A p450 enzyme mutant and its application in catalyzing dhea hydroxylation to prepare trihydroxyandrost-5-ene

Abstract

The present application relates to a kind of P450 enzyme mutant and its application in the preparation of trihydroxy androstene ketone by catalyzing DHEA hydroxylation.The present application provides four kinds of cytochrome P450 enzyme mutants, which are based on wild-type P450 enzyme, the alanine at position 112 is mutated to tyrosine, histidine, lysine and serine respectively by site-directed mutagenesis, which significantly improves the catalytic efficiency, can efficiently catalyze dehydroepiandrosterone (DHEA) to prepare product trihydroxy androstene ketone by two-step hydroxylation reaction.Compared with wild-type cytochrome P450 enzyme, these mutants show excellent catalytic performance, and have important industrial application value in the field of steroid drug synthesis.

Description

Technical Field

[0001] This invention relates to a P450 enzyme mutant capable of efficiently catalyzing the hydroxylation reaction of DHEA to produce trihydroxyandrostenone and its applications, belonging to the field of enzyme engineering. Background Technology

[0002] Steroid compounds, due to their unique physiological activities, have wide applications in the pharmaceutical field, covering multiple therapeutic areas such as anti-inflammation, diuresis, anabolic stimulants, contraception, anti-androgens, fertility enhancement, and anti-cancer agents. Originally extracted from animal tissues, a series of natural steroid compounds, including cholesterol, estrone, estradiol, and testosterone, have been isolated and identified. Studies have shown that the biological activity of steroid drugs is closely related to their molecular structure—specific structural modifications can significantly enhance their pharmacological effects. For example, the introduction of a double bond at the C1,2 position can enhance glucocorticoid activity, while the C11β-hydroxyl group is a key group for anti-inflammatory activity. However, the steroid nucleus structure contains multiple chiral centers, posing significant challenges to site-specific modification using chemical synthesis methods: not only are the reaction steps cumbersome, but there are also difficulties in controlling regioselectivity and stereoselectivity, high energy consumption, and severe environmental pollution.

[0003] Trihydroxyandrostenone (7α,15α-diOH-DHEA) is a dihydroxylated derivative of dehydroepiandrosterone (DHEA), belonging to the androstane steroidal compounds. It is poorly soluble in water but readily soluble in organic solvents such as ethanol and chloroform. In industrial production, 7α,15α-diOH-DHEA is used as an intermediate in the synthesis of drospirenone.

[0004] Drspirenone is a highly effective and low-toxicity next-generation steroidal contraceptive. Since its launch in Europe in 2000, it has become one of the most promising oral contraceptives for women worldwide. The key to achieving its large-scale industrial production lies in developing efficient, green, and economical synthetic routes. Traditional all-chemical synthesis methods involve 18 steps, resulting in high energy consumption and low yields. Currently, the most advanced synthetic process combines biocatalysis, utilizing microorganisms to hydroxylate DHEA at the 7α and 15α sites to generate the key intermediate 7α,15α-diOH-DHEA, followed by subsequent chemical synthesis. Compared to traditional methods, this strategy reduces the total reaction steps from 18 to 14, reducing environmental pollution and increasing product yield. As a crucial step in the dihydroxylation synthesis route, the efficiency of microbial conversion of DHEA to 7α,15α-diOH-DHEA constrains subsequent synthesis; therefore, improving the conversion efficiency of this process is of significant research importance.

[0005] Currently, fungi such as *Colletotrichum lini*, *Acremonium* sp., *Gibberella* sp., *Fusarium* sp., and *Nigrospora* sp. have been shown to catalyze the C7α and C15α dihydroxylation of DHEA. Among these strains, *C. lini* is one of the strains reported in recent years to be able to efficiently complete this transformation reaction, and the key enzymes for this transformation reaction have been cloned and identified.

[0006] Cytochrome P450 enzymes are widely distributed in prokaryotes and eukaryotes, with over 300,000 species belonging to more than 700 families identified to date. Cytochrome P450 enzymes possess a broad substrate spectrum, capable of converting over 250,000 substances, including steroids, terpenes, fatty acids, and alkanes, playing an irreplaceable role in in vivo drug metabolism and exogenous substance degradation. Although heterologous expression in yeast can achieve the conversion of DHEA to 7α,15α-diOH-DHEA, wild-type P450 enzymes generally suffer from low catalytic efficiency and poor substrate tolerance, severely limiting the feasibility of industrial production. Therefore, developing efficient and stable P450 enzyme mutants is crucial for improving product yield and reducing production costs.

