Bacteria-derived steroid side chain lyase and application of mutant of bacteria-derived steroid side chain lyase

Abstract

The invention discloses a bacterium-derived steroid side chain lyase and application of a mutant of the bacterium-derived steroid side chain lyase. Compared with the amino acid sequence shown in SEQ ID NO: 1, the steroid side chain lyase has amino acid residue difference at one or more sites of the 91st site, the 323rd site and the 324th site; compared with SEQ ID NO: 1, the amino acid sequence has at least 90% of sequence consistency; and the steroid side chain lyase has a steroid side chain splitting effect equivalent to or better than that of the steroid side chain lyase with the amino acid sequence shown as SEQ ID NO: 1. The steroid side chain lyase or the mutant thereof breaks through the limitation that traditional eukaryotic source P450scc is low in expression level, poor in activity and large in transformation difficulty, not only expands the species distribution range of P450scc, but also provides efficient and engineered novel enzyme resources for microbial synthesis of steroid key prodrugs (such as pregnenolone and progesterone), and has broad application prospects. The method has great application potential and economic value.

Description

Technical Field

[0001] This invention relates to the field of enzyme engineering, and more particularly to the application of a steroid side chain cleavage enzyme and its mutants in the synthesis of steroid intermediates progesterone or pregnenolone. Background Technology

[0002] Steroid drugs are among the most widely used drugs in clinical practice, employed for anti-tumor, anti-inflammatory, antimicrobial, antiviral, and antifungal purposes. Furthermore, steroid compounds have broad application prospects in agriculture and chemical industries. Therefore, the industrial production of steroids is of great significance for maintaining human health and promoting economic development. Pregnenolone and progesterone are not only essential steroid hormones in mammals but also common precursors for many important steroid drugs. Currently, pregnenolone and progesterone are obtained through traditional plant extraction or chemical synthesis methods using natural sterols as raw materials, which suffer from drawbacks such as low yield and severe environmental pollution.

[0003] Cholesterol side-chain degrading enzymes (P450scc) exist in both mammals and plants. These enzymes catalyze the cleavage of cholesterol side chains to generate pregnenolone, which is then converted to progesterone by 3β-hydroxysteroid dehydrogenase / isomerase. This one-step enzymatic reaction replaces the complex 5-7 steps of chemical synthesis, providing a simple, efficient, and highly promising microbial synthetic route for the production of important steroid intermediates such as pregnenolone and progesterone. However, the microbial expression of these two types of P450scc from higher animals and plants faces difficulties and low yields, resulting in pregnenolone conversion rates remaining at the μmol level, severely restricting the industrial application of P450scc. Therefore, identifying and characterizing bacterial P450scc with high catalytic activity and operational stability is helpful in overcoming the key technical bottlenecks in the efficient synthesis of steroid drugs via microbial transformation.

[0004] Therefore, those skilled in the art are dedicated to developing bacterial steroid side-chain lyases with high catalytic activity and further improving catalytic efficiency through enzyme engineering, thereby promoting the efficient biosynthesis of important steroid intermediates. Summary of the Invention

[0005] In view of the above-mentioned deficiencies of the prior art, the technical problem to be solved by the present invention is to provide a bacterial steroid side chain lyase and its application in the synthesis of important steroid intermediates progesterone or pregnenolone, thereby solving the problem that existing eukaryotic steroid side chain lyases are difficult to apply in industrial production.

[0006] To achieve the above objectives, the present invention provides a steroidal side-chain lyase, the amino acid sequence of which is any one of the amino acid sequences shown in (P1) to (P7): (P1) The amino acid sequence of the steroidal side chain lyase as shown in SEQ ID NO:1; (P2) An amino acid sequence in which the three key catalytic residues S91, T323 and M324 in the amino acid sequence of (P1) are replaced and / or deleted and / or added by one or more amino acid residues and have the same function. (P3) has an amino acid sequence that is more than 90% homologous to (P1) or (P2); (P4) The amino acid sequence of the fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of any amino acid sequence in (P1)-(P3); (P5) The amino acid sequence obtained by truncating or modifying 1-100 amino acids at the N-terminus of any of the proteins defined in (P1)-(P3).

