Cytochrome P450 cholesterol side chain lyase mutant, progesterone-producing recombinant saccharomyces cerevisiae and application thereof

Abstract

The invention provides a cytochrome P450 cholesterol side chain lyase mutant, recombinant saccharomyces cerevisiae for producing progesterone and application of the recombinant saccharomyces cerevisiae. The cytochrome P450 cholesterol side chain lyase mutant is obtained through ESM-2 language model screening, and the catalytic efficiency can be remarkably improved. The efficient recombinant saccharomyces cerevisiae is obtained by constructing the recombinant saccharomyces cerevisiae, optimizing the copy number of key enzymes, introducing an electron transfer system and overexpressing the key enzymes, the yield of progesterone is remarkably increased, and the recombinant saccharomyces cerevisiae is suitable for industrial production of progesterone and has important application value.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a cytochrome P450 cholesterol side-chain lyase mutant, and more specifically, to a progesterone-producing recombinant Saccharomyces cerevisiae and its applications. Background Technology

[0002] Saccharomyces cerevisiae possesses its own ergosterol synthesis pathway, and through metabolic engineering, it can be used to de novo synthesize various steroid hormones from simple and inexpensive carbon sources (such as glucose and ethanol). Currently, the biggest bottleneck in yeast de novo steroid hormone synthesis technology is the low yield, which is due to the low efficiency of the catalytic step mediated by cholesterol-side-chain cleavage enzyme (P450scc). P450scc, encoded by the CYP11A1 gene, catalyzes the formation of pregnenolone from cholesterol through three consecutive reactions: the first step catalyzes the hydroxylation of cholesterol at C22 to form 22R-hydroxycholesterol, followed by hydroxylation at C20 to generate 20R,22R-hydroxycholesterol, and finally catalyzes the cleavage of the C20-C22 chain to generate pregnenolone and 4-methylpentanal. The entire catalytic process also requires two redox chaperone proteins—flavoprotein adrenocortical reductase (AdR) and non-heme iron-sulfur protein adrenocorticotropic hormone (Adx)—to assist P450scc in gaining electrons from NAD(P)H.

[0003] Since Duport et al. began their research in Saccharomyces cerevisiae in 1998, they have achieved de novo synthesis of pregnenolone by introducing plant-derived DWF5, mature bovine (Bos taurus)-derived CYP11A1, Adx, and AdR. They further completed de novo synthesis of progesterone by introducing human (Homo sapiens)-derived 3β-hydroxysteroid dehydrogenase (3β-HSD). However, since then, significant breakthroughs have not been achieved in the synthesis of pregnenolone and progesterone using animal-derived P450scc. This phenomenon is mainly attributed to the insufficient catalytic efficiency of P450scc (Duport, C.; Spagnoli, R.; Degryse, E.; Pompon, D., Self-sufficient biosynthesis of pregnenolone and progesterone in engineered yeast. Nat Biotechnol 1998, 16 (2), 186-9). For decades, researchers both domestically and internationally have been dedicated to improving the catalytic efficiency of P450scc by optimizing the electron transfer process, achieving a series of results. For example, in 1993, Harikrishna et al. constructed a P450scc-AdR-Adx fusion protein. This design enhanced the binding probability of P450scc to its chaperone protein, increasing the catalytic Vmax from 1.7 ng / L·d to 9.1 ng / L·d. 3(Harikrishna, JA; Black, SM; Szklarz, GD; Miller, WL, Construction and function of fusion enzymes of the human cytochrome P450scc system. DNA Cell Biol1993,12(5), 371-9.). In 2019, Zhang et al. compared the combination of P450scc and Adx / AdR redox chaperone proteins from nine different plant and animal sources and found that when mature wild boar (Sus scrofa) SsCYP11A1 was co-expressed with bovine (Bostaurus) BtAdx and BtAdR in Yarrowialipolytica, the pregnenolone yield reached the highest level, with a yield of 78.0 mg / L in a 5L high-density fermenter (Zhang, R.; Zhang, Y.; Wang, Y.; Yao, M.; Zhang, J.; Liu, H.; Zhou, X.; Xiao, W.; Yuan, Y., Pregnenolone Overproduction in Yarrowialipolytica by Integrative Components Pairing of the Cytochrome P450scc System. ACS Synth Biol 2019,8 (12), 2666-2678).

