Use of a p450bm3 mutant in the synthesis of steroid drugs

Abstract

The application discloses a cytochrome P450BM3 mutant, which has an amino acid sequence of one of SEQ ID NOs:1-3, can effectively catalyze a hydroxylation reaction of a substrate methyl diketone, androstenone or 4,9 substance to generate corresponding hydroxylated steroids C7beta-hydroxymethyl diketone, C7beta-hydroxy androstenone, C11alpha-hydroxy 4,9 substance, has excellent regioselectivity, and has higher enzyme activity compared with an initial cytochrome P450BM3, and can reduce the production cost of related steroid drugs.

Description

Technical Field

[0001] This invention belongs to the field of biocatalysis technology, specifically, it relates to a cytochrome P450BM3 mutant and its application in the synthesis of steroid drugs. Background Technology

[0002] Steroid drugs are among the most valuable and widely used medications, with various steroid drugs currently widely used for a wide range of clinical diseases, including autoimmune diseases, inflammation, cancer, coronavirus infections, and osteoporosis. Introducing different functional groups, such as hydroxyl groups, into the steroid skeleton through regioselectivity and stereoselectivity is crucial for the physiological and pharmacological activity of steroid drugs.

[0003] In the synthesis of steroidal drugs, chiral hydroxylation reactions at specific sites are frequently catalyzed using enzymes or enzyme systems. Enzymes, as catalysts, are typically characterized by excellent regio or stereoselectivity to natural substrates, but may exhibit lower thermal and solvent stability. Due to their narrow substrate spectra, enzymes tend to be more specific than chemical catalysts, limiting their broad applicability. Therefore, discovering or developing an enzyme that catalyzes a specific reaction and achieving commercially viable yields and sufficient purity is a challenging task.

[0004] Cytochrome P450 belongs to the superfamily of heme-containing enzymes and is widely distributed in nature. Cytochrome P450BM3 monooxygenase is a large single-peptide protein of approximately 120 kDa. It is a single-component cytochrome P450 monooxygenase formed by the natural fusion of an oxidative domain and a reductase domain composed of NADPH-dependent FAD and FMN. The heme domain is 55 kDa, and the flavin-dependent reductase domain is 65 kDa. The natural fusion of the heme and reductase domains results in a system with high electron transfer efficiency and velocity, enhancing the catalytic activity of P450BM3. Its natural substrate turnover rate is approximately 3000 times / minute, exhibiting very high catalytic activity. However, since transformations involving non-natural substrates typically have low NADPH consumption rates and poor coupling efficiency, and exhibit low or even no activity with larger steroid substrates and smaller substrate molecules such as cyclohexane, benzene (phenol), and benzyl alcohol, the application of wild-type P450BM3 is somewhat limited.

[0005] The hydroxylation of steroids by P450BM3 has been the most widely reported. In 1997, it was reported that the P450BM3 mutant F87A exhibited C2β and C15β hydroxylation activities on testosterone, and C2β and C16β hydroxylation activities on progesterone. V. Rea, AJ Kolkman, E. Vottero, and others discovered that P450BM3 catalyzes an important steroid mutant, A82W, achieving for the first time the hydroxylation of testosterone at the C16α / β position by the P450BM3 mutant. Subsequently, based on this article, Carlos G. Acevedo-Rocha, Charles G. Gamble, and others, guided by information obtained from mutant landscapes and molecular dynamics simulations, rationally designed an iterative saturated mutant library and developed a feasible directed evolution strategy. This strategy enabled near-perfect stereo- and diastereoselective hydroxylation of five different steroids (testosterone, nandrolone, berberine, androstenedione, and norethindrone) at the C16 position with high catalytic activity. Summary of the Invention

[0006] Our research group reported in the literature (Li A, Acevedo-Rocha CG, D'Amore L, et al., Regio-and Stereoselective Steroid Hydroxylation at the C7-Position by Cytochrome P450 Monooxygenase Mutants[J]. Angewandte Chemie International Edition, 2020.) that, through DNA assembly and USER-based protein library construction technology, and using a concentrated mutagenesis strategy, we constructed a mutant library with up to 15 amino acid mutations that can be introduced simultaneously in the active pocket center using the P450BM3 triple mutant (F87G / A328G / A330W) as a template. For the first time, we screened out the P450BM3 mutant LG-23 (amino acid sequence SEQ ID NO:7) with 14 mutation sites, which has C7β-hydroxylation activity against eight steroidal compounds. It also has high activity and high selectivity, filling the gap in steroid C7β-hydroxylation. C7β-hydroxylation of steroids can be used to synthesize various steroid drugs, such as the anticancer drug fulvestridium, the cardiovascular drug eplerenone, and the diuretic spironolactone. However, when LG-23 catalyzes steroids, only a few mM of product is formed, indicating low activity that cannot meet the yield requirements for industrial production, thus limiting its industrial application.

[0007] To promote the application of LG-23 in steroid synthesis, it is essential to enhance its enzymatic activity while maintaining its regio and stereoselectivity. Therefore, we used LG-23 as the initial enzyme and continued to modify it through protein engineering and directed evolution. After multiple rounds of site-directed saturation mutagenesis, we successfully obtained some mutants with higher enzyme activity. These mutants can hydroxylate steroids such as methyl diketones and androstenediones at the C7β position and exhibit C11 hydroxylation activity for 4,9-hydroxylated compounds, showing potential for the industrial synthesis of related steroid drugs. We also used isopropanol dehydrogenase to regenerate and cycle the cofactor NADPH. By constructing a co-expression plasmid of isopropanol dehydrogenase and the P450BM3 mutant, as well as a co-expression strain containing the co-expression plasmid, we improved the catalytic activity of the P450BM3 mutant for steroids, replacing the method of adding dehydrogenase and NADP. + The present invention utilizes methods to regenerate and cycle the cofactor NADPH. Specifically, the present invention includes the following technical solutions.

[0008] The first aspect of the present invention provides a cytochrome P450BM3 mutant, which is a mutant formed by mutation of one or more, preferably two or more, of the following sites in the amino acid sequence of the initial cytochrome P450BM3 (hereinafter referred to as LG-23): Y51, G157, T260. The cytochrome P450BM3 mutant has the function of catalyzing the reaction of methyl diketone, androstenedione or 4,9-dione to generate C7β-hydroxymethyl diketone, C7β-hydroxyandrostenedione or C11α-hydroxy4,9-dione, respectively.

[0009] Preferably, it is a mutant formed by one or more mutations selected from the following groups in the amino acid sequence SEQ ID NO:7 of the initial enzyme LG-23: Y51I, G157S, T260G.

