Steroid c1,2 dehydrogenase mutant and application thereof

Abstract

The application provides a steroid C1,2 dehydrogenase mutant and application, and belongs to the technical field of biochemistry. The amino acid sequence of the steroid C1,2 dehydrogenase mutant is shown in SEQ ID NO 3 or SEQ ID NO 5. The obtained steroid C1,2 dehydrogenase mutant can be used for preparing C1,2-position dehydrogenated steroid compounds such as estrone from high-concentration substrate synthesis.

Description

Technical Field

[0001] This application relates to a steroid C1,2 dehydrogenase mutant and its application, belonging to the field of biochemical technology. Background Technology

[0002] Steroid drugs (also known as corticosteroids) have important physiological functions and wide clinical applications. As the second largest class of drugs after antibiotics, steroid drugs have anti-inflammatory, anti-allergic, anti-shock, and anti-allergic effects. Many steroid drugs contain double bonds at the 1 and 2 positions, which generally enhance their potency and reduce drug-induced salt retention. Clinically used steroid drugs mainly include three categories: adrenocortical hormones, sex hormones, and anabolic steroids. Steroid drugs occupy an important position in the chemical drug system, and market demand has remained among the highest for many years.

[0003] The synthesis of steroid drugs from steroidal compounds through chemical dehydrogenation often faces challenges related to regioselectivity, stereoselectivity, complex reaction steps, and the use of toxic reagents or solvents. Currently, microbial transformation has become an important pathway for producing new steroidal drugs, steroidal active pharmaceutical ingredients, and their key intermediates. Microbial transformation of steroidal compounds refers to the selective modification or alteration of a specific part (group) of a steroidal compound using microbial cells or their enzymes, thereby obtaining structurally similar but physiologically enhanced compounds or intermediates—i.e., steroidal drugs. Microbial transformation not only boasts advantages such as high specificity, mild reaction conditions, fewer steps, shorter cycles, fewer side reactions, and environmental friendliness, but also allows reactions to occur at almost any position on the steroid nucleus molecule, accomplishing many reactions that are difficult to achieve or perform using chemical methods. Furthermore, it exhibits high stereoselectivity and regioselectivity, such as dehydrogenation at the C1,2 position of the A ring, 11α-hydroxylation, and 11β-hydroxylation. In addition, the diverse and abundant nature of microorganisms, along with the potential for combinatorial transformation reactions within cells, gives it a greater advantage in obtaining diverse and novel products.

[0004] Steroidal C1,2 dehydrogenase (KstD) belongs to the flavoprotease class and is a key enzyme responsible for catalyzing the dehydrogenation reaction at the C1,2 position of ring A, but it suffers from low activity. Limited structural information about the KstD protein restricts its molecular modification, and there are currently few reports on molecular modifications that enhance the activity of this enzyme against 19-nor-4-androstenedione. Summary of the Invention

[0005] In view of the aforementioned deficiencies in the existing technology: The first objective of this application is to provide a steroid C1,2 dehydrogenase mutant, the nucleotide sequence of which is shown in SEQ ID NO 3.

[0006] The second objective of this application is to provide a gene encoding the above-mentioned steroid C1,2 dehydrogenase mutant, the nucleotide sequence of which is shown in SEQ ID NO 4.

[0007] Preferred, The N-terminus of the steroid C1,2 dehydrogenase mutant is linked with an MBP sequence, yielding the amino acid sequence SEQ ID NO 5, which is used to enhance protein expression and purification.

[0008] The corresponding nucleotide sequence obtained by encoding SEQ ID NO 5 is shown in SEQ ID NO 6.

[0009] A third objective of this application is to provide an expression vector containing SEQ ID NO 4 or SEQ ID NO 6.

[0010] A fourth objective of this application is to provide a bacterial strain containing the aforementioned expression vector. Preferably, the strain is *Escherichia coli*.

