3-ketosteroid-delta1-dehydrogenase mutant and application thereof in catalyzing dehydrogenation of steroid compound

Abstract

The invention discloses a 3-ketosteroid-delta1-dehydrogenase mutant and application of the 3-ketosteroid-delta1-dehydrogenase mutant in catalyzing dehydrogenation of a steroid compound, the mutant with high catalytic activity is obtained through screening, and the solubility of a substrate is increased by adding a cosolvent Tween-80, so that the biological catalytic efficiency is improved. Under the optimal condition, the mutant disclosed by the invention can improve the feeding amount conversion rate of 15g / L of (16 alpha)-methyl androstane-4, 9-diene-3, 17-diketone to 99.9%.

Description

(1) Technical Field

[0001] The present invention relates to a 3-sterone-Δ1-dehydrogenase mutant and its application in catalyzing the dehydrogenation of C1 and C2 positions of steroid compounds. (2) Background Art

[0002] Steroid drugs are a class of drugs named according to their chemical structure, referring to drugs with the "cyclopentane and polyhydrophenanthrene" parent nucleus structure in their molecular structure. Clinically, steroid hormones are the second largest class of drugs in terms of dosage, second only to antibiotics, and are an indispensable class of exogenous hormones. In clinical use, steroid drugs are mainly divided into three categories: adrenal cortical hormones, sex hormones, and anabolic hormones. Steroid drugs play an important role in the chemical drug system. Currently, more than 400 steroid drugs have been produced globally, and the market demand for steroid drugs has ranked among the top for many years.

[0003] Microbe-mediated steroid transformation involves using microbial cells or their enzyme systems to perform specific biochemical modifications on steroid compounds. This biocatalytic process specifically modifies a particular site of the steroid, generating novel compounds or intermediates with similar structures but enhanced physiological activities. Compared with traditional chemical synthesis, microbial transformation offers multiple advantages: it reduces the synthesis steps, shortens the production cycle, increases the yield, while reducing the frequency of side reactions, and the reaction conditions are milder and environmentally friendly. In addition, microbial transformation also has excellent stereoselectivity and regioselectivity, and can perform special reactions that are difficult or impossible to achieve by traditional chemical methods, such as dehydrogenation reactions at the C1,2 positions of the A ring, hydroxylation at the 11α and 11β positions, etc. With the continuous maturity of the microbial transformation process, the steroid drug synthesis production route has gradually been upgraded and transformed into the process route of "phytosterol - androstenedione - steroid drugs", and has gradually become the mainstream industrial production method in the steroid drug industry.

[0004] The dehydrogenation of the 1,2 positions of (16α)-methyl-4,9-androstadiene-3,17-dione is an important step in the synthesis of the drug dexamethasone. Dexamethasone is a glucocorticoid hormone, and its derivatives include hydrocortisone, prednisone, etc. Its pharmacological effects are mainly anti-inflammatory, anti-toxic, anti-allergic, and anti-rheumatic. It is widely used clinically, is easily absorbed from the digestive tract, and has a lower plasma protein binding rate compared to other corticosteroid drugs. It has good anti-inflammatory and immunosuppressive effects and can be used to treat various diseases such as bronchospasm, allergy, shock, ulcerative colitis, etc.

[0005] Directed evolution is also considered as irrational design and is one of the most commonly used protein engineering techniques. Directed evolution of enzymes refers to the in vitro simulation of natural evolution under experimental conditions. Through multiple rounds of repeated mutation and directed screening, new enzyme mutants that meet the expectations can be finally obtained. The two most important processes in directed evolution are: one is to introduce random mutations into the target gene to construct a mutant library; the other is to perform high-throughput screening on the constructed library. Directed evolution randomly mutates the entire sequence of the target protein, which enables the screening of even amino acids that are far from the protein active site. Compared with rational design, directed evolution does not require knowledge of the three-dimensional structure of the target protein nor relevant structure-function information. In addition, directed evolution randomly mutates the entire sequence, including amino acid sites that are far from the active site but are important for maintaining the structure, stability, catalytic properties of the active site, etc. of the entire enzyme, which has more possibilities for enzyme modification.

[0006] 3-Sterone-Δ1-dehydrogenase PrKstD from Propionibacterium sp has a length of 1557 bp and encodes 518 amino acids. Its specific activity is higher than that of other dehydrogenases and it has a broad substrate spectrum. The catalytic performance of PrKstD was characterized and it was found that it can convert 3-ketosteroid compounds substituted at C9, C10, C11 and C17 positions into inactive C6-substituted compounds through substrates without a C4-C5 double bond, such as 11β,17-dihydroxy-6α-methyl-1,4-diene-3,20-dione. In addition, PrKstD also has corresponding catalytic activities for C6 substitution and C11 substitution; through the analysis of its protein crystal structure, the active pocket of PrKstD is more open than that of other KstDs, which is convenient for the enzyme to bind to the substrate. Since the double bond of the substrate (16α)-methyl-androst-4,9-diene-3,17-dione at C9-C11 inhibits the dehydrogenase, the double bond will bind to the amino acid residues in the active pocket, which is not conducive to the release of the substrate after dehydrogenation. Therefore, the directed evolution method was used to modify the enzyme molecule to obtain mutants that can efficiently catalyze the dehydrogenation of (16α)-methyl-androst-4,9-diene-3,17-dione. (III) Summary of the Invention

[0007] The object of the present invention is to provide a 3-sterone-Δ1-dehydrogenase mutant and its application in catalyzing the dehydrogenation of C1 and C2 positions of steroid compounds. In the present invention, a mutant library of the 3-sterone-Δ1-dehydrogenase PrKstD gene is obtained by error-prone PCR technology of directed evolution, and then the mutant library is screened by high-throughput screening method to obtain a mutant that can efficiently catalyze the dehydrogenation of (16α)-methyl-androst-4,9-diene-3,17-dione to produce (16α)-16-methyl-androst-1,4,9(11)-triene-3,17-dione; the present invention also optimizes the conditions for induced culture of the mutant strain and the catalytic reaction system and conditions, further improving the stability and activity of the enzyme, thereby improving the biocatalytic efficiency, solving the problem of poor water solubility of the substrate, and opening up a green, environmental protection, safe, efficient and low-cost biocatalytic path for the dehydrogenation of (16α)-methyl-androst-4,9-diene-3,17-dione.