[0004] Previous studies have shown that heterologous expression of the chaperone protein Adx in *Saccharomyces cerevisiae* results in functional impairment due to the lack of the [2Fe-2S] cofactor, and knocking out the CCC1 gene alleviates this deficiency (CN115786152A, publication date 2023.03.14). Another study showed that although mammalian-derived P450scc is a mitochondrial type of P450 (Class I), heterologous expression in *Saccharomyces cerevisiae* can still utilize P450 reductase (CPR) for electron transfer, exhibiting superior electron transfer capacity compared to the natural Adx-AdR combination. The optimal combination is the mature form of wild boar-derived *Sus scrofa* tSsCYP11A1 and *AoCPR* derived from *Pteris vittata* (CN118909813A, publication date 2024.11.08). Despite extensive research, a significantly high-yielding engineered *Saccharomyces cerevisiae* strain remains lacking in this field.

[0005] Therefore, developing a high-yield recombinant brewer's yeast that produces progesterone is of great significance in this field. Summary of the Invention

[0006] To address the aforementioned technical issues, this invention provides a cytochrome P450 cholesterol side-chain lyase mutant, a recombinant Saccharomyces cerevisiae producing progesterone, and their applications. The mutant, obtained through screening using the ESM-2 language model, significantly improves catalytic efficiency. Based on this, a recombinant Saccharomyces cerevisiae strain was constructed, key enzyme copy numbers were optimized, the electron transport system tBtAdx-tBtAdR was introduced, and key enzymes were overexpressed to obtain the PG-PANC strain. This strain exhibits a significantly increased progesterone production, setting a new record for progesterone production in Saccharomyces cerevisiae expressing animal-derived CYP11A1, and is suitable for industrial-scale progesterone production.

[0007] On one hand, the present invention provides a mutant of cytochrome P450 cholesterol side-chain lyase, wherein the mutant, compared with the amino acid sequence of wild-type cytochrome P450 cholesterol side-chain lyase, has mutations at the following sites corresponding to the amino acid sequence shown in SEQ ID NO.2: positions 75, 118, 394, 248, 469, 274, 173, 107, 215, 369 and / or 418, wherein the wild-type cytochrome P450 cholesterol side-chain lyase is derived from pigs.

[0008] Specifically, the 75th amino acid is mutated to a non-F amino acid, such as A, R, K, V, T, H, S, I, M, E, D, L, G, Y, C, Q, W, P, N, preferably mutated to A or S.

[0009] Specifically, the 118th amino acid is mutated to a non-T amino acid, such as A, R, K, F, W, H, S, I, M, E, D, L, G, Y, C, Q, P, V, N, preferably mutated to Q.

[0010] Specifically, the 394th amino acid is mutated to a non-G amino acid, such as A, R, K, F, W, H, S, I, M, E, D, L, T, Y, C, Q, P, V, N, preferably mutated to D or N.

[0011] Specifically, the 248th amino acid is mutated to a non-W amino acid, such as A, R, K, F, T, H, S, I, M, E, D, L, G, Y, C, Q, P, V, N, preferably mutated to R or K.

[0012] Specifically, the 469th amino acid is mutated to a non-F amino acid, such as A, R, K, V, T, H, S, I, M, E, D, L, G, Y, C, Q, W, P, N, preferably mutated to S or K.

[0013] Specifically, the 274th amino acid is mutated to a non-S amino acid, such as A, R, K, V, T, H, F, I, M, E, D, L, G, Y, C, Q, W, P, N, preferably mutated to F, M, L, or I.

[0014] Specifically, the 173rd amino acid is mutated to a non-T amino acid, such as A, R, K, F, W, H, S, I, M, E, D, L, G, Y, C, Q, P, V, N, preferably mutated to C.

[0015] Specifically, the 107th amino acid is mutated to a non-G amino acid, such as A, R, K, F, W, H, S, I, M, E, D, L, T, Y, C, Q, P, V, N, preferably mutated to E.

[0016] Specifically, the 215th amino acid is mutated to a non-D amino acid, such as A, R, K, F, W, H, S, I, M, E, G, L, T, Y, C, Q, P, V, N, preferably mutated to A.

[0017] Specifically, the 369th amino acid is mutated to a non-M amino acid, such as A, R, K, F, T, H, S, I, P, E, D, L, G, Y, C, Q, W, V, N, preferably mutated to H.

[0018] Specifically, the 418th amino acid is mutated to a non-W amino acid, such as A, R, K, F, T, H, S, I, P, E, D, L, G, Y, C, Q, M, V, N, preferably mutated to F.