[0011] The fifth objective of this application is to provide a method for producing the steroid C1,2 dehydrogenase mutant described in SEQ ID NO 3 or SEQ ID NO 5, comprising the following steps: culturing the strain described in the fourth objective, collecting the culture, and obtaining the steroid C1,2 dehydrogenase mutant corresponding to SEQ ID NO 3 or SEQ ID NO 5.

[0012] The sixth aspect of this application aims to provide the application of the steroid C1,2 dehydrogenase mutant described in SEQ ID NO 3 or SEQ ID NO 5 in the preparation of a steroidal drug, wherein the steroidal drug is estrone, prepared by a C1,C2 dehydrogenation reaction using 19-nor-4-androstenedione as a substrate. Specifically: an emulsifier, an electron acceptor, and the steroid C1,2 dehydrogenase mutant described in SEQ ID NO 3 or SEQ ID NO 5 are added to a 19-nor-4-androstenedione solution and mixed to obtain estrone.

[0013] The emulsifier is at least one of PEG 200, β-cyclodextrin, Triton X-100 or Tween-80, preferably Tween-80.

[0014] The electron acceptor is at least one of phenazine methyl sulfate (PMS) or menaquinone, preferably phenazine methyl sulfate.

[0015] The 19-nor-4-androstenedione solution was a 50 mM Tris-HCl solution.

[0016] The pH of the reaction is 7.5 to 8.5.

[0017] The reaction temperature is 25~30℃.

[0018] The seventh aspect of this application is to provide the application of the steroid C1,2 dehydrogenase mutant described in SEQ ID NO 3 or SEQ ID NO 5 in the preparation of C1,2-dehydrosteroid compounds, wherein the reaction substrate of the C1,2-dehydrosteroid compound is 16α-hydroxy-4,9(11)-androsadiene-3,17-dione, 16β-hydroxy-4,9(11)-androsadiene-3,17-dione or 4,9-estradiol-3,17-dione.

[0019] Compared with the prior art, the present invention has the following beneficial effects: In this application, both the reaction system for preparing the steroid C1,2 dehydrogenase mutant and the estradiol based on the steroid C1,2 dehydrogenase mutant are aqueous systems. The reaction system is simple and mild, avoiding the use of large amounts of organic solvents and toxic reagents, making it safe, green, and environmentally friendly. The post-reaction processing is simple, using conventional solvents such as methanol and ethanol, all of which can be recycled, significantly reducing production costs. Furthermore, the feed concentration can reach 50-100 g / L, and the steroid C1,2 dehydrogenase mutant exhibits high catalytic activity, with advantages such as short conversion time (<30 h), high conversion rate (>95%), few side reactions, and low cost. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application.

[0021] Figure 1 Electrophoresis diagram of KstD site-directed mutant nucleic acid.

[0022] Figure 2 SDS-PAGE electrophoresis images of KstD wild-type and mutant.

[0023] Figure 3 The results of liquid phase detection of the reaction solution with 19-nor-4-androstenedione as the catalytic substrate for the KstD mutant and estradiol.

[0024] Figure 4 TLC plate image of the reaction solution of KstD mutant catalyzing substrate 19-nor-4-androstenedione as estradiol. Detailed Implementation

[0025] To make the technical problems, technical solutions, and beneficial effects to be solved by this application clearer, the technical solutions in the embodiments of this application will be further described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are only used to explain this application and are not intended to limit the technical solutions of this application. All other embodiments obtained by those skilled in the art based on the embodiments in this application without creative effort are within the scope of protection of this application.

[0026] In the following embodiments: The steroid C1,2 dehydrogenase (KstD) gene is derived from Mycolicibacterium goodii. The amino acid sequence of the KstD enzyme is shown in SEQ ID NO 1. The host codons for expression in E. coli were optimized based on the sequence of SEQ ID NO 1, and the optimized gene sequence is shown in SEQ ID NO 2.

