A 3-ketosteroid delta1-dehydrogenase mutant and use thereof in catalyzing dehydrogenation of steroid compounds

Abstract

The application discloses a 3-steroid ketone-delta 1-dehydrogenase mutant and application thereof in catalyzing dehydrogenation of a steroid compound, and the mutant with high catalytic activity is screened, and a cosolvent Tween-80 is added to increase solubility of a substrate and then improve biological catalytic efficiency.Under optimal conditions, the mutant can increase the conversion rate of (16alpha)-methyl androst-4,9-diene-3,17-dione 15g / L to 99.9%.

Description

(I) Technical Field

[0001] This invention relates to a 3-sterone-Δ1-dehydrogenase mutant and its application in catalyzing the dehydrogenation of steroid compounds at the C1,2 position. (II) Background Technology

[0002] Steroid drugs are a class of drugs named according to their chemical structure, referring to drugs whose molecular structure contains the "cyclopentane-polyhydrophenanthrene" core structure. Clinically, steroid hormones are the second most used class of drugs after antibiotics, and are an indispensable type of exogenous hormone. In clinical use, steroid drugs are mainly divided into three categories: adrenocortical hormones, sex hormones, and anabolic steroids. Steroid drugs occupy an important position in the chemical drug system; currently, more than 400 steroid drugs are produced globally, and the market demand for steroid drugs has ranked among the top for many years.

[0003] Microbial-mediated steroid transformation involves the specific biochemical modification of steroid compounds using microbial cells or their enzyme systems. This biocatalytic process specifically alters a particular site on the steroid, producing novel compounds or intermediates with similar structures but enhanced physiological activity. Compared to traditional chemical synthesis, microbial transformation offers several advantages: it reduces synthetic steps, shortens production cycles, increases yield, reduces the frequency of side reactions, and provides milder and more environmentally friendly reaction conditions. Furthermore, microbial transformation exhibits excellent stereoselectivity and regioselectivity, enabling the execution of specific reactions that are difficult or impossible to achieve using conventional chemical methods, such as dehydrogenation at the C1,2 position of the A ring and hydroxylation at the 11α and 11β positions. As microbial transformation processes mature, the steroid synthesis route is gradually transforming and upgrading to a "phytosterol-androstenedione-steroid drug" process, which is becoming the mainstream industrial production method in the steroid industry.

[0004] The dehydrogenation at the 1,2 positions of (16α)-methylandrost-4,9-diene-3,17-dione is a crucial step in the synthesis of the drug dexamethasone. Dexamethasone is a glucocorticoid, and its derivatives include hydrocortisone and prednisone. Its pharmacological effects are mainly anti-inflammatory, antitoxic, anti-allergic, and antirheumatic. It is widely used clinically, readily absorbed from the digestive tract, and has a lower plasma protein binding rate than other corticosteroids. It possesses good anti-inflammatory and immunosuppressive effects and can be used to treat various diseases such as bronchospasm, allergies, shock, and ulcerative colitis.

[0005] Directed evolution, also known as non-rational design, is one of the most commonly used protein engineering techniques. Directed evolution of enzymes refers to the process of simulating natural evolution in vitro under experimental conditions, undergoing multiple rounds of repeated mutations and targeted screening to ultimately obtain new enzyme mutants that meet expectations. The two most important processes in directed evolution are: first, introducing random mutations into the target gene to construct a mutant library; and second, performing high-throughput screening on the constructed library. Directed evolution involves random mutations of the entire sequence of the target protein, which allows for the screening of amino acids that are far removed from the protein's active site. Compared to rational design, directed evolution does not require knowledge of the target protein's three-dimensional structure or related structure-function information. Furthermore, directed evolution involves random mutations of the entire sequence, including amino acid sites that are far from the active site but are crucial for maintaining the overall structure, stability, and catalytic properties of the active site, thus offering greater possibilities for enzyme modification.

[0006] The 3-sterone-Δ1-dehydrogenase PrKstD, derived from *Propionibacterium sp.*, is 1557 bytes long and encodes 518 amino acids. It exhibits higher specific activity and a broader substrate spectrum compared to other dehydrogenases. Characterization of PrKstD's catalytic performance revealed that it can convert C9, C10, C11, and C17 substituted 3-ketosteroids into inactive C6-substituted compounds, such as 11β,17-dihydroxy-6α-methyl-1,4-en-3,20-dione, using substrates lacking C4-C5 double bonds. Furthermore, PrKstD also demonstrates catalytic activity for C6 and C11 substitutions. Analysis of its protein crystal structure shows that PrKstD's active pocket is more open than that of other KstDs, facilitating enzyme-substrate binding. Since the C9-C11 double bond of the substrate (16α)-methylandrost-4,9-diene-3,17-dione inhibits dehydrogenase activity, the double bond binds to amino acid residues in the active pocket, hindering substrate release after dehydrogenation. Therefore, directed evolution was used to modify the enzyme molecule to obtain mutants that can efficiently catalyze the dehydrogenation of (16α)-methylandrost-4,9-diene-3,17-dione. (III) Summary of the Invention