[0010] Furthermore, its amino acid sequence is SEQ ID NO:1, which is the T260G mutant of the initial enzyme LG-23, abbreviated as LG-23 / T260G in this article; or its amino acid sequence is SEQ ID NO:2, which is the Y51I / T260G mutant of the initial enzyme LG-23, abbreviated as LG-23 / Y51I / T260G in this article; or its amino acid sequence is SEQ ID NO:3, which is the Y51I / G157S / T260G mutant of the initial enzyme LG-23, abbreviated as LG-23 / Y51I / G157S / T260G in this article.

[0011] A second aspect of the invention is to provide a gene encoding the aforementioned cytochrome P450BM3 mutant.

[0012] Preferably, the nucleotide sequence of the gene encoding the amino acid sequence SEQ ID NO:1 of the cytochrome P450BM3 mutant, namely LG-23 / T260G, is SEQ ID NO:4; the nucleotide sequence of the gene encoding the amino acid sequence SEQ ID NO:2 of the cytochrome P450BM3 mutant, namely LG-23 / Y51I / T260G, is SEQ ID NO:5; and the nucleotide sequence of the gene encoding the amino acid sequence SEQ ID NO:3 of the cytochrome P450BM3 mutant, is SEQ ID NO:6.

[0013] A third aspect of the present invention is to provide a plasmid containing the above-described coding gene. This plasmid may be a pET vector such as pET22b, pET24a, or pET28a, or other vectors such as pSH plasmids or pRSFDuet plasmids.

[0014] A fourth aspect of the present invention is to provide a microorganism for expressing the above-described cytochrome P450BM3 mutant, preferably a transformant transformed with the above-described plasmid.

[0015] The aforementioned microorganisms can be selected from Escherichia coli, Bacillus subtilis, Pichia pastoris, and Saccharomyces cerevisiae, with Escherichia coli being preferred, and Escherichia coli BL21(DE3) being more preferred.

[0016] A fifth aspect of the present invention is to provide the use of the above-mentioned cytochrome P450BM3 mutant or the above-mentioned microorganism in the production of steroidal compounds.

[0017] As an alternative implementation, the aforementioned microorganisms may be in the form of bacterial cells or their cell fragments.

[0018] In an exemplary application, methyl diketone, androstenedione, or 4,9-hydroxyl group were used as substrates, and the hydroxylation reaction was catalyzed by the above-mentioned cytochrome P450BM3 mutant or the above-mentioned microorganisms to generate the corresponding hydroxylated steroids C7β-hydroxymethyl diketone, C7β-hydroxyandrostenedione, or C11α-hydroxyl group, respectively, with reaction formulas (1), (4), and (5):

[0019] (1)

[0020] (4)

[0021] (5)

[0022] Furthermore, the above-mentioned application methods also include using 18-methyldione, adrenaline, testosterone, or nandrolone as substrates, respectively, and using the cytochrome P450BM3 mutant or the microorganism to catalyze a hydroxylation reaction to generate the corresponding hydroxylated steroids C7β-hydroxy18-methyldione, C7β-hydroxyadrenaline, C7β-hydroxytestosterone, or C7β-hydroxynandrolone, with reaction formulas (6), (7), (8), and (9), respectively:

[0023] (6)

[0024] (7)

[0025] (8)

[0026] (9)

[0027] Optionally, the above reaction system may contain glucose dehydrogenase, glucose, and NADP. + (Nicotinamide adenine dinucleotide phosphate, coenzyme II) cofactor cycling system; or the above reaction system contains alcohol dehydrogenase, isopropanol and NAD+. + The cofactor cycle system of (nicotinamide adenine dinucleotide, i.e., coenzyme I).

[0028] Furthermore, the aforementioned cytochrome P450BM3 can be co-expressed with glucose dehydrogenase or alcohol dehydrogenase in the cofactor circulation system in the same microbial strain.

[0029] The cytochrome P450BM3 mutants LG-23 / T260G, LG-23 / Y51I / T260G, and LG-23 / Y51I / G157S / T260G obtained by genetic engineering mutations in this invention exhibit higher enzyme activity and excellent regioselectivity in catalyzing steroid hydroxylation reactions, and also have broad substrate spectrum. The constructed engineered strains can hydroxylate steroids such as methyldione and androstenedione at the C7β position, and have C11 hydroxylation activity for 4,9-hydroxylated compounds, reducing the production cost of related steroid drugs and showing promise for industrial development. Attached Figure Description

[0030] Figure 1 This is the HPLC chromatogram of the methyl diketone reaction catalyzed by LG-23 in Example 1.

[0031] Figure 2 This describes the reaction of LG-23 / T260G in different cosolvents in Example 2.

[0032] Figure 3The plasmid map (A) expressing LG23 / T260G, the plasmid map (B) co-expressing LG23 / T260G and IPADHM4, and the whole-cell polyacrylamide gel electrophoresis image (C) are shown in Example 4. Figure 3 In C, M stands for Marker; lanes 1-3 are the cell lysate supernatants of E. coli expression strains expressing IPADHM4 alone, co-expressing LG-23 / T260G+IPADHM4, and expressing LG-23 / T260G alone, respectively.

[0033] Figure 4 It represents the product titer (concentration) of the methyl diketone reaction catalyzed by LG-23 / T260G under different cofactor regeneration systems.

[0034] Figure 5 It shows C7β-hydroxymethyldiketone 1 1H NMR (DMSO-d, 400MHz) spectrum.

[0035] Figure 6 It shows C7β-hydroxymethyldiketone 13 C10 NMR (DMSO-d, 400MHz) spectrum.

[0036] Figure 7 The spectrum of C7β-hydroxymethyldiketone HSQC (DMSO-d, 400MHz) is shown.

[0037] Figure 8 The spectrum of C7b-hydroxymethyldiketone HMBC (DMSO-d, 400MHz) is shown.

[0038] Figure 9 The spectrum of C7β-hydroxymethyldiketone NOSY (DMSO-d, 400MHz) is shown. Detailed Implementation

[0039] To improve the enzyme activity of the initial enzyme LG-23, the inventors, based on a computer-simulated 3D model of the protein sequence, and through rational analysis and semi-rational design, selected amino acid sites around the active pocket for site-directed saturation mutations, resulting in some mutants with improved enzyme activity, such as (Y51I, G157S, T260G) mutants.

[0040] In this article, the terms “(enzyme activity) increase” or “enhancement” as used above mean an increase of at least 100% compared to the reference level, such as an increase of at least 1, at least 2, or at least 3, or at least 5, or at least 10, or at least 20 times compared to the reference level.