[0019] On one hand, the present invention provides a fusion protein comprising the mutant and P450 reductase, wherein the mutant and P450 reductase are directly or indirectly linked, wherein the indirect link is through a linker sequence, such as GGGS, and preferably, the P450 reductase is derived from blue acrylonitrile, and the amino acid sequence of the P450 reductase is shown in SEQ ID NO.3.

[0020] On one hand, the present invention provides a recombinant expression vector, the vector comprising a nucleic acid sequence encoding the fusion protein, preferably further comprising a promoter and a terminator, preferably, the promoter being P GAL1 The terminator is T FBA1 .

[0021] On one hand, the present invention provides a method for preparing recombinant Saccharomyces cerevisiae, the method comprising, based on a starting strain of Saccharomyces cerevisiae, overexpressing genes encoding the ergosterol synthesis pathway in Saccharomyces cerevisiae, introducing exogenous genes 3β-HSD gene, StDWF5 gene, GgDHCR24 gene, and VcCYP90B27 gene, and inserting P at the ATF2 site. GAL1 For promoter, T FBA1 The nucleic acid sequence encoding the fusion protein is used to terminate the regulation. Preferably, the starting strain is BY74742. Preferably, the genes encoding the ergosterol synthesis pathway in *Saccharomyces cerevisiae* include ERG10, ERG13, tHMG1, ERG12, ERG8, ERG19, IDI1, ERG20, ERG9, and ERG1. The SG IDs of the amino acid sequences encoding the above genes are S000005949, S000004595, S000004540, S000004821, S000004833, S000005326, S000006038, S000003703, S000001233, and SGD:S000003407. The nucleic acid sequence encoding the 3β-HSD gene is shown in SEQ ID NO.1. The amino acid sequence of the StDWF5 gene is uniprot. The amino acid sequence of the GgDHCR24 gene is M1BG0, the uniprot ID is A0A8V0ZY51, and the amino acid sequence of the VcCYP90B27 gene is shown in SEQ ID NO.6. The insertion is to knock out the entire expression frame of the gene and insert the corresponding genotype.

[0022] Specifically, the nucleic acid sequence of the fusion protein is further integrated at the GRE3, ura3, and DPP1 sites, and the nucleic acid sequence of the StDWF5 gene is inserted at the GAL10 site. The SGIDs of the amino acid sequences of the GRE3, ura3, and DPP1 genes are S000001146, S000000747, and S000002692, respectively.

[0023] Specifically, the promoter P is inserted at the YPR1 gene site. GAL10 and Termination T ADH1 The expressed tBtAdR gene sequence and promoter P GAL1 and Termination T ADH2 The expressed tBtAdx gene sequence; the CCC1 gene knocked out. The SG IDs of the amino acid sequences of the YPR1 and CCC1 genes are S000002776 and S000004210, respectively. The nucleotide sequence encoding tBtAdx is shown in SEQ ID NO.4, and the nucleotide sequence encoding tBtAdR is shown in SEQ ID NO.5.

[0024] On the one hand, the present invention also provides recombinant brewing yeast obtained using the above method.

[0025] On the one hand, the present invention also provides the use of the above-mentioned mutant, or the fusion protein, or the recombinant expression vector, or recombinant Saccharomyces cerevisiae in the production of progesterone, or in the preparation of progesterone products.

[0026] On the other hand, the present invention also provides a method for producing progesterone, comprising a step of fermenting the recombinant brewer's yeast to produce progesterone, optionally further comprising a step of separating the produced progesterone.

[0027] Those skilled in the art can identify the amino acids of the nitrile hydrolase protein 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.

[0028] 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).

[0029] As used in this article, the term "F75S" means that the amino acid F at position 75 is changed to amino acid S, for example, "S274I" means that the amino acid S at position 274 is mutated to I, and so on.

[0030] Beneficial effects:

[0031] The cytochrome P450 cholesterol side-chain lyase mutant provided by this invention significantly improves catalytic efficiency, especially the mutant tSsCYP11A1. F75S It can increase the yield of progesterone to 148.30 mg / L, an increase of 2.44 times. Several amino acid sites, such as the 75th and 274th positions, play a key regulatory role in the catalytic activity of the P450scc system and provide important targets for subsequent protein engineering.