[0007] (P6) is an amino acid sequence that has more than 90% homology with (P1) or (P2) and has the same function or better steroid side chain cleavage effect. (P7) The amino acid sequence of a protein with the same function obtained by truncating or modifying 1-100 amino acids at the N-terminus of any of the proteins defined in (P1)-(P3).

[0008] Furthermore, the amino acid at position 91 of the steroid side chain lyase is mutated to S91M or S91I; the amino acid at position 323 is mutated to T323I, T323L, T323C or T323V; and the amino acid at position 324 is mutated to M324L or M324I.

[0009] Furthermore, the steroidal side chain lyase is a mutant of the amino acid shown in SEQ ID NO:1; the mutant amino acid residues are: S91M, S91I, T323I, T323L, T323C, T323V, M324L or / and M324I.

[0010] Furthermore, the mutant amino acid residues also include mutations in combinations of amino acid residues, such as M324I and S91M, M324I and S91I, M324I and T323I, M324I and T323L, M324I and T323C, and / or M324I and T323V.

[0011] The present invention also discloses a nucleotide encoding a steroid side chain lyase, the nucleic acid sequence of which also includes a nucleic acid sequence optimized by microbial codons.

[0012] The present invention also discloses a nucleotide recombinant expression vector containing an encoding steroid side chain lyase.

[0013] The present invention also discloses an engineered bacterium containing a recombinant expression vector of a nucleotide encoding a steroid side chain lyase. The host cell used in the construction is Escherichia coli, Pseudomonas aeruginosa, or Mycobacterium, and it also contains an electron-transferring ferrooxidase and / or ferrooxidase reductase.

[0014] The present invention also discloses an engineered bacterium containing nucleotides encoding steroid side chain lysins, wherein the host cells used in the construction are Escherichia coli, Pseudomonas aeruginosa, or Mycobacterium.

[0015] Furthermore, the engineered bacteria also contain electron-transferring active ferrooxidases and / or ferrooxidase reductases.

[0016] This invention also discloses the application of a steroid side-chain cleaving enzyme, which reacts a steroid / sterol compound of Formula I with the cleaving enzyme to obtain the steroid side-chain cleavage product of Formula II;

[0017] Where R is a straight-chain or branched substituted or unsubstituted alkyl, alkenyl or ynyl group; Of the carbon atoms at positions 1 to 17, except for positions 10 and 13, there are 0 or more carbon-carbon unsaturated bonds at any of these positions. Of the carbon atoms at positions 1 to 17, excluding positions 10 and 13, any 0 or more carbon atoms are independently substituted with -OH or =O.

[0018] Compared with existing technologies, this invention has the following advantages and beneficial effects: P450scc derived from higher plants and animals faces many problems such as difficulty in microbial expression, low yield, and difficulty in modification, which limits the industrial application of P450scc. The steroidal side-chain lysin described in this invention is derived from bacteria and is easier to express and modify than P450scc derived from eukaryotes, making it suitable for large-scale industrial production. Through enzyme engineering modification, the catalytic efficiency is increased by about 6 times. The catalytic application of these enzymes will help develop a microbial synthesis system for the efficient synthesis of important steroidal intermediates such as pregnenolone and progesterone from plant sterols, thereby breaking through the bottleneck of efficient synthesis of important steroidal drugs through microbial transformation.

[0019] In a preferred embodiment 1 of the present invention, the process of obtaining steroidal side chain lyase is described in detail; In another preferred embodiment 2 of the present invention, the process of obtaining ferric oxide reductase and ferric oxide reductase is described in detail. In another preferred embodiment 3 of the present invention, the reaction process of steroid side chain cleavage catalyzed by steroid side chain cleavage enzyme is described in detail; In another preferred embodiment 4 of the present invention, the analysis process of key catalytic sites of CYP204A5 is described in detail; In another preferred embodiment 5 of the present invention, the saturation mutation and combined mutation of the key catalytic sites of CYP204A5 are described in detail. In another preferred embodiment 6 of the present invention, the construction process of recombinant engineered Escherichia coli containing steroidal side chain lysin is described in detail; In another preferred embodiment 7 of the present invention, the process of synthesizing progesterone by engineered Escherichia coli catalyzing 4-cholesten-3-one is described in detail.

