5alpha-reductase mutant and application thereof in synthesis of steroid C4, 5 hydrogenation drug intermediate

Abstract

The invention belongs to the technical field of enzyme gene engineering and enzyme engineering, and discloses a 5alpha-reductase mutant and application thereof in synthesis of steroid C4, 5 hydrogenation drug intermediates. Compared with an amino acid sequence of wild type 5 alpha-reductase, the mutant has mutation of at least two sites corresponding to the following sites of the amino acid sequence as shown in SEQ ID NO.1: the 56th site, the 109th site and / or the 144th site. The mutant has efficient catalytic activity on 3-keto steroids, a high-yield target product is obtained under high substrate feeding concentration, no by-product is generated, and the conversion rate is not lower than 99%. The invention also discloses a coding gene, a recombinant vector, an engineering bacterium and application of the mutant, the reaction conditions are mild, the process is simple and environment-friendly, the defects of the traditional method are effectively overcome, technical support is provided for green industrial production of steroid drug intermediates, and the application prospect is wide.

Description

Technical Field

[0001] This invention belongs to the field of enzyme gene engineering and enzyme engineering technology, specifically involving 5α-reductase mutants and their application in the synthesis of steroidal C4,5-position hydrogenation drug intermediates. Background Technology

[0002] Steroid compounds, also known as steroidal compounds, are widely present in the cells and tissues of living organisms, such as ergosterol in fungal steroid cells, campesterol in plants, and cholesterol in animal cells. As an important class of drugs, more than 250 types of steroid compounds have been identified. Steroid hormones are widely used due to their remarkable effects on tumors and inflammation. They are also key drugs for treating rheumatoid arthritis, bronchitis, and endocrine disorders such as Addison's disease. Steroid drugs possess strong anti-infective, anti-allergic, antiviral, and anti-shock pharmacological effects, and have become the second largest class of drugs after antibiotics, with a very broad market prospect.

[0003] The reduction of the C4,5 double bond in the A ring of steroids is a crucial step in the production of key intermediates for steroidal drugs. Compounds such as androstenedione-4-ene-3,17-dione (AD), progesterone, and testosterone, after this reduction, yield intermediates that are key precursors in the synthesis of dozens of steroid hormone drugs, including metronidazole, metronidazole, and metenolone, exhibiting significant market demand and application value. Currently, the reduction of steroidal compounds at the C4,5 position mainly involves chemical methods and microbial fermentation. Chemical reduction relies on metal catalysts and suffers from low yields, numerous byproducts, cumbersome procedures, the need for large amounts of organic solvents, and significant environmental hazards; therefore, it has been gradually replaced by biological methods. Microbial fermentation reduction, currently reported using mycobacteria, avoids the aforementioned drawbacks of chemical methods, but suffers from low substrate concentrations, long conversion times, and relatively low conversion and yield rates.

[0004] 5α-Reductase is a reducing coenzyme II (NADPH)-dependent enzyme that catalyzes the hydrogenation reduction of the carbon-carbon double bond (C=C) at the C4 and C5 positions of the A ring of 3-sterone compounds, converting it into a carbon-carbon single bond (CC) in one step, and has broad application prospects.

[0005] 5α-reductase is ubiquitous in mammalian cells but relatively rare in prokaryotes. Recent studies have shown that Zhao et al. successfully achieved the biosynthesis of 5α-AD using phytosterols as substrates by coupling 5α-reductase with glucose-6-phosphate dehydrogenase (Zhao Y, Shen Y, Ma S, et al. Production of 5α-androstene-3,17-dione from phytosterols by co-expression of 5α-reductase and glucose-6-phosphate dehydrogenase in engineered Mycobacterium neoaurum [J]. GreenChemistry, 2019, 21(7): 1809-1815.), but due to the low activity of 5α-reductase, this route suffers from problems such as low feed concentration and long conversion cycle. Given that the industrial production technology from phytosterols to AD is now quite mature, the raw material AD can be obtained cheaply and on a large scale. Against this backdrop, the one-step catalytic conversion of AD to 5α-AD using 5α-reductase has become an efficient and sustainable synthetic strategy, providing a promising new approach for the green manufacturing of steroid drugs.

[0006] Therefore, there is an urgent need in this field to screen for highly efficient 5α-reductases to meet industrial needs. Summary of the Invention

[0007] To address the problems existing in the prior art, this invention provides a 5α-reductase mutant with significantly enhanced catalytic performance and its applications. This mutant is particularly suitable for the efficient conversion of important steroidal drug intermediates such as androstenedione-4-ene-3,17-dione (AD), progesterone, and 11α-hydroxyprogesterone (11α-OH-progesterone) at substrate concentrations up to 10 g / L.

[0008] In a first aspect, the present invention provides a 5α-reductase mutant, wherein the mutant, compared with the amino acid sequence of wild-type 5α-reductase, has mutations at at least two sites corresponding to the amino acid sequence shown in SEQ ID NO.1: position 56, position 109, and / or position 114. Preferably, the amino acid sequence of the wild-type 5α-reductase is as shown in SEQ ID NO.1 or has at least 90% identity with the amino acid sequence shown in SEQ ID NO.1.

[0009] Specifically, the 56th amino acid is mutated to a non-A amino acid, such as D, R, K, F, W, H, S, I, M, E, G, L, T, Y, C, Q, P, V, N, preferably mutated to L.

[0010] Specifically, the 109th amino acid is mutated to a non-I amino acid, such as D, R, K, F, W, H, S, A, M, E, G, L, T, Y, C, Q, P, V, N, preferably mutated to M, L, or V.

[0011] Specifically, the 114th amino acid is mutated to a non-I amino acid, such as D, R, K, F, W, H, S, A, M, E, G, L, T, Y, C, Q, P, V, N, preferably mutated to M, L, or V.