[0032] Meanwhile, based on the cytochrome P450 cholesterol side-chain lyase mutant, two electron transport ligands, AoCPR and tBtAdx-tAdR, were introduced to overexpress key genes, and the constructed Saccharomyces cerevisiae PG-PANC significantly improved progesterone production. In 5L high-density fermentation, the progesterone yield reached 1426.21 mg / L, setting a new record for progesterone production in Saccharomyces cerevisiae expressing animal-derived CYP11A1. This is superior to the expression level of plant-derived enzyme systems in yeast and has the potential for industrial application. Through metabolic engineering, progesterone can be synthesized de novo from inexpensive carbon sources, and the yields in shake flasks and fermenters are significantly improved, providing a high-quality strain for the efficient industrial production of progesterone. Attached Figure Description

[0033] Figure 1 This is a diagram of the de novo progesterone synthesis pathway in Saccharomyces cerevisiae. The green box shows the pre-squalene pathway, the blue box shows the post-squalene pathway, and the red box shows the exogenous introduction pathway and a schematic diagram of electron transfer in CYP11A1.

[0034] Figure 2 The results of systematic mutation prediction of tSsCYP11A1 were obtained using the ESM-2 language model. Based on the LLR score ranking of the top 50 candidate mutants, 26 mutant types were randomly selected for experimental verification to verify their progesterone production in shake-flask fermentation.

[0035] Figure 3 The figures represent the progesterone production of strains FC113-PN-Sl3β-HSD, PG-FLE, PG-St, and PG-PANC in shake flasks. The broken line represents the progesterone production of strains PG-FLE, PG-St, and PG-PANC after marker replenishment in 5L high-density fermentation. The numbers in the broken line graph are in mg / L. Detailed Implementation

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 5L fermentation method: Select the appropriate experimental strains according to the initial OD 600 ≈0.1 was inoculated into primary (2 mL), secondary (20 mL), and tertiary seed culture (200 mL) containing YPD medium, and into a 5L fermenter (2L system) containing high-density initial medium. The high-density initial medium consisted of: glucose 20 g / L, KH2PO4 8 g / L, inositol 1 g / L, yeast extract 10 g / L, peptone 20 g / L, (NH4)2SO4 15 g / L, 8 g / L SD medium, 3 ml / L vitamins, 2 ml / L CaCl2, and 10 ml / L trace metal salts. During high-density fermentation, dissolved oxygen (DO) was controlled at 30%, pH at 5.0, temperature at 30℃, initial rotation speed at 300 rpm (maximum 1000 rpm), and initial aeration at 3.0 rpm (maximum 10.0 rpm). Once the strain has depleted its initial carbon source, specifically as dissolved oxygen levels drop to 30% and then rebound to above 50%, the dissolved oxygen rebound feeding program is activated, feeding at a rate of 10g of glucose per feeding. The initial feeding medium consists of 770g / L glucose monohydrate, 50ml / L 20x sulfate, and 9g / L KH₂PO₄. Cell OD was monitored. 600 Once the culture medium reaches 100, replace it with 50% anhydrous ethanol and add 10 g / L of galactose as an inducer. Continue culturing, and monitor the OD of the strain at all times. 600 Monitor the yield and continue cultivation until the yield reaches its maximum, at which point the cultivation process ends.

[0040] Example 1: Further improving the catalytic activity of CYP11A1 using the ESM-2 language model

[0041] (1) Constructing a progesterone-producing Saccharomyces cerevisiae engineered chassis strain with sufficient precursors

