyme ligation is carried out at 25 ℃ for 30 min; the ligation product is transformed into E. coli DH5α competent cells by heat shock method, and is coated on a hygromycin resistant plate, and is incubated at 37 ℃ overnight; the correct positive clone is screened out by colony PCR and sequencing results, that is, the plasmid PCR-hyg-crRNA required for gene knockout is obtained. After detecting the plasmid concentration, the electroporation method is used to transform the competent cells of the strain, and the kanamycin, hygromycin and dehydrated tetracycline containing resistance plate is coated, and the transformants are selected for PCR screening of in-frame deletion positive mutant strains, and the positive mutant strains are sequenced and verified, that is, the knockout strain is obtained.

[0036] S2, the single gene overexpression plasmid or the combined overexpression plasmid is electroporated into the competent cells of the genetically engineered strain constructed in step S1, and a kanamycin plate is coated to grow single colonies, and the knockout-overexpression genetically engineered strain is obtained by screening.

[0037] For example, the single gene overexpression plasmid PMS-bv2539 or the combined overexpression plasmid PMS-ksha3&ksha4&bv2539 is electroporated into mHust-ΔkstD-ΔIclR2535 competent cells; the single gene overexpression plasmid PMS-bv2539 or the combined overexpression plasmid PMS-kstd2&bv2539 is electroporated into mHust-ΔkshA-ΔIclR2535 competent cells; the single gene overexpression plasmid PMS-bv2539 is electroporated into mHust-ΔkstD-ΔkshA-ΔIclR2535 competent cells; and a kanamycin plate is coated for single colony growth. Verification by PCR amplification, if a positive band of about 1600 bp is amplified, it is the strain mHust-ΔkstD-ΔIclR2535-Obv2539 or mHust-ΔkshA-ΔIclR2535-Obv2539 or mHust-ΔkstD-ΔkshA-ΔIclR2535-Obv2539; if a positive band of about 3300 bp is amplified, it is the strain mHust-ΔkshA-ΔIclR2535-Okstd2&bv2539; and if a positive band of about 3900 bp is amplified, it is the strain mHust-ΔkstD-ΔIclR2535-Oksha3&ksha4&bv2539.

[0038] The construction of the single gene overexpression plasmid PMS-bv2539 includes the following steps:

[0039] The target fragment is obtained by PCR amplification using the Mycobacterium HK-90 genome as a template, and then the purified target gene is connected to the Mycobacterium free expression plasmid PMS; the seamless cloning technology is selected for the construction of the recombinant plasmid, which does not need to consider the restriction sites on the vector, and only needs to introduce a sequence (15-20 bp) homologous to the linearized vector end at the 5' end of the upstream and downstream primers of the target gene fragment. The steps of the seamless cloning technology are as follows: double enzyme digestion of the plasmid PMS; then the obtained target fragment and linearized vector are connected using a seamless cloning kit, and the reaction conditions are 50°C incubation for 15 min; the connection product is further transformed into E. coli DH5α competent cells, and positive clones are selected by PCR verification, and the single gene overexpression plasmid PMS-bv2539 is obtained after sequencing verification.

[0040] The construction of the combined overexpression plasmid PMS-kstd2&bv2539 or PMS-ksha3&ksha4&bv2539 includes the following steps:

[0041] For example, the recombinant plasmid PMS-kstd2&bv2539 is constructed by the following steps: the kstD2 and BV-2539 genes are amplified from the mycobacterium HK-90 genome by PCR, and then the purified kstD2 and BV-2539 genes are connected to the mycobacterium free expression plasmid PMS; the seamless cloning technology is used to construct the recombinant plasmid, the ligation product is further transformed into the E. coli DH5α competent cells, and then the positive clones are selected by PCR verification and sequencing verification.

[0042] In a second aspect, the present application provides a genetically engineered strain constructed by the method.

[0043] In a third aspect, the present application provides an application of the genetically engineered strain in the transformation of diosgenin or plant sterol to synthesize steroid drug intermediates.

[0044] Further, the steroid drug intermediates include C-19 steroid drug intermediates and C-21 steroid drug intermediates; the C-19 steroid drug intermediates include but are not limited to 9-hydroxyandrostenedione, androstenedione, androstenedione, and the C-21 steroid drug intermediates include but are not limited to 9, 16-dihydroxyprogesterone, 16-dehydroprogesterone, 9-hydroxy-16-dehydroprogesterone, and 1, 16-bishydroxyprogesterone. Specifically, the genetically engineered strain can be used to transform diosgenin to synthesize C-19 steroid drug intermediates 9-hydroxyandrostenedione, androstenedione, androstenedione, and C-21 steroid drug intermediates 9, 16-dihydroxyprogesterone, 16-dehydroprogesterone, 9-hydroxy-16-dehydroprogesterone, and 1, 16-bishydroxyprogesterone, and can be used to transform plant sterol to synthesize 9-hydroxyandrostenedione, androstenedione, androstenedione.

