A p450 enzyme mutant for steroid c11a-hydroxylation

Abstract

This invention relates to the field of biotechnology, and more particularly to a P450 enzyme mutant for steroid C11α-hydroxylation, the amino acid sequence of which is shown in SEQ ID NO:2. In a whole-cell catalytic system of *Saccharomyces cerevisiae*, this P450 enzyme mutant achieved a 14-fold increase in the conversion rate of 17α-hydroxyprogesterone compared to the wild type. Expression of this mutant in a systematically modified *Pichia pastoris* chassis strain increased the substrate conversion rate to 96.5% and the product selectivity to 99.1%. Scale-up culture at 2.5 L, with a single feed of 60 g of substrate, yielded a 11α,17α-dihydroxyprogesterone titer of 24.8 g / L. Furthermore, this mutant also exhibited good catalytic activity and C11α-hydroxylation selectivity for various steroid drugs such as progesterone and canrenone, demonstrating a broad substrate spectrum and promising prospects for industrial applications.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to a P450 enzyme mutant for steroid C11α-hydroxylation. Background Technology

[0002] Steroid drugs are hailed as "keys to life" due to their widespread use in treating rheumatism, cardiovascular diseases, lymphocytic leukemia, tumors, bacterial encephalitis, skin diseases, and endocrine disorders. Among them, C11α-hydroxylated steroid compounds not only possess bactericidal, immunomodulatory, fertility-controlling, and drug-enhancing effects, but also serve as key intermediates in the synthesis of many important drugs, demonstrating potential value in the prevention and adjuvant treatment of acquired immunodeficiency syndrome and adrenal insufficiency. For example, C11α-hydroxylated 17α-hydroxyprogesterone and progesterone are important intermediates in the synthesis of corticosteroids such as hydrocortisone acetate; the C11α-hydroxylated products of 18-methylnandrolone, nandrolone, and 18-methyldiketone are precursors to common contraceptives; and 11α-hydroxycanrenone is a key intermediate in the synthesis of the cardiovascular drug eplerenone. In addition, 11α,17α-dihydroxyprogesterone is a core intermediate in the synthesis of corticosteroids such as cortisone, hydrocortisone, prednisolone, prednisolone acetate, dexamethasone, and triamcinolone. Its efficient synthesis is of great significance for the clinical treatment of adrenocortical insufficiency, congenital adrenocortical hyperplasia, and connective tissue diseases.

[0003] Traditional chemical synthesis methods for achieving C11α-hydroxylation typically involve multiple steps and require the use of heavy metal catalysts, strong acids, and other reagents. This results in lengthy synthetic routes, demanding conditions, difficulty in controlling selectivity, and the generation of large amounts of difficult-to-treat waste, placing significant pressure on the environment and contradicting the principles of green chemistry and sustainable development.

[0004] In contrast, biocatalysis, particularly the transformation techniques utilizing microorganisms or enzymes, is considered a more ideal technical route for achieving steroid hydroxylation due to its mild reaction conditions and environmental friendliness. Among numerous biocatalysts, cytochrome P450 monooxygenases, with their powerful oxidizing capabilities, are potential tools for achieving the hydroxylation of inert CH bonds. However, the resources of naturally occurring P450 enzymes capable of efficiently and specifically catalyzing the C11α-hydroxylation of steroids are currently limited, and they often suffer from low catalytic activity, such as insufficient substrate conversion and regioselectivity, thus restricting their direct application in large-scale industrial production. Summary of the Invention

[0005] In view of this, the present invention proposes a P450 enzyme mutant for steroid C11α-hydroxylation.

[0006] The technical solution of this invention is implemented as follows:

[0007] In a first aspect, the present invention provides a P450 enzyme mutant, the amino acid sequence of which is shown in SEQ ID NO:2.

[0008] Secondly, the present invention provides a gene encoding the P450 enzyme mutant.

[0009] Thirdly, the present invention provides a recombinant vector containing a gene encoding the P450 enzyme mutant.

[0010] Fourthly, the present invention provides a transformant containing the recombinant vector.

[0011] Fifthly, the present invention provides the application of the P450 enzyme mutant in catalyzing the C11α-hydroxylation of a steroid substrate, wherein the steroid substrate is 17α-hydroxyprogesterone, progesterone, canrone, nandrolone, 18-methylnandrolone, or 18-methyldiketone.

[0012] Furthermore, the specific steps for catalyzing the C11α-hydroxylation of the steroid substrate include: culturing transformants containing the recombinant vector, adding the steroid substrate to the culture system, and conducting a whole-cell biocatalytic reaction.

[0013] Furthermore, in some specific embodiments, the concentration of the steroid substrate in the culture system is 10~100 mmol / L.

[0014] In a sixth aspect, the present invention provides a method for preparing 11α,17α-dihydroxyprogesterone, comprising a transformant containing the recombinant vector, adding 17α-hydroxyprogesterone to a culture system, and performing a whole-cell biocatalytic reaction.