[0012] Preferably, the mutant is selected from any one or more of the following:

[0013] (1) Compared with the amino acid sequence of wild-type 5α-reductase, the amino acid sequence corresponding to SEQ ID NO: 1 is mutated to M at position 109 and to V at position 114;

[0014] (2) Compared with the amino acid sequence of wild-type 5α-reductase, the amino acid sequence corresponding to SEQ ID NO: 1 is mutated to M at position 109 and position 114;

[0015] (3) Compared with the amino acid sequence of wild-type 5α-reductase, the amino acid sequence corresponding to SEQ ID NO: 1 is mutated to L at position 109 and to M at position 114;

[0016] (4) Compared with the amino acid sequence of wild-type 5α-reductase, the amino acid sequence corresponding to SEQ ID NO: 1 is mutated to V at position 109 and to M at position 114;

[0017] (5) Compared with the amino acid sequence of wild-type 5α-reductase, the amino acid sequence corresponding to SEQ ID NO: 1 is mutated to L at position 56, V at position 109, and L at position 114.

[0018] (6) Compared with the amino acid sequence of wild-type 5α-reductase, the amino acid sequence corresponding to SEQ ID NO: 1 is mutated to L at position 56 and L at position 114;

[0019] (7) Compared with the amino acid sequence of wild-type 5α-reductase, the amino acid sequence corresponding to SEQ ID NO: 1 is mutated to L at position 56 and to M at position 114;

[0020] (8) Compared with the amino acid sequence of wild-type 5α-reductase, the amino acid sequence corresponding to SEQ ID NO: 1 is mutated to L at position 56, V at position 109, and V at position 114.

[0021] (9) Compared with the amino acid sequence of wild-type 5α-reductase, the amino acid sequence corresponding to SEQ ID NO: 1 is mutated to L at position 56 and to M at position 109;

[0022] Preferably, the mutant amino acid sequence is as shown in any one of SEQ ID NO: 2-SEQ ID NO: 10, or has 90% identity with any one of the amino acid sequences shown in SEQ ID NO: 2-SEQ ID NO: 10.

[0023] Those skilled in the art will understand that the structure of a protein (in this invention, a 5α-reductase mutant) can be altered without adversely affecting its activity and function. For example, one or more conserved amino acid substitutions can be introduced into the protein's amino acid sequence without adversely affecting the protein molecule's activity and / or three-dimensional structure. Examples and implementations of conserved amino acid substitutions are familiar to those skilled in the art. Specifically, an amino acid residue can be substituted with another amino acid residue belonging to the same group as the site to be substituted, i.e., a nonpolar amino acid residue can replace another nonpolar amino acid residue, a polar uncharged amino acid residue can replace another polar uncharged amino acid residue, a basic amino acid residue can replace another basic amino acid residue, and an acidic amino acid residue can replace another acidic amino acid residue. Such substituted amino acid residues may or may not be encoded by the genetic code. Conservative substitutions, where an amino acid is replaced by another amino acid belonging to the same group, fall within the scope of this invention, as long as the substitution does not lead to inactivation of the protein's biological activity. Therefore, the mutants of this invention can contain one or more conserved substitutions in their amino acid sequences. Furthermore, this invention also covers proteins that also contain one or more other nonconservative substitutions, provided that such nonconservative substitutions do not significantly affect the desired function and biological activity of the mutants of this invention.

[0024] Conservative amino acid substitutions can occur at one or more predicted non-essential amino acid residues. “Non-essential” amino acid residues are those that can be altered (deleted, substituted, or replaced) without changing biological activity, while “essential” amino acid residues are required for biological activity. A “conservative amino acid substitution” is a substitution in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Amino acid substitutions can occur in the non-conservative regions of the aforementioned 5α-reductase mutants. Generally, such substitutions are not performed on conserved amino acid residues, or on amino acid residues located within conserved motifs, where such residues are required for protein activity. However, those skilled in the art will understand that functional variants may have fewer conserved or non-conserved alterations in conserved regions.

[0025] As is well known in the art, one or more amino acid residues can be altered (replaced, deleted, truncated, or inserted) from the N and / or C ends of a protein while retaining its functional activity. Therefore, proteins from which one or more amino acid residues have been altered from the N and / or C ends of a 5α-reductase mutant while retaining their desired functional activity are also within the scope of this invention. These alterations can include those introduced by modern molecular methods such as PCR, which includes PCR amplification that alters or lengthens the protein-coding sequence by means of oligonucleotides containing amino acid-coding sequences used in the PCR amplification.

[0026] It should be recognized that proteins can be altered in various ways, including amino acid substitutions, deletions, truncations, and insertions, and methods for such operations are generally known in the art. For example, amino acid sequence variants of the aforementioned proteins can be prepared by mutating DNA. This can also be accomplished through other forms of mutagenesis and / or directed evolution, for example, using known mutagenesis, recombination, and / or shuffling methods, combined with relevant screening methods, to perform single or multiple amino acid substitutions, deletions, and / or insertions.

[0027] Those skilled in the art will understand that these minor amino acid changes in the 5α-reductase mutants of this invention can occur (e.g., naturally occurring mutations) or be generated (e.g., using r-DNA technology) without loss of protein function or activity. If these mutations occur in the catalytic domain, active site, or other functional domains of a protein, the properties of the polypeptide may be altered, but the polypeptide may retain its activity. If the mutations are not located near the catalytic domain, active site, or other functional domains, a smaller impact can be expected.

[0028] Those skilled in the art can identify the amino acids of the 5α-reductase mutant of the present invention using methods known in the art, such as localized mutagenesis, protein evolution, or bioinformatics analysis. The catalytic domains, active sites, or other functional domains of the protein can also be determined through physical structural analysis, such as by techniques like nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, combined with mutations in presumed key site amino acids.

[0029] In this invention, amino acid residues can be represented by a single letter or by three letters, for example: alanine (Ala, A), valine (Val, V), glycine (Gly, G), leucine (Leu, L), glutamic acid (Gln, Q), phenylalanine (Phe, F), tryptophan (Trp, W), tyrosine (Tyr, Y), aspartic acid (Asp, D), asparagine (Asn, N), glutamic acid (Glu, E), lysine (Lys, K), methionine (Met, M), serine (Ser, S), threonine (Thr, T), cysteine ​​(Cys, C), proline (Pro, P), isoleucine (Ile, I), histidine (His, H), and arginine (Arg, R).