[0008] The technical solution adopted by the present invention is as follows:

[0009] In the first aspect, the present invention provides a 3-sterone-Δ1-dehydrogenase mutant, and the amino acid sequence of the mutant is as shown in SEQ ID NO: 3.

[0010] The 3-sterone-Δ1-dehydrogenase mutant of the present invention is obtained by mutating the 3-sterone-Δ1-dehydrogenase PrKstD derived from Propionibacterium sp by error-prone PCR technology and then screening by high-throughput screening. The nucleotide sequence of the gene encoding the 3-sterone-Δ1-dehydrogenase is as shown in SEQ ID NO: 1.

[0011] In the second aspect, the present invention provides a gene encoding a 3-sterone-Δ1-dehydrogenase mutant, and the nucleotide sequence of the encoding gene is as shown in SEQ ID NO: 2.

[0012] In the third aspect, the present invention provides a recombinant expression vector containing the gene encoding the 3-sterone-Δ1-dehydrogenase mutant, and the recombinant expression vector is based on the plasmid pET-21a(+).

[0013] In the fourth aspect, the present invention provides a recombinant genetic engineering bacterium prepared by transforming the recombinant expression vector, and the engineering bacterium uses Escherichia coli BL21(DE3) as the host bacterium.

[0014] In the fifth aspect, the present invention provides an application of the 3-sterone-Δ1-dehydrogenase mutant in catalyzing the dehydrogenation of C1 and C2 positions of steroid compounds. Specifically, the application is the application of catalyzing the synthesis of (16α)-16-methyl-androst-1,4,9(11)-triene-3,17-dione from (16α)-methyl-androst-4,9-diene-3,17-dione.

[0015] Furthermore, the method of the application is as follows: using the wet bacteria obtained by inducing and culturing the recombinant genetic engineering bacteria containing the 3-sterone-Δ1-dehydrogenase mutant encoding gene or the crude enzyme solution extracted by ultrasonic disruption of the wet bacteria as the catalyst, using the steroid compound (16α)-methyl-androst-4,9-diene-3,17-dione as the substrate, using phenazine methosulfate (PMS) as the proton acceptor, using flavin adenine dinucleotide (FAD) as the coenzyme, adding a cosolvent, and using a buffer solution with a pH of 6-10 as the reaction medium to construct a reaction system, reacting at 25-45°C and 180 rpm for 48-120 h (preferably 30°C and 96 h) to obtain a reaction solution of the dehydrogenated compound (16α)-16-methyl-androst-1,4,9(11)-triene-3,17-dione; the cosolvent is β-cyclodextrin, methanol, ethanol, isopropanol or Tween-80.

[0016] Furthermore, the buffer solution is preferably a 50 mM Tris-HCl buffer solution with a pH of 8.0.

[0017] Furthermore, in the reaction system, the final concentration of the substrate added is 5-25 g / L (preferably 5-15 g / L), the final concentration of phenazine methosulfate added is 1 mM, the final concentration of FAD added is 0.2 mM, the dosage of the catalyst is 10-30 g / L (preferably 20 g / L) based on the mass of the wet bacteria; the volume addition concentration of the cosolvent is 5-15% (preferably 10%).

[0018] Furthermore, the cosolvent is preferably Tween-80.

[0019] Furthermore, the catalyst is prepared by the following method:

[0020] Inoculate the recombinant genetic engineering bacteria containing the 3-sterone-Δ1-dehydrogenase mutant encoding gene into the LB liquid medium containing 50 μg / mL ampicillin, culture overnight at 37°C and 180 rpm to obtain a seed solution; inoculate the seed solution into the LB liquid medium containing 50 μg / mL ampicillin at an inoculation amount of 3% by volume, culture at 37°C and 180 rpm until the OD 600 is 0.6-0.8, add IPTG with a final concentration of 0.1 mM, induce and culture at 25°C and 180 rpm for 16 h to obtain a fermentation broth; centrifuge the fermentation broth at 8000 rpm and 4°C for 10 min, resuspend the obtained bacterial cell precipitate with physiological saline, and then centrifuge at 8000 rpm and 4°C for 10 min to collect the wet bacteria;

[0021] The wet bacterial cells were resuspended in 50 mM Tris-HCl buffer (pH = 8.0) at a concentration of 80 g / L. After the resuspended solution was placed on ice for 30 min, it was subjected to ultrasonic disruption (conditions: ultrasonic power 360 W, ultrasonic treatment for 3 s, intermittent for 7 s, ultrasonic treatment for 10 min). After ultrasonic treatment, the disrupted solution was centrifuged at 8000 rpm and 4 °C for 10 min, and the precipitate was discarded. The obtained supernatant was the crude enzyme solution.

[0022] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0023] In the present invention, directed evolution was carried out on 3-oxosteroid-Δ1-dehydrogenase PrKstD derived from Propionibacterium sp., a mutant gene with high catalytic activity towards (16α)-methyl-androst-4,9-diene-3,17-dione was screened and obtained, and it was constructed into a pET-21a(+)-PrKstD-T recombinant plasmid, and further an E. coli BL21(DE3)-pET-21a(+)-PrKstD-T recombinant engineering bacterium was constructed.

[0024] (16α)-Methyl-androst-4,9-diene-3,17-dione is insoluble in water, which reduces the production efficiency of biocatalytic preparation. To solve the problems of poor solubility of organic substrates in water and low mass transfer in the aqueous phase, the present invention adds a co-solvent Tween-80 (10%, v / v) to increase the solubility of the substrate and thus improve the biocatalytic efficiency. Under the optimal conditions, the mutant of the present invention can increase the conversion rate of the feeding amount of (16α)-methyl-androst-4,9-diene-3,17-dione at 15 g / L to 99.9%. (IV) Description of the Drawings

[0025] Figure 1 It is a schematic diagram of PrKstD catalyzing the dehydrogenation of the substrate (16α)-methyl-androst-4,9-diene-3,17-dione to synthesize (16α)-16-methyl-androst-1,4,9(11)-triene-3,17-dione.