[0045] In a fourth aspect, the present application provides a method for transforming diosgenin to synthesize steroid drug intermediates by using the genetically engineered strain, which includes the following steps: inoculating the genetically engineered strain into a culture medium for cultivation, and transforming diosgenin to obtain steroid drug intermediates.

[0046] Further, the method is in the form of fermentation transformation, and diosgenin is added for transformation during the cultivation growth process, and the obtained steroid drug intermediates include 9-hydroxyandrostenedione, androstenedione, androstenedione, 9, 16-dihydroxyprogesterone, 16-dehydroprogesterone, 9-hydroxy-16-dehydroprogesterone, or 1, 16-bishydroxyprogesterone.

[0047] The inventors found that, in the process of metabolizing dioscin by the microorganism, sterol metabolism gene cluster SCG-2 participates in and dominates the metabolism of dioscin by the microorganism, and the sterol metabolism gene cluster SCG-2 is a potential gene cluster encoding dioscin metabolism. The present application adopts the strategy of knocking out kstD and / or kshA genes to eliminate their enzyme activity in the strain to construct a genetically engineered strain for synthesizing steroidal drug intermediates, so as to block the metabolism of dioscin by the microorganism, such as the degradation of dioscin steroidal nucleus by Mycobacterium HK-90. By knocking out and mutating various kstD and kshA isozymes in Mycobacterium HK-90 and characterizing the metabolic properties of the mutant bacteria, it is found that kstD2 and kshA3 in the SCG-2 cluster are key genes in the dioscin metabolic pathway, and the gene kshA4 outside the cluster is also a key metabolic gene, and it is also found that kstD1, kstD3, kshA1 and kshA2 assist in metabolism. Therefore, the kstD1-3 and kshA1-4 genes are iteratively knocked out, and the engineering strains mHust-ΔkstD, mHust-ΔkshA and mHust-ΔkstD-ΔkshA are successfully constructed. Among them, mHust-ΔkstD metabolizes dioscin to synthesize 9-hydroxyandrostenedione through C-21 steroidal intermediate 9,16-dihydroxyprogesterone, and metabolizes plant sterols to stably synthesize 9-hydroxyandrostenedione; mHust-ΔkshA metabolizes dioscin and plant sterols to stably synthesize androstadienedione; and mHust-ΔkstD-ΔkshA metabolizes dioscin and plant sterols to stably synthesize androstenedione.

[0048] And the inventors also found that the pathway of the strain to decompose dioscin needs to pass through C-27→C-21→C-19 steroid compound transformation, through in vitro characterization of the monooxygenase BV-2539 in the SCG-2 cluster, it is found that it is an important mechanism to catalyze C-21 to C-19 steroid compound transformation in the dioscin decomposition pathway, knocking out BV-2539 makes the mutant strain metabolize dioscin to stably synthesize 16-dehydroprogesterone. Through knocking out, complementing and overexpressing IclR-2535 gene in the SCG-2 cluster, it is found that IclR-2535 is a key transcriptional negative regulator, which causes the serious obstruction of the downstream transformation of steroid intermediates in the dioscin metabolic pathway. The combined knockout of kstD1-3 and IclR-2535 obtains a genetically engineered strain mHust-ΔkstD-ΔIclR2535 which can efficiently synthesize 9-hydroxyandrostenedione from dioscin; the combined knockout of kshA1-4 and IclR-2535 obtains a genetically engineered strain mHust-ΔkshA-ΔIclR2535 which can efficiently synthesize androstenedione from dioscin; the combined knockout of kstD1-3, kshA1-4 and IclR-2535 obtains a genetically engineered strain mHust-ΔkstD-ΔkshA-ΔIclR2535 which can efficiently synthesize androstenedione from dioscin. On the basis of the above genetically engineered strains, knocking out BV-2539 completely blocks the metabolic flow of dioscin to C-19 steroid drug intermediates in the strain, so that the genetically engineered strain can efficiently synthesize C-21 steroid drug intermediates 9,16-dihydroxyprogesterone, 16-dehydroprogesterone, 9-hydroxy-16-dehydroprogesterone or 1,16-didehydroprogesterone.