[0030] As used herein, the term “A56L” indicates that amino acid A at position 56 is changed to amino acid L. For example, “I109M” indicates that amino acid 1 at position 109 is mutated to M. For example, “I109M-I114V” indicates that amino acid 1 at position 109 is mutated to M while amino acid 114 at position 114 is mutated to V, and so on.

[0031] Secondly, the present invention provides a nucleic acid molecule encoding the 5α-reductase mutant, preferably, the nucleotide sequence of the nucleic acid molecule is as shown in any one of SEQ ID NO: 11-SEQ ID NO: 19, or has 90% identity with the nucleotide sequences shown in any one of SEQ ID NO: 11-SEQ ID NO: 19.

[0032] Thirdly, the present invention provides a recombinant expression vector containing the aforementioned nucleic acid molecule. Preferably, the backbone vector of the recombinant expression vector is selected from pET-21a(+) or pPICZαA.

[0033] Fourthly, the present invention provides a host cell comprising the 5α-reductase mutant, the nucleic acid molecule, or the recombinant expression vector, preferably Escherichia coli or Pichia pastoris, preferably Escherichia coli C43 (DE3).

[0034] Fifthly, the present invention provides a cell culture comprising the aforementioned host cells.

[0035] In a sixth aspect, the present invention provides the use of the 5α-reductase mutant, or the nucleic acid molecule, or the recombinant expression vector, or the host cell, or the cell culture in the preparation of steroidal drugs, preferably in the catalytic reduction reaction of 3-ketosteroid compounds at the C4,5 position.

[0036] Specifically, the 3-ketosteroid compound is selected from androst-4-en-3,17-dione (AD), 17β-hydroxyandrost-4-en-3-one (testosterone), 11α-hydroxyandrost-4-en-3,17-dione (11α-OH-AD), 19-norandrost-4-en-3,17-dione (19-nor-AD), and 19-hydroxyandrost-4-en-3,17-dione (19OH). At least one of the following: -AD), 20-hydroxymethylpregn-4-en-3-one (BA), DL-18-methyl-4-estren-3,17-dione (DL-ethyldione), pregn-4-en-3,20-dione (progesterone), androstened-1,4-dien-3,17-dione (ADD), and 11α-hydroxypregn-4-en-3,20-dione (11α-OH-progesterone).

[0037] In a seventh aspect, the present invention provides a method for preparing steroidal compounds, using a 3-ketosteroidal compound as a substrate, adding the 5α-reductase mutant or the host cell or the cell culture, and carrying out a catalytic reaction at pH 8.0 and a temperature of 37°C to obtain a steroidal drug intermediate. Preferably, the catalytic reaction system further contains glucose, glucose dehydrogenase, and NADP. + And a co-solvent, preferably, the co-solvent is selected from at least one of methanol, ethanol, and DMSO.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] This invention provides a 5α-reductase mutant with significantly enhanced catalytic performance, exhibiting high catalytic activity for various 3-ketosteroid compounds, especially achieving an AD conversion rate of 99%, thus solving the problems of low activity and poor substrate tolerance of existing 5α-reductases. The mutant can efficiently convert target substrates at high substrate concentrations of 10 g / L, shortening the conversion cycle and meeting the needs of industrial production. The reaction does not require large amounts of organic solvents, avoiding the environmental hazards of chemical methods and the low efficiency of microbial fermentation methods, resulting in a simple and environmentally friendly process. Furthermore, the mutant construction and preparation methods are mature and easy to scale up, providing strong technical support for the green and efficient synthesis of steroid drug intermediates, significantly reducing production costs, and showing good prospects for industrial application. Attached Figure Description

[0040] Figure 1 These are the GC spectra of 5α-AD and 5β-AD standards.

[0041] Figure 2 This is a GC diagram of AD transformation by C43-(DE3)-9 recombinant bacteria;

[0042] Figure 3 This is the GC chromatogram of a progesterone standard.

[0043] Figure 4 This is a GC diagram of progesterone conversion by recombinant C43-(DE3)-9 bacteria. Detailed Implementation

[0044] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result of the description. However, these embodiments are merely illustrative and do not constitute any limitation on the scope of protection defined by the claims of the present invention.

[0045] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0046] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0047] Example 1: Preparation of wild-type 5α-reductase recombinant expression plasmid and recombinant expression transformant

[0048] Gene synthesis and vector construction: A possible C4,5(5α) reductase sequence from Prevotellaceae bacterium (NCBI accession number WP_002667090.1) was discovered using gene methods. The gene was synthesized by codon optimization based on the protein sequence (its amino acid sequence is shown in SEQ ID NO: 1) and constructed into the pET21a expression vector. The gene insertion sites were Nde I and Hind III.

[0049] Transformation of the recombinant plasmid: E. coli C43(DE3) competent cells were prepared using the calcium chloride method. 10 μL of the recombinant plasmid was added to 50 μL of competent cells and incubated on ice for 30 minutes. After heat shock at 42°C for 45 seconds, the cells were quickly placed on ice for 2 minutes. 600 μL of LB broth was added, and the cells were incubated at 37°C with shaking for 1 hour. 200 μL of the bacterial culture was spread onto LB agar plates containing 100 μg / mL ampicillin and incubated overnight at 37°C. Single colonies were picked, and positive clones were obtained through colony PCR and double enzyme digestion of the plasmid, namely the recombinant expression transformant E. coli C43(DE3) / pET21a-5α-reductase (hereinafter referred to as C43(DE3)-9).