[0026] Figure 2 It is the nucleic acid electrophoresis map of colony PCR in step 3 of Example 1. Lane1, 2, 3, 4, 5 represent the PCR products of 5 randomly selected colonies.

[0027] Figure 3 It is the DCPIP standard curve equation of Example 1.

[0028] Figure 4 It is the column chart of the relative enzyme activity of PrKstD at different induction temperatures in Example 2.

[0029] Figure 5 It is the column chart of the relative enzyme activity of PrKstD at different concentrations of IPTG in Example 3.

[0030] Figure 6 Bar graph of the relative enzyme activity of PrKstD at different induction times in Example 4.

[0031] Figure 7 Electrophoresis pattern of the error-prone PCR amplification product and the double-digested product of plasmid pET-21a(+) in Example 5; among them, Lane1, 2, and 3 are the error-prone PCR amplification products, and Lane4, 5, 6, 7, and 8 are the double-digested products of plasmid pET-21a(+).

[0032] Figure 8 Substrate conversion rates of the 10 mutants obtained by primary screening in Example 7 for amplified rescreening. (V) Specific implementation manners

[0033] The present invention will be further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0034] Composition of the culture medium used in the embodiments of the present invention:

[0035] Composition of LB liquid medium: NaCl 10 g / L, peptone 10 g / L, yeast extract 5 g / L, solvent is water, pH 7.0.

[0036] Composition of LB solid medium: NaCl 10 g / L, peptone 10 g / L, yeast extract 5 g / L, agar 20 g / L, solvent is water, pH 7.0.

[0037] Composition of TB medium: tryptone 11.8 g / L, yeast extract 23.6 g / L, K2HPO4 9.4 g / L, KH2PO4 2.2 g / L, glycerol 4 mL / L, solvent is water.

[0038] Unless otherwise specified in the embodiments of the present invention, the solvent of the solution is double-distilled water.

[0039] Example 1: Determination of the enzyme activity of 3-oxosteroid-Δ1-dehydrogenase PrKstD

[0040] 1. Obtaining the target gene of 3-oxosteroid-Δ1-dehydrogenase PrKstD

[0041] Screen the steroid compound C1,2 dehydrogenase by referring to the literature, and select the C1,2 dehydrogenase PrKstD that has a high catalytic potential for (16α)-methyl-androst-4,9-diene-3,17-dione and good enzyme stability. Then, through retrieval in the NCBI database, select the gene sequence of 3-oxosteroid-Δ1-dehydrogenase PrKstD derived from Propionibacterium sp (NCBI accession number: NLT29951.1). Hand over the obtained gene sequence to Beijing Tsingke Biotechnology Co., Ltd., Hangzhou Branch for synthesis. The nucleotide sequence is as shown in SEQ ID NO: 1, and the synthesized gene has a His-tag at the end.

[0042] SEQ ID NO: 1:

[0043]

[0044] 2. Synthesis of Recombinant Plasmid pET-21a(+)-PrKstD

[0045] Entrusted Hangzhou Branch of Beijing Tsingke Biotechnology Co., Ltd. to insert the PrKstD target gene into two restriction enzyme sites, HindIII and BmaHI, of the expression vector pET-21a(+), to obtain the recombinant plasmid pET-21a(+)-PrKstD.

[0046] 3. Construction and Identification of Recombinant Genetic Engineering Bacteria

[0047] Introduce the pET-21a(+)-PrKstD plasmid into competent E. coli BL21(DE3) cells. The specific method is as follows: After thawing the competent E. coli BL21(DE3) cells on ice for 20 min, add 10 μL of the recombinant plasmid pET-21a(+)-PrKstD to 100 μL of competent E. coli BL21(DE3) cells, gently pipette and mix well, then incubate on ice for 30 min, followed by heat shock at 42 °C in a water bath for 90 s. After incubating on ice for 2 min, add 0.9 mL of LB liquid medium without antibiotics, and resuscitate at 37 °C and 180 rpm for 1 h. Centrifuge the resuscitated bacterial solution in a centrifuge, discard 900 μL of the supernatant, pipette and mix well the remaining 100 μL of the bacterial solution with the precipitate, and spread it on an LB solid medium containing 50 μg / mL ampicillin, and culture overnight at 37 °C.

[0048] Randomly pick 5 single colonies from the overnight culture plate, pipette and mix them well in the corresponding numbered 30 μL of ddH2O respectively. Take 10 μL of each mixed solution and transfer it to the corresponding numbered LB liquid medium containing 50 μg / mL ampicillin, culture overnight at 37 °C and 180 rpm. Boil the remaining 20 μL of each mixed solution for 10 min respectively, cool it, and use it as a template for colony PCR, and then verify it by 0.9% nucleic acid agarose gel electrophoresis ( Figure 2 ), to obtain the transformed positive clones.

[0049] The total volume of the colony PCR reaction system is 25 μL: 1 μL of boiled bacterial solution template, 1 μL of primer F, 1 μL of primer R, 12.5 μL of 2×Hieff PCR Master Mix, and 9.5 μL of ddH2O. The PCR amplification program is as follows: ① 94 °C for 5 min, ② 94 °C for 30 s, ③ 55 °C for 30 s, ④ 72 °C for 1 min, ⑤ 72 °C for 10 min, and cycles ②③④ are repeated 30 times. 2×Hieff PCR Master Mix is purchased from Beijing Tsingke Biotechnology Co., Ltd. Primers F and R are designed according to the gene sequence of 3-ketosteroid-Δ1-dehydrogenase PrKstD and synthesized by Hangzhou Branch of Beijing Tsingke Biotechnology Co., Ltd.

[0050] Primer F: 5’-GCGGATCCATGACTCAGACTTGG-3’,

[0051] Primer R: 5’-GTGATGGTGGTTCTTCGCCATATCC-3’.