[0049] In addition, the inventors also found that single gene overexpression or combined overexpression of the key genes kstD2, kshA3, kshA4 and BV-2539 which catalyze the synthesis of C-19 steroid drug intermediates in the dioscin pathway can significantly improve the accumulation of steroid drug intermediates. Therefore, the present application first creatively constructs a series of genetically engineered strains which can transform dioscin to synthesize steroid drug intermediates by engineering dioscin-decomposing microorganisms such as Mycobacterium HK-90. The steroid drug intermediates synthesized by the genetically engineered strains of the present application can be widely used in the production of androgen and corticosteroid hormones and other steroid drugs. The present application uses genetically engineered strains to produce steroid drug intermediates, which greatly reduces the environmental pollution problem in the production process of steroid drug intermediates, improves the conversion rate of steroid drug intermediates, simplifies the production steps of steroid drugs, helps to improve the production efficiency of steroid drugs and reduce costs, has good social value and economic value, and is suitable for industrial production.

[0050] Compared with the prior art, the present application has the following advantages:

[0051] The present application is based on genetic engineering and metabolic engineering technology, by blocking the product degradation pathway of the microorganism which can decompose diosgenin, overexpressing key metabolic genes, thereby obtaining a series of genetically engineered strains for converting diosgenin to synthesize steroid drug intermediates. The present application uses genetically engineered strains to produce steroid drug intermediates, replaces the multi-step chemical degradation of diosgenin by biotransformation method, for the first time realizes the green manufacturing of genetically engineered strains converting diosgenin to synthesize steroid drug intermediates, greatly reduces the environmental pollution problem, improves the conversion rate of steroid drug intermediates, simplifies the production steps of steroid drugs, significantly improves the production efficiency of steroid drugs, and greatly reduces the production cost. BRIEF DESCRIPTION OF DRAWINGS

[0052] Figure 1 PCR verification results of knocking out genes kstD1, kstD2 or kstD3, respectively;

[0053] Figure 2 PCR verification results of knocking out genes kshA1, kshA2, kshA3 or kshA4, respectively;

[0054] Figure 3 PCR verification results of knocking out genes BV-2539 or IclR-2535, respectively;

[0055] Figure 4 Mass spectrum of steroid drug intermediate 9-hydroxyandrostenedione;

[0056] Figure 5 Mass spectrum of steroid drug intermediate androstenedione;

[0057] Figure 6 Mass spectrum of steroid drug intermediate androstenedione;

[0058] Figure 7 Mass spectrum of steroid drug intermediate 16-dehydroprogesterone;

[0059] Figure 8 Liquid chromatogram of fermenting and synthesizing steroid drug intermediate 9-hydroxyandrostenedione;

[0060] Figure 9 Liquid chromatogram of fermenting and synthesizing steroid drug intermediate androstenedione;

[0061] Figure 10 Liquid chromatogram of fermenting and synthesizing steroid drug intermediate androstenedione;

[0062] Figure 11 Liquid chromatogram of fermenting and synthesizing steroid drug intermediate 16-dehydroprogesterone;

[0063] Figure 129-hydroxyandrostenedione, androstenedione, androstenediol, 9,16-dihydroxyprogesterone, 16-dehydroprogesterone, 9-hydroxy-16-dehydroprogesterone, and 1,16-bishydrodehydroprogesterone. DETAILED DESCRIPTION

[0064] The technical solutions of the present application will be described clearly and completely below. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0065] The present application provides a construction method of a genetically engineered strain for converting diosgenin to produce a steroid pharmaceutical intermediate, comprising the following steps:

[0066] 1) selecting a microorganism capable of decomposing and metabolizing diosgenin;

[0067] 2) single gene knockout inactivation or combined knockout inactivation is performed on key genes for degradation of steroid compound mother nucleus, key genes for catalyzing C-21 to C-19 steroid compound conversion, and key negative transcriptional regulatory factors in the microorganism; wherein the key genes for degradation of steroid compound mother nucleus include kstD and / or kshA, the kstD includes kstD1, kstD2 and kstD3, and the kshA includes kshA1, kshA2, kshA3 and kshA4; the key genes for catalyzing C-21 to C-19 steroid compound conversion include BV-2539; and the key negative transcriptional regulatory factors include IclR-2535. In the following specific embodiments, the nucleotide sequences of kstD1, kstD2 and kstD3 are respectively represented by SEQ ID No: 1-3, the nucleotide sequences of kshA1, kshA2, kshA3 and kshA4 are respectively represented by SEQ ID No: 4-7, the nucleotide sequence of BV-2539 is represented by SEQ ID No: 8, and the nucleotide sequence of IclR-2535 is represented by SEQ ID No: 9.

[0068] In some examples, the microorganism capable of decomposing and metabolizing diosgenin in step 1) includes a non-pathogenic microorganism in the genus Mycobacterium, Mycolicibacterium, Mycolicibacillus, Mycolicibacter or Mycobacteroides.

