17beta-hydroxysteroid dehydrogenase mutant, engineering bacterium and application

Abstract

The invention discloses a 17 beta-hydroxysteroid dehydrogenase mutant, an engineering bacterium and application, methyl diketone is used as a substrate, and nandrolone is prepared by biotransformation of the 17 beta-hydroxysteroid dehydrogenase mutant. Wherein the 17beta-hydroxysteroid dehydrogenase is 17beta-hydroxysteroid dehydrogenase (17beta-HSDcl) derived from fungus Cochliobolus lunatus, and the mutant of the 17beta-hydroxysteroid dehydrogenase is a single mutant obtained by mutating histidine His at the 164 site of the 17beta-hydroxysteroid dehydrogenase into valine Val, a single mutant obtained by mutating threonine Thr at the 151 site of the 17beta-hydroxysteroid dehydrogenase into alanine Ala, or a double mutant obtained by jointly mutating the 164 site and the 151 site of the 17beta-hydroxysteroid dehydrogenase. According to the invention, pET28a is used as an expression plasmid, and heterologous expression of a 17 beta-HSDcl mutant in E.coli BL21 (DE3) is realized. After 24-hour whole-cell transformation of the engineering bacteria, the nandrolone yields of a single mutant H164V, a single mutant T151A and a double mutant H164V / T151A are respectively 1.27 times, 1.30 times and 1.34 times that of the single mutant H164V, the single mutant T151A and the double mutant H164V / T151A before mutation. The invention provides a foundation for industrial production of nandrolone by microorganisms.

Description

Technical Field

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

[0002] Steroid hormone drugs are a class of drugs that contain a cyclopentane polyhydrogen phenanthrene nucleus structure in their molecules. Currently, there are more than 400 steroid hormone drugs produced worldwide, mainly including adrenal cortical hormones and sex hormones. They are widely used in the fields of anti-tumor, anti-inflammatory, anti-allergic, immune regulation and treatment of reproductive developmental diseases. They are 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 phosphorus deposition, and promotes bone tissue growth. Nandrolone's protein anabolic effect is 12 times that of testosterone, and it can also inhibit glucocorticoids. Low doses of Nandrolone can significantly increase nitrogen storage in muscles, increase mineral content in bones, and enhance collagen synthesis. It is mostly used in medicine to treat muscle injuries and osteoporosis, and can also be used as a sports nutrition supplement.

[0004] At present, the synthesis of nandrolone mostly adopts chemical methods, involving multiple steps such as etherification and reductive hydrolysis. This method has low selectivity, many by-products, pollutes the environment, and involves highly toxic organic reagents and compounds. However, the microbial transformation method can achieve highly specific synthesis of nandrolone, and the process is simpler and greener.

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

[0006] Therefore, studying and modifying 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 purpose of the present invention is to provide a 17β-hydroxysteroid dehydrogenase mutant, an engineered bacterium and its application. Specifically, the highly active mutants 17β-HSDcl-H164V, 17β-HSDcl-T151A and 17β-HSDcl-H164V / T151A were obtained by analyzing the active site of the enzyme, and the enzymatic properties of the mutants were studied; the mutants were heterologously expressed in Escherichia coli, and the engineered strains E.coli BL21(DE3) / pET28a-17β-HSDcl-H164V, E.coliBL21(DE3) / pET28a-17β-HSDcl-T151A and E.coliBL21(DE3) / pET28a-17β-HSDclH164V / T151A were successfully constructed, and the engineered strains were used to realize the conversion from methyl diketone to nandrolone, which provides useful guidance for the industrial production of nandrolone by 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 a starting amino acid sequence as shown in SEQ ID NO.1 into valine, or mutating the threonine at position 151 into alanine, or mutating the histidine at position 164 into valine and the threonine at position 151 into 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 by chemical transformation;

[0023] (3) The colonies grown on the LB and kanamycin pressure plates were picked, cultured in shake flasks, and the plasmids were extracted for single and double enzyme digestion verification to obtain the 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 the use of the 17β-hydroxysteroid dehydrogenase mutant, or the vector, or the recombinant engineering strain microorganism to transform methyl diketone 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 methyl diketone 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 methyl diketone 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 or SEQ ID NO.5 or SEQ ID NO.7.

