17beta-hydroxysteroid dehydrogenase mutant, engineered bacteria and application thereof

Abstract

The application discloses a 17beta-hydroxysteroid dehydrogenase mutant, an engineering bacterium and application, and uses the 17beta-hydroxysteroid dehydrogenase mutant to biotransform methyl diketone as a substrate to prepare nandrolone. The 17beta-hydroxysteroid dehydrogenase is 17beta-hydroxysteroid dehydrogenase (17beta-HSDcl) from a fungus Cochliobolus lunatus, and the mutant is a single mutant in which histidine His at the 164th position is mutated into valine Val, a single mutant in which threonine Thr at the 151st position is mutated into alanine Ala, or a double mutant in which the 164th position and the 151st position are jointly mutated. The application uses pET28a as an expression plasmid, realizes heterologous expression of the 17beta-HSDcl mutant in E.coli BL21 (DE3), and through 24h whole cell transformation of the engineering bacterium, the nandrolone yield of the single mutant H164V, the single mutant T151A and the double mutant H164V / T151A is 1.27 times, 1.30 times and 1.34 times that of before mutation, respectively. The application provides a basis for industrialized production of nandrolone by microorganisms.

Description

Technical Field

[0001] The present invention belongs to the technical field of enzyme engineering and biocatalysis, and particularly relates to a 17β-hydroxysteroid dehydrogenase mutant, an engineered bacterium and applications. Background Art

[0002] Steroid hormones are a class of drugs containing a cyclopentanepolyhydrophenanthrene nucleus. Currently, over 400 steroid hormones are produced globally, primarily comprising adrenocortical hormones and sex hormones. They are widely used in anti-tumor, anti-inflammatory, anti-allergic, immunomodulatory, and reproductive and developmental disorders, making them the second largest class of drugs after antibiotics.

[0003] Nandrolone (17β-hydroxy-19-norandrost-4-ene-3-one) is an androgenic anabolic steroid that promotes protein synthesis, inhibits protein breakdown, increases calcium and phosphate deposition, and promotes bone growth. Nandrolone's protein anabolic activity is 12 times that of testosterone and can inhibit glucocorticoids. Even low doses of nandrolone can significantly increase nitrogen storage in muscle, increase bone mineral content, and enhance collagen synthesis. It is primarily used medically to treat muscle injuries and osteoporosis, and is also used as a sports nutrition supplement.

[0004] Currently, the synthesis of nandrolone is mostly achieved through chemical methods involving multiple steps, such as etherification and reductive hydrolysis. These methods have low selectivity, produce numerous byproducts, pollute the environment, and involve highly toxic organic reagents and compounds. Microbial transformation, on the other hand, allows for highly specific synthesis of nandrolone, with a simpler, more environmentally friendly process.

[0005] 17β-Hydroxysteroid dehydrogenase (17β-HSD) is a key enzyme for the interconversion between the C-17 carbonyl and hydroxyl groups of steroid compounds. In the final step of steroid biosynthesis, it promotes the interconversion between active and inactive forms of steroid hormones and requires the cofactor NAD(P)H / NAD(P). + 17β-HSD plays an indispensable role in the synthesis of steroid drugs and is currently mainly used in the production of testosterone.

[0006] Therefore, researching and modifying a more efficient 17β-HSD and applying it to the biosynthesis of nandrolone is of great significance for its green industrial production. Summary of the Invention

[0007] Purpose of the Invention: The present invention provides a 17β-hydroxysteroid dehydrogenase mutant, engineered bacteria, and applications. Specifically, through analysis of the enzyme's active site, highly active mutants 17β-HSDcl-H164V, 17β-HSDcl-T151A, and 17β-HSDcl-H164V / T151A were obtained, and the enzymatic properties of the mutants were studied. The mutants were heterologously expressed in Escherichia coli, resulting in the successful construction of engineered strains E. coli BL21(DE3) / pET28a-17β-HSDcl-H164V, E. coli BL21(DE3) / pET28a-17β-HSDcl-T151A, and E. coli BL21(DE3) / pET28a-17β-HSDclH164V / T151A. The engineered strains achieved the conversion of methyldiketone to nandrolone, providing valuable guidance for the industrial production of nandrolone via microbial fermentation.