[0020] The following will further explain the concept, specific structure, and technical effects of the present invention in conjunction with the accompanying drawings, so as to fully understand the purpose, features, and effects of the present invention. Attached Figure Description

[0021] Figure 1 This is an electrophoretic image of CYP204A5 expression detected by 12% sodium dodecyl sulfonate polyacrylamide gel (SDS-PAGE) electrophoresis of a preferred embodiment 1 of the present invention. Figure 2 This is a schematic diagram of the process of steroidal side chain cleavage enzyme catalyzing 4-cholesten-3-one in a preferred embodiment 3 of the present invention; Figure 3 This is a diagram showing the in vitro catalytic characterization results of a steroidal side-chain lyase according to a preferred embodiment 3 of the present invention. Figure 4 This is a preferred embodiment 4 of the present invention, showing the docking model of the CYP204A5 simulated structure with the substrate 4-cholesten-3-one and a schematic diagram of the key amino acids around the substrate binding pocket. Figure 5 This is a preferred embodiment 4 of the present invention, showing the biochemical results of an alanine scanning mutant of amino acid residues within a 5 Å range of the interaction between CYP204A5 and the substrate cholesterolone. Figure 6 This is the analysis result of the relative catalytic efficiency of CYP204A5 and its mutant in the conversion of 4-cholestyren-3-one to progesterone in a preferred embodiment 5 of the present invention; Figure 7 This is an UPLC diagram of the synthesis of progesterone from 4-cholestyren-3-one by CYP204A5 wild-type and mutant engineered bacteria, which is a preferred embodiment of the present invention, in Example 7. Detailed Implementation

[0022] The following description, with reference to the accompanying drawings, illustrates several preferred embodiments of the present invention to make its technical content clearer and easier to understand. The present invention can be embodied in many different forms, and the scope of protection of the present invention is not limited to the embodiments mentioned herein.

[0023] Example 1: Obtaining steroid side chain lyase

[0024] (1) Obtaining the gene encoding steroid side chain cleavage enzyme

[0025] This invention utilizes non-patent literature 3 ( MBio CYP204A5, which possesses steroid side-chain cleavage activity, was obtained by screening for P450 enzymes in the genomes of potential steroid metabolizing strains reported in 2016, 7(2), 10-1128. . This enzyme is derived from the genus *Sphingosine Box*. Sphingopyxis sp.)H115. Based on the codon preference of E. coli, the nucleic acid sequence encoding CYP204A5 was optimized, and then sent to a company for synthesis and insertion between the NdeI and XhoI restriction sites of the expression vector pET28a to obtain the recombinant plasmid pCYP204A5.

[0026] The amino acid sequence of the steroidal side chain lyase CYP204A5 is shown in SEQ ID NO. 1, and the corresponding optimized sequence encoding the nucleic acid is shown in SEQ ID NO. 2.

[0027] (2) Obtaining engineered bacteria expressing steroid side chain lyase

[0028] Transform E. coli BL21(DE3) competent cells with the pET28a plasmid carrying the gene encoding steroidal side chain lyase into E. coli BL21(DE3) competent cells. Spread the cells on LB selective plates containing 50 μg / mL kanamycin and incubate upside down at 37 °C for about 12 hours. Transformants that grow on LB selective plates containing kanamycin are the transformants with the recombinant plasmid, which are the engineered bacteria expressing steroidal side chain lyase.

[0029] (3) Fermentation culture of engineered bacteria

[0030] Single colonies were picked from LB selective culture plates and inoculated into 5 mL of LB liquid medium containing 50 μg / mL kanamycin, and cultured at 37 °C and 220 rpm for 12 h. Subsequently, the culture was transferred at a 1:100 inoculation ratio to 500 mL of LB medium containing 50 μg / mL kanamycin, and cultured at 37 °C and 220 rpm until OD (dose elapsed). 600 The culture medium was brought to a final concentration of 0.8–1.0. The culture medium was then cooled on ice for 10 min to below 18 °C. Isopropyl-β-D-thiogalactopyranoside (IPTG) (100 μM), 5-aminolevulinic acid (500 μM), and Fe2+ (200 μM) were added. 2+ Expression was induced at 18 °C and 220 rpm for 20 h.