[0052] 4. Preparation of wet cells and crude enzyme solution

[0053] Inoculate the successfully transformed positive clones in step 3 into 50 mL of LB liquid medium containing 50 μg / mL ampicillin, culture overnight at 37 °C and 180 rpm to obtain a seed solution. Inoculate the seed solution into 150 mL of LB liquid medium containing 50 μg / mL ampicillin at an inoculation amount of 3% (v / v), culture at 37 °C and 180 rpm until the OD 600 is 0.6 - 0.8, add IPTG with a final concentration of 0.1 mM, induce and culture at 25 °C and 180 rpm for 16 h to obtain a fermentation broth. Centrifuge the fermentation broth at 8000 rpm and 4 °C for 10 min, resuspend the obtained cell precipitate with physiological saline, then centrifuge at 8000 rpm and 4 °C for 10 min to collect wet cells.

[0054] Resuspend the wet cells at a ratio of 80 g / L with 50 mM Tris-HCl buffer (pH = 8.0), place the resuspended solution on ice for 30 min, then perform ultrasonic disruption (conditions: ultrasonic power 360 W, ultrasonic for 3 s, intermittent for 7 s, ultrasonic for 10 min). After ultrasonic disruption, centrifuge the disrupted solution at 8000 rpm and 4 °C for 10 min, discard the precipitate, and the obtained supernatant is the crude enzyme solution.

[0055] 5. Enzyme activity detection

[0056] The principle is that 3-oxosteroid-Δ1-dehydrogenase PrKstD reacts with the substrate to remove two hydrogens at its C1,2 positions to form a double bond. The coenzyme PMS will receive the removed hydrogen ions to form PMSH2 to ensure the cyclic progress of the reaction, and PMSH2 will transfer the hydrogen ions to DCPIP to form DCPIPH2. DCPIPH2 has no absorbance at 600 nm. Detecting the consumption of DCPIP can indirectly reflect the enzyme activity of PrKstD.

[0057] Use a spectrophotometer to measure the activity of 3-oxosteroid-Δ1-dehydrogenase PrKstD according to the following method:

[0058] The reaction system for determining the activity of PrKstD enzyme is 2 mL in final concentration composition: 0.5 mM (16α)-methyl-androst-4,9-diene-3,17-dione, 5% methanol (v / v), 150 μM 2,6-dichlorophenol indophenol (DCPIP), 1.5 mM phenazine methosulfate (PMS), 50 mM Tris-HCl buffer (pH 8.0) made up to 2 mL. After incubating at 30 °C for 1 minute, an appropriate amount of crude enzyme solution prepared by the above method is added, quickly mixed, the change in absorbance at 600 nm is detected, and the content of DCPIP is calculated according to the DCPIP standard curve.

[0059] The DCPIP standard curve is prepared under the enzyme activity detection conditions, with the DCPIP concentration as the abscissa and the absorbance at 600 nm as the ordinate, Figure 3 as shown.

[0060] The enzyme activity unit is expressed as U, where 1 U is defined as the amount of enzyme required to reduce 1 μmol of DCPIP per minute at 30 °C and pH 8.0.

[0061] Example 2: Optimization of the induction temperature of E. coli BL21(DE3)-pET-21a(+)-PrKstD recombinant bacteria

[0062] Optimize the induction temperature of E. coli BL21(DE3)-pET-21a(+)-PrKstD. Change the induction temperatures in step 4 of Example 1 to 16, 20, 25, 30, and 37 °C respectively, and keep other operations the same. Crude enzyme solutions induced at each temperature are prepared. Use the same method as in step 5 of Example 1 to measure the enzyme activity of PrKstD at different induction temperatures, and calculate the relative enzyme activity at other temperatures with the highest enzyme activity as 100%. The results are shown in Figure 4 .

[0063] It can be seen from Figure 4 that the induction temperature has a great influence on the expression of the target protein by the engineered bacteria. At low temperatures, the protein synthesis rate slows down, and the newly generated protein chains have more time to fold correctly, so the formation of inclusion bodies can be reduced, and finally the protein activity is relatively high. At high temperatures, although the protein synthesis rate increases, the folding rate of its protein chains also accelerates, which may lead to misfolding or aggregation of proteins, forming insoluble inclusion bodies, thus affecting the total yield and quality of the protein, and finally the protein activity decreases. When the induction temperature is below 25 °C, the relative enzyme activity of the enzyme increases with the increase of the induction temperature. When the induction temperature is above 25 °C, the relative enzyme activity of the enzyme decreases with the increase of the induction temperature. Therefore, 25 °C is selected as the optimal induction temperature.

[0064] Example 3: Optimization of the concentration of inducer (IPTG) for the recombinant strain E. coli BL21(DE3)-pET-21a(+)-PrKstD

[0065] The concentration of inducer (IPTG) for E. coli BL21(DE3)-pET-21a(+)-PrKstD was optimized. The final concentrations of IPTG in step 4 of Example 1 were changed to 0.05, 0.1, 0.2, 0.5, and 1.0 mM respectively, and other operations were the same. The crude enzyme solutions induced at each concentration were prepared. The enzyme activity of PrKstD at different inducer concentrations was measured by the same method as in step 5 of Example 1. The relative enzyme activity under other concentrations of induction was calculated with the highest enzyme activity as 100%, and the results are shown in Figure 5 .

[0066] From Figure 5 it can be seen that the inducer (IPTG) has a great influence on the expression of the target protein by the engineered bacteria. When the concentration of the inducer is low, the enzyme production is low, and at the same time, the inducer has a certain toxicity to the cells. When the concentration is too high, it will affect the cell growth. Therefore, it is necessary to select an appropriate concentration of the inducer. When the IPTG concentration is below 0.1 mM, the relative enzyme activity of the enzyme increases with the increase of the IPTG concentration. When the IPTG concentration is above 0.1 mM, the relative enzyme activity of the enzyme decreases with the increase of the IPTG concentration. Therefore, 0.1 mM IPTG concentration is selected as the optimal inducer concentration.