[0008] The first object of the present invention is to provide a 17β-hydroxysteroid dehydrogenase mutant, the amino acid sequence of the mutant is shown in SEQ ID NO.3, or SEQ ID NO.5, or SEQ ID NO.7.

[0009] In one embodiment of the present invention, the 17β-hydroxysteroid dehydrogenase mutant is obtained by mutating the histidine at position 164 of the 17β-hydroxysteroid dehydrogenase 17β-HSDcl having the starting amino acid sequence as shown in SEQ ID NO.1 to valine, or mutating the threonine at position 151 to alanine, or mutating the histidine at position 164 to valine and the threonine at position 151 to alanine.

[0010] In one embodiment of the present invention, the 17β-hydroxysteroid dehydrogenase gene (GenBank: AAR04485.1) is derived from the fungus Cochliobolus lunatus.

[0011] In one embodiment of the present invention, the nucleotide sequence of the gene encoding the 17β-hydroxysteroid dehydrogenase is shown as SEQ ID NO.2.

[0012] The second object of the present invention is to provide a gene encoding the mutant, whose nucleotide sequence is any one of the following: a) a nucleotide sequence as shown in SEQ ID NO.4 or SEQ ID NO.6 or SEQ ID NO.8; b) a nucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO.3 or SEQ ID NO.5 or SEQ ID NO.7 or a nonsense mutation sequence thereof.

[0013] In one embodiment of the present invention, the nucleotide sequence encoding the 17β-hydroxysteroid dehydrogenase mutant gene is shown as SEQ ID NO.4 or SEQ ID NO.6 or SEQ ID NO.8.

[0014] The third object of the present invention is to provide a vector (or expression plasmid) comprising (or carrying) the 17β-hydroxysteroid dehydrogenase mutant gene.

[0015] In one embodiment of the present invention, the expression plasmid comprises pET28a.

[0016] The fourth object of the present invention is to provide a genetically engineered strain (or recombinant engineered strain) expressing the 17β-hydroxysteroid dehydrogenase mutant.

[0017] In one embodiment of the present invention, the nucleotide sequence encoding the 17β-hydroxysteroid dehydrogenase mutant gene is shown as SEQ ID NO.4 or SEQ ID NO.6 or SEQ ID NO.8.

[0018] In one embodiment of the present invention, the recombinant expression plasmid is pET28a-17β-HSDcl-H164V or pET28a-17β-HSDcl-T151A or pET28a-17β-HSDcl-H164V / T151A.

[0019] In one embodiment of the present invention, the host bacteria includes Escherichia coli BL21 (DE3).

[0020] In one embodiment of the present invention, the specific method for constructing the genetically engineered strain comprises the following steps:

[0021] (1) Select the restriction sites BamHI and HindIII on the pET28a plasmid and insert the mutant gene into the corresponding position of the plasmid to obtain the recombinant plasmid pET28a-17β-HSDcl-H164V or pET28a-17β-HSDcl-T151A or pET28a-17β-HSDcl-H164V / T151A;

[0022] (2) The recombinant plasmid pET28a-17β-HSDcl-H164V or pET28a-17β-HSDcl-T151A or pET28a-17β-HSDcl-H164V / T151A was transformed into Escherichia coli for expression;

[0023] (3) Colonies grown on LB and kanamycin pressure plates were picked and cultured in shake flasks. Plasmids were extracted and verified by single and double enzyme digestion to obtain recombinant engineered strains E. coli BL21 (DE3) / pET28a-17β-HSDcl-H164V or E. coli BL21 (DE3) / pET28a-17β-HSDcl-T151A or E. coli BL21 (DE3) / pET28a-17β-HSDcl-H164V / T151A.