[0041] The term "mutation" includes, but is not limited to, the substitution, deletion, insertion, or chemical modification of amino acid residues, preferably a positive mutation, i.e., a mutation that increases enzyme activity. The substitution can be a non-conservative substitution, a conserved substitution, or a combination of both. A "conservative" amino acid substitution or mutation refers to the interchangeability of residues with similar side chains, and therefore generally includes the substitution of amino acids in a polypeptide with amino acids from the same or similar amino acid definition class. However, as used herein, if a conserved mutation can alternatively be an aliphatic to aliphatic, nonpolar to nonpolar, polar to polar, acidic to acidic, basic to basic, aromatic to aromatic, or restriction residue to restriction residue substitution, then a conserved mutation does not include hydrophilic to hydrophilic, hydrophobic to hydrophobic, hydroxyl-containing to hydroxyl-containing, or small residue to small residue substitution. As is known in the art, common examples of conservative substitutions include: substitutions between aromatic amino acids F, W, and Y; substitutions between hydrophobic amino acids L, I, and V; substitutions between polar amino acids Q and N; substitutions between basic amino acids K, R, and H; substitutions between acidic amino acids D and E; and substitutions between hydroxyl amino acids S and T. Furthermore, A, V, L, or I can be conservatively mutated to another aliphatic residue or another nonpolar residue. Exemplary conservative substitutions can be performed according to the table below, wherein amino acids belonging to the same partition in the second column can be substituted for each other, and preferably, amino acids in the same row in the third column can be substituted for each other:

[0042]

[0043] "Non-conservative substitution" refers to the substitution or mutation of an amino acid in a polypeptide with an amino acid having significantly different side chain properties. Non-conservative substitution can be performed between, rather than within, the amino acids defined above. In one embodiment, a non-conservative mutation affects (a) the structure of the peptide backbone in the substituted region (e.g., proline replacing glycine), (b) charge or hydrophobicity, or (c) side chain volume.

[0044] "Deletion" refers to a modification of a peptide by removing one or more amino acids from a reference peptide. Deletion can include the removal of one or more amino acids, two or more amino acids, five or more amino acids, ten or more amino acids, fifteen or more amino acids, or twenty or more amino acids, up to 10% of the total number of amino acids constituting the reference enzyme, while preserving enzyme activity and / or the modified properties of the engineered aldolase. Deletion can target the interior and / or ends of the peptide. In several embodiments, the deletion can comprise a continuous segment or can be discontinuous.

[0045] "Insertion" refers to a modification of a polypeptide by adding one or more amino acids to a reference polypeptide. In some embodiments, modified engineered aldolases include inserting one or more amino acids into a naturally occurring aldolase and inserting one or more amino acids into other modified aldolase polypeptides. The insertion can be internal to the polypeptide, or at the carboxyl terminus or amino terminus. Insertions as used herein include fusion proteins as known in the art. The insertion can be a continuous amino acid segment or separated by one or more amino acids in a naturally occurring polypeptide.

[0046] To perform site-directed mutagenesis, a three-dimensional model of cytochrome P450BM3 was constructed using bioinformatics techniques. This model was used to infer its enzyme catalytic active center, active pocket, substrate / product entry / exit channels, rigid / flexible structural regions, etc. Potential structurally influential sites were selected, and saturation mutagenesis was attempted.

[0047] In this article, the terms "initial (type)," "initial (monooxygenase)," and "initial type (monooxygenase)" have the same meaning, all referring to the cytochrome P450BM3 mutant LG-23 with the amino acid sequence SEQ ID NO:7. For the sake of convenience, initial cytochrome P450BM3 and its mutants are collectively referred to as "cytochrome P450BM3" in this article.

[0048] The amino acid sequence of the cytochrome P450BM3 mutant of the present invention is well known, so those skilled in the art can easily obtain its encoding gene, expression cassettes and plasmids containing these genes, and transformants containing the plasmids.

[0049] To optimize the expression of cytochrome P450BM3 or its mutants in E. coli, the most commonly used gene for genetic engineering, codon optimization was performed on the expression genes of these enzymes.

[0050] Codon optimization is a technique used to maximize protein expression in an organism by increasing the translation efficiency of genes of interest. Different organisms often exhibit a particular preference for one of a set of codons encoding the same amino acid due to mutational predisposition and natural selection. For example, in fast-growing microorganisms such as *E. coli*, optimized codons reflect the composition of their respective genomic tRNA repertoires. Thus, in fast-growing microorganisms, low-frequency codons for amino acids can be used for high-frequency codon substitutions of the same amino acid. Consequently, the expression of optimized DNA sequences is improved in fast-growing microorganisms.

[0051] For example, in order to express cytochrome P450BM3 in Escherichia coli, the coding gene of the codon-optimized mutant LG-23 / T260G can be SEQ ID NO:4; the coding gene of the mutant LG-23 / Y51I / T260G can be SEQ ID NO:5; and the coding gene of the mutant LG-23 / Y51I / T260G can be SEQ ID NO:6.

[0052] These genes, expression cassettes, plasmids, and transformants can be obtained through genetic engineering construction methods well known to those skilled in the art.

[0053] The host for the aforementioned transformant can be any microorganism suitable for expressing cytochrome P450BM3, including bacteria and fungi. Preferred microorganisms are Bacillus subtilis, Pichia pastoris, Saccharomyces cerevisiae, or Escherichia coli, with Escherichia coli being preferred, and Escherichia coli BL21(DE3) being more preferred.

[0054] When used as a biocatalyst, the cytochrome P450BM3 of the present invention can be in the form of an enzyme or in the form of a bacterial cell. The enzyme form includes free enzymes and immobilized enzymes, including purified enzymes, crude enzymes, fermentation broth, enzymes immobilized on a carrier, etc.; the bacterial cell form includes live cells, dead cells, immobilized cells, etc.

[0055] As an alternative implementation, microbial cells expressing the aforementioned cytochrome P450BM3 can be used as biocatalysts for enzyme-catalyzed reactions. The microorganisms can exist as cells or fragments of their cells. Cellular forms include both live and dead cells. When microorganisms such as Bacillus subtilis, Pichia pastoris, Saccharomyces cerevisiae, or Escherichia coli cease fermentation and proliferation and are instead used for enzyme-catalyzed reactions, they are themselves naturally immobilized enzymes. Furthermore, they do not require disruption or even extraction and purification; they can be used as enzyme preparations for catalytic reactions. Since both the reaction substrate and products are small molecules, they can easily cross the cell membrane, the biological barrier of the microorganisms, thus eliminating the need for cell disruption, which is economically advantageous.

[0056] Even more advantageously, many microbial cells contain coenzymes such as NADP. + (nicotinamide adenine dinucleotide phosphate, coenzyme II) or NAD + Nicotinamide adenine dinucleotide, also known as coenzyme I, can effectively promote redox reactions without requiring or reducing the need to add expensive coenzymes to the enzyme-catalyzed reaction system.