[0050] Example 2: Construction and Screening of 5α-Reductase Mutants

[0051] 1. Construction of the C43(DE3)-9 mutant

[0052] The structure of 5α-reductase C43(DE3)-9 was predicted using Alphafold 3.0. Non-conserved residues in its substrate binding pocket and amino acids in its substrate channel were selected and subjected to saturation mutations. Degenerate codon site-directed mutagenesis was used to design mutation primers. The recombinant expression vector pET21a-C43(DE3)-9 was used as a template, and PCR was performed using high-fidelity polymerase FastPfu. The PCR reaction conditions were as follows: First round of PCR: In a PCR reaction system with a total volume of 50 μL, 10-50 ng of template, 10 μL of 5× buffer, 4 μL of dNTP (2.5 mM), 1 μL (10 μM) of each of the two mutation primers were added, and sterile distilled water was added to a final volume of 50 μL. PCR reaction program: (1) denaturation at 98℃ for 10 sec, (2) annealing at 58℃ for 20 sec, (3) extension at 72℃ for 40 sec. Steps (1) to (3) were performed for a total of 30 cycles.

[0053] Second round PCR: In a PCR reaction system with a total volume of 50 μL, add 10-50 ng of template, 10 μL of 5× buffer, 4 μL of dNTP (2.5 mM), 1 μL of MgSO4 (50 mM), 2 μL of mutation primer (first round PCR product), and add sterile distilled water to a final volume of 50 μL. PCR reaction procedure: (1) denaturation at 98℃ for 10 sec, (2) annealing at 58℃ for 20 sec, (3) extension at 72℃ for 4 min. Perform steps (1) to (3) for a total of 30 cycles. Store the product at 4℃.

[0054] After verification by agarose gel electrophoresis, the PCR products were digested with the limiting enzyme DpnI at 37°C for 2 hours. The digested products were then transformed into E. coli C43(DE3) competent cells and plated on plates containing ampicillin antibiotics, and incubated statically at 37°C for about 12 hours.

[0055] 2. High-throughput screening

[0056] Single clones were selected and induced to express on a small scale in 96-well deep-well plates. Cells were collected by centrifugation, lysed, and NADPH consumption rate was monitored at 340 nm using AD as a substrate for initial screening. Clones with significantly faster NADPH consumption rates than wild-type were selected for sequencing verification.

[0057] 3. Combination mutations of 5α-reductase (C43(DE3)-9)

[0058] Combinatorial mutations were constructed based on the results of saturation mutagenesis. The resulting monoclonal colonies were picked and cultured in Erlenmeyer flasks containing 30 mL of LB medium, and the activity of the expressed protein was detected.

[0059] Enzyme activity assay system (200 μL): Total reaction volume 0.2 mL, 100 mM Tris-HCl pH 8.0, 10 μL 1 mM AD, 10 μL 1 mM NAD pH, appropriate concentration of enzyme solution, detection started at 30℃, and the change in absorbance at 340 nm was measured. The relative reaction rate of each mutant was calculated using the enzyme activity of wild-type C43(DE3)-9 as a baseline (set to 1.0). The results are shown in Table 1.

[0060] Table 1. Relative reaction rates of AD C4,5-position reduction catalyzed by each mutant

[0061]

[0062] The results showed that mutants M2 to M9 significantly enhanced AD catalytic activity, with mutant M5 showing the most significant enhancement.

[0063] The mutant M2 has an amino acid sequence that, relative to the wild-type 5α-reductase, has a mutation at position 109 (I) of isoleucine (M) to methionine (M) and position 114 (I) of isoleucine (V) to valine (V). The mutated amino acid sequence is shown in SEQ ID NO: 2, and the nucleotide sequence of the gene it encodes is shown in SEQ ID NO: 11.

[0064] The mutant M3, relative to the amino acid sequence of the wild-type 5α-reductase, has the following amino acid sequence: isoleucine (I) at position 109 is mutated to methionine (M), and isoleucine (I) at position 114 is mutated to methionine (M). The specific amino acid sequence after the mutation is shown in SEQ ID NO: 3, and the nucleotide sequence of the gene it encodes is shown in SEQ ID NO: 12.

[0065] The mutant M4, relative to the amino acid sequence of the wild-type 5α-reductase, has the following mutations: isoleucine (I) at position 109 is mutated to leucine (L), and isoleucine (I) at position 114 is mutated to methionine (M). The specific amino acid sequence after the mutation is shown in SEQ ID NO: 4, and the nucleotide sequence of the gene it encodes is shown in SEQ ID NO: 13.

[0066] The mutant M5, relative to the amino acid sequence of the wild-type 5α-reductase, has the following mutations: isoleucine (I) at position 109 is mutated to valine (V), and isoleucine (I) at position 114 is mutated to methionine (M). The specific amino acid sequence after the mutation is shown in SEQ ID NO: 5, and the nucleotide sequence of the gene it encodes is shown in SEQ ID NO: 14.

[0067] The amino acid sequence of mutant M6, relative to the amino acid sequence of wild-type 5α-reductase, has the following mutations: alanine (A) at position 56 is mutated to leucine (L), isoleucine (I) at position 109 is mutated to valine (V), and isoleucine (I) at position 114 is mutated to leucine (L). The specific amino acid sequence after the mutation is shown in SEQ ID NO: 6, and the nucleotide sequence of the gene it encodes is shown in SEQ ID NO: 15.

[0068] The mutant M7, relative to the amino acid sequence of the wild-type 5α-reductase, has a mutation at position 56 (A) of alanine (L) to leucine (L) and position 114 (I) of isoleucine (L) to leucine (L). The specific amino acid sequence after the mutation is shown in SEQ ID NO: 7, and the nucleotide sequence of the gene it encodes is shown in SEQ ID NO: 16.

[0069] The mutant M8, relative to the amino acid sequence of the wild-type 5α-reductase, has the following mutations: at position 56, alanine (A) is mutated to leucine (L), and at position 114, isoleucine (I) is mutated to methionine (M). The specific amino acid sequence after the mutation is shown in SEQ ID NO: 8, and the nucleotide sequence of the gene it encodes is shown in SEQ ID NO: 17.