[0007] With advancements in molecular biology techniques, significant breakthroughs have been achieved in the heterologous expression and molecular modification of fungal P450 enzymes. Using expression systems such as yeast or *E. coli*, not only has the efficient expression of various fungal P450 enzymes been realized, but their catalytic activity, substrate specificity, and regioselectivity have also been enhanced through protein engineering. However, a large number of P450 genes with unknown functions still exist in the fungal genome, and only a few have been successfully deciphered and applied to steroid transformation. Summary of the Invention

[0008] The purpose of this invention is to obtain a cytochrome P450 enzyme mutant that can catalyze the efficient synthesis of trihydroxyandrostenone (7α,15α-diOH-DHEA) from dehydroepiandrosterone (DHEA).

[0009] The wild-type cytochrome P450 enzyme of this invention is derived from the cytochrome P450 enzyme CYP-c13 of *Colletotrichum flabellulatum* in the Genebank database. The accession number of this enzyme in Genebank is ASF83124. The protein sequence was submitted to Suzhou Genewise Biotechnology Co., Ltd. for codon optimization, and the optimized gene sequence was subcloned into the EcoRI-Not I restriction endonuclease site of the *Saccharomyces cerevisiae* expression vector pPIC9K.

[0010] This invention provides four cytochrome P450 enzyme mutants, which, compared with wild-type cytochrome P450 enzymes, exhibit improved molar conversion of trihydroxyandrostenone.

[0011] This invention provides amino acid sequences of cytochrome P450 enzyme mutants as shown in SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, and SEQ ID NO:9. The cytochrome P450 enzyme mutant is based on the parental sequence SEQ ID NO:2, with alanine at position 112 mutated to tyrosine, histidine, lysine, and serine.

[0012] The present invention provides nucleotide sequences encoding catalytic reactions of DHEA to produce trihydroxyandrostenone, as shown in SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, and SEQ ID NO:10.

[0013] This invention provides four expression vectors, each containing the above four mutant nucleotide sequences, with pYES2 as the expression vector.

[0014] This invention provides four genetically engineered bacteria, each containing the above-mentioned expression vector, and the strain is constructed using Saccharomyces cerevisiae.

[0015] This invention provides a method for converting DHEA into trihydroxyandrostenone using the above-mentioned cytochrome P450 enzyme mutant and genetically engineered bacteria.

[0016] This invention involves the rational design and directed evolution of cytochrome P450 enzymes derived from *C. lini*, enhancing their catalytic efficiency and substrate adaptability to achieve efficient DHEA conversion. The mutant strain obtained through precise modification achieves a synergistic improvement in catalytic efficiency and selectivity, accelerating the synthesis of the target product and reducing byproduct accumulation by regulating the hydroxylation reaction step. Compared to the wild type, the mutant strain provided by this invention exhibits significantly enhanced 15α-hydroxylation efficiency, efficiently converting the intermediate 7α-OH-DHEA into the target product 7α,15α-diOH-DHEA while reducing intermediate accumulation. This is of great significance for the industrial production of 7α,15α-diOH-DHEA, significantly improving production efficiency and product purity, reducing downstream separation costs, and demonstrating promising application prospects. This invention also provides key technological support for the green biomanufacturing of 7α,15α-diOH-DHEA, promoting the sustainable development of steroid drug production. Attached Figure Description

[0017] Figure 1This is a thin-layer chromatogram of the fermentation products of wild-type cytochrome P450 enzyme and its mutant DHEA72 h in the embodiments of the present invention.

[0018] Figure 2 High-performance liquid chromatograms of fermentation products from wild-type and mutant strains after 72 h.

[0019] Figure 3 The molar conversion rates of 7α,15α-diOH-DHEA and 7α-OH-DHEA at different sites are for wild type and different sites. Detailed Implementation

[0020] The present invention will be described in detail below with reference to the embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0021] 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. 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.