[0069] In some examples, step 2) comprises a combined knockout inactivation of kstD1, kstD2 and kstD3; or a combined knockout inactivation of kshA1, kshA2, kshA3 and kshA4; or a combined knockout inactivation of kstD1, kstD2, kstD3, kshA1, kshA2, kshA3 and kshA4; or a combined knockout inactivation of kstD1, kstD2, kstD3 and IclR-2535; or a combined knockout inactivation of kshA1, kshA2, kshA3, kshA4 and IclR-2535; or a combined knockout inactivation of kstD1, kstD2, kstD3, kshA1, kshA2, kshA3, kshA4 and IclR-2535; or a combined knockout inactivation of kstD1, kstD2, kstD3, BV-2539 and IclR-2535; or a combined knockout inactivation of kshA1, kshA2, kshA3, kshA4, BV-2539 and IclR-2535; or a combined knockout inactivation of kstD1, kstD2, kstD3, kshA1, kshA2, kshA3, kshA4, BV-2539 and IclR-2535.

[0070] The method further comprises a step of: 3) single gene overexpression or combined overexpression of key genes catalyzing the synthesis of C-19 steroid drug intermediates in the microorganism diosgenin pathway; wherein the key genes catalyzing the synthesis of C-19 steroid drug intermediates include kstD2, kshA3, kshA4, BV-2539.

[0071] In some examples, step 3) comprises single gene overexpression of BV-2539; or combined overexpression of kshD2 and BV-2539; or combined overexpression of kshA3, kshA4 and BV-2539.

[0072] In some examples, the steroid drug intermediates include C-19 steroid drug intermediates 9-hydroxyandrostenedione, androstenedione, and C-21 steroid drug intermediates 9,16-dihydroxyprogesterone, 16-dehydroprogesterone, 9-hydroxy-16 dehydroprogesterone, 1,16-didehydroprogesterone (chemical structures of steroid drug intermediates are shown as Figure 12

[0073] The experimental conditions in the following specific examples are in accordance with conventional experimental conditions or in accordance with the experimental conditions recommended by the manufacturers; the materials, reagents and the like used, if not specifically stated, are reagents and materials obtained from commercial channels.

[0074] ​The host bacteria used in the following specific examples: Mycolicibacterium sp. HK-90 was isolated and screened by the inventors, and the preservation number is CCTCC M2024221. For details, please refer to patent CN118126872A. The genome has been uploaded to GeneBank, and the sequence number is CP129613.

[0075] The culture medium involved in the following specific examples is as follows:

[0076] LB medium (1 L): 10 g of tryptone, 5 g of yeast extract powder, 10 g of NaCl, pH 7.0; the solid medium is based on this and 15-20 g of agar powder is added.

[0077] Seed medium (1 L): 16 g of tryptone, 10 g of yeast extract powder, 6 g of NaCl, pH 7.2; the solid medium is based on this and 15-20 g of agar powder is added.

[0078] Fermentation medium (1 L): 20 g of glucose, 10 g of tryptone, 6 g of yeast extract powder, 6 g of sodium chloride, 3 g of (NH4)2HPO4, 0.4 g of MgSO4·7H2O, 5 × 10 -4 g of MnCl2.

[0079] The detection of the steroid drug intermediate product in the following specific examples is as follows:

[0080] Extraction: After the fermentation is completed, add three times the volume of ethyl acetate to the fermentation liquid, shake well and ultrasonic, then centrifuge at 6000 rpm for 5 min, collect the upper ethyl acetate extract, evaporate the reagent in a rotary evaporator, and redissolve the metabolites with chromatographic grade methanol for detection and analysis.

[0081] High performance liquid chromatography (HPLC) detection: HPLC is used to detect the products of the strain metabolizing diosgenin or phytosterol, the sample volume is 10 μL, the reverse phase chromatographic column is Ecosil C18 (250 mm × 4.6 mm, 5 μm particle size), the column temperature is 30℃, the ultraviolet detection wavelength is 204 nm and 254 nm, the mobile phase is A (10 mM NH4Ac aqueous solution) and B (methanol), and the metabolites are detected by gradient elution program, and the mobile phase program is as follows: 0 to 30 min, 5% B to 90% B; 30 min to 55 min, 90% B. In addition, LC-MS / MS is used to further analyze the metabolites.