[0015] Furthermore, the host cell of the transformant is *Saccharomyces cerevisiae* or *Pichia pastoris*.

[0016] When the host cell is Saccharomyces cerevisiae, in some specific embodiments, the recombinant vector is obtained by linking the gene encoding the P450 enzyme mutant with the pYES2 plasmid.

[0017] When the host cell is Pichia pastoris, in some specific embodiments, the recombinant vector is obtained by linking the gene encoding the P450 enzyme mutant with the pPICZA plasmid.

[0018] The Pichia pastoris host cell is a conventional host in the art. The host cell must meet the basic requirements of stable replication of the recombinant expression vector and effective expression of the exogenous P450 enzyme mutant gene. Pichia pastoris X33 and a series of optimized chassis cells derived from it are preferred. In some specific embodiments, the optimization of Pichia pastoris X33 chassis cells includes the following steps:

[0019] (1) Overexpression of the endogenous HEM1 gene in the chassis cells (GenBank: XM_002491600.1).

[0020] (2) Knock out the endogenous HMX1 gene in the chassis cells (GenBank: XM_002492302.1).

[0021] (3) The exogenous Metarhizium anisopliae cytochrome b5 gene (the amino acid sequence encoded by it is shown in SEQ ID NO:3) is integrated and expressed in the chassis cells.

[0022] (4) The exogenous Metarhizium anisopliae ABC transporter gene (the amino acid sequence encoded by it is shown in SEQ ID NO:4) is integrated and expressed in the chassis cells.

[0023] the term:

[0024] Gene: A nucleic acid sequence that encodes a polypeptide chain or functional RNA. It may contain a coding sequence and a regulatory sequence that is operatively linked to it.

[0025] Vectors, recombinant vectors: Vectors are nucleic acid molecules that can introduce a target nucleic acid sequence into a host cell. Vectors containing the target nucleic acid sequence are called recombinant vectors.

[0026] Plasmid: When expressing a gene or the protein encoded by a gene, a "vector" can be a plasmid. Unless otherwise specified, the terms "vector" and "plasmid" are used interchangeably in this invention.

[0027] Import / Transduction: This refers to the process of introducing a foreign nucleic acid molecule into a host cell. This nucleic acid molecule can exist in an extrachromosomal form or be integrated into the host cell's genome. Importation can be achieved through methods known in the art, such as transformation, transfection, and transduction. Host cells containing the imported nucleic acid molecule can be called transgenic, recombinant, transformed, or engineered host cells.

[0028] Transformant: A host cell containing exogenous nucleic acid molecules. These exogenous nucleic acid molecules may exist in an extrachromosomal form or be integrated into the host cell's genome.

[0029] The beneficial effects of the present invention include at least the following:

[0030] The P450 enzyme mutant provided by this invention, in a whole-cell catalytic system of Saccharomyces cerevisiae, with 10 mM 17α-hydroxyprogesterone as substrate, reacted for 72 h and its conversion rate was 14 times that of the wild-type P450 enzyme (CYP68N3_ma).

[0031] This invention utilizes a modified Pichia pastoris chassis to obtain a mutant strain. Expressing the P450 enzyme mutant on this chassis yields a 96.5% conversion rate for the target steroid and a 99.1% product selectivity. Scale-up culture in a 5 L fermenter (2.5 L), with a single feed of 60 g of substrate, achieves a product titer of 24.8 g / L. This P450 enzyme mutant exhibits excellent catalytic activity and C11α-hydroxylation selectivity for various steroid drugs, including progesterone and canrenone, demonstrating a broad substrate spectrum and significant industrial application potential. Attached Figure Description

[0032] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0033] Figure 1 The figures show the high-performance liquid chromatography (HPLC) analysis results of 17α-hydroxyprogesterone standards, 11α,17α-dihydroxyprogesterone standards, and the products obtained from the reaction of 17α-hydroxyprogesterone catalyzed by each group of recombinant Saccharomyces cerevisiae strains. The strains in the figure are: empty vector recombinant bacteria without the cyp68n3_ma gene (CK, as negative control), transformant containing the wild-type cyp68n3_ma gene (strain number N3), and transformants containing the corresponding mutant genes (see Table 2); S and P correspond to 17α-hydroxyprogesterone standards and 11α,17α-dihydroxyprogesterone standards, respectively.

[0034] Figure 2 The figure shows the substrate conversion rate of 17α-hydroxyprogesterone catalyzed by each group of recombinant Saccharomyces cerevisiae strains after 72 h of reaction. The strain groupings in the figure are defined as follows: Figure 1 Same as above;

[0035] Figure 3 The figures show the HPLC analysis results of 17α-hydroxyprogesterone standards, 11α,17α-dihydroxyprogesterone standards, and the products obtained from the reaction of 17α-hydroxyprogesterone catalyzed by each group of recombinant Pichia pastoris strains. The strains in each group are: empty recombinant strain (CK, as negative control), strain X33, strain H, strain HB, and strain HBT (see Table 4); S and P correspond to 17α-hydroxyprogesterone standards and 11α,17α-dihydroxyprogesterone standards, respectively.