[0022] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0023] Unless otherwise specified, the experimental methods described in the following examples are generally performed under conventional conditions or as recommended by the respective manufacturers. Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention employ conventional techniques in molecular biology, biochemistry, analytical chemistry, cell culture, recombinant DNA technology, and related fields. Unless otherwise specified, the reagents and materials mentioned in the examples are all commercially available products.

[0024] Example 1: Construction of recombinant Saccharomyces cerevisiae integrating wild-type P450 enzymes

[0025] A protein sequence from *Colletotrichum flavomarginata* that encodes the P450 enzyme was submitted to Suzhou Genewise Biotechnology Co., Ltd. The company optimized the codons based on the protein sequence. The optimized codons can be expressed in *Saccharomyces cerevisiae*. The protein sequence of the P450 enzyme is shown in SEQ ID NO: 1. The optimized sequence was cloned into the yeast expression vector pPIC9K, and the company subcloned the vector into *E. coli* Top10 strain.

[0026] Plasmids were extracted using a plasmid extraction kit. Using the plasmid as a template, PCR was performed with the primers shown in Table 1 and the PCR system shown in Table 2 to obtain the P450 enzyme gene fragment. This gene fragment was then cloned into the EcoRI and NotI restriction endonuclease sites of the pYES2 vector to construct the pYES2-P450 recombinant vector. Positive clones were screened on ampicillin-resistant plates, and the transformants were sent to Genewiz Biotechnology Co., Ltd. for sequencing to confirm their correctness. Genetically engineered bacteria with correct sequencing results were extracted and stored at -80 °C.

[0027] Table 1: Primer sequences used in this invention

[0028]

[0029] Table 2 Reverse PCR system:

[0030]

[0031] Reverse PCR amplification conditions: 95 ℃ pre-denaturation for 5 min; 95 ℃ denaturation for 30 s, 54 ℃ annealing for 30 s, 72 ℃ extension for 5 min, 25 cycles; incubation at 12 ℃.

[0032] Example 2: Screening of recombinant Saccharomyces cerevisiae

[0033] Plasmids with correct sequencing results were extracted and transformed into *Saccharomyces cerevisiae* INVSC1 competent cells using lithium acetate conversion. Positive transformants were screened on SC plates. Single colonies were picked, inoculated on YPD plates, and cultured at 30 °C for 24 h before plasmid extraction and PCR verification.

[0034] Example 3: Substrate transformation of recombinant brewer's yeast

[0035] The recombinant bacteria obtained in Example 2 were regenerated on YPD medium to enrich the bacterial cell volume, and then used for steroidal transformation fermentation experiments. 50 mL of YPD liquid medium was added to a 250 mL Erlenmeyer flask, ensuring the same inoculum size as the wild-type and mutant recombinant bacteria. Inoculation was performed at a liquid culture volume ratio of 4-6%, and the initial OD was controlled. 600At 0.6, using a wild-type Saccharomyces cerevisiae strain as a control, after culturing on a shaker at 30 ℃ and 180 r / min for 24 h, 3.75 mL of 20% galactose was added to an Erlenmeyer flask as an inducer, followed by the addition of DHEA substrate at a concentration of 3 g / L, with methanol as the co-solvent. 1 mL samples were taken every 24 h after substrate addition for TLC and HPLC analysis. The shaker fermentation process lasted for 72 h.

[0036] Table 3 Fermentation reaction system

[0037]

[0038] Example 4: TLC Analysis

[0039] The procedure for thin-layer chromatography is as follows: Spot 2 μL of sample onto a GF254 high-performance silica gel plate, using DHEA, 7α-DHEA, and 7α,15α-diOH-DHEA standards as controls. After thoroughly drying the spotted silica gel plate, place it in a chromatography lamp (developing solvent: chloroform:ethanol 10:1) for full development. Remove the silica gel plate and dry it with a hairdryer. Evenly spray the developing agent (50% concentrated sulfuric acid ethanol solution). After drying, purple-red and blue spots will appear.

[0040] Thin-layer chromatograms of the fermentation products of wild-type enzyme and mutant enzyme DHEA72 h are shown below. Figure 1 As shown, within 72 hours of fermentation, the substrate of the mutant strain was almost completely converted into the product. Furthermore, the proportion of 7α,15α-diOH-DHEA in the product was significantly higher than that of 7α-OH-DHEA. The amount of 7α,15α-diOH-DHEA produced by the mutant recombinant strain was significantly increased.