[0024] The fifth object of the present invention is to provide an application of the 17β-hydroxysteroid dehydrogenase mutant, the vector, or the recombinant engineered microbial strain for converting methyldiketone into nandrolone.

[0025] In one embodiment of the present invention, the recombinant engineered strain expresses a 17β-hydroxysteroid dehydrogenase mutant.

[0026] In one embodiment of the present invention, the substrate is methyldiketone and the catalyst is a 17β-hydroxysteroid dehydrogenase mutant.

[0027] The sixth object of the present invention is to provide a method for producing nandrolone through bioconversion, wherein methyldiketone is used as a substrate and converted into nandrolone under the action of a 17β-hydroxysteroid dehydrogenase mutant, wherein:

[0028] The amino acid sequence of the 17β-hydroxysteroid dehydrogenase mutant is shown in SEQ ID NO.3, SEQ ID NO.5 or SEQ ID NO.7.

[0029] In one embodiment of the present invention, the specific conversion steps for producing nandrolone by bioconversion are as follows: adding IPTG at a final concentration of 0.2-0.7 mmol / L to LB medium containing a genetically recombinant engineered strain for induction, adding a substrate methyl diketone at a final concentration of 0.5-1 g / L for conversion, the substrate cosolvent is DMSO at a final concentration of 10-16% (v / v), the conversion temperature is 20-30°C, the conversion pH is 7-8, and the conversion time is 12-24 hours, wherein the genetically engineered strain contains a gene encoding the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.3 or SEQ ID NO.5 or SEQ ID NO.7.

[0030] Preferably, in one embodiment of the present invention, the conversion temperature is 23-27°C, the conversion pH is 7.3-7.7, and the conversion time is 12-24h.

[0031] Further preferably, in one embodiment of the present invention, the conversion temperature is 25° C., the conversion pH is 7.5, and the conversion time is 24 h.

[0032] In one embodiment of the present invention, the conversion rate of the substrate methyldiketone is higher than 50%.

[0033] In one embodiment of the present invention, the culture method of the recombinant engineered strain is to inoculate the genetically engineered strain into 5-10 mL LB medium, culture for 10-14 h, and then inoculate 1-2% of the inoculum into a 250 mL container containing 50 mL LB medium, and culture at 35-37 ° C until the OD 600 =0.6-0.8, and then transferred to 20-25℃ for induction culture for 12-24h.

[0034] Preferably, in one embodiment of the present invention, the culture method of the recombinant engineered strain is to inoculate the genetically engineered strain into 10 mL of LB medium, culture for 12 h, and then inoculate it into a 250 mL container containing 50 mL of LB medium at a 2% inoculum size, and culture at 37°C until the OD 600 =0.8, and then transferred to 25℃ for induction culture for 24h.

[0035] Beneficial Effects: This study identified the amino acids in the active center of 17β-HSDcl through molecular docking simulations and performed site-directed mutagenesis of key amino acid residues, enhancing the catalytic activity of 17β-HSDcl. The modified 17β-HSDcl mutant was heterologously expressed in Escherichia coli BL21(DE3) and successfully used to convert methyldiketone to produce nandrolone. After 24 hours of whole-cell transformation of the engineered bacteria, the single mutant H164V, single mutant T151A, double mutant H164V / T151A and wild-type 17β-hydroxysteroid dehydrogenase can convert 1g / L of methyldiketone into 511.11mg / L, 525.76mg / L, 540.72mg / L and 403.52mg / L of nandrolone, respectively. The yields of the single mutant H164V, single mutant T151A and double mutant H164V / T151A are 1.27 times, 1.30 times and 1.34 times that before mutation, respectively, providing a basis for the industrial production of nandrolone by microorganisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1This is a diagram for the verification of the expression of recombinant Escherichia coli 17β-HSDcl-H164V / T151A protein (A: SDS-PAGE analysis of 17β-HSDcl-H164V / T151A; M: protein marker; 1: blank crude enzyme solution; 2: blank crushed precipitate; 3: 17β-HSDcl-H164V / T151A crude enzyme solution; 4: 17β-HSDcl-H164V / T151A crushed precipitate; B: WB analysis of 17β-HSDcl-H164V / T151A; M: protein marker; 1: 17β-HSDcl-H164V / T151A crude enzyme solution; 2: 17β-HSDcl-H164V / T151A crushed precipitate).