[0036] Figure 4The results of high-density fermentation of Pichia pastoris HBT strain in Example 2 are shown, specifically the product titer and optical density (OD) during the conversion of 17α-hydroxyprogesterone to 11α,17α-dihydroxyprogesterone in a 5 L bioreactor. 600 The graph shows the changes over time; in the graph: A: time point for glycerol feeding during Pichia pastoris fermentation; B: time point for methanol induction in Pichia pastoris; C: time point for substrate feeding.

[0037] Figure 5 The figure shows the HPLC analysis results of the product obtained by Pichia pastoris HBT strain catalyzing progesterone in Example 3; in the figure, S1: progesterone standard, P1: 11α-hydroxyprogesterone standard;

[0038] Figure 6 The figure shows the HPLC analysis results of the product obtained by Pichia pastoris HBT strain catalyzing canrone in Example 3; in the figure, S2: canrone standard, P2: 11α-hydroxycanrone standard;

[0039] Figure 7 The figure shows the HPLC analysis results of the product obtained by Pichia pastoris HBT strain catalyzing nandrolone in Example 3; in the figure, S3: nandrolone standard, P3: 11α-hydroxynandrolone standard;

[0040] Figure 8 The figure shows the HPLC analysis results of the product obtained by Pichia pastoris HBT strain catalyzing 18-methylnandrolone in Example 3; in the figure, S4: 18-methylnandrolone standard, P4: 11α-hydroxy-18-methylnandrolone standard;

[0041] Figure 9 The figure shows the HPLC analysis results of the product obtained by the Pichia pastoris HBT strain catalyzing 18-methyldiketone in Example 3; in the figure, S5: 18-methyldiketone standard, P5: 11α-hydroxy-18-methyldiketone standard;

[0042] Figures 5 to 9 The strains are described below:

[0043] CK: This strain was created by transforming the Pichia pastoris host HBT into the empty expression vector pPICZA, and its genome does not contain the exogenous cyp68n3_ma gene or its mutant; N3M3: Pichia pastoris HBT strain containing the N3M3 gene. Detailed Implementation

[0044] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. Reagents or instruments used, unless otherwise specified, are all commercially available products. Experimental methods in the following embodiments without specific conditions are generally performed under conventional conditions, such as those described in *Molecular Cloning: A Laboratory Manual (Fourth Edition)* published by Cold Spring Harbor Laboratory, or according to the manufacturer's recommendations. The sequencing work in the sequence synthesis and expression vector construction processes in the following embodiments was performed by Suzhou Genewiz Biotechnology Co., Ltd. Furthermore, it should be noted that, unless otherwise defined, the scientific and technical terms used in the context of this invention should have the meanings commonly understood by those skilled in the art.

[0045] Table 1

[0046]

[0047]

[0048] Example 1

[0049] Previous studies have identified a P450 enzyme from *Metarhizium anisopliae* EEG016 capable of site-directed 11α-hydroxylation of 17α-hydroxyprogesterone. Its amino acid sequence is shown in SEQ ID NO:1, and it has been named CYP68N3_ma. However, CYP68N3_ma suffers from low conversion efficiency for high concentrations of steroidal substrates. In a whole-cell catalytic system of *Saccharomyces cerevisiae* with a reaction time of 72 h and a substrate concentration of 10 mmol / L 17α-hydroxyprogesterone, CYP68N3_ma exhibited 100% selectivity for C11α-hydroxylation of 17α-hydroxyprogesterone, but the substrate conversion rate was only 1.6%.

[0050] 1. Site prediction, construction and screening of site-directed saturation mutant libraries

[0051] (1) Method

[0052] This invention uses AlphaFold2 to construct the structural model of CYP68N3_ma, then uses LEDOCK to complete the docking of the heme prosthetic group (heme is embedded in the active site of the P450 enzyme, forming the catalytic center), and uses YASARA to dock the substrate 17α-hydroxyprogesterone to the protein catalytic center. Amino acid residues within a 5 Å radius around the substrate are selected, and an NNK saturation mutagenesis strategy is used for catalytic selectivity screening using 96-well plates. The PCR product obtained from the saturation mutagenesis is digested with DpnI enzyme (8.5 µL PCR product, 1 µL Cutsmart Buffer, and 0.5 µL enzyme), and transformed into E. coli BL21(DE3) competent cells. The cells are plated on solid LB plates containing 100 μg / mL Amp resistance and incubated overnight at 37°C. All single colonies were washed off with sterile water and collected. Plasmids were extracted and transformed into Saccharomyces cerevisiae INVSC1 competent cells. The cells were then spread on uracil-free SC solid medium (with 2% more agar added to the liquid medium) and incubated upside down in a 30°C incubator for 48 hours to construct a site-directed saturated mutant library.