[0041] Example 5: HPLC Analysis

[0042] HPLC conditions were as follows: Column type: Agilent XSD-C18, 4.6 × 250 mmol·L⁻¹ -1 5 μm; Mobile phase: acetonitrile / 0.1% phosphoric acid solution; Column temperature: 30 °C; Detection wavelength: 210 nm; Flow rate: 1 mL / min; Injection volume: 20 μL; Detection time: 25 min.

[0043] The molar conversion rates of 7α-OH-DHEA and 7α,15α-diOH-DHEA in wild-type and mutant strains were quantitatively calculated based on HPLC analysis results. Figure 3As shown in the figure, the molar conversion rate of 7α,15α-diOH-DHEA by the wild-type strain at 72 h was 33.5%, and the byproduct 7α-OH-DHEA was also present in the conversion products, with a molar conversion rate of 7.8%. The mutant strain A112Y, after 72 h, showed a molar yield of 55.9% for 7α,15α-diOH-DHEA, while the molar conversion rate of 7α-OH-DHEA was only 2.21%; the mutant strain A112H, after 72 h, showed a molar yield of 45.5% for 7α,15α-diOH-DHEA, while the molar conversion rate of 7α-OH-DHEA was only 2.18%; the mutant strain A112K, after 72 h, showed a molar yield of 44.8% for 7α,15α-diOH-DHEA, while the molar conversion rate of 7α-OH-DHEA was only 1.48%; and the mutant strain A112S, after 72 h, showed a molar yield of 47.8% for 7α,15α-diOH-DHEA, while the molar conversion rate of 7α-OH-DHEA was only 1.58%. This indicates that the 15α-hydroxylation efficiency of the mutant strain was greatly enhanced, enabling it to efficiently convert the intermediate product 7α-OH-DHEA into the target product 7α,15α-diOH-DHEA, while reducing the accumulation of intermediate products.

[0044] Through precise modification, the mutant strain achieved a synergistic improvement in catalytic efficiency and selectivity, accelerating the synthesis of the target product and reducing byproduct accumulation by regulating the hydroxylation reaction step. This characteristic is of great significance for the industrial production of 7α,15α-diOH-DHEA, as it can significantly improve production efficiency and product purity, reduce downstream separation costs, and demonstrate promising application prospects.

[0045] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A cytochrome P450 enzyme mutant, characterized in that, The cytochrome P450 enzyme mutant is obtained by mutating alanine at position 112 of the amino acid sequence shown in SEQ ID NO: 1, and the amino acid sequence of the mutant is any one of the following: (1) Mutated to tyrosine, the amino acid sequence of which is shown in SEQ ID NO:3; (2) The mutation is to histidine, and its amino acid sequence is shown in SEQ ID NO:5; (3) It is mutated to lysine, and its amino acid sequence is shown in SEQ ID NO:7; (4) It is mutated to serine, and its amino acid sequence is shown in SEQ ID NO:

9.

2. The gene encoding the cytochrome P450 enzyme mutant of claim 1.

3. The encoding gene according to claim 2, characterized in that, The nucleotide sequence of the encoding gene is as follows: (1) The nucleotide sequence encoding the amino acid sequence SEQ ID NO:3 is shown in SEQ ID NO:4; (2) The nucleotide sequence encoding the amino acid sequence SEQ ID NO:5 is shown in SEQ ID NO:6; (3) The nucleotide sequence encoding the amino acid sequence SEQ ID NO:7 is shown in SEQ ID NO:8; (4) The nucleotide sequence encoding the amino acid sequence SEQ ID NO:9 is shown in SEQ ID NO:

10.

4. A recombinant vector, characterized in that, The recombinant vector contains the coding gene as described in claim 2 or 3.

5. A genetically engineered bacterium expressing the cytochrome P450 enzyme mutant of claim 1, characterized in that, The genetically engineered bacteria comprises the recombinant vector as described in claim 4.

6. The application of the cytochrome P450 enzyme mutant of claim 1 in catalyzing the hydroxylation of dehydroepiandrosterone to generate trihydroxyandrostenone.

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