[0037] Figure 2 This is the optimum temperature for 17β-HSDcl-H164V / T151A.

[0038] Figure 3 This is the optimal pH for 17β-HSDcl-H164V / T151A.

[0039] Figure 4 This is the HPLC analysis of the substrate methyldiketone and the product nandrolone in the recombinant Escherichia coli whole cell transformation culture medium.

[0040] Figure 5 This is the mass spectrometry analysis of nandrolone produced in the whole cell transformation broth of recombinant Escherichia coli.

[0041] Figure 6 This is the hydrogen spectrum analysis of the product nandrolone in the recombinant Escherichia coli whole cell transformation solution.

[0042] Figure 7 This is the carbon spectrum analysis of the product nandrolone in the recombinant Escherichia coli whole cell transformation solution. DETAILED DESCRIPTION

[0043] Example

[0044] The present invention will be further described below with reference to specific examples so that those skilled in the art can better understand the present invention. The specific material ratios, process conditions and results described in the examples are only for illustrating the present invention and cannot be used as limiting the content or scope of the present invention.

[0045] The culture medium and detection method involved in the following examples are as follows:

[0046] LB medium: 5 g / L yeast powder, 10 g / L peptone, 10 g / L sodium chloride (2% agar powder is added to the solid medium).

[0047] HPLC detection conditions: chromatographic column Agilent TC-C18, 4.6×250mmol / L, 5μm; mobile phase: V 乙腈 :V 水 =7:3 (V / V); column temperature: 30°C; UV detection wavelength: 254 nm; flow rate: 0.5 mL / min; injection volume: 10 μL.

[0048] Example 1: Crystal structure and molecular docking analysis of 17β-HSDcl

[0049] The 17β-hydroxysteroid dehydrogenase gene (GenBank: AAR04485.1) is derived from the fungus Cochliobolus lunatus. Its amino acid sequence is shown in SEQ ID NO. 1, and the nucleotide sequence encoding the amino acid sequence is shown in SEQ ID NO. 2.

[0050] The crystal structure of 17β-HSDcl (PDB ID: 4FJ2) was obtained from the PDB database. The ligand and water molecules were removed in Pymol to isolate the protein monomer. Molecular docking of the protein receptor with the methyldiketone small molecule ligand was performed using Autodock Vina. Analysis and screening revealed that histidine at position 164 and threonine at position 151 were the key active sites of 17β-HSDcl in this study.

[0051] Example 2: Construction of Escherichia coli BL21(DE3) / pET28a-17β-HSDcl-H164V, BL21(DE3) / pET28a-17β-HSDcl-T151A and BL21(DE3) / pET28a-17β-HSDcl-H164V / T151A strains

[0052] (1) Using a site-directed mutagenesis kit, primers were designed as shown in Table 1. PCR was performed using pET28a-17β-HSDcl as the template. PCR conditions were as follows: 95°C for 3 min, 34 cycles (95°C for 30 s, 58°C for 30 s, 72°C for 1.5 min), and 72°C for 10 min. PCR amplification system consisted of 1 μL template, 1 μL each of upstream and downstream primers, 20 μL Prime Star Max (Premix) DNA, and 17 μL ddH2O.

[0053] (2) Purify and recover the PCR product using a gel recovery kit and set aside.

[0054] (3) Take out the competent E. coli BL21 (DE3) stored at -20℃ and place it on ice for 10 minutes to dissolve.

[0055] (4) Add 10 μL of recovered product, mix well, and let stand on ice for 30 min.