[0053] After transforming the site-directed saturated mutant library plasmid into competent Saccharomyces cerevisiae INVSC1 cells and producing single colonies, each colony was picked and cultured in 3 mL of uracil-free SC liquid medium. Then, 1 mL of the bacterial culture was transferred to 50 mL of uracil-free SC medium and cultured at 28 °C and 220 rpm until OD500 was reached. 600 The values ​​were 5-6. Next, the uridine-free SC medium was replaced with uridine-free SC-galactose induction medium. The mixture was centrifuged at 4000 rpm at 4°C to remove the supernatant. The *Saccharomyces cerevisiae* cells were resuspended in an equal volume of uridine-free SC-galactose induction medium and induced at 28°C for 12 h. Then, 10 mmol / L 17α-hydroxyprogesterone was added to initiate a whole-cell reaction of *Saccharomyces cerevisiae*. The samples after 72 h of reaction were analyzed by HPLC.

[0054] (2) Culture medium formulation:

[0055] Uracil-free SC liquid basal medium: 0.67% (0.67g / 100mL) amino-free yeast nitrogen source, 0.01% amino acid mixture a (lysine, arginine, leucine, threonine, cysteine, tryptophan, adenine), 0.005% amino acid mixture b (valine, histidine, methionine, aspartic acid, proline, serine, phenylalanine, tyrosine, isoleucine), 2% glucose, add pure water to the required volume, stir until completely dissolved, and then autoclave for later use.

[0056] Uracil-free SC-galactose induction medium: The above-mentioned basic medium is used as the stock solution, except that the carbon source is replaced with 2% galactose, and the other components and preparation methods are exactly the same.

[0057] (3) Results

[0058] The results showed that in the whole-cell catalytic system of *Saccharomyces cerevisiae*, under the conditions of 72 h of reaction and 10 mmol / L substrate 17α-hydroxyprogesterone, three amino acid residues (F111, T115, and E374) produced mutants with increased C11α-hydroxylation products, namely F111A, T115I, and E374H. These three mutants maintained the C11α-hydroxylation selectivity for 17α-hydroxyprogesterone (remaining 100%) while enhancing the substrate conversion rate, which was 13.9% (F111A) and 5.8% (T115I and E374H), respectively. Compared with the substrate conversion rate of only 1.6% of wild-type CYP68N3_ma, the catalytic activity was significantly enhanced.

[0059] 2. Mutant Combinations and Whole-Cell Biocatalytic Reactions

[0060] The designs for pairwise and triple mutation combinations of F111A, T115I, and E374H are as follows:

[0061] Table 2

[0062]

[0063] The corresponding gene sequences were codon-optimized, and gene synthesis was outsourced to a third party. The wild-type cyp68n3_ma gene and its mutant genes were constructed into the pYES2 vector, respectively, and transformed into *Saccharomyces cerevisiae* to catalyze the reaction with the substrate 17α-hydroxyprogesterone. The specific steps are as follows: the amplification primers for the cyp68n3_ma gene (or mutant) are shown in Table 1 (SEQ ID NO: 5-6). The amplification primers for the pYES2 vector are shown in Table 1 (SEQ ID NO: 7-8). The target gene fragment was recovered, and the recovered fragment and the pYES2 plasmid vector fragment were ligated using T5 exonuclease. The ligation was then performed into *E. coli* DH5α competent cells and plated on solid LB agar plates with a final ampicillin concentration of 50 µg / mL. After colonies grew on the plates, single colonies were picked and cultured at 37°C for plasmid extraction. The resulting plasmids were the expression vectors for pYES2-CYP68N3_ma and its mutants. Colony PCR was used to verify the successful construction of the vectors.

[0064] The successfully constructed recombinant plasmid was transformed into *Saccharomyces cerevisiae* INVSC1 competent cells. Single colonies were picked and cultured in 3 mL of uracil-free SC liquid medium for 48 h. After cell growth, 1 mL of the bacterial culture was transferred to 50 mL of uracil-free SC medium and cultured at 28 ℃ and 220 rpm until OD500 was reached. 600 The values ​​were 5-6. Next, the uridine-free SC medium was replaced with uridine-free SC-galactose induction medium. The mixture was centrifuged at 4000 rpm at 4°C to remove the supernatant. The *Saccharomyces cerevisiae* cells were resuspended in an equal volume of uridine-free SC-galactose induction medium and induced at 28°C for 12 h. Then, 10 mmol / L 17α-hydroxyprogesterone was added to initiate a whole-cell reaction of *Saccharomyces cerevisiae*. The samples after 72 h of reaction were analyzed by HPLC.