[0007] The purpose of this invention is to provide a 3-sterone-Δ1-dehydrogenase mutant and its application in catalyzing the dehydrogenation of steroidal compounds at the C1,2 position. This invention obtains a mutant library of the 3-sterone-Δ1-dehydrogenase PrKstD gene using error-prone PCR technology based on directed evolution, and then screens the mutant library using a high-throughput screening method to obtain a mutant that can efficiently catalyze the dehydrogenation of (16α)-methylandrost-4,9-diene-3,17-dione to (16α)-16-methylandrost-1,4,9(11)-triene-3,17-dione. This invention also optimizes the conditions for inducing 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 substrate water solubility, and opening up a green, environmentally friendly, safe, efficient and low-cost biocatalytic pathway for the dehydrogenation of (16α)-methylandrost-4,9-diene-3,17-dione.

[0008] The technical solution adopted in this invention is:

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

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

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

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

[0013] Fourthly, the present invention provides a recombinant genetically engineered bacterium prepared by transformation of the recombinant expression vector, wherein the engineered bacterium uses Escherichia coli BL21(DE3) as the host bacterium.

[0014] Fifthly, the present invention provides an application of the 3-sterone-Δ1-dehydrogenase mutant in catalyzing the dehydrogenation of steroidal compounds at the C1,2 position. Specifically, the application is the catalytic synthesis of (16α)-16-methylandrost-1,4,9(11)-triene-3,17-dione from (16α)-methylandrost-4,9-diene-3,17-dione.

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

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

[0017] Furthermore, in the reaction system, the final concentration of the substrate is 5-25 g / L (preferably 5-15 g / L), the final concentration of phenazine methyl sulfate is 1 mM, the final concentration of FAD is 0.2 mM, the amount of catalyst is 10-30 g / L (preferably 20 g / L) based on the mass of wet bacterial cells, and the volumetric concentration of the cosolvent is 5-15% (preferably 10%).

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

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

[0020] 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 seed culture. The seed culture was then inoculated into LB liquid medium containing 50 μg / mL ampicillin at a volume concentration of 3% and cultured at 37°C and 180 rpm until OD... 600 The concentration of the culture medium was 0.6-0.8. IPTG was added to a final concentration of 0.1 mM, and the culture was induced at 25℃ and 180 rpm for 16 h to obtain the fermentation broth. The fermentation broth was centrifuged at 8000 rpm and 4℃ for 10 min. The resulting bacterial precipitate was resuspended in physiological saline and centrifuged at 8000 rpm and 4℃ for 10 min to collect the wet bacterial cells.

[0021] The wet bacterial cells were resuspended in 50 mM Tris-HCl buffer (pH = 8.0) at a concentration of 80 g / L. The resuspended solution was placed on ice for 30 min and then sonicated (conditions: sonication power 360 W, sonication for 3 s, interval 7 s, sonication for 10 min). The sonicated solution was centrifuged at 8000 rpm and 4℃ for 10 min and the precipitate was discarded. The resulting supernatant was the crude enzyme solution.

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

[0023] This invention involves directed evolution of the 3-sterone-Δ1-dehydrogenase PrKstD derived from Propionibacterium sp., screening for mutant genes with high catalytic activity against (16α)-methylandrost-4,9-diene-3,17-dione, and constructing it into the pET-21a(+)-PrKstD-T recombinant plasmid. Further, the E. coli BL21(DE3)-pET-21a(+)-PrKstD-T recombinant engineered bacterium was constructed.

[0024] (16α)-Methylandrost-4,9-diene-3,17-dione is poorly soluble in water, which reduces the production efficiency of biocatalytic preparation. To address the problem of poor water solubility and low mass transfer of the organic substrate in the aqueous phase, this invention increases the solubility of the substrate and thus improves the biocatalytic efficiency by adding the co-solvent Tween-80 (10%, v / v). Under optimal conditions, the mutant of this invention can increase the conversion rate of (16α)-methylandrost-4,9-diene-3,17-dione from a feed amount of 15 g / L to 99.9%. (iv) Description of the attached drawings

[0025] Figure 1 A schematic diagram of the dehydrogenation synthesis of (16α)-16-methylandrost-1,4,9(11)-triene-3,17-dione from the PrKstD catalytic substrate (16α)-methylandrost-4,9-diene-3,17-dione.