[0031] (4) Purification of steroid side chain lyase

[0032] In step (3), the cultured bacterial culture was centrifuged at 4 °C and 13,500 xg for 10 min to collect the bacterial cells. The bacterial cells were resuspended in an appropriate amount of lysis buffer (Lysis Buffer, 50 mM 4-hydroxyethylpiperazine ethanesulfonic acid (Hepes), 300 mM NaCl, 5 mM imidazole, 10% glycerol, pH 7.5); the resuspended bacterial culture was sonicated until it was homogeneous and clear; then it was centrifuged at 4 °C and 13,500 xg for 60 min, and the supernatant was collected. The cell lysis supernatant was mixed with 2 mL of Ni-IDA Beads that had been washed with deionized water and lysis buffer, and the mixture was incubated at low temperature for 30 min to ensure that the protein and Ni NTABeads were fully bound. The mixture was transferred to an empty affinity chromatography column fitted with a gasket. Nickel beads containing bound proteins were collected under gravity. The column was first washed with 10 column volumes of Wash Buffer (50 mM Hepes, 300 mM NaCl, 40 mM imidazole, 10% glycerol, pH 7.5) to remove non-specifically adsorbed proteins. Elution was then performed with 10 column volumes of Elution Buffer (50 mM Hepes, 300 mM NaCl, 300 mM imidazole, 10% glycerol, pH 7.5). The eluent was collected, and protein size and purity were determined using a 12% SDS-PAGE gel. The results are shown below. Figure 1 As shown, M represents a low molecular weight protein standard (Marker), and the others, from left to right, are Marker and CYP204A5, respectively. Eluents with a protein purity greater than 90% were combined and recovered. The purified steroidal side-chain lyase was purified using a PD-10 gel column. The protein buffer was replaced with protein storage buffer (50 mM Heps, 100 mM NaCl, 10% glycerol, pH 7.5), then concentrated using a 30 kDa ultrafiltration tube (Millipore). Finally, the mixture was pipetted and dispensed into 50-100 μL tubes, which were then flash-frozen in liquid nitrogen at -80 °C. The target steroidal side-chain lyase was then analyzed for protein concentration using a NanoDrop OneC (Thermo Scientific).

[0033] Example 2: Obtaining ferric oxidoreductase and ferric oxidoreductase

[0034] Derived from Spleen-long Synechococcus ( Synechococcus elongatusThe redox chaperones of PCC 7942 (ferredoxin Fdx / ferredoxin reductase FdR) were optimized according to the codon preference of *E. coli*, and synthesized by a company. These chaperones were then cloned into the NdeI and XhoI restriction sites of the expression vector pET28a to obtain recombinant plasmids pFdx and pFdR. The recombinant plasmids were transformed into competent *E. coli* BL21(DE3) cells. Transformants were picked and cultured in LB medium containing the corresponding antibiotics. Expression was induced with 100 μM IPTG, and the enzymes were purified with Ni-IDA. The optimized nucleotide sequences encoded by Fdx and FdR are shown in SEQ ID NO. 3-4.

[0035] Example 3: The reaction of steroid side chain cleavage catalyzed by steroid side chain lyase

[0036] The reaction system consisted of 100 μL of a catalytic buffer (50 mM TES buffer, sodium 2-[(tris(hydroxymethyl)methyl)amino]-1-ethanesulfonate) (pH = 7.5), containing 10 μM of the steroidal side-chain lyase obtained in Example 1, 40 μM of ferredoxin (Fdx), 10 μM of ferredoxin reductase (FdR), 500 μM of steroidal substrate, and an NADPH regeneration system comprising 1 mM glucose, 1 mM glucose dehydrogenase (GDH), and 2 mM NADP. + The mixture was placed at 18 °C and allowed to stand for 12 hours. After the reaction was complete, an equal volume of methanol was added to quench the reaction. The mixture was then centrifuged at 13,500 x g for 10 min, and the supernatant was collected for LC-MS analysis. The LC-MS conditions were as follows: Instrument: Shimadzu LC-MS2020; Chromatographic column: Shimadzu Shim-pack GIST C18 column (2 μm, 2.1 × 100 mm); Mobile phase: Mobile phase A: water (with 0.01% formic acid added), Mobile phase B: acetonitrile); Flow rate: 0.2 mL / min; Detection wavelength: UV absorbance at 254 nm; Gradient conditions: t = 0 min, 30% B; t = 8 min, 100% B; t = 20 min, 100% B; t = 21 min, 30% B; t = 25 min, 30% B.