[0027] The mutant V304L / W305G with high activity was obtained by modifying the wild-type KstD enzyme. The corresponding amino acid sequence is shown in SEQ ID NO 3. The host codons of E. coli expression were optimized according to the sequence of SEQ ID NO 3. The optimized gene sequence is shown in SEQ ID NO 4.

[0028] Example 1

[0029] This embodiment demonstrates the expression of wild-type KstD enzyme, and the process is as follows: Based on the amino acid composition of the wild-type KstD enzyme, codon optimization was performed on its expression system in *E. coli*. The optimized KstD enzyme nucleotide sequence was then fully synthesized, and the target gene was incorporated into the expression vector pET28a(+). The constructed expression vector was transformed into competent cells of the expression strain BL21(DE3). Correct single colonies were picked and inoculated into 5 mL of LB broth containing kanamycin, and cultured overnight at 37°C with shaking. The next day, the cells were transferred at a 2% inoculation rate to 50 mL of LB broth containing kanamycin, and cultured at 37°C with shaking for approximately 2.5 h. After measuring the OD600 of the bacterial culture to approximately 0.6–0.8, isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 0.3 mM. The cells were cultured at 20°C with shaking for 20 h, and then centrifuged at 5000 rpm for 5 min to collect the cells. The collected cells were then transferred to 2 mL of 50 mM pH... Resuspend the enzyme in 8.0% Tris-HCl buffer and mix well. Sonicate the resuspended enzyme for 10 min and centrifuge at 12000 r / min for 10 min to collect the supernatant.

[0030] Example 2

[0031] This embodiment involves the modification of the KstD enzyme, and the process is as follows: The wild-type KstD enzyme was structurally modeled using AlphaFold. The resulting model was then molecularly docked using AutoDuck molecular docking software. The docking results were analyzed to determine the active site of the KstD enzyme and the relevant sites in the substrate pocket. Several mutants were designed, including mutants W124G, H141S, T205F, S290V, V304L, W305A, W305G, and G498Y. The mutant primers are shown in Table 1.

[0032] The PCR reaction conditions were: 95℃ for 3 min, 30 cycles (95℃ for 30 s, 58℃ for 30 s, 72℃ for 4 min), and 72℃ for 5 min.

[0033] PCR amplification system: 1 µL template, 1 µL each of forward and reverse primers, 20 µL DNA polymerase mixture, and 17 µL ddH2O.

[0034] PCR products were purified and recovered using a gel extraction kit, and the concentration of the recovered products was verified using a nucleic acid micro-detector. The products were transformed into competent *E. coli* cells, plated on kanamycin LB agar plates, and positive colonies were picked for verification. After overnight incubation at 37°C on a shaker, plasmids were extracted and then transformed into *E. coli* BL21 to obtain a site-directed mutagenesis-positive recombinant strain. Inoculate 5 mL of LB liquid medium containing kanamycin and culture overnight at 37°C with shaking. The next day, transfer 2% of the culture to 50 mL of LB liquid medium containing kanamycin and culture at 37°C with shaking for about 2.5 h. After sampling and measuring the OD600 of the bacterial culture to approximately 0.6-0.8, add isopropyl-β-D-thiogalactoside (IPTG) to a final concentration of 0.3 mM. Culture at 20°C with shaking for 20 h. Collect the bacterial cells by centrifugation at 5000 r / min for 5 min. Resuspend the collected bacterial cells in 2 mL of 50 mM pH 8.0 Tris-HCl buffer and mix well. Sonicate the resuspended solution for 10 min and centrifuge at 12000 r / min for 10 min to collect the supernatant enzyme solution.

[0035] Table 1: Primer sequences .