[0042] Based on the metabolic engineering strategy outlined in Chinese patent CN118909813A (publication date 2024.11.08), this study overexpressed enzymes in the ergosterol synthesis pathway of *Saccharomyces cerevisiae*, involving 10 key genes (ERG10, ERG13, tHMG1, ERG12, ERG8, ERG19, IDI1, ERG20, ERG9, and ERG1) in the squalene synthesis pathway. These genes are derived from *Saccharomyces cerevisiae* S288C, and their specific sequences can be obtained from the SDG website: https: / / www.yeastgenome.org. The catalytic pathway is described in [link to catalytic pathway description]. Figure 1 This effectively enhanced the supply of precursors and introduced a tomato source (Solanum lycopersicum) Sl3β-HSD with better catalytic activity, the nucleotide sequence of which is shown in SEQ ID NO.1 (Lee, HJ; Nakayasu, M.; Akiyama, R.; Kobayashi, M.; Miyachi, H.; Sugimoto, Y.; Umemoto, N.; Saito, K.; Muranaka, T.; Mizutani, M., Identification of a 3β-Hydroxysteroid Dehydrogenase / 3-Ketosteroid Reductase Involved in α-Tomatine Biosynthesis in Tomato. Plant Cell Physiol 2019, 60 (6)). (1304-1315), strain FC113-PN-Sl3β-HSD was constructed, and the progesterone shake-flask yield was increased to 60.85 mg / L. The specific genotypes of the strains are shown in Table 1. The corresponding amino acid sequences of the genes involved are listed in Table 2 from the Saccharomyces cerevisiae website https: / / www.yeastgenome.org / . StDWF5 (amino acid sequence number: M1B6G0) and GgDHCR24 (amino acid sequence number: A0A8V0ZY51) are from the website: https: / / www.uniprot.org / . The general principle for promoters and terminators is to use the first 800 bp or the last 500 bp of the corresponding gene. GAL10 / 1 This means that GAL10 and GAL1 exist in the gene cluster in opposite directions of expression extension, and the sequence between the two genes is a promoter that can bidirectionally drive expression. GAL10 / 1 The sequence is as follows: the positive driver is the promoter P. GAL1 The reverse drive is the promoter P. GAL10 .

[0043] Table 1. Names and genotypes of all strains involved in the experiment.

[0044]

[0045] Genotype of strain FC113-PN-Sl3β-HSD: Based on strain BY4742, the TRP1 gene was knocked out, and a promoter P was inserted at the leu2 site. PGK1 and Termination T ADH1 The gene tHMG1, which is truncated 516 amino acids at its N-terminus, is expressed by the promoter P. PDC1 and Termination T ADH2 The expressed gene is ERG12, and the promoter is P. ENO2 and Termination T PDC1 The expressed gene is IDI1, and the promoter is P. PYK1 and Termination T PGI1 The expressed gene is ERG19, and the promoter is P. FBA1 and Termination T TDH2 The expressed gene is ERG13, and the promoter is P. TDH3 and Termination T TPI1 The expressed gene ERG8 and promoter P TEF1 and Termination T CYC1 The gene ERG10 is expressed; an insertion at the NDT80 site is used to activate the promoter P. PGK1 and Termination T ADH1 The expressed gene is ERG20, and the promoter is P. TDH3 and Termination T TPI1 The expressed gene is ERG1, and the promoter is P. TEF1 and Termination T CYC1 The gene ERG9 is expressed; a promoter P is inserted at the ERG5 gene locus. GAL7 and Termination T SPG5 The expressed gene is StDWF5, and the promoter is P. GAL10 and Termination T CWP2 The expressed gene GgDHCR24 and promoter P GAL10 and Termination T PRM9 The gene VcCYP90B27 was expressed; the GAL1 gene was knocked out; and a promoter P was inserted at the ATF2 site. GAL1 and Termination T FBA1 The gene sequence of the expressed fusion protein tSsCYP11A1-GGGS-AoCPR; an insertion at the his3 site to activate the promoter P GAL1 and Termination T CYC1 The expressed gene is 3β-HSD.

[0046] The genotype of strain PG-FLE is similar to that of strain FC113-PN-Sl3β-HSD, except that tSsCYP11A1 at the ATF2 site is replaced with tSsCYP11A1. F75S The gene sequence.

[0047] Genotype of strain PG-St: Based on strain PG-FLE, a promoter P was inserted at the ura3 gene locus. GAL1 and Termination T FBA1 The expressed fusion protein is tSsCYP11A1 F75S The gene sequence of -GGGS-AoCPR and the HIS3 gene; the promoter P was inserted at the GRE3 gene locus. GAL1 and Termination T FBA1 The expressed fusion protein is tSsCYP11A1 F75S -GGGS-AoCPR gene sequence; insert promoter P at the DPP1 gene site. GAL1 and Termination T FBA1 The expressed fusion protein is tSsCYP11A1 F75S The gene sequence of -GGGS-AoCPR was used to insert the StDWF5 gene sequence at the GAL10 site.

[0048] Genotype of strain PG-PANC: Based on strain PG-St, the promoter P was inserted at the YPR1 gene locus. GAL10 and Termination T ADH1 The expressed tBtAdR gene sequence and promoter P GAL1 and Termination T ADH2 The expressed tBtAdx gene sequence; CCC1 gene knockout.