[0026] Figure 2 This is an electrophoresis image of colony PCR nucleic acid from step 3 of Example 1. Lanes 1, 2, 3, 4, and 5 represent the PCR products of five randomly selected colonies.

[0027] Figure 3 The equation for the DCPIP standard curve is shown in Example 1.

[0028] Figure 4 This is a bar graph showing the relative enzyme activity of PrKstD at different induction temperatures in Example 2.

[0029] Figure 5 This is a bar graph showing the relative enzyme activity of PrKstD at different concentrations of IPTG in Example 3.

[0030] Figure 6 This is a bar graph showing the relative enzyme activity of PrKstD at different induction times in Example 4.

[0031] Figure 7 The image shows the electrophoresis diagrams of the error-prone PCR amplification products and the double enzyme digestion products of plasmid pET-21a(+) in Example 5; where Lane 1, 2, and 3 are error-prone PCR amplification products, and Lane 4, 5, 6, 7, and 8 are double enzyme digestion products of plasmid pET-21a(+).

[0032] Figure 8 The substrate conversion rate of the 10 mutants obtained from the initial screening in Example 7 was obtained through a scale-up rescreening. (V) Detailed Implementation

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

[0034] The culture medium used in the embodiments of this invention consists of:

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

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

[0037] TB culture medium composition: 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, the solvent for the solutions in this invention is double-distilled water.

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

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

[0041] By reviewing literature and screening for steroidal C1,2 dehydrogenases, PrKstD, a C1,2 dehydrogenase with high catalytic potential and good stability for (16α)-methylandrost-4,9-diene-3,17-dione, was selected. A search of the NCBI database revealed the gene sequence of the 3-sterone-Δ1-dehydrogenase PrKstD derived from *Propionibacterium* sp. (NCBI accession number: NLT29951.1). The obtained gene sequence was synthesized by Beijing Qingke Biotechnology Co., Ltd., Hangzhou Branch. The nucleotide sequence is shown in SEQ ID NO: 1, and the synthesized gene ends with a His-tag.

[0042] SEQ ID NO: 1:

[0043]

[0044] 2. Synthesis of recombinant plasmid pET-21a(+)-PrKstD

[0045] We commissioned Beijing Qingke Biotechnology Co., Ltd. Hangzhou Branch to insert the PrKstD target gene into the HindIII and BmaHI restriction sites of the expression vector pET-21a(+), resulting in the recombinant plasmid pET-21a(+)-PrKstD.

[0046] 3. Construction and identification of recombinant genetically engineered bacteria

[0047] The pET-21a(+)-PrKstD plasmid was introduced into E. coli BL21(DE3) competent cells. The specific method was as follows: After thawing E. coli BL21(DE3) competent cells on ice for 20 min, 10 μL of the recombinant plasmid pET-21a(+)-PrKstD was added to 100 μL of E. coli BL21(DE3) competent cells. The cells were gently mixed by pipetting and incubated on ice for 30 min, followed by heat shock at 42℃ for 90 s, and then on ice for 2 min. 0.9 mL of LB antibiotic-free liquid medium was added, and the cells were incubated at 37℃ and 180 rpm for 1 h. After centrifugation, 900 μL of the supernatant was discarded. The remaining 100 μL of the bacterial culture was mixed with the precipitate by pipetting and then spread onto LB solid medium containing 50 μg / mL ampicillin and incubated overnight at 37℃.

[0048] Five single colonies were randomly selected from the overnight culture plates and mixed by pipetting in 30 μL of ddH2O corresponding to their respective numbers. 10 μL of each mixture was transferred to LB broth containing 50 μg / mL ampicillin, and incubated overnight at 37°C and 180 rpm. The remaining 20 μL of each mixture was boiled for 10 min, cooled, and used as templates for colony PCR. The results were then verified by 0.9% nucleic acid agarose gel electrophoresis. Figure 2 ), and obtained positive clones of transformation.

[0049] The total volume of the colony PCR reaction system was 25 μL: 1 μL of boiled bacterial 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 was: ① 94℃ for 5 min, ② 94℃ for 30 s, ③ 55℃ for 30 s, ④ 72℃ for 1 min, ⑤ 72℃ for 10 min, with cycles ②, ③, and ④ repeated 30 times. The 2×Hieff PCR Master Mix was purchased from Beijing Qingke Biotechnology Co., Ltd. Primers F and R were designed based on the gene sequence of 3-sterone-Δ1-dehydrogenase PrKstD and synthesized by the Hangzhou branch of Beijing Qingke Biotechnology Co., Ltd.