[0037] The process by which steroid side-chain cleavage enzymes catalyze 4-cholesten-3-one is as follows: Figure 2 As shown.

[0038] The in vitro catalytic characterization analysis results of steroid side chain cleavage enzymes are as follows: Figure 3 As shown, Part A is the LC-MS analysis spectrum of the substrate 4-cholesten-3-one catalyzed by steroidal side chain lyase; Part B is the LC-MS analysis spectrum of the side chain degradation of the substrates cholesterol, sterol and β-sitosterol catalyzed by CYP204A5.

[0039] The results showed that CYP204A5 had good catalytic effects on 4-cholesten-3-one, cholesterol, sterol and β-sitosterol. Therefore, further research on CYP204A5 will be conducted below.

[0040] Example 4: Analysis of key catalytic sites in CYP204A5

[0041] (1) Simulation of CYP204A5 crystal structure and docking with substrate 4-cholesten-3-one

[0042] A three-dimensional model of the protein CYP204A5 was constructed using Alphafold 3, and then an interaction model between the protein and its substrate 4-cholesten-3-one was established via molecular docking using SwissDock. The relevant catalytic sites around its catalytic pocket, such as..., were analyzed. Figure 4 The image shows 22 associated amino acid residues around the substrate binding pocket of CYP204A5, namely 77S, 78M, 81F, 90Y, 91S, 177S, 180M, 250L, 253W, 254A, 257E, 258T, 320V, 321A, 322F, 323T, 324M, 325I, 326R, 429A, 430G, and 431T.

[0043] (2) Mutate the 22 amino acids around the substrate pocket to alanine or glycine.

[0044] Using the previously constructed plasmid pCYP204A5 as a template, rolling circle PCR was performed. 2 μL of the PCR product was examined by agarose gel electrophoresis to verify band size. The PCR product with verified size was digested with Dpn I enzyme for 3 hours, and the original plasmid was removed before transformation into *E. coli* DH5α. The extracted plasmid was sequenced and confirmed to be correct, then transformed into *E. coli* BL21(DE3) for expression.

[0045] 3) Functional verification of mutant proteins

[0046] A series of mutants were expressed and purified according to the method in Example 1. Using 4-cholesten-3-one as a substrate, the mutant proteins were functionally validated according to the method in Example 3. The reaction solution was analyzed by HPLC. Alanine scanning was performed to detect the amino acid residues within 5 Å of the interaction between CYP204A5 and the substrate cholesterone. The analytical results are as follows. Figure 5 As shown in the figure. The results show that the catalytic activity of most mutant proteins is significantly reduced or even disappears, proving that most amino acids in the catalytic pocket affect the catalytic activity of the protein.

[0047] Example 5: Saturation Mutations and Combinatorial Mutations of Key Catalytic Sites in CYP204A5

[0048] (1) Construction of mutants

[0049] Using the previously constructed plasmid pCYP204A5 as a template, the mutant expression plasmid was constructed and validated according to the steps in Example 4.

[0050] (2) Functional verification of mutants

[0051] A series of mutants were expressed and purified according to the method in Example 1. Using 4-cholesten-3-one as a substrate, the mutant proteins were functionally verified according to the method in Example 3. UPLC analysis of the reaction solution was performed. The relative catalytic efficiency of CYP204A5 and its mutants in catalyzing the conversion of 4-cholesten-3-one to progesterone is shown in the following results. Figure 6 As shown in the figure. The results showed that compared with wild-type CYP204A5, the mutants S91M, S91I, T323V, T323L, T323C, T323I, M324L, M324I and the combined mutants M324I-S91M, M324I-S91I, M324I-T323V, M324I-T323L, M324I-T323C, and M324I-T323I significantly improved the catalytic activity for the substrate 4-cholesten-3-one.