[0070] The mutant M9, relative to the amino acid sequence of the wild-type 5α-reductase, has the following mutations: at position 56, alanine (A) is mutated to leucine (L); at position 109, isoleucine (I) is mutated to valine (V); and at position 114, isoleucine (I) is mutated to valine (V). The specific amino acid sequence after the mutation is shown in SEQ ID NO: 9, and the nucleotide sequence of the gene it encodes is shown in SEQ ID NO: 18.

[0071] The mutant M10, relative to the amino acid sequence of the wild-type 5α-reductase, has the following mutations: at position 56, alanine (A) is mutated to leucine (L), and at position 109, isoleucine (I) is mutated to methionine (M). The specific amino acid sequence after the mutation is shown in SEQ ID NO: 10, and the nucleotide sequence of the gene it encodes is shown in SEQ ID NO: 19.

[0072] Example 3: Transformation of 3-ketosteroid compounds by recombinant bacteria and mutants of 5α-reductase (C43(DE3)-9).

[0073] General method:

[0074] Seed culture: Recombinant bacteria containing 5α-reductase (C43(DE3)-9) and its mutants were picked with an inoculation loop and inoculated into LB medium containing ampicillin. The culture was carried out overnight at 37°C and 200 rpm.

[0075] Fermentation induction culture: The overnight cultured seed culture was transferred to the fermentation medium at an inoculum rate of 1%, and cultured at 37°C and 200 rpm until OD200 reached. 600At a concentration of approximately 0.6-1.0 nm, 0.1 mM IPTG was added, and the mixture was incubated at 25°C and 200 rpm for 10-12 hours to obtain the fermentation broth. The cells were collected by centrifugation (6000 rpm), resuspended in 100 mM Tris-HCl buffer (pH 8.0), and homogenized under high pressure to obtain the crude enzyme solution. The substrate (concentrations are shown in the following experiments, all final concentrations), 100 mM glucose, 3-6 U / mL glucose dehydrogenase, 0.1-0.5 g / L NADP+, and 5-15% (v / v) solubilizer were added to the crude enzyme solution to initiate the reaction. After stirring at 37°C for a certain time, the product was extracted with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, concentrated, and the conversion rate was calculated by GC analysis. The GC spectra of the 5α-AD and 5β-AD standards are shown below. Figure 1 As shown.

[0076] In the catalytic system of recombinant bacteria and mutants of 5α-reductase (C43(DE3)-9) described in this invention, the role of the co-solvent is to improve the solubility of the hydrophobic steroid substrate, ensuring sufficient contact between it and the enzyme molecules, thereby improving reaction efficiency. Methanol, ethanol, DMSO, and other co-solvents were used in the following experiments, and all were proven effective. To simplify variables and highlight the correspondence between enzyme and substrate, DMSO, which has excellent solubility and good enzyme compatibility, was uniformly used as the standard co-solvent in subsequent comparative experiments.

[0077] 1. Transformation of AD by recombinant bacteria containing 5α-reductase (C43(DE3)-9)

[0078] Take 500 mL of crude enzyme solution, centrifuge and resuspend in 500 mL of Tris-HCl buffer (pH 8.0, 100 mM), homogenize by high pressure, add AD to the above enzyme solution at a concentration of 10 g / L, add 100 mM glucose, 3 U / mL glucose dehydrogenase, and NADP. + 0.2 g / L; 10% methanol as a co-solvent; reaction was carried out with stirring at 37 °C. After 6 h of reaction, the mixture was extracted with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The results are as follows. Figure 2 As shown, the conversion rate of the GC detection reaction was 99%.

[0079] 2. The conversion of progesterone by recombinant bacteria containing 5α-reductase (C43(DE3)-9)

[0080] Centrifuge 500 mL of crude enzyme solution and resuspend it in 500 mL of Tris-HCl buffer (pH 8.0, 100 mM). Homogenize the solution using a high-pressure autoclave. Add 11-progesterone to the enzyme solution at a concentration of 10 g / L, along with 100 mM glucose, 3 U / mL glucose dehydrogenase, and NADP. +0.2 g / L; 10% ethanol as a co-solvent; reaction at 37 °C with stirring. After 6 h of reaction, extract with ethyl acetate, combine the organic phases, dry with anhydrous sodium sulfate, and remove the solvent under reduced pressure. The GC chromatogram of the brass standard is shown below. Figure 3 As shown, the GC diagram of progesterone transformation by recombinant C43-(DE3)-9 is as follows. Figure 4 As shown, the conversion rate of the GC detection reaction was 85%.

[0081] 3. The conversion of 11α-OH-progesterone by recombinant bacteria containing 5α-reductase (C43(DE3)-9)

[0082] Take 500 mL of crude enzyme solution, centrifuge and resuspend in 500 mL of Tris-HCl buffer (pH 8.0, 100 mM), homogenize by high pressure, add 11-hydroxyprogesterone to the above enzyme solution at a concentration of 10 g / L, add 100 mM glucose, 3 U / mL glucose dehydrogenase, and NADP. + 0.2 g / L; 10% isopropanol as a co-solvent; reaction was stirred at 37 °C. After 6 h of reaction, the mixture was extracted with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. GC analysis showed the conversion rate to be 53%.

[0083] 4. The conversion of 11-carbonyl-progesterone by recombinant bacteria containing 5α-reductase (C43(DE3)-9)

[0084] Centrifuge 500 mL of crude enzyme solution and resuspend it in 500 mL of Tris-HCl buffer (pH 8.0, 100 mM). Homogenize the solution using a high-pressure autoclave. Add 11-carbonyl-progesterone to the enzyme solution at a concentration of 10 g / L. Add 100 mM glucose, 3 U / mL glucose dehydrogenase, and NADP. + 0.2 g / L; 10% DMSO as a co-solvent; reaction was carried out with stirring at 37 °C. After 6 h of reaction, the mixture was extracted with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. GC analysis showed that the conversion rate was 42%.