[0049] Adding back the marker: Taking BY4742 as an example, it lacks the gene URA3 for synthesizing uracil, the gene LEU2 for synthesizing LEU protein, the gene LYS2 for synthesizing LYS protein, and the gene HIS3 for synthesizing HIS protein. These genes are used as life-or-death selection tags. During genetic modification, life-or-death selection is used to initially screen for correct transformants. However, during high-density fermentation, the lack of these genes will affect the growth of the strain. When these genes are added back, it becomes a "wild-type" strain, which can utilize carbon sources to synthesize all the necessary nutrients. Specifically, the genotype involves inserting the HIS3-TRP1-URA3-LEU2-LYS2 gene sequence at the Delta15 gene locus. In addition, strain PG-PANC-AC, based on the added marker, additionally added the ERG5 gene sequence at the ROX1 gene locus for further growth recovery.

[0050] The insertion principle described in this experiment is to knock out the entire gene expression frame and insert the corresponding genotype.

[0051] Table 2. SGD IDs of the amino acid sequences of the gene involved in this invention.

[0052]

[0053] Genetic manipulation of the strain, exogenous gene sequence information (StDWF5, GgDHCR24, VcCYP90B27, tSsCYP11A1, tBtAdx, tBtAdR, P) GAL10 / 1 For details regarding product processing and analysis methods, please refer to Table 2 and patents CN118909813A (publication date 2024.11.08), CN115786152A (publication date 2023.03.14), and application number 202411169852.3. Strain construction method:

[0054] All genes were derived from *Saccharomyces cerevisiae* S288C, or synthesized from GenScipt (Nanjing, China) after optimization using *Saccharomyces cerevisiae* codon preferences. The CRISPR-Cas9 plasmid used for genetic modification in this study was named pURA3-CRISPR (GenBank: PQ816245.1), composed of two plasmids: p414-TEF1p-Cas9-CYC1t (#43802, addgene) and p426-SNR52p-gRNA.CAN1.Y-SUP4t (#43803, addgene). This optimized plasmid retained the ura3 selection marker and replaced the original SNR52 promoter controlling gRNA expression with the endogenous tRNAGly promoter. When editing the targeted gene, only the N20 sequence of the sgRNA in the plasmid needed to be replaced using seamless cloning technology (Seamless Cloning Kit #D7010S, BeyotimeBiotechnology, Shanghai, China). Donor sequence fragment PCR amplification was performed using a High-fidelity PrimeSTAR GXL (TaKaRa, Kyoto, Japan). To ensure effective homologous recombination, the donor sequence was designed with homologous arms at both ends of at least 50 bp from the target site in the *Saccharomyces cerevisiae* genome. More than 100 ng of the donor DNA fragment and a CRISPR-Cas9 recombinant plasmid were co-transformed into 60 μL of competent yeast cells using electroporation. The transformation products were plated on SD-URA synthesis-deficient medium selection plates and incubated at 30°C for 48–72 h until single colonies formed. To verify the correct transformants, colony PCR was performed using a High-fidelity PrimeSTAR MAX (TaKaRa, Kyoto, Japan).

[0055] (2) Screening for progesterone-producing mutants using the ESM-2 language model

[0056] The unsupervised learning protein language model ESM-2, with a parameter scale of 15 billion, can achieve direct protein prediction without relying on multiple sequence alignments or external data. Through a self-supervised learning method using random masking training, it can effectively capture the evolutionary conservation and structure-function relationships of protein sequences. As an open-source platform, ESM-2 provides an efficient tool for rational protein design. We introduced this model to perform evolutionary-scale mutation analysis on tSsCYP11A1 and ranked the obtained LLR scores. The amino acid sequence of the wild-type tSsCYP11A1 is shown in SEQ ID NO. 2.