[0065] The results are as follows Figure 1 and Figure 2 As shown, the recombinant Saccharomyces cerevisiae transformant reacted with 17α-hydroxyprogesterone during whole-cell biotransformation, generating a product peak with the same elution time as the product standard. The substrate conversion rate of the mutant CYP68N3_ma-F111A / E374H (named N3M2) was 9 times that of the wild type. The substrate conversion rate of the mutant CYP68N3_ma-F111A / E374H / T115I (named N3M3) was 14 times that of the wild type, and the C11α-hydroxylation selectivity remained unaffected, remaining at 100%.

[0066] Example 2

[0067] Heterologous expression of N3M3 in Pichia pastoris and its application in steroidal whole-cell biotransformation

[0068] 1. Construction of heterologous expression vectors

[0069] The N3M3 gene (i.e., CYP68N3_ma-F111A / E374H / T115I) was ligated to the pPICZA plasmid to obtain the recombinant plasmid pPICZA-N3M3. The primers for amplifying the N3M3 gene are shown in Table 1 (SEQ ID NO: 9-10). The primers for amplifying the pPICZA vector are shown in Table 1 (SEQ ID NO: 11-12). The constructed pPICZA-N3M3 plasmid was transformed into *E. coli* DH5α competent cells and plated on LB agar plates containing 35 µg / mL bleomycin. Single colonies were picked, cultured, and plasmid extracted; the resulting plasmid was pPICZA-N3M3. Colony PCR was used to verify the successful construction of the vector.

[0070] To further enhance the catalytic performance of Pichia pastoris chassis cells, we conducted systematic genetic modification. Specific strategies included: optimizing the endogenous heme synthesis pathway (overexpressing the HEM1 gene and knocking out the HMX1 gene), and targeted integration of the Cyt b5 gene from Metarhizium anisopliae with the ABC transporter gene. The operational steps are as follows:

[0071] 2. Optimization of heme synthesis pathway

[0072] Heme is an essential cofactor for P450 enzyme activity. The HEM1 gene encodes 5-aminolevulinic acid synthase (ALAS), a key rate-limiting enzyme in the heme biosynthesis pathway. Overexpression of HEM1 relieves the rate-limiting enzyme, thereby increasing heme supply. The HMX1 gene encodes heme oxygenase; knockout of HMX1 blocks the heme degradation pathway. Through these two steps, Pichia pastoris strains with HEM1 overexpression and HMX1 gene knockout were obtained, synergistically optimizing the heme biosynthesis process in Pichia pastoris. The specific editing methods for the HEM1 and HMX1 genes are as follows:

[0073] 2.1 Construction of HEM1 gene overexpression donor vector

[0074] Using Pichia pastoris X33 genomic DNA as a template, primer pairs HEM1-F / HEM1-R, UP2-F / UP2-R, and DN2-F / DN2-R (Table 3) were used to amplify the HEM1 coding sequence (with pPICZ vector homologous sequences at the 5' and 3' ends), the upstream homologous arm UP2 and the downstream homologous arm DN2 of the PNSI-2 neutral insertion site from the Pichia pastoris genome.

[0075] Using pPICZ plasmid as a template, primer pPICZ-T5 was used. 通用 -F、pPICZ-T5 通用 -R (Table 3) was used for PCR amplification to obtain a linearized pPICZ vector backbone containing the complete AOX1 promoter and AOX1 terminator.

[0076] The HEM1 coding sequence was ligated to the linearized pPICZ vector backbone using T5 seamless cloning, transformed into E. coli, and sequenced to verify correctness, resulting in the recombinant vector pPICZ-T5. HEM1 .

[0077] pPICZ-T5 HEM1 Using the UP2 homologous arm as the backbone, the DN2 homologous arm was connected upstream of the AOX1 promoter and downstream of the AOX1 terminator via T5 seamless cloning, thus constructing a HEM1 targeted overexpression donor vector with the structure "UP2-AOX1 promoter-HEM1-AOX1 terminator-DN2".

[0078] 2.2 Construction of CRISPR-Cas9 editing plasmid targeting the PNSI-2 site

[0079] Using the pCAI-Cas9 vector as a backbone, a gRNA expression cassette fragment targeting the PNSI-2 site was amplified using primers gPNSI-2-F and gPNSI-2-R (Table 3). This fragment was then inserted into the pCAI-Cas9 vector via T5 seamless cloning, transformed into E. coli, and sequenced to verify its correctness. The resulting edited plasmid pCAI-Cas9-gPNSI-2 was then extracted for later use.