[0082] Example 1 Construction of genetically engineered strain mHust-ΔkstD

[0083] The inventors found that there are multiple isozymes of KstD in Mycobacterium HK-90. In this embodiment, the CRISPR-Cas12a-NHEJ gene editing technology was first used to independently knock out the kstD gene in wild-type Mycobacterium HK-90. The genotypes of the mutant strains were verified by colony PCR and sequencing, and the results of kstD gene knockout verification are shown in Figure 1 Due to the NHEJ mechanism, the repair of genomic cuts causes deletions of different sizes of gene fragments, resulting in randomness in the mutation types of the strains. To avoid interference from other genes, only the mutant types with in-frame deletions of the target genes were screened. The metabolic characteristics of the kstD knockout mutant strains were found to be that only when kstD2 in the SCG-2 cluster was knocked out, the mutant strain could synthesize the steroid intermediate from diosgenin. This fully confirms that the specific SCG-2 is a potential gene cluster encoding diosgenin catabolism. In Mycobacterium HK-90, kstD2 is the main contributor to the KstD enzyme activity in the diosgenin metabolic pathway, and the steroid intermediates produced in the metabolic pathway can activate the steroid metabolic enzymes distributed in other positions of the genome, so that kstD1 and kstD3 genes assist the strain in metabolizing diosgenin. Taking these factors into account, the genetically engineered strain mHust-ΔkstD with combined knockout of the kstD1, kstD2 and kstD3 genes was constructed in this embodiment.

[0084] Example 2 Construction of genetically engineered strain mHust-ΔkshA

[0085] The inventors found that there are four KshA isozymes in Mycobacterium HK-90. In this embodiment, single gene knockout mutant strains were first constructed, and the results of PCR verification of the genotypes of the mutant strains are shown in Figure 2 After inoculating the mutant strains for fermentation, it was found that in the kshA single gene knockout strains, except for the mutant strain m-ΔkshA3 obtained by knocking out kshA3, which could metabolize diosgenin to enrich C-27 intermediate C 27 H 38O6 and trace amounts of C-19 steroidal intermediates androstandione, no other steroidal intermediates were detected in the fermentation broth of all other mutants, and when the fermentation time of mutant strain m-ΔkshA3 was extended, all steroidal intermediates were completely degraded, thus, other KshA isozymes in mycobacterium HK-90 also play a key role in sterol metabolism. In this embodiment, the mutant strain m-ΔkshA3 was further disrupted to eliminate KshA enzyme activity; through iterative gene knockout, double gene knockout mutant strains m-ΔkshA3,1, m-ΔkshA3,2 and m-ΔkshA3,4 were constructed; the mutant strains were inoculated in a fermentation medium containing diosgenin, and sampling and monitoring showed that the double gene knockout of kshA1, kshA2 or kshA4 and kshA3 could promote the accumulation of intermediate C 27 H 38 O6 content, but the mutant strain m-ΔkshA3,4 could obviously enrich androstandione. On the other hand, it was found through comparison that the enrichment product capacity of the mutant strains m-ΔkshA3,1 and m-ΔkshA3,2 was much lower than that of m-ΔkshA3,4, which indicated that kshA4 might be another key gene for the metabolism of steroidal compounds in mycobacterium HK-90.

[0086] The double gene knockout mutant strains were incubated in a medium added with androstandione substrate, and the stability of the mutant strains in enriching intermediate metabolites was quantitatively characterized by detecting the residual amount of androstandione in the fermentation broth; the HPLC detection results showed that only when kshA4 gene was inactivated on the basis of m-ΔkshA3, the mutant strain could reduce the degradation of the intermediate product and retain more androstandione content; at the same time, compared with the blank control, the androstandione content of the mutant strain m-ΔkshA3,4 could delay the decrease rate of androstandione content in the medium, but it might still have hydroxylation activity at the C9 position, thereby causing the degradation of androstandione, which was consistent with the situation in the late stage of diosgenin fermentation by the strain, and the results showed that kshA1 and kshA3 in the strain might also participate in the degradation metabolism of sterols. Therefore, all kshA genes in the strain are involved in the metabolism of sterols, in which kshA3 and kshA4 are the main contributors to the KshA enzyme activity of the strain, and kshA1 and kshA2 assist diosgenin metabolism. Based on these factors, a genetically engineered strain mHust-ΔkshA with combined knockout of kshA1, kshA2, kshA3 and kshA4 genes was constructed in this embodiment.

[0087] Example 3 Construction of genetically engineered strain mHust-ΔkstD-ΔkshA

[0088] Androstenedione, as an upstream intermediate of 9-hydroxyandrostenedione and androstadienedione, is a necessary path of steroid metabolism; when the enzyme activities of kstD and kshA are eliminated at the same time in the strain, this important intermediate of steroid drugs can be obtained in theory. Therefore, this embodiment continues to knock out the genes kshA1, kshA2, kshA3 and kshA4 on the basis of the genetically engineered strain mHust-ΔkstD in Embodiment 1, and constructs the seven-gene knockout strain mHust-ΔkstD-ΔkshA which blocks the degradation of the steroidal nucleus of dioscin.