[0036] The enzyme activity of the wild-type KstD enzyme in Example 1 and the mutant in Example 2 was measured as follows: The reaction mixture included 50 mM Tris-HCl, pH 8.0, 1.5 mM PMS, 40 µm DCPIP, substrates of different concentrations (2.5 µm to 400 µm) dissolved in 2% methanol, and appropriate amounts of supernatant enzyme solution collected in Examples 1 and 2. The measurement wavelength was 600 nm, the reaction temperature was 30 °C, and the reduction rate of DCPIP per minute was measured using a microplate reader (ε600nm = 18.7 × 10⁻⁶). 3 cm -1 ·M -1 Since the amount of dehydrogenation of C1(2) of the steroid substrate can be approximated by the amount of DCPIP reduced, this method can be used to determine the enzyme activity of different KstD mutants. The enzyme activity unit is expressed as U, where 1 U is defined as the reduction of 1 µmol of DCPIP per minute at 30 °C and pH 8.0. The results are shown in Table 2.

[0037] Table 2: Enzyme activity of KstD enzyme and its mutants .

[0038] All mutants were screened, and the results showed that mutant V304L / W305G (denoted as KstD) was the most effective. V304L / W305G The amino acid sequence SEQ ID NO:3 increased the catalytic activity of the substrate by 2.5 times.

[0039] Electrophoresis images of site-directed mutant nucleic acids of each KstD enzyme are shown below. Figure 1 As shown: from left to right, the nucleic acid electrophoresis of single-point mutant Pcr samples are W124G, H141S, T205F, S290V, V304L, W305A, W305G, and G498Y.

[0040] Example 3

[0041] This embodiment performs MBP / KstD V304L / W305G Construction and expression of fusion enzymes, including MBP / KstD V304L / W305G Fusion enzymes refer to the KstD enzyme mutant KstD V304L / W305G The N-terminus of the gene is linked with an MBP sequence, as follows: Using the KstD mutant plasmid from Example 2, and the KstD mutant plasmid and the MBP gene sequence as templates, the MBP gene sequence with homologous arms at both ends and the KstD mutant plasmid were amplified by PCR. The MBP / KstD structure was then constructed using homologous recombination. V304L / W305G Fusion enzyme plasmid pET-28a(+)-MBP-KstD V304L / W305G The recombinant plasmid was introduced into Escherichia coli BL21(DE3) to obtain the fusion enzyme mutant recombinant strain BL21-pET-28a(+)-MBP-KstD. V304L / W305G .

[0042] Pick a single colony and incubate it overnight at 37°C in 5 ml of LB broth. Then, inoculate it at a 1% inoculum into 50 ml of LB broth and incubate at 37°C until OD reaches 100%. 600 =0.6-0.8, add 0.3mM IPTG, and continue culturing at 20℃ for 20h.

[0043] After induction, the cells were collected by centrifugation at 5000 rpm for 5 min. The collected cells were resuspended in 2 ml of 50 mM pH 8.0 Tris-HCl buffer and mixed well. The resuspended solution was sonicated for 10 min and centrifuged at 12000 rpm for 10 min to collect the supernatant enzyme solution.

[0044] The collected supernatant enzyme solution was analyzed by SDS-PAGE protein electrophoresis. The results are as follows: Figure 2 As shown: Target protein MBP / KstD V304L / W305G The theoretical size is approximately 96 kDa. From Figure 2 As can be seen, the correct band appears at the corresponding position, proving that the enzyme protein MBP / KstD... V304L / W305G It can be expressed in soluble form in Escherichia coli.