[0057] In applications where cytochrome P450BM3 mutants are used to catalyze the synthesis of steroid compounds, a cofactor regeneration system can be added to the reaction system. As a preferred embodiment, when cytochrome P450BM3 and glucose dehydrogenase (GDH) are used for co-catalysis, glucose can be added to the reaction system as a substrate for glucose dehydrogenase. During the reaction, glucose dehydrogenase catalyzes the oxidation of glucose, while simultaneously converting NADP... + NAD + It is reduced to (NADPH)NADH, while cytochrome P450BM3 and NAD(P)H catalyze the hydroxylation of the substrate. The amount of glucose dehydrogenase and glucose added can be determined by simple experiments.

[0058] As an alternative implementation, when using a cytochrome P450BM3 mutant and alcohol dehydrogenase for co-catalysis, isopropanol can be added to the reaction system as a substrate for alcohol dehydrogenase. During the reaction, alcohol dehydrogenase catalyzes the oxidation of isopropanol, while simultaneously converting NADP... + NAD + It is reduced to (NADPH)NADH, while cytochrome P450BM3 and NAD(P)H catalyze the hydroxylation of the substrate. The amounts of alcohol dehydrogenase and isopropanol added can be determined by simple experiments.

[0059] Those skilled in the art will readily understand that the glucose dehydrogenase and alcohol dehydrogenase described above can be provided either in enzyme form or in the form of expressed microbial cells.

[0060] In an alternative implementation, cytochrome P450BM3 can be co-expressed with glucose dehydrogenase (GDH) or alcohol dehydrogenase (ADH) in the same strain, thus eliminating the need to add the two enzymes or expressing cells in proportion to the catalytic reaction system.

[0061] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0062] Example

[0063] This article involves the addition amount, content and concentration of various substances. Unless otherwise specified, the percentage content mentioned refers to the mass percentage.

[0064] Materials and methods

[0065] The whole genome synthesis, primer synthesis and sequencing in the embodiments were outsourced to Sangon Biotech (Shanghai) Co., Ltd.

[0066] The molecular biology experiments in the examples included plasmid construction, enzyme digestion, ligation, preparation of competent cells, transformation, and culture medium preparation, etc., mainly referring to "Molecular Cloning: A Laboratory Manual," 3rd edition (J. Sambrook and DW Russell, eds., translated by Huang Peitang et al., Science Press, Beijing, 2002). Specific experimental conditions could be determined through simple experiments if necessary.

[0067] PCR amplification experiments should be performed according to the reaction conditions provided by the plasmid or DNA template supplier or the kit instructions. Adjustments can be made through simple experiments if necessary.

[0068] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.2. (LB solid medium with an additional 20 g / L agar powder.)

[0069] TB medium: 24 g / L yeast extract, 12 g / L tryptone, 16.43 g / L K₂HPO₄·3H₂O, 2.31 g / L KH₂PO₄, 5 g / L glycerol, pH 7.0-7.5. (TB solid medium with an additional 20 g / L agar powder.)

[0070] Methyldione, androstenedione, 4,9-methyldione, 18-methyldione, adrenocorticosteroid, testosterone, nandrolone, and other compounds were all purchased from Sigma-Aldrich.

[0071] HPLC detection methods for substrates and products:

[0072] All samples were analyzed by Shimadzu high-performance liquid chromatography (LC-2030 or LC-2030C). An Agilent ZORBAX SB C18 (250×4.6 mm) column was used with methanol, acetonitrile, and ultrapure water as the mobile phase. Methyl diketones, androstenediones, and compounds 4 and 9 were analyzed using the following method: the flow rate was set to 1.5 mL / min, and the mobile phase was methanol:acetonitrile:ulpure water, 1 min (15:15:70), 1–2 min (20:20:60), 2–7 min (35:35:30), 7–10 min (15:15:70). The detection wavelength was 250 nm, and the detection wavelength for compounds 4 and 9 was 310 nm.

[0073] The identification of the reaction products C7β-hydroxymethyldione, C7β-hydroxyandrostenedione, C7β-hydroxy18-methyldione, C7β-hydroxyadrenergic steroid, C7β-hydroxytestosterone, and C7β-hydroxynandrolone is reported in the literature Li A, et al., Regio-and Stereoselective Steroid Hydroxylation at the C7-Position by Cytochrome P450 Monooxygenase Mutants [J]. Angewandte Chemie International Edition, 2020.; the identification of C11α-hydroxy4,9 is reported in the literature Peng Yaqin, Gao Chenghua, Zhang Zili, et al. A Chemoenzymatic Strategy for the Synthesis of Steroid Drugs Enabled by P450 Monooxygenase-Mediated Steroidal Core Modification [J]. ACS Catalysis, 2022, 12(5): 2907-14.

[0074] It should be noted that, for the sake of convenience, in the embodiments, the strain number, plasmid number, enzyme number, and enzyme-encoding gene number may share the same number. This is easily understood by those skilled in the art, that is, the same number can refer to different biological forms in different environments.

[0075] In the examples, plasmids expressing cytochrome P450BM3, such as pRSFDuet-LG-23, and plasmids used for gene editing operations were constructed and preserved by Professor Li Aitao's research group at the School of Life Sciences, Hubei University. Any unit or individual can obtain these plasmids to verify the present invention, but they may not be used for other purposes, including development, scientific research, and teaching, without the permission of Hubei University.

[0076] Example 1: Site-directed saturation mutagenesis and functional verification of the P450 BM3 enzyme mutant LG-23

[0077] 1.1 Site-directed saturation mutagenesis of mutant LG-23

[0078] This embodiment, based on the P450BM3 enzyme mutant LG-23 (Li A, et al., Regio-and Stereoselective Steroid Hydroxylation at the C7-Position by Cytochrome P450 Monooxygenase Mutants[J]. Angewandte Chemie International Edition, 2020.) with steroid 7β-hydroxylation activity, used the plasmid vector pRSFDuet-LG-23 expressing the mutant LG-23 as a template. Amino acid sites around the active pocket in the enzyme model were selected, and site-directed saturation mutagenesis of the LG-23 gene was performed using primer PCR mutagenesis. The plasmid vector pRSFDuet-LG-23 was constructed by our research group (ibid.), and its structural diagram is shown below. Figure 3 As shown in Figure A. Some primers are shown in Table 1 below, with the screening of the T260 mutant as an example.

[0079] Table 1: Primer sequences for LG-23 site-directed saturation mutagenesis

[0080]

[0081]

[0082] Note: The suffix "F" in primer names indicates forward; "R" indicates reverse.

[0083] The PCR system (20 μL) consisted of: 0.1–1 ng template, 1 μL (10 μM) of each of a pair of mutant primers, 5 μL of PrimeSTARMax DNA polymerase, and sterile distilled water to a final volume of 20 μL.