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

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

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

[0053] The successfully transformed positive clones from step 3 were inoculated into 50 mL of LB liquid medium containing 50 μg / mL ampicillin and cultured overnight at 37°C and 180 rpm to obtain seed culture. The seed culture was then inoculated into 150 mL of LB liquid medium containing 50 μg / mL ampicillin at a volume concentration of 3% and cultured at 37°C and 180 rpm until OD... 600 The concentration of the culture medium was 0.6-0.8. IPTG was added to a final concentration of 0.1 mM, and the mixture was induced and cultured at 25°C and 180 rpm for 16 h to obtain the fermentation broth. The fermentation broth was centrifuged at 8000 rpm and 4°C for 10 min. The resulting bacterial pellet was resuspended in physiological saline and centrifuged at 8000 rpm and 4°C for 10 min to collect the wet bacterial cells.

[0054] The wet bacterial cells were resuspended in 50 mM Tris-HCl buffer (pH = 8.0) at a concentration of 80 g / L. The resuspended solution was placed on ice for 30 min and then sonicated (conditions: sonication power 360 W, sonication for 3 s, interval 7 s, sonication for 10 min). The sonicated solution was centrifuged at 8000 rpm and 4℃ for 10 min and the precipitate was discarded. The resulting supernatant was the crude enzyme solution.

[0055] 5. Enzyme activity detection

[0056] The principle is that the 3-sterone-Δ1-dehydrogenase PrKstD reacts with the substrate to remove two hydrogens at the C1 and C2 positions to form a double bond. The coenzyme PMS accepts the removed hydrogen ions to form PMSH2 to ensure the cyclical progress of the reaction. PMSH2 then transfers hydrogen ions to DCPIP to form DCPIPH2. DCPIPH2 has no absorbance at 600 nm. The amount of DCPIP consumed can indirectly reflect the enzyme activity of PrKstD.

[0057] The activity of 3-sterone-Δ1-dehydrogenase PrKstD was determined using a spectrophotometer as follows:

[0058] The final concentration of the 2 mL reaction system for the PrKstD enzyme activity assay consisted of: 0.5 mM (16α)-methylandrost-4,9-diene-3,17-dione, 5% methanol (v / v), 150 μM 2,6-dichlorophenolindophenol (DCPIP), 1.5 mM methyl phenazine sulfate (PMS), and 50 mM Tris-HCl buffer (pH 8.0). The solution was brought to a final volume of 2 mL, incubated at 30°C for 1 minute, and then a suitable amount of crude enzyme solution prepared as described above was added. The mixture was quickly mixed, and the absorbance at 600 nm was measured. The DCPIP content was calculated based on the DCPIP standard curve.

[0059] The DCPIP standard curve was constructed using enzyme activity detection conditions, with DCPIP concentration on the x-axis and absorbance at 600 nm on the y-axis. Figure 3 As shown.

[0060] Enzyme activity is expressed in units of 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 induction temperature for E. coli BL21(DE3)-pET-21a(+)-PrKstD recombinant strain

[0062] The induction temperature of E. coli BL21(DE3)-pET-21a(+)-PrKstD was optimized by changing the induction temperature in step 4 of Example 1 to 16, 20, 25, 30, and 37 °C, respectively, while keeping other operations the same, to prepare crude enzyme solutions at each induction temperature. The enzyme activity of PrKstD at different induction temperatures was measured using the same method as in step 5 of Example 1. The relative enzyme activity at other temperatures was calculated with the highest enzyme activity as 100%. The results are shown in […]. Figure 4 .

[0063] from Figure 4 The results show that induction temperature has a significant impact on the expression of the target protein by engineered bacteria. At low temperatures, protein synthesis slows down, allowing newly generated protein chains more time for proper folding, thus reducing inclusion body formation and ultimately resulting in higher protein activity. At high temperatures, although protein synthesis speed increases, protein chain folding speed also accelerates, which may lead to protein misfolding or aggregation, forming insoluble inclusion bodies, thereby affecting the total protein yield and quality, ultimately resulting in decreased protein activity. Below 25℃, the relative enzyme activity increases with increasing induction temperature; above 25℃, the relative enzyme activity decreases with increasing induction temperature. Therefore, 25℃ is selected as the optimal induction temperature.

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

[0065] The concentration of the inducing agent (IPTG) in *E. coli* BL21(DE3)-pET-21a(+)-PrKstD was optimized. The final IPTG concentration in step 4 of Example 1 was changed to 0.05, 0.1, 0.2, 0.5, and 1.0 mM, respectively, while maintaining the same other operations. Crude enzyme solutions were prepared after induction at each concentration. The enzyme activity of PrKstD at different inducing agent concentrations was determined using the same method as in step 5 of Example 1. The relative enzyme activity at other induction concentrations was calculated with the highest enzyme activity as 100%. The results are shown in […]. Figure 5 .