[0085] 5. Transformation of 19-OH-AD by recombinant bacteria containing 5α-reductase (C43(DE3)-9)

[0086] Take 500 mL of crude enzyme solution, centrifuge and resuspend in 500 mL of Tris-HCl buffer (pH 8.0, 100 mM), homogenize by high pressure, add 19-OH-AD to the above enzyme solution at a concentration of 10 g / L, add 100 mM glucose, 3 U / mL glucose dehydrogenase, and NADP. + 0.2 g / L; 10% methanol as a co-solvent; reaction was carried out with stirring at 37 °C. After the reaction was completed, the mixture was extracted with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. GC analysis showed that the conversion rate was 71%.

[0087] 6. The conversion of ethyl diketone by recombinant bacteria containing 5α-reductase (C43(DE3)-9)

[0088] Take 500 mL of crude enzyme solution, centrifuge and resuspend in 500 mL of Tris-HCl buffer (pH 8.0, 100 mM), homogenize by high pressure, add ethyl diketone to the above enzyme solution at a concentration of 10 g / L, add 100 mM glucose, 3 U / mL glucose dehydrogenase, and NADP. + 0.2 g / L; 10% DMF as a co-solvent; reaction was carried out with stirring at 37°C. After the reaction was completed, the mixture was extracted with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. GC analysis showed that the conversion rate was 64%.

[0089] 7. The conversion of androstenediones (4,9-3,17-diones) by recombinant bacteria containing 5α-reductase (C43(DE3)-9).

[0090] Take 500 mL of crude enzyme solution, centrifuge and resuspend in 500 mL of Tris-HCl buffer (pH 8.0, 100 mM), homogenize by high pressure, add androstened 4,9-3,17-dione to the above enzyme solution at a concentration of 10 g / L, add 100 mM glucose, 3 U / mL glucose dehydrogenase, and NADP. + 0.2 g / L; 10% DMSO as a co-solvent; reaction was carried out with stirring at 37 °C. After the reaction was completed, the mixture was extracted with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. GC analysis showed that the conversion rate was 58%.

[0091] 8. The conversion of testosterone by recombinant bacteria containing 5α-reductase (C43(DE3)-9)

[0092] Take 500 mL of crude enzyme solution, centrifuge and resuspend in 500 mL of Tris-HCl buffer (pH 8.0, 100 mM), homogenize by high pressure, add testosterone to the above enzyme solution at a concentration of 10 g / L, add 100 mM glucose, 3 U / mL glucose dehydrogenase, and NADP. + 0.2 g / L; 10% ethanol as a co-solvent; reaction was carried out with stirring at 37 °C. After the reaction was completed, the mixture was extracted with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. GC analysis showed that the conversion rate was 47%.

[0093] 9. Transformation of 11α-OH-AD by recombinant bacteria containing 5α-reductase (C43(DE3)-9)

[0094] Take 500 mL of crude enzyme solution, centrifuge and resuspend in 500 mL of Tris-HCl buffer (pH 8.0, 100 mM), homogenize by high pressure, add 11α-OH-AD to the above enzyme solution at a concentration of 10 g / L, add 100 mM glucose, 3 U / mL glucose dehydrogenase, and NADP. + 0.2 g / L; 10% DMF as a co-solvent; reaction was carried out with stirring at 37 °C. After the reaction was completed, the mixture was extracted with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. GC analysis showed that the conversion rate was 39%.

[0095] 10. Transformation of 19-nor-AD by recombinant bacteria containing 5α-reductase (C43(DE3)-9)

[0096] Take 500 mL of crude enzyme solution, centrifuge and resuspend in 500 mL of Tris-HCl buffer (pH 8.0, 100 mM), homogenize by high pressure, add 19-nor-AD to the above enzyme solution at a concentration of 10 g / L, add 100 mM glucose, 3 U / mL glucose dehydrogenase, and 0.2 g / L NADP. + The reaction was carried out with 10% DMSO as a co-solvent at 37°C under stirring. After the reaction was complete, the mixture was extracted with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. GC analysis showed that the conversion rate was 17%.

[0097] 11. Transformation of BA by recombinant 5α-reductase (C43(DE3)-9) bacteria

[0098] Take 500 mL of crude enzyme solution, centrifuge and resuspend in 500 mL of Tris-HCl buffer (pH 8.0, 100 mM), homogenize by high pressure, add BA to the above enzyme solution at a concentration of 10 g / L, add 100 mM glucose, 3 U / mL glucose dehydrogenase, and 0.2 g / L NADP. + The reaction was carried out with 10% 1,4-dioxane as a co-solvent at 37°C under stirring. After the reaction was complete, the mixture was extracted with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. GC analysis showed that the conversion rate was 13%.

[0099] Table 2. Transformation effects of recombinant 5α-reductase (C43(DE3)-9) bacteria on different substrates.

[0100]

[0101] The results are shown in Table 2. The 5α-reductase (C43(DE3)-9) recombinant bacteria showed high conversion rates for different 3-ketosteroid compounds, especially AD, which achieved a conversion rate of 99%.

[0102] Example 4: The conversion effect of 5α-reductase mutants M5 and M8 on AD.

[0103] Take 100 mL of crude enzyme solution (M5, M8) respectively, centrifuge and resuspend in Tris-HCl buffer (pH 8.0, 100 mM), homogenize by high pressure, and add AD to the above enzyme solution at a concentration of 10 g / L, along with 100 mM glucose, 3 U / mL glucose dehydrogenase, and NADP. + 0.2 g / L; 10% DMSO as a co-solvent, 100 mL of mixture was stirred at 37 °C. After 6 h of reaction, the mixture was extracted with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. GC analysis showed that the conversion rate of AD by both mutants reached 99%.

[0104] Example 5: Comparison of the catalytic efficiency of various 5α-reductase mutants for progesterone.

[0105] To verify the improved catalytic efficiency of the mutant on progesterone, parallel comparative experiments were conducted using wild-type and multiple mutants.