[0067] Example 4: Optimization of the induction time for the recombinant strain E. coli BL21(DE3)-pET-21a(+)-PrKstD

[0068] The induction time for E. coli BL21(DE3)-pET-21a(+)-PrKstD was optimized. The induction times in step 4 of Example 1 were changed to 8, 12, 16, 20, and 24 h respectively, and other operations were the same. The crude enzyme solutions at each induction time were prepared. The enzyme activity of PrKstD at different induction times was measured by the same method as in step 5 of Example 1. The relative enzyme activity after induction at other times was calculated with the highest enzyme activity as 100%, and the results are shown in Figure 6 .

[0069] From Figure 6 it can be seen that the induction time has a great influence on the expression of the target protein by the engineered bacteria. With the increase of the induction time, the relative enzyme activity of PrKstD gradually increases; when the induction time reaches 16 h, the relative enzyme activity of PrKstD no longer increases but remains unchanged. Therefore, 16 h induction time is the most suitable.

[0070] Example 5: Construction and optimization of a random mutant library of 3-oxosteroid-Δ1-dehydrogenase PrKstD

[0071] (1) Primer design

[0072] Error-prone PCR primers F and R were designed based on the gene sequence of 3-oxosteroid-Δ1-dehydrogenase PrKstD (SEQ ID NO: 1) and synthesized by Beijing Tsingke Biotechnology Co., Ltd., Hangzhou Branch.

[0073] Primer F: 5’-agcaaatgggtcgcggatccGTGACCGACCAGAACAACATCAC-3’,

[0074] Primer R: 5’-tcgagtgcggccgcaagcttTTAGGTGTGACCTGCAGCG-3’.

[0075] (2) Error-prone PCR amplification

[0076] Using the plasmid pET-21a(+)-PrKstD obtained in step 1 of Example 1 as a template, error-prone PCR amplification of the target gene PrKstD was performed using primers F and R in step (1).

[0077] First, the error-prone PCR system was optimized to obtain an appropriate mutation frequency. By changing the concentrations of Mg 2+ and Mn 2+ , the mutation frequency of error-prone PCR was optimized. Specifically, the concentration gradients of Mg 2+ were: 2, 3, 4 mM; the concentration gradients of Mn 2+ were: 0.05, 0.1, 0.2, 0.3 mM, a total of 12 groups. After PCR amplification, it was verified by 0.9% nucleic acid agarose gel electrophoresis, and then recovered and purified using a Sangon column DNA gel recovery kit. After sequencing, the mutation frequencies of 3‰ - 6‰ were selected: Mg 2+ concentration was 2 mM, Mn 2+ concentration was 0.1 mM (Table 1).

[0078] Secondly, the amplification products of the optimized error-prone PCR system were detected by 0.9% nucleic acid agarose gel electrophoresis, as shown in Figure 7 Lane 1, 2, 3. The target fragment was recovered and purified using a Sangon column DNA gel recovery kit and denoted as PrKstD-T.

[0079] Table 1. Mutation frequencies corresponding to different Mg 2+ and Mn 2+ concentrations

[0080]

[0081] Error-prone PCR amplification system: The total reaction volume is 50 μL, including 0.5 μL of plasmid template, 2 μL of primer F, 2 μL of primer R, 5 μL of 10× Taq Buffer, 1 μL of dNTPs, 0.5 μL of Taq enzyme, 4 μL of 25 mM MgCl2, 0.5 μL of 10 mM MnCl2, and 34.5 μL of ddH2O.

[0082] The error-prone PCR amplification program is as follows: ① 95°C for 5 min, ② 95°C for 15 s, ③ 55°C for 15 s, ④ 72°C for 2 min, ⑤ 72°C for 5 min, and cycles ②③④ are repeated 35 times. Taq enzyme, 10× Taq Buffer, and dNTPs were purchased from Nanjing Novoprotein Scientific Inc.

[0083] (3) Double digestion of pET-21a(+) vector:

[0084] The pET-21a(+) vector was double-digested with Takara restriction enzyme BamHI and Takara restriction enzyme HindIII, and the digestion system was reacted at 37°C for 3 h. After double digestion, it was verified by 0.9% nucleic acid agarose gel electrophoresis ( Figure 7 in Lanes 4, 5, 6, 7, 8), and the digested vector was recovered and purified using a Sangon column DNA gel extraction kit.

[0085] The double digestion reaction system of pET-21a(+) vector is: 2 μL of BamHI, 2 μL of HindIII, 10 μL of 10× K Buffer, and 70 μL of pET-21a(+).

[0086] (4) Construction of mutant library

[0087] The target fragment PrKstD-T in step (2) and the digested vector in step (3) were ligated according to the corresponding ligation system. After the ligation system was reacted at 50°C for 5 min, the obtained pET-21a(+)-PrKstD-T recombinant plasmid was transferred into E. coli BL21 competent cells (purchased from Beijing Tsingke Biotechnology Co., Ltd.), and spread on an LB solid plate containing 50 μg / mL ampicillin and cultured overnight at 37°C.

[0088] The ligation system of the target gene PrKstD-T and the digested vector pET-21a(+) is: 5 μL of 2× Clon ExpressMix, 3.5 μL of vector pET-21a(+), and 1.5 μL of PrKstD-T. Among them, 2× Clon Express Mix was purchased from Nanjing Novoprotein Scientific Inc.

[0089] The method for introducing the pET-21a(+)-PrKstD-T recombinant plasmid into competent E. coli BL21 cells is as follows: After thawing the competent E. coli BL21 cells on ice for 20 min, add 10 μL of the recombinant plasmid to 100 μL of competent E. coli BL21 cells. Gently pipette to mix evenly and then incubate on ice for 30 min. Then, perform a heat shock at 42 °C in a water bath for 90 s. After incubating on ice for 2 min, add 0.9 mL of LB liquid medium without antibiotics and resuscitate at 37 °C and 180 rpm for 1 h. Centrifuge the resuscitated bacterial solution in a centrifuge and discard 900 μL of the supernatant. Pipette the remaining 100 μL of the bacterial solution and the precipitate to mix evenly, and then spread it on an LB solid medium containing 50 μg / mL ampicillin and incubate overnight at 37 °C.