[0066] from Figure 5 The results show that the inducer (IPTG) has a significant impact on the expression of the target protein in engineered bacteria. Lower inducer concentrations result in lower enzyme production, and the inducer also exhibits some toxicity to cells. Excessively high concentrations can negatively affect cell growth; therefore, selecting an appropriate inducer concentration is crucial. Below 0.1 mM, the relative enzyme activity increases with increasing IPTG concentration; above 0.1 mM, the relative enzyme activity decreases with increasing IPTG concentration. Therefore, 0.1 mM IPTG is selected as the optimal inducer concentration.

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

[0068] The induction time of E. coli BL21(DE3)-pET-21a(+)-PrKstD was optimized by changing the induction time in step 4 of Example 1 to 8, 12, 16, 20, and 24 h, respectively, while keeping other operations the same, to prepare crude enzyme solutions for each induction time. The enzyme activity of PrKstD at different induction times was determined using 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%. The results are shown in […]. Figure 6 .

[0069] from Figure 6 The results show that induction time has a significant impact on the expression of the target protein by engineered bacteria. As the induction time increases, the relative enzyme activity of PrKstD gradually increases; however, after 16 hours of induction, the relative enzyme activity of PrKstD no longer increases but remains constant. Therefore, an induction time of 16 hours is the most suitable.

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

[0071] (1) Primer design

[0072] Based on the gene sequence of 3-sterone-Δ1-dehydrogenase PrKstD (SEQ ID NO: 1), error-prone PCR primers F and R were designed and synthesized by the Hangzhou branch of Beijing Qingke Biotechnology Co., Ltd.

[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, the target gene PrKstD was amplified by error-prone PCR using primers F and R in step (1).

[0077] First, the error-prone PCR system was optimized to obtain a suitable mutation frequency by changing the Mg content. 2+ and Mn 2+ The concentration of Mg was used to optimize the mutation frequency of error-prone PCR, specifically: 2+ Concentration gradient: 2, 3, 4 mM; Mn 2+ The concentration gradients were 0.05, 0.1, 0.2, and 0.3 mM, for a total of 12 groups. After PCR amplification, the results were verified by 0.9% agarose gel electrophoresis, followed by purification using a Sangon Biotech column-based DNA gel extraction kit. Sequencing was then performed to select mutation frequencies within the 3‰-6‰ range: Mg... 2+ Concentration of 2mM, Mn 2+ The 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, see [link to relevant data]. Figure 7 Lanes 1, 2, and 3 were used to recover and purify the target fragment using a Sangon DNA gel recovery kit, denoted as PrKstD-T.

[0079] Table 1. Different Mg 2+ and Mn 2+ Frequency of mutations corresponding to concentration

[0080]

[0081] Error-prone PCR amplification system: Total reaction volume 50 μL, including 0.5 μL plasmid template, 2 μL primer F, 2 μL primer R, 5 μL 10×Taq Buffer, 1 μL dNTPs, 0.5 μL Taq enzyme, 4 μL 25 mM MgCl2, 0.5 μL 10 mM MnCl2, and 4.5 μL ddH2O3.

[0082] The error-prone PCR amplification program is as follows: ① 95℃ for 5 min, ② 95℃ for 15 s, ③ 55℃ for 15 s, ④ 72℃ for 2 min, ⑤ 72℃ for 5 min, with ②, ③, and ④ repeated 35 times. Taq enzyme, 10×Taq Buffer, and dNTPs were purchased from Nanjing Novizan Biotechnology Co., Ltd.

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

[0084] The pET-21a(+) vector was double-digested with Takara restriction enzymes BamHI and HindIII, respectively, and the digestion was carried out at 37°C for 3 h. After double digestion, the results were verified by 0.9% nucleic acid agarose gel electrophoresis. Figure 7 The digested vectors were recovered and purified using a bio-column DNA gel recovery kit (lanes 4, 5, 6, 7, and 8).

[0085] The double enzyme digestion reaction system for pET-21a(+) vector was as follows: BamHI 2μL, HindIII 2μL, 10×K Buffer 10μL, pET-21a(+) 70μL.

[0086] (4) Construction of mutant libraries

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

[0088] The enzyme ligation system for the target gene PrKstD-T with the digested vector pET-21a(+) was as follows: 5 μL of 2×Clon Express Mix, 3.5 μL of vector pET-21a(+), and 1.5 μL of PrKstD-T. The 2×Clon Express Mix was purchased from Nanjing Novizan Biotechnology Co., Ltd.