[0089] Embodiment 4 Construction of genetically engineered strains mHust-ΔkstD-ΔIclR2535, mHust-ΔkshA-ΔIclR2535 and mHust-ΔkstD-ΔkshA-ΔIclR2535

[0090] The inventors found through research that the gene IclR-2535 in the key sterol metabolism gene cluster SCG-2 of Mycobacterium HK-90 plays an important regulatory role in the dioscin metabolic pathway. In this embodiment, the recombinant plasmid PMS-IclR-2535 is transformed into the genetically engineered strain mHust-ΔkshA to overexpress the gene IclR-2535; at the same time, the IclR-2535 gene is knocked out in the genetically engineered strain mHust-ΔkstD-ΔkshA which synthesizes androstenedione. The research results show that overexpression of the gene IclR-2535 has a great influence on the genetically engineered strain mHust-ΔkshA metabolizing dioscin to produce androstadienedione, and the synthesis amount of androstadienedione is reduced by 46.6%; after knocking out the gene IclR-2535 in the genetically engineered strain mHust-ΔkstD-ΔkshA (PCR verification results are shown in Figure 3 ), the ability of the mutant strain to synthesize steroidal drug intermediates from dioscin is enhanced, which is about 1.22 times higher than that of the control strain. Combined with the coding prediction of the gene IclR-2535 and the knocking out and overexpression of the gene, the high and low yield strain phenotypes are converted, and it is concluded that the gene IclR-2535 is a key negative transcriptional regulator affecting the strain to metabolize dioscin, and the existence of the regulator causes the conversion of intermediates to downstream to be blocked. Therefore, on the basis of the genetically engineered strains in Embodiments 1, 2 and 3, this embodiment continues to knock out the key negative transcriptional regulator IclR-2535, and constructs the genetically engineered strains mHust-ΔkstD-ΔIclR2535, mHust-ΔkshA-ΔIclR2535 and mHust-ΔkstD-ΔkshA-ΔIclR2535 which efficiently synthesize C-19 steroidal drug intermediates 9-hydroxyandrostenedione, androstadienedione and androstenedione from dioscin.

[0091] Example 5 Construction of genetically engineered strains mHust-ΔkstD-ΔIclR2535-ΔBV2539, mHust-ΔkshA-ΔIclR2535-ΔBV2539, mHust-ΔkstD-ΔkshA-ΔIclR2535-ΔBV2539

[0092] The inventors found that the gene BV-2539 in the key sterol metabolism gene cluster SCG-2 in Mycobacterium HK-90 is a key gene catalyzing the conversion of dioscin to C-19 steroidal compounds. Therefore, this example further knocks out the key gene BV-2539 (PCR verification results are shown in Figure 3 ) based on the genetically engineered strains in Example 4, completely blocks the metabolic flow of the strain converting dioscin to C-19 steroidal drug intermediates, and constructs genetically engineered strains mHust-ΔkstD-ΔIclR2535-ΔBV2539, mHust-ΔkshA-ΔIclR2535-ΔBV2539, mHust-ΔkstD-ΔkshA-ΔIclR2535-ΔBV2539 (mHust-C21) that convert dioscin to high-efficiency C-21 steroidal drug intermediates 9-hydroxy-16-dehydroyohimbine, 1,16-didehydroyohimbine and 16-dehydroyohimbine.

[0093] Example 6 Construction of genetically engineered strains overexpressing genes kstD2 or kshA3, kshA4 or BV-2539

[0094] The inventors found that the genes kstD2, kshA3, kshA4 and BV-2539 in Mycobacterium HK-90 are key genes in the dioscin metabolic pathway, and single gene overexpression or combined overexpression of kstD2, kshA3, kshA4 and BV-2539 can further improve the genetically engineered strain in converting dioscin to C-19 steroidal drug intermediates 9-hydroxyandrostenedione, androstadienedione and androstenedione. Therefore, this example constructs single gene overexpression plasmid PMS-bv2539 or combined overexpression plasmid PMS-kstd2&bv2539, PMS-ksha3&ksha4&bv2539, and transforms the recombinant plasmids into the genetically engineered strains constructed in Example 4 by electroporation, and screens positive strains on kanamycin-resistant plates. The strains containing the recombinant plasmids are inoculated in liquid medium containing kanamycin, and the OD 600When the growth reached 0.8-1.0, a solution of acetamide with a final concentration of 30 mM was added to induce gene expression, and the Diosgenin substrate was added, and the metabolic characteristics of the genetically engineered strains mHust-ΔkstD-ΔIclR2535-Obv2539, mHust-ΔkshA-ΔIclR2535-Obv2539, mHust-ΔkstD-ΔkshA-ΔIclR2535-Obv2539, mHust-ΔkshA-ΔIclR2535-Okstd2&bv2539 and mHust-ΔkstD-ΔIclR2535-Oksha3&ksha4&bv2539 were characterized by fermentation.