[0084] The PCR reaction program was as follows: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 10 sec, 56℃ annealing for 15 sec, 72℃ extension for 80 sec, 29 cycles; 72℃ extension for 5 min. PCR products were stored at 8℃.

[0085] The amplified PCR product was detected by 0.7% agarose gel electrophoresis. A band of similar length to the pRSFDuet-LG-23 plasmid was observed, indicating that the gene encoding the target mutant had been amplified. Therefore, restriction endonuclease Dpn I was added to the PCR product, and after digestion at 37°C for 3–5 h, it was transformed into E. coli BL21(DE3) competent cells. After recovery with culture medium for 1 h, the cells were evenly spread onto solid LB agar plates containing 50 μl / mg kanamycin. Using an eight-slot electric pipette (Gilson, P8x1200M), 400 μL of liquid LB (50 μg / mL, Kansin) was added to a sterilized 96-well deep-well plate. + For the culture medium, pick single clones of the initial-type strain and place them in well A1, with well A2 serving as a blank control. Pick single clones from the transformation plates and transfer them to 96-well plates. Transfer 94 single clones from each plate to other wells in a 96-well deep-well plate (each 96-well plate should contain only a single NNK saturation mutant site). Incubate at 37°C and 800 rpm for 10-12 hours. Then, transfer 200 μL aliquots to 200 μL of 50% (v / v) glycerol stock plates and store at -80°C. Add 800 μL of TB medium (pre-added to a final concentration of 0.2 mM IPTG and 50 μg / mL kanamycin sulfate) to the remaining 200 μL of culture and induce expression for 16-18 hours in a high-frequency shaking incubator. The bacterial culture induced for expression for 16-18 h was centrifuged for 10 minutes at 4000 rpm and 8 °C to obtain cell pellet. The pellet was resuspended in 400 μL potassium phosphate buffer (100 mM, pH 8.0), washed once, and the supernatant was discarded. Then, 200 μL potassium phosphate buffer (100 mM, pH 8.0) was added for resuspending. The pellet was then flash-frozen in liquid nitrogen and stored in a -80 °C freezer until it was used as a cell catalyst.

[0086] Remove the 96-well deep-well plate containing the saturation mutation site of the P450BM3 mutant from the -80°C freezer and thaw at room temperature. Prepare a solution containing 6% w / v glucose, 10% v / v glycerol, and 0.4 mM NADP. + All cells were resuspended in 200 μL phosphate buffer (100 mM, pH 8.0) with 2 U / mL glucose dehydrogenase (GDH) to make the final reaction system contain 3% w / v glucose, 10% v / v glycerol, and 0.2 mM NADP. +400 μL of phosphate-buffered saline (phosphate buffer) cell resuspension containing 1 U / mL GDH was added. A specific concentration of substrate was added. The reaction was initiated at 25°C and 800 rpm on a high-speed shaking incubator and continued for 12-18 hours. 400 μL of methanol was added to extract the reaction mixture, and the mixture was centrifuged at 4000 rpm and 4°C for 10 minutes. The supernatant was centrifuged using a wwPTFE 96-well plate filter (Pall, VWR, Germany) to remove solid particles, and then filtered into new 200 μL 96-well high-performance liquid chromatography (HPLC) plates. The 96-well plates were sealed with silicone caps and analyzed by HPLC. All samples were analyzed by Shimadzu HPLC (LC-2030 or LC-2030C). Mutant strains exhibiting increased activity relative to LG-23 were identified, and these strains were subsequently screened.

[0087] The reaction formulas for the C7β hydroxylation of methyl diketones catalyzed by the P450 BM3 mutants LG-23 and LG-23 / T260G are shown in (1).

[0088] (I)

[0089] Figure 1 The HPLC chromatogram of the initial enzyme LG-23 cell-catalyzed methyldiketone reaction is shown.

[0090] Figures 5-9 The C7β-hydroxymethyl diketone was shown separately. 1 1H NMR (DMSO-d, 400MHz) spectrum 13 CNMR (DMSO-d, 400MHz) spectrum, HSQC (DMSO-d, 400MHz) spectrum, HMBC (DMSO-d, 400MHz) spectrum, and NOSY (DMSO-d, 400MHz) spectrum.

[0091] The NMR data for C7β-hydroxymethyldione are as follows:

[0092] 1H NMR(DMSO-d6,400MHz): δ5.75(1H,s,H-4),4.76(1H,d,6.4Hz,7-OH),3.33(1H,m,H-7),2.62(1H,dd,13.8,4.6Hz,H-6a),2.34(1H,dd,19.2,8.7Hz,H-16 a),2.25(1H,overlapped,H-16a),2.24(1H,overlapped,H-6b),2.21(2H,overlapped,H-2a,H-2b),2.18(1H,overlapped,H-1b),2.15(1H,overlapped ,H-10),1.97(1H,dd,19.2,9.0Hz,H-16b),1.84(1H,m,H-11a),1.77(1H,m, H-15b),1.64(1H,dt,12.7,3.2Hz,H-12a),1.51(1H,q,10.0Hz,H-8),1.43(1 H,dd,11.6,5.5Hz,H-14),1.39(1H,m,H-1b),1.28(1H,td,12.7,3.8Hz,H-11 b),1.15(1H,td,12.7,4.1Hz,H-12b),0.86(3H,s,H3-18),0.84(1H,m,H-9);

[0093] 13 C NMR(DMSO-d6,100MHz): δ220.3(C-17),198.5(C-3),164.4(C-5),124.2(C-4),72.4(C-7),49.2(C-14),47.7(C-13),46.1(C-8) ,46.0(C-9),45.2(C-6),40.9(C-10),36.1(C-2),35.5(C-16),31.1(C-12),26.1(C-1),25.1(C-11),24.6(C-15),13.7(C-18).

[0094] 1.2 Verification of the rescreening function

[0095] Streaking *E. coli* containing the above mutant onto solid LB agar plates containing 50 μg / mL kanamycin and incubating overnight at 37°C. Single colonies were selected and transferred to 2 mL of liquid LB medium containing 50 μg / mL kanamycin, and incubated overnight at 37°C with shaking. 500 μL of the bacterial culture was then inoculated into a 100 mL Erlenmeyer flask containing 50 mL of TB medium and incubated at 37°C with shaking at 220 rpm. The culture was analyzed when the absorbance OD of the culture medium reached a certain value. 600When the concentration reached 0.8, IPTG was added to a final concentration of 0.2 mM to induce expression at 25 °C for 16–20 hours. The culture medium was centrifuged at 4000 rpm and 4 °C for 10 min, the cells were collected, and washed once with 100 mM potassium phosphate buffer (pH 8.0). The cells were then stored at -80 °C.