[0090] Randomly pick out 5 single colonies from the overnight culture plates respectively, pipette and mix them evenly in the corresponding numbered 30 μL of ddH2O. Transfer 10 μL of each mixed solution into the corresponding numbered LB liquid medium containing 50 μg / mL ampicillin and culture overnight at 37 °C and 180 rpm. Boil the remaining 20 μL of each mixed solution for 10 min respectively, cool it down and use it as a template, and perform colony PCR with the primers and reaction system in step 3 of Example 1, and then verify it by 0.9% nucleic acid agarose gel electrophoresis.

[0091] After verification by colony PCR, each positive clone single colony in the plate is the random mutation library.

[0092] Example 6: Screening of the Random Mutant Library of 3-Ketosteroid-Δ1-Dehydrogenase PrKstD

[0093] (1) Use a sterile toothpick to pick the random mutation library obtained in Example 5 into a 96-well deep-well plate (master plate). 1000 μL of TB medium containing 50 μg / mL ampicillin has been added to each well of the 96-well deep-well plate in advance. Seal the inoculated 96-well deep-well plate with a sealing film and place it in an incubator at 37 °C and 180 rpm for overnight culture. Then, take 10 μL of the overnight culture bacterial solution from each well and add it to another 96-well deep-well culture plate containing 1000 μL of TB medium with a final concentration of 0.1 mM IPTG and 50 μg / mL ampicillin in each well. Place the inoculated 96-well culture plate in a shaker at 25 °C and 220 rpm for induction for 16 h. This new 96-well deep-well plate is named the replica plate. Store the master plate in a 4 °C refrigerator.

[0094] (2) The operation of library screening is as follows: Put the replicated plate after induction into a 96-well plate centrifuge, centrifuge at 3000 rpm for 15 min at 4°C, and discard the supernatant; Use a multi-channel pipette to aspirate 500 μL of 50 mM Tris-HCl buffer (pH = 8.0) to resuspend the cells; Centrifuge the 96-well plate after resuspension at 3000 rpm for 10 min at 4°C, and discard the supernatant; Use a multi-channel pipette to aspirate 200 μL of 50 mM Tris-HCl buffer (pH = 8.0) to resuspend the cells; Put the 96-well plate into an -80°C refrigerator for 2 h, take it out and thaw at room temperature, then add lysozyme with a final concentration of 0.5 mg / mL, incubate for 30 min, and centrifuge to obtain the crude enzyme solution.

[0095] (3) Add 200 μL of the high-throughput screening reaction system to the enzyme-linked immunosorbent assay (ELISA) plate respectively, place it in an ELISA reader with an absorbance of 600 nm, and react at 37°C; Measure and record the change in absorbance every 2 min, and measure continuously for 5 times. Record the position on the master plate corresponding to the crude enzyme solution with a faster decrease in absorbance than that of PrKstD before mutation.

[0096] The final concentration composition of the high-throughput screening reaction system is: 20 μL of 15 mM methyl viologen sulfate aqueous solution (PMS), 20 μL of 4 mM 2,6-dichlorophenol indophenol aqueous solution (DCPIP), 20 μL of 5 mM (16α)-methyl-androst-4,9-diene-3,17-dione methanol solution in water, 130 μL of 50 mM Tris-HCl (pH = 8.0) buffer, and 10 μL of crude enzyme solution. Mix them evenly to form a 200 μL reaction system.

[0097] (4) According to the position recorded in (3), preserve the bacterial liquid on the corresponding master plate. Take 500 μL of the bacterial liquid and 500 μL of 40% glycerol each into a cryotube for cells, and store them in a -20°C refrigerator, which are the dominant mutants.

[0098] Example 7: Rescreening of the PrKstD mutant of 3-sterone-Δ1-dehydrogenase

[0099] Perform rescreening on the 10 dominant mutants obtained from the primary screening in Example 6 to prevent false positives. Expand and induce the culture of the 10 mutants according to the method in Example 1 to obtain resting cells. Catalyze the reaction of the resting cells with the substrate (16α)-methyl-androst-4,9-diene-3,17-dione, and screen to obtain the best dominant mutants through liquid chromatography analysis.

[0100] The total volume of the reaction system is 5 mL: 5 g / L substrate (16α)-methyl-androst-4,9-diene-3,17-dione, 1 mM PMS, 0.2 mM FAD, the final concentration of wet bacterial cells added is 20 g / L, the volume concentration of methanol is 10%, and the reaction medium is 50 mM Tris-HCl buffer (pH = 8.0) to make up to 5 mL. The reaction is carried out at 30 °C and 180 rpm for 96 h. After the reaction is completed, the reaction solution is extracted 3 times with an equal volume of ethyl acetate and then combined. After evaporation to dryness, it is redissolved in acetonitrile and filtered through a 0.22 μm filter membrane. The substrate conversion rate of the filtrate is detected by liquid chromatography, and the substrate conversion rate is shown in Figure 8 shown.

[0101] The liquid chromatograph used is the LC-20A high-performance liquid chromatograph (purchased from Shimadzu Corporation, Japan), equipped with a C18 chromatographic column (Ultimate, 5 μm, 250 mm × 4.6 mm), at 30 °C, and a 240 nm ultraviolet detector. The mobile phase is acetonitrile: water = 50:50, by volume ratio, the flow rate is 1.0 mL / min, and the injection volume is 20 μL.

[0102] It can be seen from Figure 8 that mutant 3 has the best dehydrogenation conversion effect on the substrate. Mutant 3 is named E. coli BL21(DE3)-pET-21a(+)-PrKstD-T. Its mutant amino acid sequence is shown in SEQ ID NO: 3, and the nucleotide sequence is shown in SEQ ID NO: 2. The method of Example 1 is used for induction expression to prepare wet bacterial cells as resting cells for subsequent experiments.