[0089] The method for introducing the pET-21a(+)-PrKstD-T recombinant plasmid into E. coli BL21 competent cells is as follows: After thawing E. coli BL21 competent cells on ice for 20 min, 10 μL of the recombinant plasmid is added to 100 μL of E. coli BL21 competent cells. The cells are gently mixed by pipetting and incubated on ice for 30 min, followed by heat shock at 42℃ for 90 s, then on ice for 2 min. 0.9 mL of LB antibiotic-free liquid medium is added, and the cells are incubated at 37℃ and 180 rpm for 1 h. After centrifuging the incubated culture, 900 μL of supernatant is discarded. The remaining 100 μL of culture is mixed with the precipitate by pipetting and spreading onto LB solid medium containing 50 μg / mL ampicillin, and incubated overnight at 37℃.

[0090] Five single colonies were randomly selected from the overnight culture plates and mixed by pipetting in 30 μL of ddH2O corresponding to the plate number. 10 μL of each mixture was transferred to LB liquid medium containing 50 μg / mL ampicillin and cultured overnight at 37°C and 180 rpm. The remaining 20 μL of each mixture was boiled for 10 min and cooled. The resulting mixture was used as a template for colony PCR using the primers and system from step 3 of Example 1. The results were then verified by 0.9% nucleic acid agarose gel electrophoresis.

[0091] After colony PCR verification, each positive clone colony in the plate is a random mutant library.

[0092] Example 6: Screening of a random mutant library of 3-sterone-Δ1-dehydrogenase PrKstD

[0093] (1) The random mutant library obtained in Example 5 was picked up with a sterile toothpick and transferred to a 96-well plate (mother plate). Each well of the 96-well plate had 1000 μL of TB medium containing 50 μg / mL ampicillin added beforehand. The inoculated 96-well plate was sealed with a sealing film and incubated overnight at 37°C and 180 rpm. Then, 10 μL of the overnight culture was added to another 96-well plate containing 1000 μL of TB medium with a final concentration of 0.1 mM IPTG and 50 μg / mL ampicillin in each well. The inoculated 96-well plate was incubated at 25°C and 220 rpm for 16 h. This new 96-well plate was named the replication plate. The mother plate was stored at 4°C.

[0094] (2) The library screening procedure is as follows: After the induction is completed, place the plate into a centrifuge at 4°C and 3000 rpm for 15 min, and discard the supernatant; use a pipette to aspirate 500 μL of 50 mM Tris-HCl buffer (pH = 8.0) and resuspend the bacterial cells; centrifuge the resuspended 96-well plate at 4°C and 3000 rpm for 10 min, and discard the supernatant; use a pipette to aspirate 200 μL of 50 mM Tris-HCl buffer (pH = 8.0) and resuspend the bacterial cells; place the 96-well plate in a -80°C freezer for 2 h, take it out and thaw at room temperature, add lysozyme to 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 high-throughput screening reaction system to each microplate, place it in a microplate reader with an absorbance of 600 nm, and react at 37 °C; measure and record the change in absorbance every 2 min, and perform 5 consecutive measurements. Record the position of the mother plate corresponding to the crude enzyme solution whose absorbance decreases faster than that of PrKstD before mutation.

[0096] The final concentration composition of the high-throughput screening reaction system was as follows: 20 μL of 15 mM methyl phenazine sulfate aqueous solution (PMS), 20 μL of 4 mM 2,6-dichlorophenolindophenol aqueous solution (DCPIP), 20 μL of 5 mM (16α)-methylandrost-4,9-diene-3,17-dione methanol aqueous solution, 130 μL of 50 mM Tris-HCl (pH=8.0) buffer, and 10 μL of crude enzyme solution. The mixture was thoroughly mixed to form a 200 μL reaction system.

[0097] (4) Based on the position recorded in (3), preserve the bacterial culture on the corresponding mother plate, take 500 μL of bacterial culture and 50% glycerol into a cell cryopreservation tube, and store it in a -20℃ freezer. This is the dominant mutant.

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

[0099] The 10 dominant mutants obtained from the initial screening in Example 6 were re-screened to prevent false positives. The 10 mutants were expanded and induced to form resting cells according to the method in Example 1. The resting cells were then subjected to a catalytic reaction with the substrate (16α)-methylandrost-4,9-diene-3,17-dione, and the optimal dominant mutant was selected by liquid chromatography analysis.