[0106] Experimental Methods: Engineered bacteria expressing wild-type 5α-reductase (C43(DE3)-9) and mutants M2-M10 were cultured separately. Cells were induced and collected using standard methods. All cells were resuspended and lysed in the same volume of 100 mM Tris-HCl buffer (pH 8.0) to prepare crude enzyme solutions. The total protein concentration of each crude enzyme solution was determined using the Bradford method, and all solutions were uniformly diluted to the same concentration (1.0 mg / mL).

[0107] Reaction system (1 mL): 500 μL standardized crude enzyme solution, progesterone (concentration 10 g / L), glucose (50 mM), glucose dehydrogenase (3 U / mL), NADP + (0.2 g / L), 10% DMSO as a co-solvent, and Tris-HCl buffer were added to a final volume of 1 mL. The reaction was carried out at 37 °C and 300 rpm with shaking for 30 minutes. After the reaction was complete, the mixture was immediately extracted with ethyl acetate and analyzed by GC to calculate the initial reaction rate. The relative reaction rates of each mutant were calculated using the wild-type reaction rate as a baseline (set as 1.00), and the results are shown in Table 3.

[0108] Table 3. Relative reaction rates of progesterone C4,5-position reduction catalyzed by each mutant

[0109]

[0110] Example 6: The conversion of progesterone by the 5α-reductase mutant M5

[0111] Take 100 mL of crude enzyme solution, centrifuge and resuspend in 100 mL of Tris-HCl buffer (pH 8.0, 100 mM), homogenize by high pressure, add progesterone to the above enzyme solution at a concentration of 10 g / L, add 100 mM glucose, 3 U / mL glucose dehydrogenase, and NADP. + 0.2 g / L; 10% DMSO as a co-solvent; reaction was carried out with stirring at 37 °C. After 6 h of reaction, the mixture was extracted with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. GC analysis showed that the conversion rate was 85%.

[0112] Example 7: Comparison of catalytic efficiencies of various 5α-reductase mutants on 11α-OH-progesterone

[0113] To verify the enhanced catalytic efficiency of the mutant on 11α-OH-progesterone, parallel comparative experiments were conducted using wild-type and multiple mutants.

[0114] Experimental Methods: Engineered bacteria expressing wild-type 5α-reductase C43(DE3)-9 and mutants M2-M10 were cultured separately. Cells were induced and collected using standard methods. All cells were resuspended and lysed in the same volume of 100 mM Tris-HCl buffer (pH 8.0) to prepare crude enzyme solutions. The total protein concentration of each crude enzyme solution was determined using the Bradford method, and all solutions were uniformly diluted to the same concentration (1.0 mg / mL).

[0115] Reaction system (1 mL): 500 μL standardized crude enzyme solution, 11α-OH-progesterone (concentration 10 g / L), glucose (50 mM), glucose dehydrogenase (3 U / mL), NADP + (0.2 g / L), 10% DMSO as a co-solvent, and Tris-HCl buffer were added to a final volume of 1 mL. The reaction was carried out at 37 °C and 300 rpm with shaking for 30 minutes. After the reaction was complete, the mixture was immediately extracted with ethyl acetate and analyzed by GC to calculate the initial reaction rate. The relative reaction rates of each mutant were calculated using the wild-type reaction rate as a baseline (set as 1.00), and the results are shown in Table 4.

[0116] Table 4. Relative reaction rates of the reduction of 11α-OH-progesterone at the C4,5 position catalyzed by each mutant.

[0117]

[0118] Example 8: Conversion of 11α-OH-progesterone by the 5α-reductase mutant M5

[0119] Take 100 mL of crude enzyme solution (M5), centrifuge and resuspend in Tris-HCl buffer (pH 8.0, 100 mM), homogenize by high pressure, add 11α-OH-progesterone to the above enzyme solution at a concentration of 10 g / L, add 100 mM glucose, 3 U / mL glucose dehydrogenase, and NADP. + 0.2 g / L; 10% DMSO as a co-solvent, stirred at 37°C for 100 mL. After 6 h of reaction, the mixture was extracted with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. GC analysis showed the conversion rate to be 53%.

[0120] Example 9: Comparison of catalytic efficiencies of various 5α-reductase mutants for 11-carbonyl-progesterone

[0121] To verify the improvement of the catalytic efficiency of the mutant on 11-carbonyl-progesterone, parallel comparative experiments were conducted using wild-type and multiple mutants.

[0122] Experimental Methods: Engineered bacteria expressing wild-type 5α-reductase (C43(DE3)-9) mutants M2-M10 were cultured, and bacterial cells were induced and collected using standard methods. All bacterial cells were resuspended and lysed in the same volume of 100 mM Tris-HCl buffer (pH 8.0) to prepare crude enzyme solutions. The total protein concentration of each crude enzyme solution was determined using the Bradford method, and all solutions were uniformly diluted to the same concentration (1.0 mg / mL).

[0123] Reaction system (1 mL): 500 μL standardized crude enzyme solution, progesterone (concentration 10 g / L), glucose (50 mM), glucose dehydrogenase 3 U / mL, NADP + (0.2 g / L), 10% DMSO as a co-solvent, and Tris-HCl buffer were added to a final volume of 1 mL. The reaction was carried out at 37 °C and 300 rpm with shaking for 30 minutes. After the reaction was complete, the mixture was immediately extracted with ethyl acetate and analyzed by GC to calculate the initial reaction rate. The relative reaction rates of each mutant were calculated using the wild-type reaction rate as a baseline (set as 1.00), and the results are shown in Table 5.

[0124] Table 5. Relative reaction rates of catalytic reduction of 11-carbonyl-progesterone at C4,5 position by each mutant.