[0056] (5) Add 1 mL of LB medium and shake at 37°C for 45 minutes to revive.

[0057] (6) Centrifuge at 6000 rpm for 1 min, discard 1 mL of supernatant, mix the remaining 110 μL, and apply it to a kanamycin-resistant plate.

[0058] (7) The colonies grown on the kanamycin-resistant plates were picked and cultured in shake flasks. The plasmids were extracted for single and double enzyme digestion verification. The correct transformants were inoculated into liquid LB medium. The single-site mutant Escherichia coli BL21(DE3) / pET28a-17β-HSDcl-H164V strain, Escherichia coli BL21(DE3) / pET28a-17β-HSDcl-T151A strain, and double-site mutant Escherichia coli BL21(DE3) / pET28a-17β-HSDcl-H164V / T151A strain were successfully constructed.

[0059] Table 1 Mutation primers

[0060]

[0061] Example 3: Expression and purification of 17β-HSDcl-H164V, 17β-HSDcl-T151A and 17β-HSDcl-H164V / T151A in Escherichia coli

[0062] After activation, the recombinant E. coli was cultured in 50 mL of liquid LB at 37°C and 220 rpm until the OD 600 0.8-1.0, 0.5mmol·L -1 IPTG was added to a final concentration and expression was induced at 25°C, 220 rpm for 12 h. The cells were harvested by centrifugation at 8000 rpm for 5 min at 4°C. The cells were resuspended in 5 mL of PBS and disrupted by freeze-grinding. The supernatant (crude enzyme solution) was isolated by centrifugation at 12000 rpm for 5 min. The 6×His-tagged protein was purified using a kit (Shanghai Sangon Biotechnology Co., Ltd., China).

[0063] (1) Take 1mL of 50% BeyoGold TM His-tag Purification Resin (reduction-resistant chelating agent) was centrifuged at 1000 g for 10 s at 4°C. The storage solution was discarded. One column volume of non-denaturing lysis buffer was added to the gel and mixed to equilibrate the gel. The gel was centrifuged at 1000 g for 10 s at 4°C. The liquid was discarded. The equilibration was repeated 1-2 times and the liquid was discarded.

[0064] (2) Add 4 mL of crude enzyme solution to every 0.5 mL of gel, mix well, and shake slowly on a shaker at 4°C for 60 min to fully bind the His-tagged target protein.

[0065] (3) Mix the crude enzyme solution and BeyoGold TM The mixture of His-tag Purification Resin (reduction-resistant chelating agent) is loaded into an appropriate empty column tube, and the cover at the bottom of the purification column is opened to allow the flow-through to flow out. Part of the flow-through can be collected for subsequent analysis.

[0066] (4) Add 1-2 column volumes of non-denaturing washing buffer and wash the column 5 times.

[0067] (5) Use a non-denaturing eluent for elution, and the collected eluate is the purified target protein sample.

[0068] Example 4: Enzymatic Properties Analysis of 17β-HSDcl-H164V, 17β-HSDcl-T151A, and 17β-HSDcl-H164V / T151A

[0069] The purified target protein sample obtained in Example 3 was tested for protease activity by NADPH consumption. A 200 μL reaction system included 0.5 mmol / L NADPH, 0.2 mmol / L methyldiketone dissolved in 2% methanol, 0.1 mmol / L purified 17β-HSDcl, and a buffer solution to complete the system. The OD was measured by a microplate reader. 340 changes.

[0070] (1) Determination of the optimal reaction temperature: 200 μL of the reaction system was placed in a shaker at different temperatures, ranging from 15 to 40°C. Three parallel experiments were set at each temperature, and a blank control was also set to measure the effect of temperature on enzyme activity.

[0071] The optimal reaction temperature of 17β-HSDcl is shown in Figure 2 As shown, it has a good reaction effect at 20-30°C, especially better at 23-27°C, and its optimal reaction temperature is 25°C.