[0096] Cells were rinsed with 10 mL of 100 mM potassium phosphate buffer (pH 8.0) containing 5% w / v glucose, 5% v / v glycerol, and 0.2 mM NADP. + 10 U GDH was resuspended in a 50 mL centrifuge tube and immediately flash-frozen in liquid nitrogen. The suspension was then thawed in water at room temperature. 5 mL of the thawed suspension was transferred to a 50 mL Erlenmeyer flask, and 5 g / L of methyl diketone, androstenedione, or 5 g / L of 4,9-diketone (prepared as a 200 mM stock solution with DMF) was added. The mixture was reacted at 25 °C and 220 rpm for 24 hours. Samples were taken at intervals, extracted with methanol, centrifuged at high speed for 1 min, and filtered through a 0.22 μm filter into a sample vial. The conversion rate and product distribution were detected by HPLC. After 24 hours of reaction, the substrate conversion was 43%, at which point the titer (concentration) of C7β-hydroxymethyl diketone was 2.2 g / L, and the selectivity for C7β-hydroxylation was 97%. The sequencing company confirmed that the mutant was LG-23 / T260G, meaning that the threonine at position 260 was mutated to glycine. Its amino acid sequence is shown in SEQ ID NO: 1, and its nucleotide sequence is shown in SEQ ID NO: 4.

[0097] Following the method described above, LG-23 / T260G expression was induced by immersing cells in 10 mL of 100 mM potassium phosphate buffer (pH 8.0) containing 5% w / v glucose, 5% v / v glycerol, and 0.2 mM NADP. + 10 U GDH was resuspended in a 50 mL centrifuge tube and immediately flash-frozen in liquid nitrogen. The suspension was then thawed in water at room temperature. 5 mL of the thawed suspension was transferred to a 50 mL Erlenmeyer flask, and 5 g / L androstenedione or 5 g / L 4,9-dimethylformamide (prepared as a 200 mM stock solution in DMF) was added. After 24 h of reaction, the substrate conversion was 43%. At this point, the titer of C7β-hydroxyandrostenedione was 2.3 g / L, with a C7β-hydroxylation selectivity of 97%; the titer of C11α-hydroxy4,9-dimethylformamide was 1.3 g / L, with a C11α-hydroxylation selectivity of 95%.

[0098] Example 2: Functional verification of mutant LG-23 / T260G in different cosolvents

[0099] Dimethyl sulfoxide (DMSO), methanol, acetone, olive oil, and hydroxypropyl-β-cyclodextrin were selected as co-solvents to dissolve the substrate methyldiketone. When using hydroxypropyl-β-cyclodextrin as a co-solvent, cyclodextrin was weighed at a mass ratio of methyldiketone:hydroxypropyl-β-cyclodextrin = 1:8 (final cyclodextrin concentration 22% w / v), and dissolved in an appropriate amount of 100 mM pH 8.0 potassium phosphate buffer. Then, Tween 80 (final concentration 0.1% w / v) and the substrate were added. Finally, an ultrasonic probe was used to further break down the methyldiketone crystals and to thoroughly mix the solution to promote dissolution, yielding a 100 mM pH 8.0 methyldiketone cyclodextrin solution.

[0100] Using GDH and glucose as cofactor cycling systems, a whole-cell catalytic reaction system was constructed according to the method described in Example 1. Methyl diketones at 5 g / L were added with different co-solvents for the reaction, and samples were taken at 2 h and 6 h. The catalytic reaction of LG-23 / T260G in different co-solvents is shown below. Figure 2 As shown, hydroxypropyl-β-cyclodextrin was found to be the best reaction solvent, with the conversion rate of methyl diketones catalyzed by LG-23 / T260G reaching 83% and the titer of C7β-hydroxymethyl diketone reaching 3.43 g / L. Compared with using DMF as a solvent, the product titer was increased by 1.4 times, and twice that of using acetone as a solvent.

[0101] Example 3: Functional verification of mutant LG-23 / T260G in different cofactor regeneration systems

[0102] Glucose dehydrogenase (GDH) is derived from *Priestia megaterium*. The GDH gene is expressed independently, and the crude enzyme from the lysate supernatant catalyzes the regeneration and cycling of the cofactor NADPH. The catalytic equation is as follows: Glucose + NADP... + → Gluconolactone + NADPH.

[0103] Alcohol dehydrogenase TbADH (derived from *Thermoanaerobacter brockii*) and isopropanol dehydrogenase mutant IPADHM4 (derived from *Brucella suis* ATCC 23445) were selected as candidate dehydrogenases for the cofactor cycle system. Both ADHs can utilize isopropanol as an alcohol donor to produce acetone without affecting the pH of the reaction system. Furthermore, to reduce the influence of organic reagents on the reaction system, 1% v / v isopropanol was added to the reaction system as an alcohol donor. The catalytic equation is: isopropanol + NADP⁻. + → Acetone + NADPH.

[0104] Following the method described above, strains E. coli BL21(DE3)-pRSFDuet-TbADH and E. coli BL21(DE3)-pRSFDuet-IPADHM4 were induced to express their contents. The cell pellet obtained after harvesting the bacteria was adjusted to OD using 100 mM pH 8.0 potassium phosphate buffer. 600 =20% cell suspension. Cells were disrupted using a cell sonicator, and the enzyme supernatant obtained after centrifugation was used to determine its activity. NADPH production was determined using a spectrophotometer according to the NADPH measurement method. The activity of the enzyme supernatant of E. coli BL21(DE3)-pRSFDuet-TbADH against NADP was measured. + The activity of the enzyme supernatant of E. coli BL21(DE3)-pRSFDuet-IPADHM4 against NADP+ was 77.6 U / mL, and the activity of the enzyme supernatant against NADP+ was 4300 U / mL.

[0105] The effects of three dehydrogenases—GDH, TbADH, and IPADHM4—on whole-cell catalysis were compared using the mutant strain LG-23 / T260G. Each strain was reacted with 10 g / L of methyl diketone solution (dissolved in cyclodextrin). With 1 U / mL of enzyme, the highest product titer was achieved using GDH and glucose as a cofactor regeneration system, reaching a C7β-hydroxymethyl diketone titer (concentration) of 4.4 g / L. However, the reaction ceased after 8 hours, at which point the pH was 5.6. Using ADH and isopropanol as cofactor regeneration systems, C7β-hydroxymethyl diketone titers reached 3.6 g / L and 3.4 g / L, respectively. However, after 8 hours, the P450BM3 mutant was still active, at which point the pH was 7.8, showing no significant change from pH 8.0. When the added glucose dehydrogenase and isopropanol dehydrogenase reached enzyme activities of 5 U / mL and 10 U / mL in the reaction system, respectively, the IPADHM4 and isopropanol cofactor regeneration system performed best, with the C7β-hydroxymethyl diketone titer reaching 6.8 g / L, which is 1.6 times that of the cofactor regeneration system using GDH and glucose. Furthermore, the pH value did not change significantly, thus demonstrating the feasibility of using ADH and isopropanol as a cofactor regeneration system.