[0100] The total volume of the reaction system was 5 mL: 5 g / L substrate (16α)-methylandrost-4,9-diene-3,17-dione, 1 mM PMS, 0.2 mM FAD, wet cell addition to a final concentration of 20 g / L, methanol concentration of 10%, and 50 mM Tris-HCl buffer (pH = 8.0) to a final volume of 5 mL. The reaction was carried out at 30 °C and 180 rpm for 96 h. After the reaction was complete, the reaction solution was extracted three times with equal volumes of ethyl acetate, the extracts were combined, evaporated to dryness, redissolved in acetonitrile, and filtered through a 0.22 μm filter membrane. The substrate conversion rate of the filtrate was determined by liquid chromatography. The substrate conversion rate is shown in the figure below. Figure 8 As shown.

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

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

[0103] SEQ ID NO: 3

[0104] MTQTWDEEYDVVVIGAGGGSLTGALVAAREGLKVLVAEATDRFGGTTAYSGGGLWWPNNQALKRAGVEDTPEAAAQYYHGIVGDDSPRELQEAYLAGGPALVKYLEDNGLMEFLIYPWPDYFGKEPTA HNEGGRTMMPMYFPAEEMGDLRDQVRSGLPAERRGEPLPDMMIGGQALIGRLVLNLSKEPNVTMRRNAEGRKLIMEDGRVAGVIVSIDGQDKAIKATKGVLVAAGGFEQNQEMREKYGVPGHARDTMGA PRNFGLVQQSAIELGADTALMDQAWWSPGLTHPDGSSTFSLWFTGGIFVNNHGERFVNESWAYDKLGRAIIDLVDEGRMTLPYWMVYDNRAGERVPCNTTSVPMVETEEYREAGLWHTADTLEELADK IGVPADKLVATVERFNEFAANEKDEHFDRGGEAYDRSFSEGKSPLVPITEGPFHAAQFGLSDLGTKGGLKTDVDARVLDTGGNVIPGLYAAGNSMAPASGKVYPGGGNPIGSSMVFSYLAALDMAKN*.

[0105] Example 8: Effect of reaction temperature on dehydrogenation reaction

[0106] The catalytic activity of PrKstD-T was detected at different temperatures to investigate the effect of different temperatures on the activity of PrKstD-T and to design a reaction system.

[0107] The reaction system (5 mL) contained 20 g / L PrKstD-T resting cells, 1 mM PMS, 5 g / L substrate (16α)-methylandrost-4,9-diene-3,17-dione, 0.2 mM FAD, 10% (v / v) methanol, and 50 mM Tris-HCl buffer (pH = 8.0) to a final volume of 5 mL. The reaction was carried out at 25, 30, 35, 40, and 45 °C at 180 rpm for 96 h. After the reaction, the reaction solution was extracted three times with the same volume of ethyl acetate. The combined ethyl acetate layers were evaporated to dryness, reconstituted with acetonitrile, and filtered through a 0.22 μm filter membrane. The substrate conversion rate of the filtrate was determined using the method in Example 7, and the results are shown in Table 2.

[0108] Table 2. Effect of different reaction temperatures on the dehydrogenation reaction

[0109]

[0110] As shown in Table 2, the enzyme exhibits the highest catalytic activity at 30℃. Although increasing the temperature within the low-temperature range increases the number of collisions between the enzyme and substrate molecules, thereby increasing the catalytic rate, the enzyme activity begins to decline when the temperature exceeds 30℃. This may be because excessively high temperatures cause the enzyme to absorb a large amount of energy, resulting in the destruction of hydrogen bonds that maintain its structure, altering the protein's spatial morphology and preventing it from performing its effective catalytic function. Therefore, controlling the reaction at 30℃ is most conducive to catalysis.

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

[0112] The final concentration of the reaction system (5 mL) consisted of: 20 g / L PrKstD-T resting cells, 1 mM PMS, 5 g / L substrate (16α)-methylandrost-4,9-diene-3,17-dione, 0.2 mM FAD, and 10% (v / v) methanol, brought to a final volume of 5 mL with buffer solutions of pH 6.0, 7.0, 8.0, 9.0, and 10.0 (6.0 and 7.0 were 50 mM PB, 8.0 and 9.0 were 50 mM Tris-HCl, and 10.0 was 25 mM glycine-NaOH buffer). The reaction was carried out at 30 °C and 180 rpm for 96 h. After the reaction, the reaction solution was extracted three times with the same volume of ethyl acetate. The combined ethyl acetate layers were evaporated to dryness, reconstituted with acetonitrile, and filtered through a 0.22 μm filter membrane. The substrate conversion rate of the filtrate was determined using the method in Example 7, and the results are shown in Table 3.