[0052] The nucleic acid sequence encoding the mutant S91M is shown in SEQ ID NO. 5; The nucleic acid sequence encoding the mutant S91I is shown in SEQ ID NO. 6; The nucleic acid sequence encoding the mutant T323V is shown in SEQ ID NO. 7; The nucleic acid sequence encoding the mutant T323L is shown in SEQ ID NO. 8; The nucleic acid sequence encoding the mutant T323C is shown in SEQ ID NO. 9; The nucleic acid sequence encoding the mutant T323I is shown in SEQ ID NO. 10; The nucleic acid sequence encoding mutant M324L is shown in SEQ ID NO. 11; The nucleic acid sequence encoding mutant M324I is shown in SEQ ID NO. 12; The nucleic acid sequence encoding the mutant M324I-S91M is shown in SEQ ID NO. 13; The nucleic acid sequence encoding the mutant M324I-S91I is shown in SEQ ID NO. 14; The nucleic acid sequence encoding the mutant M324I-T323V is shown in SEQ ID NO. 15; The nucleic acid sequence encoding the mutant M324I-T323L is shown in SEQ ID NO. 16; The nucleic acid sequence encoding the mutant M324I-T323C is shown in SEQ ID NO. 17; The nucleic acid sequence encoding the mutant M324I-T323I is shown in SEQ ID NO. 18; Example 6: Construction of recombinant engineered Escherichia coli containing steroidal side-chain lyase The construction of engineered bacteria involves the following two steps: 1) Construction of plasmids containing nucleotide sequences encoding ferrooxidase / ferrooxidase reductase The gene encoding the iron redox protein Fdx from Synechococcus elongatus was amplified using primers 1 (5'-GGATCCGAATTCGATGGCAACCTACAAGGTTACGCTCG-3') and 2 (5'-GGCCGCAAGCTTTTAGTAGAGGTCTTCTTCTTTGTGG-3') and inserted between the EcoRI-HindIII restriction sites of the first multiple cloning site of the vector pCDFDuet. The plasmid with the correct site selected for sequencing was Fdx-pCDFDuet. The gene encoding the ferrooxidase reductase FdxR of Synechococcus elongatus was amplified using primers 3: 5'-GATATACATATGTTGAATGCGAGTGTGGCTGGCG-3' and 4: 5'-ACCAGACTCGAGGGCTGAACTAGTAGGTTTCAACATG-3', and inserted between the NdeI-XhoI restriction sites at the second multiple cloning site of plasmid Fdx-pCDFDuet. The plasmid Fdx-FdxR-pCDFDuet was constructed by sequencing the selected sites correctly.

[0053] 2) Transformation of co-expression plasmids

[0054] Take one tube of BL21(DE3) competent cells, add 5 μL of the expression plasmid of the steroidal side chain lyase obtained in Example 1 or the expression plasmid of the steroidal side chain lyase mutant obtained in Example 5, and the plasmid Fdx-FdxR-pCDFDuet obtained above. After incubating on ice for 30 min, heat shock at 42 °C for 90 s, incubate on ice for 2 min, add 1 mL of LB liquid medium, incubate at 37 °C for 1 h, spread on a plate containing dual resistance to kanamycin and streptomycin (50 μg / mL), incubate upside down at 37 °C overnight, pick single colonies and culture in LB liquid medium containing dual resistance to kanamycin / streptomycin for 12 h to obtain recombinant engineered Escherichia coli containing steroidal side chain lyase.

[0055] Example 7: Engineered Escherichia coli catalyzes the synthesis of progesterone from 4-cholesten-3-one

[0056] Specifically, it consists of the following 3 steps: (1) Induction culture: The engineered Escherichia coli strain from Example 6 was inoculated into LB liquid medium containing 50 μg / mL kanamycin and 50 μg / mL streptomycin dual resistance, and transferred at a 1% inoculum to 50 mL of LB liquid medium containing the corresponding antibiotics. The culture was carried out at 37 °C and 220 rpm until OD. 600 Once the pH reaches 0.8–1.0, cool the culture medium on ice for 10 min to lower the temperature to below 18 °C. Add isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 100 μM, 5-aminolevulinic acid (5-aminolevulinic acid) at 400 μM, and Fe2+ at 200 μM. 2+ The cells were induced to express the gene at 18 °C and 220 rpm for 20 h. After the induction was completed, the cells were collected by centrifugation at 7,000 xg for 10 min.