[0106] Example 4: Construction and functional verification of a strain co-expressing mutant LG-23 / T260G and isopropanol dehydrogenase

[0107] 4.1 Construction method of recombinant expression vector co-expressing LG-23 / T260G enzyme and IPADHM4 enzyme

[0108] Using the pRSFDuet-LG-23 / T260G mutant plasmid as a template, the plasmid was back-amplified using primers containing the RBS (ribosome binding site) and a 20bp homologous arm to obtain fragment 1 containing the RBS and homologous arm. Using the pRSFDuet-IPADHM4 plasmid as a template, the IPADHM4 gene was amplified using a primer containing the RBS (with a 20bp homologous arm) upstream and a primer containing the T7 terminator (with a 20bp homologous arm) downstream to obtain fragment 2. The primers used are shown in Table 2.

[0109] The PCR system (20 μL) consisted of: 0.1–1 ng template, 1 μL (10 μM) of each of a pair of mutant primers, 5 μL of PrimeSTARMax DNA polymerase, and sterile distilled water to a final volume of 20 μL.

[0110] Table 2: Primers used to construct recombinant expression vectors co-expressing LG-23 / T260G and IPADHM4 enzymes

[0111]

[0112] Note: The suffix "F" in primer names indicates forward; "R" indicates reverse. The same applies below.

[0113] Fragment 1 and fragment 2 were detected and recovered by gel electrophoresis, and their concentrations were measured using a micro-spectrophotometer. Following the T5 exonuclease ligation system, the two PCR fragments with homologous ends were ligated by cutting the 5' ends of the homologous arms with T5 exonuclease. The reaction mixture was placed on ice and reacted for 4 min. Then, 4 μL of the reaction mixture was chemically transformed into 100 μL of LE.coli DH5α, and then plated entirely on LB (50 μg / mL Kansas). + The plates were then incubated overnight at 37°C. Single colonies were picked from the plates and inoculated into 2 mL of LB medium (Kan). + The bacterial culture was prepared in 50 μg / mL solution and numbered. After incubation at 37℃ and 220 rpm for 6 h, a small amount of bacterial culture was aspirated for colony PCR verification. For strains containing the target band, 1 mL of bacterial culture was sent for sequencing. For strains with correct sequencing and successful construction of the target plasmid, the plasmid was extracted using a plasmid mini-extraction kit and then transformed into E. coli BL21(DE3) by electroporation to obtain the target recombinant E. coli expression strain, which was named E. coli-pRSFDuet-LG-23 / T260G_IPADHM4. See also Figure 3A is the plasmid map of LG23 / T260G expression, B is the plasmid map of co-expressing LG23 / T260G and IPADHM4, and C is the SDS gel electrophoresis image of the supernatant of co-expressing cell lysate.

[0114] 4.2 Co-expression function verification

[0115] The cell culture method is described in the functional verification section of Example 1. During the reaction, the co-expressing cells were removed from the -80°C freezer, thawed, centrifuged, and resuspended in a 50 mL centrifuge tube using a 27.2 g / L methyl diketone solution (substrate dissolved in cyclodextrin) at pH 8.0. The cell concentration was adjusted to OD0.0. 600 =60. Then, take 5 mL of the bacterial suspension and place it in a 50 mL Erlenmeyer flask, add 1% v / v isopropanol and 10 μL of 50 mM NADP. + The reaction was carried out at 25℃ and 220 rpm for 24 hours. Samples were taken at intervals, extracted with methanol, centrifuged at high speed for 1 min, and filtered through a 0.22 μm filter into sample vials. The conversion rate and product distribution were detected by HPLC. The titer of C7β-hydroxymethyl diketone reached 2.1 g / L after 20 min of reaction; after 24 h, the titer reached 12.9 g / L. Furthermore, without the addition of NADP... + In the same situation, it has the same effect.

[0116] Example 5: Site-directed mutagenesis and functional verification of mutant LG-23 / T260G

[0117] 5.1 Site-directed mutagenesis of LG-23 / T260G

[0118] Site-directed mutagenesis was performed on several key amino acids at the substrate channel inlet in the enzyme model. The plasmid vector pRSFDuet-LG-23 / T260G_IPADHM4, which expresses the mutant LG-23 / T260G and isopropanol dehydrogenase, was used as a template. The gene of the mutant LG-23 / T260G was mutagenized at specific sites using primer PCR mutagenesis technology, and W47, Y51 and I401 were mutated to the target amino acids. The primers used are shown in Table 3.

[0119] The PCR system (20 μL) consisted of: 0.1–1 ng template, 1 μL (10 μM) of each of a pair of mutant primers, 5 μL of PrimeSTARMax DNA polymerase, and sterile distilled water to a final volume of 20 μL.

[0120] The amplified PCR products were detected by 0.7% agarose gel electrophoresis. A band of similar length to the pRSFDuet-LG-23 / T260G_IPADHM4 plasmid was observed, indicating that the gene encoding the target mutant had been amplified. Therefore, restriction endonuclease Dpn I was added directly to the PCR products, and after digestion at 37°C for 3–5 h, the cells were transformed into E. coli BL21(DE3) competent cells. After recovery with culture medium for 1 h, the cells were evenly spread onto solid LB plates containing 50 μl / mg kanamycin. After overnight culture at 37°C, single clones were selected and cultured in 3 mL of LB liquid medium containing 50 μl / mg kanamycin. The clones were then sent to a sequencing company for sequencing to obtain the correct mutants, which were named. The obtained mutants are listed in Table 3, and the PCR primers used are listed in Table 4.

[0121] Table 3: Mutants constructed using LG-23 / T260G as a template

[0122]

[0123] Table 4: Site-directed mutagenesis primers for mutant LG-23 / T260G

[0124]

[0125] 5.2 Functional Verification

[0126] The cell culture method is described in the functional verification section of Example 1. During the reaction, cells from each strain were removed from a -80°C freezer, allowed to thaw, centrifuged, and resuspended in 50 mL centrifuge tubes using a 27.2 g / L methyl diketone solution (substrate dissolved in cyclodextrin) at pH 8.0. The cell concentration was adjusted to OD0.0. 600 =60. Then, 5 mL of the bacterial suspension was transferred to a 50 mL Erlenmeyer flask, and 1% v / v isopropanol was added. The mixture was reacted at 25 °C and 220 rpm for 24 hours. Samples were taken at intervals, extracted with methanol, centrifuged at high speed for 1 min, and filtered through a 0.22 μm filter into sample vials. HPLC was used to detect the conversion rate and product distribution of each strain. The results showed that, compared with LG-23 / T260G, its mutant LG-23 / Y51I / T260G had further improved catalytic activity. After 24 h of reaction, the titer of C7β-hydroxymethyl diketone reached 16.7 g / L. Its amino acid sequence is shown in SEQ ID NO: 2, and its nucleotide sequence is shown in SEQ ID NO: 5.