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

[0114]

[0115] As shown in Table 3, PrKstD-T exhibits the highest activity at pH 8. Both excessively high and low pH values ​​can alter the charged state of substrate and enzyme molecules, causing unfolding and exposure of internal structures, leading to loss of enzyme activity and thus affecting enzyme-substrate binding. Besides significantly impacting enzyme activity, pH also greatly affects enzyme stability. Excessively high or low pH values ​​can alter the conformation of the enzyme's active site, or even change the entire enzyme molecule's structure, causing denaturation and inactivation. Therefore, PrKstD-T is best suited for reaction in a Tris-HCl buffer solution at pH 8.

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

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

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

[0119]

[0120] Table 4 shows that the conversion rate of PrKstD-T enzyme gradually increases with increasing reaction time, but the dehydrogenation reaction begins to slow down after 96 hours. Therefore, to save reaction time, the optimal reaction time for the dehydrogenation reaction is 96 hours.

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

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

[0123] The final composition of the reaction system (5 mL) was: 20 g / L PrKstD-T resting cells, 1 mM PMS, substrate (16α)-methylandrost-4,9-diene-3,17-dione (final concentrations of 5, 10, 15, 20, and 25 g / L, respectively), 0.2 mM FAD, 10% (v / v) methanol, and 50 mM Tris-HCl buffer (pH = 8.0) to a final volume of 5 mL. The reaction was carried out at 30 °C and 180 rpm for 96 h. After the reaction, the reaction solution was extracted three times with the same volume of ethyl acetate. The combined ethyl acetate layers were evaporated to dryness, reconstituted with acetonitrile, and filtered through a 0.22 μm filter membrane. The substrate conversion rate of the filtrate was determined using the method in Example 7, and the results are shown in Table 5.

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

[0125]

[0126] As shown in Table 5, the enzyme activity of the system is affected by the increase of the concentration of the substrate (16α)-methylandrost-4,9-diene-3,17-dione. When the substrate concentration is higher than 15 g / L, the conversion rate of the substrate decreases rapidly, indicating that high concentrations of substrate have an inhibitory effect on the reaction.

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

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

[0129] Table 6. Effect of cosolvents 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 is less toxic to proteins, thus promoting the forward progress of the dehydrogenation reaction. Therefore, Tween-80 is selected as the most suitable cosolvent.

Claims

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

3.

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

3. The application of the 3-sterone-Δ1-dehydrogenase mutant of claim 1 in catalyzing the dehydrogenation at the C1,2 position of steroid compounds, characterized in that, The application is to catalyze the synthesis of (16α)-16-methylandrost-4,9-diene-3,17-dione from (16α)-methylandrost-1,4,9(11)-triene-3,17-dione.

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

5. The application as described in claim 4, characterized in that, The buffer solution was a 50 mM Tris-HCl buffer solution with a pH of 8.

0.

6. The application as described in claim 4, characterized in that, In the reaction system, the final concentration of the substrate is 5-25 g / L, the final concentration of phenazine methyl sulfate is 1 mM, the final concentration of the coenzyme is 0.2 mM, the amount of catalyst is 10-30 g / L based on the mass of wet bacterial cells, and the volumetric concentration of the cosolvent is 5-15%.

7. The application as described in claim 4, characterized in that, The co-solvent is Tween-80.

8. The application as described in claim 4, characterized in that, The catalyst is prepared according to the following method: Recombinant genetically engineered bacteria containing the gene encoding the 3-sterone-Δ1-dehydrogenase mutant were inoculated into LB liquid medium containing 50 mg / mL ampicillin and cultured overnight at 37 ℃ and 180 rpm to obtain seed culture. The seed culture was then inoculated into LB liquid medium containing 50 mg / mL ampicillin at a volume concentration of 3% and cultured at 37 ℃ and 180 rpm until OD... 600 The concentration of the enzyme was 0.6-0.

8. IPTG was added to a final concentration of 0.1 mM, and the culture was induced at 25 ℃ and 180 rpm for 16 h to obtain the fermentation broth. The fermentation broth was centrifuged at 8000 rpm and 4 ℃ for 10 min. The resulting bacterial pellet was resuspended in physiological saline and centrifuged at 8000 rpm and 4 ℃ for 10 min to collect the wet bacterial cells. The wet bacterial cells were resuspended in 50 mM Tris-HCl buffer at pH 8.0 at a concentration of 80 g / L. The resuspended solution was placed on ice for 30 min and then sonicated at 360 W for 10 min, with a 3 s sonication interval and a 7 s interval. The sonicated lysate was centrifuged at 8000 rpm and 4 ℃ for 10 min and the pellet was discarded. The resulting supernatant was the crude enzyme solution.

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