[0080] Table 3

[0081]

[0082] 2.3 Construction of HMX1 gene knockout donor vector

[0083] Using Pichia pastoris X33 genomic DNA as a template, primers UP were used respectively. HMX1 -F / UP HMX1 -R、DN HMX1 -F / DN HMX1 -R (Table 4) was used for PCR amplification to obtain the upstream homologous arm UP of the HMX1 gene coding region. HMX1 Downstream homologous arm DN HMX1 Gel recovery and purification. Using the pPICZ linearized backbone as a vector, UP was seamlessly cloned via T5. HMX1 With DN HMX1 By directly connecting the homologous arms, a "UP" structure can be constructed. HMX1 -DN HMX1 "The donor vector was knocked out, and the gel was purified after linearization."

[0084] 2.4 Construction of CRISPR-Cas9 editing plasmid targeting the HMX1 site

[0085] Using the pCAI-Cas9 vector as a backbone, primers and gRNA were used. HMX1 -F, gRNA HMX1 -R amplification yielded a gRNA expression cassette fragment targeting the coding region of the HMX1 gene, which was then seamlessly inserted into the pCAI-Cas9 vector via T5 cloning. After sequencing verification, the edited plasmid pCAI-Cas9-gRNA was obtained. HMX1 Extract plasmids for later use.

[0086] 2.5 Pichia pastoris co-transformation

[0087] Linearized HEM1 overexpression donor vectors and HMX1 knockout donor vectors were combined with pCAI-Cas9-gPNSI-2 and pCAI-Cas9-gRNA. HMX1The edited plasmids were co-electrotransformed into wild-type Pichia pastoris X33 competent cells, and selection was performed using solid YPD plates containing 300 μg / mL bleomycin resistance. Single colonies were picked, and colony PCR and sequencing were used to verify the sequence correctness, resulting in Pichia pastoris strains that overexpressed HEM1 and knocked out HMX1.

[0088] 3. Cyt b5 gene targeted integration

[0089] Based on a Pichia pastoris strain with HEM1 overexpression and HMX1 knockout, the Cytb5 coding sequence from Metarhizium anisopliae was targeted and integrated into the PNSI-6 site. The electron transport-assisted function of Cytb5 was utilized to optimize strain performance. The specific procedures are as follows:

[0090] 3.1 Construction of Cyt b5 gene overexpression donor vector

[0091] Using *Metarhizium anisopliae* strain EEG016 cDNA as a template, PCR amplification was performed using primers Cytb5-F and Cytb5-R to obtain the full-length coding sequence of Cyt b5 containing homologous sequences from the pPICZ vector at both the 5' and 3' ends. Using the linearized pPICZ vector backbone as a template, the Cyt b5 coding sequence was seamlessly ligated via T5 cloning. After sequencing verification, the recombinant vector pPICZ-T5 was obtained. Cytb5 .

[0092] Using Pichia pastoris X33 genomic DNA as a template, PCR amplification was performed using primers UP6-F / UP6-R and DN6-F / DN6-R, respectively, to obtain the upstream homologous arm UP6 and the downstream homologous arm DN6 of the PNSI-6 neutral insertion site, which were then purified by gel extraction.

[0093] pPICZ-T5 Cytb5 Using the UP6 homologous arm as the backbone, the UP6 homologous arm was connected upstream of the AOX1 promoter and the DN6 homologous arm was connected downstream of the AOX1 terminator via T5 seamless cloning, thus constructing a targeting donor vector with the structure "UP6-AOX1 promoter-Cytb5-AOX1 terminator-DN6". After linearization, the vector was purified by gel extraction for later use.

[0094] 3.2 Construction of CRISPR-Cas9 editing plasmid targeting the PNSI-6 site

[0095] Using the pCAI-Cas9 vector as a backbone, a gRNA expression cassette fragment targeting the PNSI-6 site was amplified using primers gPNSI-6-F and gPNSI-6-R. The fragment was then seamlessly inserted into the pCAI-Cas9 vector using T5 cloning. After sequencing verification, the edited plasmid pCAI-Cas9-gPNSI-6 was obtained and extracted for later use.

[0096] 3.3 Pichia pastoris transformation and identification of positive strains

[0097] The linearized Cyt b5 overexpression donor vector and the pCAI-Cas9-gPNSI-6 editing plasmid were co-electrotransformed into competent cells of a Pichia pastoris strain that overexpressed HEM1 and knocked out HMX1. Selection was performed using solid YPD plates containing 300 μg / mL bleomycin resistance. Single colonies were picked, and after verifying sequence correctness through colony PCR and sequencing, Pichia pastoris strains with HEM1 overexpression, HMX1 gene knockout, and Cyt b5_ma gene insertion were obtained.

[0098] 4. Targeted integration of ABC transporter genes

[0099] Based on strains with HEM1 overexpression, HMX1 gene knockout, and Cyt b5_ma gene insertion, the ABC transporter coding sequence from Metarhizium anisopliae was targeted and integrated into the Pichia pastoris PNSI-1 neutral insertion site, thus solving the bottleneck problem of exogenous steroid transfer in yeast.