[0127] Example 6: Site-directed mutagenesis and functional verification of mutant LG-23 / Y51I / T260G

[0128] 6.1 Site-directed saturation mutagenesis of LG-23 / Y51I / T260G

[0129] To target the substrate molecule exit channel in the enzyme model, the following amino acids surrounding the LG-23 / Y51I / T260G channel were selected for saturation-directed mutagenesis: F107, I153, G157, and F405. Using the plasmid vector pRSFDuet-LG-23 / Y51I / T260G and isopropanol dehydrogenase as templates, the gene of the mutant LG-23 / Y51I / T260G was subjected to site-directed mutagenesis using primer PCR mutagenesis. The amino acids F107, I153, G157, and F405 were selected for saturation-directed mutagenesis. The primers used are shown in Table 5.

[0130] Table 5: Site-directed saturation mutagenesis primers for mutant LG-23 / Y51I / T260G

[0131]

[0132] PCR amplification and mutant strain screening were performed according to the method in Example 1. Mutant strains with significantly increased C7β hydroxylation enzyme activity compared to LG-23 / Y51I / T260G were selected for rescreening and functional verification.

[0133] 6.2 Functional Verification

[0134] Functional verification of each mutant strain was performed according to the method described in Example 5.2.

[0135] During the reaction, cells from each strain were removed from a -80°C freezer, allowed to thaw, centrifuged, and the supernatant was discarded. Cells were resuspended in 50 mL centrifuge tubes using a 27.2 g / L methyl diketone solution (pH 8.0, substrate dissolved in cyclodextrin), and the cell concentration was adjusted to OD0.0. 600 =60. Then, 5 mL of the bacterial suspension was transferred to a 50 mL Erlenmeyer flask, and 1% v / v isopropanol was added. The mixture was reacted at 25 °C and 220 rpm for 24 hours. Samples were taken at intervals, extracted with methanol, centrifuged at high speed for 1 min, and filtered through a 0.22 μm filter membrane into sample vials. HPLC was used to detect the conversion rate and product distribution of each strain. Among all the rescreened strains, the mutant LG-23 / Y51I / G157S / T260G was found to have higher catalytic activity. After 24 h of reaction, the titer of C7β-hydroxymethyl diketone reached 22.4 g / L. Its amino acid sequence is shown in SEQ ID NO: 3, and its nucleotide sequence is shown in SEQ ID NO: 6.

[0136] Example 7: Investigation of the catalytic activity of cytochrome P450BM3 mutant against other steroid compounds

[0137] Following the method described in Example 1, the co-expressing strains expressing mutants LG-23, LG-23 / T260G, LG-23 / Y51I / T260G, and LG-23 / Y51I / G157S / T260G and isopropanol dehydrogenase were induced to express their respective mutants.

[0138] The catalytic ability and position specificity of these strains in catalyzing the hydroxylation of substrates methyldione, androstenedione, 4,9-dione, 18-methyldione, adrenocorticosteroids, testosterone, and nandrolone were investigated.

[0139] During the reaction, the cells were removed from the -80°C freezer, allowed to thaw, and then centrifuged. The cells were resuspended in 50 mL centrifuge tubes using different steroidal compounds at pH 8.0, and the cell concentration was adjusted to OD0.05. 600 =60. All steroidal substrates were dissolved using cyclodextrin and Tween, following the same method as in Example 1. Then, 5 mL of a bacterial suspension containing 30 g / L of steroidal compound was transferred to a 50 mL Erlenmeyer flask, and 2% v / v isopropanol was added. The mixture was reacted at 25°C and 220 rpm for 24 hours. Samples were taken at intervals, extracted with methanol, centrifuged at high speed for 1 min, and filtered through a 0.22 μm filter into a sample vial. The conversion rate and product distribution were detected by HPLC. The transformation of the substrates by different mutant strains are shown in Table 6.

[0140] Table 6: Catalytic activity of P450BM3 mutant against different steroidal compounds

[0141]

[0142]

[0143] The results showed that the mutants LG-23 / T260G, LG-23 / Y51I / T260G, and LG-23 / Y51I / G157S / T260G could not only catalyze position-specific hydroxylation reactions of methyl diketones, androstenediones, and 4,9-hydroxylated substrates, but also catalyze hydroxylation reactions of other steroid substrates such as 18-methyl diketones, adrenocorticosteroids, testosterone, and nandrolone, while maintaining high position specificity. This demonstrates that these mutants have a broad substrate spectrum and show potential for industrial application.

[0144] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A cytochrome P450BM3 mutant, characterized in that, Its amino acid sequence is SEQ ID NO:2 or SEQ ID NO:

3.

2. The gene encoding the cytochrome P450BM3 mutant of claim 1.

3. The gene according to claim 2, characterized in that, The nucleotide sequence of the gene encoding the amino acid sequence of cytochrome P450BM3 mutant SEQ ID NO:2 is SEQ ID NO:5, and the nucleotide sequence of the gene encoding the amino acid sequence of cytochrome P450BM3 mutant SEQ ID NO:3 is SEQ ID NO:

6.

4. A plasmid, characterized in that, It contains the gene described in claim 3.

5. A microorganism expressing the cytochrome P450BM3 mutant of claim 1, characterized in that, The microorganism is a transformant that has transformed the plasmid described in claim 4.

6. The microorganism according to claim 5, characterized in that, The microorganisms were selected from Escherichia coli, Bacillus subtilis, Pichia pastoris, and Saccharomyces cerevisiae.

7. The microorganism according to claim 6, characterized in that, The microorganism in question is Escherichia coli BL21(DE3).

8. The use of the cytochrome P450BM3 mutant of claim 1 or the microorganism of claim 6 in the production of steroid compounds.

9. The application according to claim 8, characterized in that, Using methyl diketone, androstenedione, or 4,9-hydroxy compounds as substrates, the hydroxylation reaction is catalyzed by the cytochrome P450BM3 mutant or the microorganism to generate the corresponding hydroxylated steroids C7β-hydroxymethyl diketone, C7β-hydroxyandrostenedione, or C11α-hydroxy 4,9-hydroxy compounds. Alternatively, using 18-methyldione, adrenaline, testosterone, or nandrolone as substrates, the hydroxylation reaction catalyzed by the cytochrome P450BM3 mutant or the microorganisms can be used to generate the corresponding hydroxylated steroids C7β-hydroxy18-methyldione, C7β-hydroxyadrenaline, C7β-hydroxytestosterone, or C7β-hydroxynandrolone.

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