[0045] Example 4

[0046] This embodiment analyzes the transformation of the steroid compound 19-nor-4-androstenedione, and the process is as follows: Weigh 10g of 19-nor-4-androstenedione, 0.03g of PMS, 0.3g of TW-80, and 1g of β-cyclodextrin into a 250mL round-bottom flask. Add 15mL of the supernatant enzyme solution collected in Example 3. Make up the volume to 100mL with 50mM pH 8.0 Tris-HCl solution, adjusting the pH to approximately 8.0. Place the system in a 30℃ constant-temperature magnetically stirred water bath. Replace the reaction system with pure oxygen three times, then place the entire system under pure oxygen protection. Start stirring to begin the reaction. At 16h, add 7.5mL of the supernatant enzyme solution collected in Example 3 and 0.015g of PMS. Samples were taken at 16h, 20h, 22h, 24h, and 26h. The conversion rate reached 98% at 24h and remained stable thereafter. After the reaction, the solid material was collected by direct filtration. The collected crude product was dried to constant weight, and 100 mL of dichloromethane solution was added. The mixture was stirred until the material was completely dissolved, and the filtrate was collected by filtration. The collected filtrate was concentrated under reduced pressure until a large amount of solid precipitated. The mixture was cooled to room temperature, and 300 mL of water was added with stirring for water precipitation. The estrone was collected by filtration, dried at 60 °C, and the liquid chromatography of the finished product was sent for analysis. The results are shown in the figure. Figure 3 , Figure 4The substrate 19-nor-4-androstenedione had a peak elution time of 8.888 min, and the product estrone had a peak elution time of 9.808 min. The product purity was 98.3%, and the yield was 91.8%. This demonstrates that the mutant provided in this application can efficiently convert the steroid compound 19-nor-4-androstenedione in a short time.

[0047] Example 5

[0048] This embodiment is set up the same as that of embodiment 4, except that the steroid compound 19-nor-4-androstenedione is replaced with substrates of the structures of formula (1), formula (2), and formula (3), respectively, and the mutant enzyme of this application is used to convert other high-concentration substrates.

[0049] .

[0050] Table 3: Conversion rates of different substrates .

[0051] As shown in Table 3, the mutant enzyme of this application can also perform C1,2-position dehydrogenation on other substrates at a concentration of 100 g / L. Compared with the wild type, after the same reaction time of 16 h, the substrate concentration after transformation by the mutant enzyme is significantly lower, while the concentration of the corresponding C1,2-position dehydrogenated steroidal compound product is significantly higher. These results also confirm that the mutant enzyme of this application exhibits substrate adaptability diversity.

[0052] The above-described embodiments are merely illustrative of several feasible implementations of the present invention, and their descriptions are relatively specific and detailed. However, they should not be construed as limiting the scope of the present invention, nor are the embodiments intended to limit the scope of protection in the claims of the present invention. For those skilled in the art, various modifications and improvements can be made without departing from the concept of the present invention. All equivalent implementations or changes that do not depart from the present invention should be included in the technology of the present invention.

Claims

1. A steroid C1,2 dehydrogenase mutant, characterized in that: The amino acid sequence of the steroid C1,2 dehydrogenase mutant is SEQ ID NO 3.

2. A gene encoding a gene, characterized in that: The nucleotide sequence of the encoding gene is SEQ ID NO 4.

3. A steroid C1,2 dehydrogenase mutant, characterized in that: The amino acid sequence of the steroid C1,2 dehydrogenase mutant is SEQ ID NO 5.

4. A gene encoding a gene, characterized in that: The nucleotide sequence of the encoding gene is SEQ ID NO 6.

5. An expression vector, characterized in that: The expression vector contains SEQ ID NO 4 or SEQ ID NO 6.

6. A strain comprising the expression vector of claim 5.

7. The strain according to claim 6, characterized in that: The strain in question is Escherichia coli.

8. The use of the steroid C1,2 dehydrogenase mutant of claim 1 or 3 in the preparation of a steroidal drug, wherein the steroidal drug is estradiol.

9. The application according to claim 8, characterized in that: The substrate for preparing the steroidal drug is 19-nor-4-androstenedione.

10. The use of the steroid C1,2 dehydrogenase mutant of claim 1 or 3 in the preparation of C1,2-dehydrosteroid compounds, wherein the reaction substrate is 16α-hydroxy-4,9(11)-androsadiene-3,17-dione, 16β-hydroxy-4,9(11)-androsadiene-3,17-dione or 4,9-estradiol-3,17-dione.

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