[0072] (2) Determination of the optimal reaction pH: Different pH reaction buffers (pH 4.0-6.0: sodium hydrogen phosphate-citric acid buffer, pH 7.0-8.0: phosphate buffer, pH 9.0-10.0: sodium carbonate-sodium bicarbonate buffer) were used in a 200 μL reaction system. The OD was immediately measured after the reaction at 25°C for 30 min. 340 The effect of different pH on enzyme activity was determined.

[0073] The optimal reaction pH of 17β-HSDcl is as follows Figure 3 As shown, it has a good reaction effect between pH 7-8, especially between pH 7.3-7.7, and its optimal reaction pH is 7.5.

[0074] Example 5: Recombinant E. coli catalyzes the production of nandrolone by methyldiketone

[0075] The E. coli BL21(DE3) / pET28a-17β-HSDcl-H164V, BL21(DE3) / pET28a-17β-HSDcl-T151A and BL21(DE3) / pET28a-17β-HSDcl-H164V / T151A strains constructed in Example 2 were inoculated into 10 mL of LB medium and cultured at 37°C for 12 h. A 2% (v / v) inoculum was inoculated into 50 mL of LB medium and cultured at 37°C until the OD 600 = about 0.8. The final concentration is 0.5mmol·L -1 The reaction was induced by IPTG, and the substrate methyldiketone was added at a final concentration of 1 g / L for conversion. The substrate cosolvent was DMSO at a final concentration of 12% (v / v). The conversion temperature was 25° C., the conversion pH was 7.5, and the conversion time was 24 h.

[0076] Take 1mL of the transformed fermentation broth in a centrifuge tube, add 1mL of ethyl acetate, and break the cells by freeze-grinding to extract the product. Evaporate the ethyl acetate, re-dissolve in acetonitrile, and analyze the product by HPLC. The results are as follows: Figure 4 shown.

[0077] The substrate conversion rate of the unmutated group BL21(DE3) / pET28a-17β-HSDcl was: 1 g / L methyldione could be converted into 403.52 mg / L nandrolone;

[0078] The substrate conversion rates of the mutant groups BL21(DE3) / pET28a-17β-HSDcl-H164V, BL21(DE3) / pET28a-17β-HSDcl-T151A and BL21(DE3) / pET28a-17β-HSDcl-H164V / T151A were: 1 g / L methyldione could be converted into 511.11 mg / L, 525.76 mg / L and 540.72 mg / L nandrolone, which were 1.27, 1.30 and 1.34 times that before mutation, respectively.

[0079] The above results indicate that 17β-hydroxysteroid dehydrogenase 17β-HSDcl can be used for biotransformation to synthesize nandrolone, and its mutants 17β-HSDcl-H164V, 17β-HSDcl-T151A and 17β-HSDcl-H164V / T151A have higher biotransformation efficiency.

[0080] Example 6: Isolation and purification of the product nandrolone

[0081] Take 50 mL of the fermentation broth from Example 5, extract the product with ethyl acetate and concentrate to 1-2 mL. Apply pre-TLC to separate and purify the product, the developing solvent is n-hexane: acetone = 2:1, scrape the silica gel at the target band, extract the product with ethyl acetate, and sonicate for 30 minutes. Remove the silica gel by suction, evaporate the solvent, and send it for nuclear magnetic resonance. The hydrogen spectrum results are as follows Figure 6 As shown, the δ H 5.71 (s, 1H) is the hydrogen proton signal of the olefin bond; δ H 4.47 (d, J = 4.8 Hz, 1H) is the active hydrogen proton signal of the hydroxyl group, which is split by the hydrogen of the oxygen-linked methine, and the peak type is d peak; δ H 3.45 (td, J = 8.5, 4.7 Hz, 1H) is the signal of the hydrogen proton of the oxygen-linked methylene, which is split by the active hydrogen of the hydroxyl group and the adjacent methylene hydrogen, and the peak type is td peak; δ H 0.69 (s, 3H) is the signal of methyl hydrogen proton. Figure 7 As shown, the δ C 198.39 is the ketone carbonyl carbon signal; δ C 166.85, 123.72 are both olefinic carbon signals; δ C 79.94 is the signal of a tertiary carbon connected to oxygen. Combining the above H- and C-spectrum data and comparing them with the H- and C-spectrum of a nandrolone standard, it can be confirmed that the product is nandrolone.