[0029] In one embodiment of the present invention, the specific transformation steps for producing nandrolone by bioconversion are as follows: adding IPTG with a final concentration of 0.2-0.7 mmol / L to the LB medium containing the genetically recombinant engineered strain for induction, adding the substrate methyl diketone with a final concentration of 0.5-1 g / L for transformation, the substrate co-solvent is DMSO with a final concentration of 10-16% (v / v), the transformation temperature is 20-30°C, the transformation pH is 7-8, and the transformation time is 12-24h, wherein the genetically engineered strain contains a gene encoding an amino acid sequence as 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 24h.

[0032] In one embodiment of the present invention, the conversion rate of the substrate methyl diketone 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 it into a 250 mL container containing 50 mL LB medium at an inoculum of 1-2%, and culture at 35-37 ° C until the OD 600 =0.6-0.8, 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, and culture at 37°C until the OD 600 =0.8, and then transferred to 25℃ for induction culture for 24h.

[0035] Beneficial effects: The present invention determines the amino acids in the active center of 17β-HSDcl through molecular docking simulation, and performs site-directed mutation on key amino acid residues, thereby improving the catalytic activity of 17β-HSDcl. The modified 17β-HSDcl mutant is heterologously expressed in Escherichia coli BL21 (DE3) and successfully applied to the conversion of methyl diketone 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 methyl diketone into 511.11mg / L, 525.76mg / L, 540.72mg / L and 403.52mg / L 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 It is the optimum temperature of 17β-HSDcl-H164V / T151A.

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

[0039] Figure 4 HPLC analysis of substrate methyldione and product nandrolone in the whole cell transformation solution of recombinant Escherichia coli.

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

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

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

[0043] Example

[0044] The present invention is further described below in conjunction with 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 used to illustrate 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: 254nm; flow rate: 0.5mL / min; injection volume: 10μL.

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

[0049] The gene of 17β-hydroxysteroid dehydrogenase (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 was obtained from the PDB database (PDB ID: 4FJ2). The ligand and water molecules were removed in Pymol to separate the protein monomer structure. AutodockVina was used to perform molecular docking between the protein receptor and the methyl diketone small molecule ligand, and the histidine at position 164 and the threonine at position 151 were selected as 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 the site-directed mutagenesis kit, the primers were designed as shown in Table 1, and PCR was performed using pET28a-17β-HSDcl as the template. The 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: 1 μL template, 1 μL upstream and downstream primers each, 20 μL Prime Star Max (Premix) DNA, ddH 2 O 17μL.

[0053] (2) Purify and recover the PCR product using a gel recovery kit for later use.

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

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

[0056] (5) Add 1 mL of LB medium and shake at 37°C for 45 min 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 resistance plate.

[0058] (7) Pick the colonies grown on the kanamycin-resistant plate, culture in shake flasks, extract the plasmids for single and double enzyme digestion verification, and inoculate the correct transformants into liquid LB culture 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 the final concentration and induced for 12 h at 25 °C and 220 rpm. The cells were harvested by centrifugation at 8000 rpm for 5 min at 4 °C. 5 mL of PBS was added to resuspend the cells, and the recombinant E. coli was broken by freezing and grinding, and the supernatant (i.e., crude enzyme solution) was separated by centrifugation at 12000 rpm for 5 min. The protein with 6×His tag was purified according to the kit (Shanghai Sangon Biotechnology Co., Ltd., China).

[0063] (1) Take 1 mL of 50% BeyoGold TM His-tag Purification Resin (reducing-resistant chelating agent), centrifuge at 1000g for 10s at 4°C, discard the storage solution, add one column volume of non-denaturing lysis buffer to the gel and mix well to balance the gel, centrifuge at 1000g for 10s at 4°C, discard the liquid, repeat the balance 1-2 times, and discard the liquid.