[0103] SEQ ID NO: 3

[0104] MTQTWDEEYDVVVIGAGGGSLTGALVAAREGLKVLVAEATDRFGGTTAYSGGGLWWPNNQALKRAGVEDTPEAAAQYYHGIVGDDSPRELQEAYLAGGPALVKYLEDNGLMEFLIYPWPDYFGKEPTAHNEGGRTMMPMYFPAEEMGDLRDQVRSGLPAERRGEPLPDMMIGGQALIGRLVLNLSKEPNVTMRRNAEGRKLIMEDGRVAGVIVSIDGQDKAIKATKGVLVAAGGFEQNQEMREKYGVPGHARDTMGAPRNFGLVQQSAIELGADTALMDQAWWSPGLTHPDGSSTFSLWFTGGIFVNNHGERFVNESWAYDKLGRAIIDLVDEGRMTLPYWMVYDNRAGERVPCNTTSVPMVETEEYREAGLWHTADTLEELADKIGVPADKLVATVERFNEFAANEKDEHFDRGGEAYDRSFSEGKSPLVPITEGPFHAAQFGLSDLGTKGGLKTDVDARVLDTGGNVIPGLYAAGNSMAPASGKVYPGGGNPIGSSMVFSYLAALDMAKN*。

[0105] Example 8: Influence of Reaction Temperature on Dehydrogenation Reaction

[0106] Detect the catalytic activity of PrKstD-T at different temperatures, explore the influence of different temperatures on the activity of PrKstD-T, and design the reaction system.

[0107] The reaction system (5 mL) contains 20 g / L of PrKstD-T resting cells, 1 mM PMS, 5 g / L of substrate (16α)-methyl-4,9-androstadiene-3,17-dione, 0.2 mM FAD, 10% (v / v) methanol, and 50 mM Tris-HCl buffer (pH = 8.0) to make up to 5 mL. React at 25, 30, 35, 40, 45 °C under 180 rpm for 96 h. After the reaction is completed, the reaction solution is extracted 3 times with the same volume of ethyl acetate. The combined ethyl acetate layer is evaporated to dryness and redissolved in acetonitrile, and then filtered through a 0.22 μm filter membrane. The substrate conversion rate of the filtrate is detected by the method of Example 7, and the results are shown in Table 2.

[0108] Table 2. Influence of Different Reaction Temperatures on Dehydrogenation Reaction

[0109]

[0110] As can be seen from Table 2, the catalytic activity of the enzyme is the highest when the reaction is carried out at 30°C. Although increasing the temperature within the low-temperature range will increase the number of collisions between the enzyme and substrate molecules, thereby increasing the catalytic rate, when the temperature is greater than 30°C, the activity of the enzyme begins to decline. This may be because too high a temperature will cause the enzyme to absorb a large amount of energy, resulting in the hydrogen bonds maintaining its structure being broken, changing the spatial conformation of the protein and making it unable to exert an effective catalytic function. Therefore, the reaction is preferably controlled at 30°C for the most favorable catalysis.

[0111] Example 9: Effect of reaction pH on dehydrogenation reaction

[0112] The final concentration composition of the reaction system (5 mL): 20 g / L of PrKstD-T resting cells, 1 mM PMS, 5 g / L of substrate (16α)-methyl-androst-4,9-diene-3,17-dione, 0.2 mM FAD, 10% (v / v) methanol, and the buffer solution with pH values of 6.0, 7.0, 8.0, 9.0, and 10.0 (where 6.0 and 7.0 are 50 mM PB, 8.0 and 9.0 are 50 mM Tris-HCl, and 10.0 is 25 mM glycine-NaOH buffer solution) is added to make up to 5 mL. The reaction is carried out at 30°C and 180 rpm for 96 h. After the reaction is completed, the reaction solution is extracted 3 times with the same volume of ethyl acetate. The combined ethyl acetate layer is evaporated to dryness and redissolved in acetonitrile, and then filtered through a 0.22 μm filter membrane. The substrate conversion rate of the filtrate is detected by the method of Example 7, and the results are shown in Table 3.

[0113] Table 3. Effect of different pH values on dehydrogenation reaction

[0114]

[0115] As can be seen from Table 3, PrKstD-T has the highest activity at pH 8. Too high or too low pH can change the charged states of substrate molecules and enzyme molecules, causing unfolding and exposure of the internal structure, resulting in the loss of enzyme activity and thus affecting the binding of the enzyme and substrate; in addition to having a great impact on enzyme activity, pH also has a great impact on enzyme stability. Too high or too low pH will change the conformation of the active center of the enzyme, or even change the structure of the entire enzyme molecule and cause it to denature and inactivate. Therefore, PrKstD-T is suitable for reaction in a Tris-HCl buffer solution with pH 8.

[0116] Example 10: Effect of reaction time on dehydrogenation reaction

[0117] The final concentration composition of the reaction system (5 mL) is as follows: 20 g / L of PrKstD-T resting cells, 1 mM PMS, 5 g / L of the substrate (16α)-methyl-androst-4,9-diene-3,17-dione, 0.2 mM FAD, 10% (v / v) methanol, and the volume is made up to 5 mL with 50 mM Tris-HCl buffer (pH = 8.0). The reactions were carried out at 30 °C and 180 rpm for 48, 72, 96, and 120 h respectively. After the reaction was completed, the reaction solution was extracted 3 times with the same volume of ethyl acetate. The combined ethyl acetate layers were evaporated to dryness and redissolved in acetonitrile, and then filtered through a 0.22 μm filter membrane. The substrate conversion rate of the filtrate was detected by the method of Example 7, and the results are shown in Table 4.

[0118] Table 4. Effects of different reaction times on the dehydrogenation reaction

[0119]

[0120] It can be found from Table 4 that as the reaction time prolongs, the conversion rate of PrKstD-T enzyme gradually increases. After 96 h, the dehydrogenation reaction begins to slow down. Therefore, in order to save reaction time, the optimal reaction time for the dehydrogenation reaction is selected as 96 h.

[0121] Example 11: Effects of substrate concentration on the dehydrogenation reaction

[0122] To meet the needs of industrial production, it is necessary to increase the substrate concentration in the reaction system as high as possible while ensuring the product yield and high enantioselectivity. Therefore, a reaction was designed to explore the product conversion rate at different substrate concentrations.