[0082] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A 17β-hydroxysteroid dehydrogenase mutant, characterized in that The amino acid sequence is shown in SEQ ID NO.3, or the amino acid sequence is shown in SEQ ID NO.5, or the amino acid sequence is shown in SEQ ID NO.

7.

2. A 17β-hydroxysteroid dehydrogenase mutant according to claim 1, characterized in that: The 17β-hydroxysteroid dehydrogenase mutant is obtained by mutating the histidine at position 164 of the 17β-hydroxysteroid dehydrogenase 17β-HSDcl as shown in SEQ ID NO.1 to valine, or mutating the threonine at position 151 to alanine, or mutating the histidine at position 164 to valine and the threonine at position 151 to alanine.

3. The 17β-hydroxysteroid dehydrogenase mutant according to claim 1, characterized in that: The 17β-hydroxysteroid dehydrogenase is derived from the fungus Cochliobolus lunatus.

4. A gene encoding a 17β-hydroxysteroid dehydrogenase mutant, characterized in that: Its nucleotide sequence is any one of the following: a) a nucleotide sequence as shown in SEQ ID NO.4, or SEQ ID NO.6, or SEQ ID NO.8; b) a nucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO.3, or SEQ ID NO.5, or SEQ ID NO.7 or a nonsense mutation sequence thereof.

5. A carrier, characterized in that Contains the coding gene as claimed in claim 4.

6. A genetically engineered strain, characterized in that: The genetically engineered strain is integrated with the coding gene according to claim 4, or contains the vector according to claim 5.

7. The genetically engineered strain according to claim 6, characterized in that The host microorganism of the engineering strain includes Escherichia coli BL21 (DE3).

8. Use of the 17β-hydroxysteroid dehydrogenase mutant according to any one of claims 1 to 3, or the vector according to claim 5, or the genetically engineered strain according to claim 6 or 7 in the preparation of nandrolone.

9. The use according to claim 8, characterized in that The preparation of nandrolone is carried out by using methyldiketone as a substrate and converting it into nandrolone.

10. A method for producing nandrolone by biotransformation, characterized in that: Using methyldione as substrate, under the action of 17β-hydroxysteroid dehydrogenase mutant, it is converted into nandrolone, among which, The amino acid sequence of the 17β-hydroxysteroid dehydrogenase mutant is shown in SEQ ID NO.3, or SEQ ID NO.5, or SEQ ID NO.

7.

11. The method according to claim 10, characterized in that The method specifically comprises the following steps: adding IPTG at a final concentration of 0.2-0.7 mmol / L to an LB medium containing a genetically engineered strain for induction, adding a substrate methyldiketone at a final concentration of 0.5-1 g / L for conversion, the substrate cosolvent being dimethyl sulfoxide (DMSO) at a final concentration of 10-16% (v / v), the conversion temperature being 20-30°C, the conversion pH being 7-8, and the conversion time being 12-24 hours, wherein the genetically engineered strain contains a gene encoding an amino acid sequence as shown in SEQ ID NO.3, SEQ ID NO.5, or SEQ ID NO.

7.

12. The method according to claim 11, characterized in that The conversion temperature is 23-27°C, the conversion pH is 7.3-7.7, and the conversion time is 12-24h.

13. The method according to claim 12, characterized in that The conversion temperature was 25°C, the conversion pH was 7.5, and the conversion time was 24 h.

14. The method according to any one of claims 10 to 13, characterized in that: The conversion rate of the substrate methyldiketone was higher than 50%.

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