[0057] (2) Biotransformation: The collected Escherichia coli cells were resuspended in 5 mL of fresh LB medium, and 4-cholesten-3-one was added to a final concentration of 100 mg / L. Biotransformation was carried out at 18 °C, 220 rpm, for 12 h.

[0058] (3) Sample processing: After biotransformation, the biotransformation system was extracted three times with an appropriate amount of ethyl acetate. The ethyl acetate phases were combined, dissolved in methanol under reduced pressure, and filtered through a 0.22 μM organic filter membrane before UPLC detection. UPLC identification and comparison with standards confirmed that the constructed *E. coli* engineered bacteria could catalyze the synthesis of progesterone from 4-cholesten-3-one, such as... Figure 7 As shown, various mutants of CYP204A5 can almost completely convert 4-cholesten-3-one into progesterone.

[0059] Preferred embodiments of the present invention are described below. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. A steroidal side-chain lyase, characterized in that, The amino acid sequence of the steroidal side-chain lyase is any one of the amino acid sequences shown in (P1) to (P5): (P1) The amino acid sequence of the steroidal side chain lyase as shown in SEQ ID NO:1; (P2) An amino acid sequence in which the three key catalytic residues S91, T323 and M324 in the amino acid sequence of (P1) are replaced and / or deleted and / or added by one or more amino acid residues and have the same function. (P3) is an amino acid sequence that has more than 90% homology with the amino acid sequence of (P1) or (P2); (P4) The amino acid sequence of the fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of any amino acid sequence in (P1)-(P3); (P5) An amino acid sequence obtained by truncating or modifying 1-100 amino acids at the N-terminus of any of the proteins defined in (P1)-(P3).

2. The steroidal side-chain lyase according to claim 1, characterized in that, The amino acid at position 91 of the steroid side chain lyase amino acid sequence is mutated to S91M or S91I; the amino acid at position 323 is mutated to T323I, T323L, T323C or T323V; and the amino acid at position 324 is mutated to M324L or M324I.

3. The steroidal side-chain lyase according to claim 2, characterized in that, The steroidal side chain lyase is a mutant of the amino acid shown in SEQ ID NO:1; the mutant amino acid residues are: S91M, S91I, T323I, T323L, T323C, T323V, M324L or / and M324I.

4. The steroidal side-chain lyase according to claim 3, characterized in that, The mutant amino acid residues also include mutations in combinations of amino acid residues, wherein the combinations of amino acid residues are M324I and S91M, M324I and S91I, M324I and T323I, M324I and T323L, M324I and T323C, or / and M324I and T323V.

5. A nucleotide encoding a steroidal side-chain lyase as described in any one of claims 1-4, characterized in that, The nucleic acid sequence also includes nucleic acid sequences optimized with microbial codons.

6. A recombinant expression vector containing the nucleotides as described in claim 5.

7. An engineered bacterium containing a recombinant expression vector of the nucleotides as described in claim 5, characterized in that, The engineered bacteria are constructed using Escherichia coli, Pseudomonas aeruginosa, or Mycobacterium; the engineered bacteria also contain electron-transferring active ferrooxidases and / or ferrooxidase reductases.

8. An engineered bacterium containing the nucleotides as described in claim 5, characterized in that, The host cells used in the construction of the engineered bacteria are Escherichia coli, Pseudomonas aeruginosa, or Mycobacterium.

9. The engineered bacteria as described in claim 8, characterized in that, The engineered bacteria also contain electron-transferring active ferrooxidases and / or ferrooxidase reductases.

10. The application of the lysin according to any one of claims 1-5, characterized in that, The application involves reacting the steroid / sterol compound of Formula I with the cleavage enzyme to obtain the steroid side chain cleavage product of Formula II; Where R is a straight-chain or branched substituted or unsubstituted alkyl, alkenyl or ynyl group; Of the carbon atoms at positions 1 to 17, except for positions 10 and 13, there are 0 or more carbon-carbon unsaturated bonds at any of these positions. Of the carbon atoms at positions 1 to 17, excluding positions 10 and 13, any 0 or more carbon atoms are independently substituted by -OH or =O.

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