[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 to 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 wash 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: Analysis of Enzymatic Properties 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 methyl diketone dissolved in 2% methanol, 0.1 mmol / L purified 17β-HSDcl, and a buffer solution to complete the system. The OD was measured by an ELISA 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 determine 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 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 measured immediately 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 methyl diketone

[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 LB medium and cultured at 37°C for 12 h. The inoculum was inoculated into 50 mL LB medium at a 2% (v / v) inoculum and cultured at 37°C until OD 600 = about 0.8. The final concentration is 0.5mmol·L -1 The reaction mixture was induced by IPTG, and the substrate methyl diketone was added at a final concentration of 1 g / L for transformation. The substrate cosolvent was DMSO at a final concentration of 12% (v / v). The transformation temperature was 25° C., the transformation pH was 7.5, and the transformation time was 24 h.

[0076] Take 1 mL of the fermentation broth after transformation into a centrifuge tube, add 1 mL of ethyl acetate, break the cells by freezing and grinding, and 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 methyl diketone 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: 1g / L methyl diketone could be converted into 511.11mg / L, 525.76mg / L and 540.72mg / 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 synthesis of nandrolone, and its mutants 17β-HSDcl-H164V, 17β-HSDcl-T151A and 17β-HSDcl-H164V / T151A have higher biotransformation efficiency.

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

[0081] Take 50 mL of the fermentation broth in 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 ultrasonicate for 30 minutes. Remove the silica gel by suction filtration, 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 methylene group, and the peak type is d peak; δ H 3.45 (td, J = 8.5, 4.7 Hz, 1H) is the signal of the oxygen-linked methylene hydrogen proton, 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 carbon spectrum C 198.39 is the signal of ketone carbonyl; δ C 166.85, 123.72 are both olefinic carbon signals; δ C 79.94 is the signal of tertiary carbon connected to oxygen. Combining the above hydrogen spectrum and carbon spectrum data and comparing with the hydrogen and carbon spectrum of the standard nandrolone, it can be determined 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 principle 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, and / or the amino acid sequence is shown in SEQ ID NO.5, and / 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 into valine, or mutating the threonine at position 151 into alanine, or mutating the histidine at position 164 into valine and the threonine at position 151 into alanine. Preferably, the 17β-hydroxysteroid dehydrogenase is derived from the fungus Cochliobolus lunatus.

3. 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.

4. A carrier, characterized in that Contains the coding gene as claimed in claim 3.

5. A genetically engineered strain, characterized in that: The genetically engineered strain is integrated with the coding gene as claimed in claim 3, or contains the vector as claimed in claim 4.

6. The genetically engineered strain according to claim 5, characterized in that The host microorganism of the engineering strain includes Escherichia coli BL21 (DE3).

7. Use of the 17β-hydroxysteroid dehydrogenase mutant according to claim 1 or 2, or the vector according to claim 4, or the genetically engineered strain according to claim 5 or 6 in the preparation of nandrolone Preferably, the preparation of nandrolone is to use methyl diketone as a substrate and convert it into nandrolone.

8. A method for producing nandrolone by biotransformation, characterized in that: Using methyl diketone 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.

9. The method according to claim 8, characterized in that Specifically comprising the following steps: adding IPTG with a final concentration of 0.2-0.7 mmol / L to induce in LB medium containing genetically engineered strains, adding substrate methyl diketone with a final concentration of 0.5-1 g / L for transformation, the substrate cosolvent is dimethyl sulfoxide DMSO with a final concentration of 10-16% (v / v), the transformation temperature is 20-30°C, the transformation pH is 7-8, and the transformation time is 12-24h, wherein the genetically engineered strain contains a gene encoding an amino acid sequence as shown in SEQ ID NO.1, or SEQ ID NO.3, or SEQ ID NO.5, or SEQ ID NO.7, preferably, the transformation temperature is 23-27°C, the transformation pH is 7.3-7.7, and the transformation time is 12-24h, Preferably, the conversion temperature is 25° C., the conversion pH is 7.5, and the conversion time is 24 h.

10. The method according to claim 8 or 9, characterized in that: The conversion of the substrate methyl diketone was higher than 50%.

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