[0100] The specific procedure is similar to the "Cyt b5 gene targeted integration" method, except that the primers for target gene amplification are ABC. trans -F and ABC trans -R, the target integration site is PNSI-1, with corresponding upstream homologous arm UP1 amplification primers UP1-F and UP1-R, and downstream homologous arm DN1 amplification primers DN1-F and DN1-R; the final constructed target donor vector structure is "UP1-AOX1 promoter-ABC transporter-AOX1 terminator-DN1". The matching CRISPR-Cas9 editing plasmid is pCAI-Cas9-gPNSI-1, in which the gRNA expression cassette amplification primers targeting the PNSI-1 site are gPNSI-1-F and gPNSI-1-R; finally, a Pichia pastoris strain with HEM1 overexpression, HMX1 gene knockout, and insertion of Cyt b5_ma and homologous ABC transporter genes was obtained.

[0101] Table 4

[0102]

[0103]

[0104] 5. Transformation and Pichia pastoris whole-cell reaction

[0105] According to the design in Table 5, the successfully constructed recombinant plasmids were transformed into the corresponding Pichia pastoris competent cells. Single colonies were picked and cultured in 3 mL of liquid YPD medium containing 300 µg / mL bleomycin (composition: 1% Yeast extract, 2% Tryptone, and 2% Glucose) for 48 h. After cell growth, 1 mL of the bacterial culture was transferred to 20 mL of BMGY medium (1% Yeast extract, 2% Tryptone, and 1.34% ammonia-free yeast nitrogen source (YNB), dissolved thoroughly in pure water, and then 1% glycerol and 10% 1 M pH 6.0 potassium phosphate buffer were added, and the culture was carried out again at 28 ℃ and 220 rpm for 48 h. The supernatant was removed by centrifugation, and the Pichia pastoris cells were resuspended in 10 mL of BMMY induction medium (composition: 1% yeast extract, 2% Tryptone, and 1.34% ammonia-free yeast nitrogen source YNB, dissolved thoroughly in pure water, and then supplemented with 1% methanol and 10% 1 M pH 6.0 potassium phosphate buffer). Induction was performed at 28 °C for 72 h. During induction, methanol (1% v / v) was added every 24 h. After induction, 10 mmol / L of 17α-hydroxyprogesterone was added to initiate a whole-cell reaction of Pichia pastoris. HPLC analysis was performed after 72 h of reaction.

[0106] Table 5

[0107]

[0108] Table 6. 72-h transformation of steroidal substrates by strain HBT

[0109]

[0110] The results are as follows Figure 3 As shown in Table 6, the HBT Pichia pastoris transformant transformed with N3M3 produced a product peak with the same elution time as the product standard, and the conversion rate reached 96.5%, with a C11α-hydroxylation selectivity of 99.1%.

[0111] 6. High-density fermentation

[0112] The process of synthesizing 11α,17α-dihydroxyprogesterone from 17α-hydroxyprogesterone by the engineered strain Pichia pastoris HBT was scaled up and verified in a 5L bioreactor. The specific operation is as follows:

[0113] Culture medium formulation:

[0114] PTM1 solution: Weigh 0.6 g copper sulfate pentahydrate, 0.008 g potassium iodide, 0.3 g manganese sulfate monohydrate, 0.03 g sodium molybdate dihydrate, 0.002 g boric acid, 0.05 g cobalt chloride hexahydrate, 2 g zinc chloride and 6.5 g ferrous sulfate heptahydrate. After dissolving them completely in a small amount of pure water, slowly pour in 0.5 mL of concentrated sulfuric acid while stirring to dissipate heat. Finally, bring the volume to 100 mL with sterile water and filter to sterilize.

[0115] BSM medium: Weigh 2.325 g calcium sulfate, 45.5 g potassium sulfate, 37.25 g magnesium sulfate and 10.325 g potassium hydroxide, add some pure water to dissolve them completely, then slowly pour in 66.75 mL of phosphoric acid while stirring to dissipate heat. Then add 100 g glycerol, and finally bring the volume to 2.5 L with sterile water.

[0116] The initial loading volume of the 5L bioreactor was 2.5 L of BSM basal salt culture medium, with 1-2 mL of antifoaming agent added. After single colonies grew, they were inoculated into 200 mL of BMGY medium and cultured in a constant temperature shaker at 30 ℃ and 220 rpm for 48 h. A stirrer was then attached to the fermenter, and the pH was set to 5.5 and the temperature to 28 ℃. After all values ​​stabilized, dissolved oxygen electrode calibration was performed, using the DO value at this point as 100%. During inoculation, 200 mL of the seed culture was poured into the 5L fermenter containing 2.5 L of BSM basal salt culture medium after igniting an alcohol swab, along with 10.875 mL of PTM1 solution. The temperature was stabilized at 28 ℃, and the rotation speed was maintained at a constant 500 rpm until feeding began. The pH was controlled at around 5.5 using ammonia; DO control was not necessary initially, but the dissolved oxygen change curve was continuously monitored. The DO level may drop slightly immediately after seed culture, to around 90%.