[0095] Example 7 Fermentation of genetically engineered strains to convert Diosgenin and phytosterol to high-efficiency synthesis of steroid drug intermediates

[0096] The genetically engineered strain stored in a -80 ℃ glycerol tube was inoculated into 20 mL of seed culture medium and incubated at 30 ℃, 200 rpm for 72 h; the seed culture solution was taken and added to fresh seed culture medium as the second level seed growth solution at a 2% inoculation amount, and incubated at 30 ℃, 200 rpm for 24 h; the second level seed was then added to a 500 mL fermentation medium (2 L fermentation flask) at a 5% inoculation ratio, and 50 g / L Diosgenin or 30 g / L phytosterol and 5% hydroxypropyl-β-cyclodextrin were added, and the fermentation was cultured and sampled to monitor the metabolic characteristics of the genetically engineered strain in converting Diosgenin.

[0097] Fermentation characterization showed that the genetically engineered strain mHust-ΔkstD metabolized phytosterol to stably synthesize C-19 steroid drug intermediate 9-hydroxyandrostenedione, and metabolized Diosgenin to C-21 steroid drug intermediate 9,16-dihydroxyprogesterone to 9-hydroxyandrostenedione; the genetically engineered strains mHust-ΔkstD-ΔIclR2535, mHust-ΔkstD-ΔIclR2535-Obv2539 and mHust-ΔkstD-ΔIclR2535-Oksha3&ksha4&bv2539 metabolized Diosgenin and phytosterol to high-efficiency stably synthesize 9-hydroxyandrostenedione (mass spectrum is shown in Figure 4 , and the liquid chromatogram of 9-hydroxyandrostenedione synthesized by fermentation is shown in Figure 8 ).

[0098] The genetically engineered strains mHust-ΔkshA, mHust-ΔkshA-ΔIclR2535, mHust-ΔkshA-ΔIclR2535-Obv2539 and mHust-ΔkshA-ΔIclR2535-Okstd2&bv2539 metabolize diosgenin and phytosterol to efficiently and stably synthesize C-19 steroid drug intermediate androstenedione (mass spectrum is shown in Figure 5 , and the liquid phase diagram of fermentation to synthesize androstenedione is shown in Figure 9 ).

[0099] The genetically engineered strains mHust-ΔkstD-ΔkshA, mHust-ΔkstD-ΔkshA-ΔIclR2535 and mHust-ΔkstD-ΔkshA-ΔIclR2535-Obv2539 metabolize diosgenin and phytosterol to efficiently and stably synthesize C-19 steroid drug intermediate androstenedione (mass spectrum is shown in Figure 6 , and the liquid phase diagram of fermentation to synthesize androstenedione is shown in Figure 10 ).

[0100] The genetically engineered strains mHust-ΔkstD-ΔIclR2535-ΔBV2539, mHust-ΔkshA-ΔIclR2535-ΔBV2539 and mHust-C21 metabolize diosgenin to efficiently and stably synthesize C-21 steroid drug intermediates 9-hydroxy-16-dehydropregnenolone, 1,16-bishydroxyprogesterone and 16-dehydropregnenolone (mass spectrum is shown in Figure 7 , and the liquid phase diagram of fermentation to synthesize 16-dehydropregnenolone is shown in Figure 11 ); wherein the genetically engineered strain mHust-C21 converts 50 g / L diosgenin to synthesize 33.80 g / L 16-dehydropregnenolone, and the molar yield is as high as 89.7%, which is the highest yield reported so far for biosynthesis of C-21 steroid drug intermediates.

[0101] In summary, the genetically engineered strains are constructed by single gene knockout inactivation or combined knockout inactivation of key genes kstD and / or kshA in the degradation of the steroid compound nucleus in microorganisms, key gene BV-2539 catalyzing the conversion of C-21 to C-19 steroid compounds, and key negative transcriptional regulator IclR-2535, and by single gene overexpression or combined overexpression of key genes kstD2, kshA3, kshA4 and BV-2539 catalyzing the synthesis of C-19 steroid drug intermediates in the diosgenin pathway of microorganisms. The green manufacturing of genetically engineered strains to convert diosgenin to synthesize steroid drug intermediates is realized for the first time, the production steps of steroid drugs are simplified, the conversion rate of steroid drug intermediates and the production efficiency of steroid drugs are greatly improved, and the genetically engineered strains have good social and economic values and are conducive to industrial production.

[0102] The above detailed description describes the embodiments of the present application in detail, but the present application is not limited to the specific details in the above embodiments. Within the scope of the claims and technical concepts of the present application, various simple modifications and changes can be made to the technical solutions of the present application, and these simple modifications all belong to the protection scope of the present application.