[0123] The final concentration composition of the reaction system (5 mL) is as follows: 20 g / L of PrKstD-T resting cells, 1 mM PMS, the substrate (16α)-methyl-androst-4,9-diene-3,17-dione (final concentrations are 5, 10, 15, 20, 25 g / L respectively), 0.2 mM FAD, 10% (v / v) methanol, and the volume is made up to 5 mL with 50 mM Tris-HCl buffer. The reaction was carried out at 30 °C and 180 rpm for 96 h. After the reaction was completed, the reaction solution was extracted 3 times with the same volume of ethyl acetate. The combined ethyl acetate layers were evaporated to dryness and redissolved in acetonitrile, and then filtered through a 0.22 μm filter membrane. The substrate conversion rate of the filtrate was detected by the method of Example 7, and the results are shown in Table 5.

[0124] Table 5. Effects of substrate concentration on the dehydrogenation reaction

[0125]

[0126] As can be seen from Table 5, the increase in the concentration of the substrate (16α)-methyl-4,9-androstadiene-3,17-dione will have a certain impact on the enzyme activity of the system. When the substrate concentration is higher than 15 g / L, the conversion rate of the substrate in the system decreases rapidly, indicating that the high concentration of the substrate inhibits the reaction.

[0127] Example 12: Effect of cosolvent on dehydrogenation reaction

[0128] The final concentration composition of the reaction system (5 mL): 20 g / L of PrKstD-T resting cells, 1 mM PMS, 15 g / L of the substrate (16α)-methyl-4,9-androstadiene-3,17-dione, 0.2 mM FAD. DMSO, methanol, ethanol, isopropanol, and Tween-80 with a volume concentration of 10% are used as cosolvents respectively, and 50 mM Tris-HCl buffer (pH = 8.0) is added to make up to 5 mL. The reaction is carried out at 30 °C and 180 rpm for 96 h. After the reaction is completed, the reaction solution is extracted 3 times with the same volume of ethyl acetate. The combined ethyl acetate layer is evaporated to dryness and redissolved in acetonitrile, and then filtered through a 0.22 μm filter membrane. The conversion rate of the substrate in the filtrate is detected by the method of Example 7, and the results are shown in Table 6.

[0129] Table 6. Effect of cosolvent on dehydrogenation reaction

[0130]

[0131] As can be seen from Table 6, the cosolvent Tween-80 has the best catalytic effect on the dehydrogenation reaction, and also has less toxicity to proteins, promoting the forward progress of the dehydrogenation reaction. Therefore, Tween-80 is selected as the optimal cosolvent.

Claims

1. A 3-oxosteroid-Δ1-dehydrogenase mutant, characterized in that, The mutant amino acid sequence is as shown in SEQ ID NO:

3.

2. A recombinant genetically engineered bacterium containing the gene encoding the 3-sterone-Δ1-dehydrogenase mutant as claimed in claim 1.

3. Use of the 3-sterone-Δ1-dehydrogenase mutant as claimed in claim 1 in the dehydrogenation of the C1 and C2 positions of a steroid compound.

4. The application according to claim 3, characterized in that The use is to catalyze the synthesis of (16α)-16-methyl-androst-1,4,9(11)-triene-3,17-dione from (16α)-methyl-androst-4,9-diene-3,17-dione.

5. The application according to claim 4, characterized in that The method of the use is as follows: using the wet cells obtained by inducing and culturing the recombinant genetically engineered bacterium containing the gene encoding the 3-sterone-Δ1-dehydrogenase mutant or the crude enzyme solution extracted by ultrasonic disruption of the wet cells as a catalyst, using the steroid compound (16α)-methyl-androst-4,9-diene-3,17-dione as a substrate, using phenazine methosulfate as a proton acceptor, using flavin adenine dinucleotide as a coenzyme, adding a cosolvent, using a buffer solution with a pH of 6-10 as a reaction medium to construct a reaction system, reacting at 25-45 °C and 180 rpm for 48-120 h to obtain a reaction solution of the dehydrogenated compound (16α)-16-methyl-androst-1,4,9(11)-triene-3,17-dione; the cosolvent is β-cyclodextrin, methanol, ethanol, isopropanol or Tween-80.

6. The application according to claim 5, characterized in that, The buffer solution is a 50 mM Tris-HCl buffer solution with a pH of 8.

0.

7. The application according to claim 5, characterized in that In the reaction system, the final concentration of the substrate added is 5-25 g / L, the final concentration of phenazine methosulfate added is 1 mM, the final concentration of the coenzyme added is 0.2 mM, the dosage of the catalyst is 10-30 g / L based on the mass of the wet cells; the volume addition concentration of the cosolvent is 5-15%.

8. The application according to claim 5, characterized in that The cosolvent is Tween-80.

9. The application according to claim 5, characterized in that, The catalyst is prepared by the following method: The recombinant genetically engineered bacteria containing the gene encoding the 3-sterone-Δ1-dehydrogenase mutant were inoculated into LB liquid medium containing 50 μg / mL ampicillin and cultured overnight at 37 °C and 180 rpm to obtain a seed solution; the seed solution was inoculated into LB liquid medium containing 50 μg / mL ampicillin at an inoculation volume concentration of 3%, and cultured at 37 °C and 180 rpm until the OD 600 was 0.6 - 0.

8. IPTG with a final concentration of 0.1 mM was added, and induced culture was carried out at 25 °C and 180 rpm for 16 h to obtain a fermentation broth; the fermentation broth was centrifuged at 8000 rpm and 4 °C for 10 min. The obtained cell precipitate was resuspended with physiological saline and then centrifuged at 8000 rpm and 4 °C for 10 min to collect wet cells; the wet cells were resuspended with 50 mM, pH 8.0 Tris-HCl buffer at a quantity of 80 g / L. After the resuspended solution was placed on ice for 30 min, it was sonicated at a sonication power of 360 W for 10 min, with sonication for 3 s and an interval of 7 s. After centrifugation of the sonicated cell disruption solution at 8000 rpm and 4 °C for 10 min, the precipitate was discarded, and the obtained supernatant was the crude enzyme solution.

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