[0117] Approximately 24 hours after inoculation in the fermenter, once the glycerol in the BSM medium is depleted, glycerol is replenished. Previously, dissolved oxygen (DO) may have dropped to a few tenths of a percent and remained so for some time; after glycerol depletion, DO will begin to rise, stabilizing at around 60%, followed by carbon source acidification and pH increase. In a clean bench, 6 mL of PTM1 is added to 500 mL of 50% glycerol, and then glycerol is replenished. During glycerol replenishment, the feeding rate is manually controlled to maintain dissolved oxygen levels between 20% and 50%. OD is measured at least three times daily. 600 The changes. Approximately 24 hours later, the OD... 600When the dissolved oxygen (DO) reaches approximately 200, feeding is stopped, and the fermenter is starved without a carbon source for 0.5-1 h. After starvation, the DO in the fermenter begins to rise. When the pH starts to rise, it indicates that glycerol is depleted, and methanol can be added to induce Pichia pastoris expression. After connecting the feeding tube, methanol is first added slowly to allow the Pichia pastoris to adapt gradually. After a period of time, the methanol feeding rate is slowly increased, and the dissolved oxygen is kept stable between 20% and 30%. The DO and stirring speed are correlated, the feeding rate is set to automatic, and the temperature is slowly adjusted from 28℃ to 25℃. During the fermentation induction process, samples are taken 2-3 times a day to record the OD of the Pichia pastoris growth. 600 Numerical values. Substrate can be added after induction for 24-120 h. Extract with an equal volume of ethyl acetate, centrifuge at 12000 rpm, collect the organic phase supernatant, dry it, and prepare it for HPLC analysis.

[0118] HPLC results showed that the average conversion rate of the system reached 43.7 g·L⁻¹ in the first 4 hours after substrate addition. -1 ·d -1 At 72 hours of conversion, the highest potency of 11α,17α-dihydroxyprogesterone reached 24.8 g / L, and the substrate conversion selectivity remained above 99% throughout the process. After extraction, recrystallization and purification, the total product yield was 83.8%. Figure 4 ).

[0119] Example 3

[0120] Application of strain HBT in catalyzing C11α-hydroxylation of different steroid substrates

[0121] Pichia pastoris strain HBT was inoculated into 3 mL of liquid YPD medium containing 300 µg / mL bleomycin and cultured for 48 h. The supernatant was removed by centrifugation, and the Pichia pastoris cells were resuspended in 25 mL of BMMY induction medium and induced at 28 ℃ for 72 h. During this period, methanol (1% v / v) was added every 24 h. After induction, a whole-cell reaction of Pichia pastoris was performed using 10 mmol / L steroid substrate (progesterone / canrenone / nandrolone / 18-methylnandrolone / 18-methyldiketone). HPLC analysis was performed after 72 h of reaction. The results are shown below. Figures 5-9 As shown in Figure 6 and Table 7.

[0122] Table 7. 72-h transformation of steroidal substrates by strain HBT

[0123]

[0124]

[0125] The results showed that the Pichia pastoris strain HBT exhibited good transformation activity for progesterone, canrone, nandrolone, 18-methylnandrolone, and 18-methyldiketone, with efficient transformation of all substrates (85.0–100%). The Pichia pastoris strain HBT also demonstrated excellent regioselectivity (87.4%–88.9%) for the C11α-hydroxylation of progesterone and canrone.

[0126] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A mutant of a P450 enzyme, characterized in that, The amino acid sequence is shown in SEQ ID NO:

2.

2. The gene encoding the P450 enzyme mutant as described in claim 1.

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

4. A transformant, characterized in that, The transformant contains the recombinant vector as described in claim 3.

5. The application of the P450 enzyme mutant as described in claim 1 in catalyzing the C11α-hydroxylation of steroid substrates, characterized in that, The steroid substrate is 17α-hydroxyprogesterone, progesterone, canrone, nandrolone, 18-methylnandrolone, or 18-methyldiketone.

6. The application as described in claim 5, characterized in that, The transformant as described in claim 4 is cultured, and a steroid substrate is added to the culture system to carry out a whole-cell biocatalytic reaction.

7. The application as described in claim 6, characterized in that, In the culture system, the concentration of steroid substrate is 10~100 mmol / L.

8. A method for preparing 11α,17α-dihydroxyprogesterone, characterized in that, The transformant as described in claim 4 was cultured, and 17α-hydroxyprogesterone was added to the culture system to carry out a whole-cell biocatalytic reaction.

9. The method as described in claim 8, characterized in that, The host cell for the transformant is either Saccharomyces cerevisiae or Pichia pastoris.

10. The method as described in claim 8, characterized in that, In the culture system, the concentration of 17α-hydroxyprogesterone is 10~100 mmol / L.

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