Claims

1. A method for constructing a genetically engineered strain for transforming diosgenin into an intermediate for the production of steroidal drugs, characterized in that, Includes the following steps: Select microorganisms that can decompose and metabolize diosgenin; Key genes involved in the degradation of steroidal compounds from their parent nucleus in microorganisms, key genes catalyzing the transformation of C-21 to C-19 steroidal compounds, and key negative transcription factors are inactivated by single-gene knockout or combined knockout. Specifically, the key genes involved in the degradation of steroidal compounds from their parent nucleus include kstD and / or kshA; the key genes catalyzing the transformation of C-21 to C-19 steroidal compounds include BV-2539; and the key negative transcription factors include IclR-2535.

2. The method for constructing a genetically engineered strain for converting diosgenin into an intermediate for producing steroidal drugs according to claim 1, characterized in that, The method further includes the step of: overexpressing a single gene or a combination of genes that catalyze the synthesis of C-19 steroidal drug intermediates in the microbial diosgenin pathway; wherein the key genes that catalyze the synthesis of C-19 steroidal drug intermediates include kstD2, kshA3, kshA4, and BV-2539.

3. The method for constructing a genetically engineered strain for converting diosgenin into an intermediate for producing steroidal drugs according to claim 1, characterized in that, The microorganisms that can decompose and metabolize diosgenin include non-pathogenic microorganisms from the genera Mycobacterium, Mycolicibacterium, Mycolicibacillus, Mycolicibacter, or Mycobacteroides.

4. The method for constructing a genetically engineered strain for converting diosgenin into an intermediate for producing a steroidal drug, as described in claim 1, is characterized in that... The kstD includes kstD1, kstD2, and kstD3, and the kshA includes kshA1, kshA2, kshA3, and kshA4; the nucleotide sequences of kstD1, kstD2, and kstD3 are the sequences shown in SEQ ID No:1-3, or the nucleotide sequences of kstD1, kstD2, and kstD3 are orthologous gene sequences of the sequences shown in SEQ ID No:1-3; the nucleotide sequences of kshA1, kshA2, kshA3, and kshA4 are the sequences shown in SEQ ID No:4-7, or the nucleotide sequences of kshA1, kshA2, kshA3, and kshA4 are orthologous gene sequences of the sequences shown in SEQ ID No:4-7.

5. The method for constructing a genetically engineered strain for converting diosgenin into an intermediate for producing a steroidal drug according to claim 4, characterized in that, This includes a combined knockout inactivation of kstD1, kstD2, and kstD3; Alternatively, a combination of knockouts can be performed to deactivate kshA1, kshA2, kshA3, and kshA4; Alternatively, a combination of knockouts can be performed to deactivate kstD1, kstD2, kstD3, kshA1, kshA2, kshA3, and kshA4; Alternatively, a combination of knockout inactivation of kstD1, kstD2, kstD3, and IclR-2535 may be performed; Alternatively, a combination of knockout inactivation of kshA1, kshA2, kshA3, kshA4, and IclR-2535 may be performed; Alternatively, a combination of knockout inactivation can be performed on kstD1, kstD2, kstD3, kshA1, kshA2, kshA3, kshA4 and IclR-2535; Alternatively, a combination of knockout inactivation of kstD1, kstD2, kstD3, BV-2539, and IclR-2535 may be performed; Alternatively, a combination of knockout inactivation can be performed on kshA1, kshA2, kshA3, kshA4, BV-2539, and IclR-2535; Alternatively, a combination of knockout inactivation can be performed on kstD1, kstD2, kstD3, kshA1, kshA2, kshA3, kshA4, BV-2539, and IclR-2535.

6. The method for constructing a genetically engineered strain for converting diosgenin into an intermediate for producing a steroidal drug according to claim 1, characterized in that, The nucleotide sequence of BV-2539 is the sequence shown in SEQ ID No:8, or the orthologous gene sequence of the sequence shown in SEQ ID No:8; the nucleotide sequence of IclR-2535 is the sequence shown in SEQ ID No:9, or the orthologous gene sequence of the sequence shown in SEQ ID No:

9.

7. The method for constructing a genetically engineered strain for converting diosgenin into an intermediate for producing a steroidal drug according to claim 2, characterized in that, This includes single-gene overexpression of BV-2539; Alternatively, combined overexpression of kshD2 and BV-2539 may be performed; Alternatively, combined overexpression of kshA3, kshA4, and BV-2539 may be performed.

8. Genetically engineered strains constructed using the method described in any one of claims 1-7.

9. The use of the genetically engineered strain according to claim 8 in the synthesis of steroidal drug intermediates from diosgenin or phytosterols.

10. The method for synthesizing steroidal drug intermediates by transforming diosgenin with the genetically engineered strain according to claim 8, characterized in that, The process includes the following steps: inoculating the genetically engineered strain of claim 8 into a culture medium and culturing it to transform diosgenin to obtain a steroidal drug intermediate.

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