[0125]

[0126] As shown in Tables 3, 4, and 5, compared with the wild type, the preferred mutants M4, M5, M6, M7, M8, and M9 provided in this invention significantly improved the catalytic efficiency of progesterone, 11α-OH-progesterone, and 11-carbonyl-progesterone, with relative reaction rates increasing by 16% to 35%. This result is consistent with the trend of these mutants exhibiting high activity with AD as a substrate (see Table 1), demonstrating that combined mutations at these sites can generally improve the enzyme's catalytic ability towards 3-keto-4-ene steroid substrates. In particular, mutant M5 exhibited the highest catalytic activity for AD and progesterone, 11α-OH-progesterone, and 11-carbonyl-progesterone, making it a candidate enzyme with great potential for industrial application.

[0127] Example 10: Conversion of 11-carbonyl-progesterone by the 5α-reductase mutant M5

[0128] Take 100 mL of crude enzyme solution, centrifuge and resuspend in Tris-HCl buffer (pH 8.0, 100 mM), homogenize by high pressure, add 11-carbonyl-progesterone to the above enzyme solution at a concentration of 10 g / L, add 100 mM glucose, 3 U / mL glucose dehydrogenase, and NADP. + 0.2 g / L; 10% DMSO as a co-solvent, stirred at 37°C for 100 mL. After 6 h of reaction, the mixture was extracted with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. GC analysis showed the conversion rate to be 42%.

[0129] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A 5α-reductase mutant, characterized in that, Compared with the amino acid sequence of wild-type 5α-reductase, the mutant has mutations at at least two sites corresponding to the amino acid sequence shown in SEQ ID NO: 1: position 56, position 109, and / or position 114; the amino acid sequence of wild-type 5α-reductase is as shown in SEQ ID NO: 1 or has at least 90% identity with the amino acid sequence shown in SEQ ID NO: 1; the amino acid at position 56 is mutated to L, the amino acid at position 109 is mutated to M, L, or V, and the amino acid at position 114 is mutated to M, L, or V.

2. The 5α-reductase mutant according to claim 1, characterized in that, The mutant is selected from one or more of the following: (1) Compared with the amino acid sequence of wild-type 5α-reductase, the amino acid sequence corresponding to SEQ ID NO: 1 is mutated to M at position 109 and to V at position 114; (2) Compared with the amino acid sequence of wild-type 5α-reductase, the amino acid sequence corresponding to SEQ ID NO: 1 is mutated to M at position 109 and position 114; (3) Compared with the amino acid sequence of wild-type 5α-reductase, the amino acid sequence corresponding to SEQ ID NO: 1 is mutated to L at position 109 and to M at position 114; (4) Compared with the amino acid sequence of wild-type 5α-reductase, the amino acid sequence corresponding to SEQ ID NO: 1 is mutated to V at position 109 and to M at position 114; (5) Compared with the amino acid sequence of wild-type 5α-reductase, the amino acid sequence corresponding to SEQ ID NO: 1 is mutated to L at position 56, V at position 109, and L at position 114. (6) Compared with the amino acid sequence of wild-type 5α-reductase, the amino acid sequence corresponding to SEQ ID NO: 1 is mutated to L at position 56 and L at position 114; (7) Compared with the amino acid sequence of wild-type 5α-reductase, the amino acid sequence corresponding to SEQ ID NO: 1 is mutated to L at position 56 and to M at position 114; (8) Compared with the amino acid sequence of wild-type 5α-reductase, the amino acid sequence corresponding to SEQ ID NO: 1 is mutated to L at position 56, V at position 109, and V at position 114. (9) Compared with the amino acid sequence of wild-type 5α-reductase, the amino acid sequence corresponding to SEQ ID NO: 1 is mutated to L at position 56 and to M at position 109.

3. A nucleic acid molecule, characterized in that, Encodes the 5α-reductase mutant as described in claim 1 or 2.

4. The nucleic acid molecule as described in claim 3, characterized in that, The nucleotide sequence of the nucleic acid molecule is as shown in any one of SEQ ID NO: 11-SEQ ID NO: 19, or has 90% identity with any one of the nucleotide sequences shown in SEQ ID NO: 11-SEQ ID NO:

19.

5. A recombinant expression vector, characterized in that, The recombinant expression vector contains the nucleic acid molecule as described in claim 3 or 4.

6. A host cell, characterized in that, It comprises the 5α-reductase mutant of claim 1 or 2, or the nucleic acid molecule of claim 3 or 4, or the recombinant expression vector of claim 5.

7. The host cell as described in claim 6, characterized in that, The host cell is either Escherichia coli or Pichia pastoris.

8. The use of the 5α-reductase mutant of claim 1 or 2, or the nucleic acid molecule of claim 3 or 4, or the recombinant expression vector of claim 5, or the host cell of claim 6 or 7 in the preparation of steroidal drugs, wherein the process is achieved by catalyzing the hydrogenation reduction reaction at the C4,5 position of a 3-ketosteroid compound.

9. The application according to claim 8, characterized in that, The 3-ketosteroid compound is selected from at least one of androst-4-ene-3,17-dione, 17β-hydroxyandrost-4-ene-3-one, 11α-hydroxyandrost-4-ene-3,17-dione, 19-norandrost-4-ene-3,17-dione, 19-hydroxyandrost-4-ene-3,17-dione, 20-hydroxymethylpregn-4-ene-3-one, DL-18-methyl-4-estren-3,17-dione, pregn-4-ene-3,20-dione, androst-1,4-diene-3,17-dione, and 11α-hydroxypregn-4-ene-3,20-dione.

10. A method for preparing steroidal compounds, characterized in that, Using a 3-ketosteroid compound as a substrate, the 5α-reductase mutant of claim 1 or 2 or the host cell of claim 6 or 7 is added, and a catalytic reaction is carried out at pH 8.0 and temperature 37°C to obtain a steroidal drug intermediate.

11. The method as described in claim 10, characterized in that, The catalytic reaction system also contains glucose, glucose dehydrogenase, and NADP. + And a co-solvent; the co-solvent is selected from at least one of methanol, ethanol, DMSO, 1,4-dioxane, DMF, and isopropanol.

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