[0057] Twenty-six mutants were randomly selected from the Top 50 candidate mutants for experimental verification. All mutants were named tSsCYP11A1. The -GGGS-AoCPR fusion protein was heterologously expressed in the recombinant strain FC113-Sl3β-HSD (this strain has the same genetic background as FC1113-PN-Sl3β-HSD, but does not integrate tSsCYP11A1). -GGGS-AoCPR expression module The specific mutation sites of different mutants of tSsCYP11A1 are shown, with the amino acid sequence of AoCPR as shown in SEQ ID NO.3. FC1113-PN-Sl3β-HSD was used as a control strain. The yield of progesterone produced during shake-flask fermentation was used as the evaluation index, and the results showed... Figure 2 ,tSsCYP11A1 F75S and tSsCYP11A1 S274L Two mutants significantly improved the catalytic efficiency of P450scc: compared with the control strain FC1113-PN-Sl3β-HSD (shake flask yield 60.85 mg / L), the mutant tSsCYP11A1... F75S It can increase progesterone production to 148.30 mg / L, an increase of 2.44 times, p<0.0001; mutant tSsCYP11A1 S274L This study increased the progesterone yield in shake flasks to 124.92 mg / L, a 2.05-fold increase (p < 0.01). This result indicates that amino acid residues at positions 75 and 274 of the CYP11A1 gene play a crucial regulatory role in the catalytic activity of the P450scc system, and also provides an important target for subsequent protein engineering. The mutant tSsCYP11A1 was also selected. F75S Further modifications were made, and the engineered strain containing this mutant was named PG-FLE. The specific genotypes are shown in Table 1.

[0058] Example 2: Further increase in progesterone production and validation through 5L high-density fermentation.

[0059] Based on strain PG-FLE, by optimizing the copy number of the key enzyme combination (StDWF5 and tSsCYP11A1F75S-GGGS-AoCPR), the shake-flask yield of strain PG-St (specific genotypes are shown in Table 1) reached 152.18 mg / L. Subsequently, the modification strategy of patent CN115786152A (publication date 2023.03.14) was adopted, inserting a promoter P at the YPR1 gene locus. GAL10 and Termination T ADH1 The expressed tBtAdR gene sequence and promoter P GAL1 and Termination TADH2 The expressed tBtAdx gene sequence was obtained; the CCC1 gene was knocked out. The SG IDs of the amino acid sequences of the YPR1 and CCC1 genes are S000002776 and S000004210, respectively. The nucleotide sequence encoding tBtAdx is shown in SEQ ID NO.4, and the nucleotide sequence encoding tBtAdR is shown in SEQ ID NO.5. The finally constructed PG-PANC strain (specific genotypes are shown in Table 1) increased the yield to 194.12 mg / L (shake flask yield results are shown in Table 1). Figure 3 )

[0060] After auxotrophic tagging and replenishment of the above-mentioned engineered strains, high-density fermentation was carried out in a 5L fermenter, employing the 1% oleic acid addition strategy described in patent application number 202411169852.3. The results showed that the progesterone yields of strains PG-FLE, PG-St, and PG-PANC reached 705.88 mg / L, 837.98 mg / L, and 1426.21 mg / L, respectively. Notably, the PG-PANC strain achieved the highest progesterone production record to date for Saccharomyces cerevisiae expressing animal-derived CYP11A1, surpassing previously reported heterologous expression levels of the plant CYP87As family in yeast (Li, R.; Guo, S.; Wang, D.; Yang, T.; Li, W.; Wang, J.; Huang, L.; Zhang, X.; Dai, Z., Elucidation of the plant progesterone biosynthetic pathway and its application in a yeast cell factory. MetabEng 2025, 90, 197-208).

[0061] Genotype of strain PG-PANC: Based on strain PG-St, the promoter P was inserted at the YPR1 gene locus. GAL10 and Termination T ADH1 The expressed tBtAdR gene sequence and promoter P GAL1 and Termination T ADH2 The expressed tBtAdx gene sequence; CCC1 gene knockout.

[0062] 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 mutant of cytochrome P450 cholesterol side-chain lyase, characterized in that, The mutant, compared with the amino acid sequence of wild-type cytochrome P450 cholesterol side-chain lyase, has mutations at the following sites corresponding to the amino acid sequence shown in SEQ ID NO.2: positions 75, 118, 394, 248, 469, 274, 173, 107, 215, 369, and / or 418, wherein the wild-type cytochrome P450 cholesterol side-chain lyase is derived from pigs.

2. The mutant according to claim 1, characterized in that, The amino acid at position 75 is mutated to a non-S amino acid, the amino acid at position 118 is mutated to a non-T amino acid, the amino acid at position 394 is mutated to a non-G amino acid, the amino acid at position 248 is mutated to a non-W amino acid, the amino acid at position 469 is mutated to a non-F amino acid, the amino acid at position 274 is mutated to a non-S amino acid, the amino acid at position 173 is mutated to a non-T amino acid, the amino acid at position 107 is mutated to a non-G amino acid, the amino acid at position 215 is mutated to a non-D amino acid, the amino acid at position 369 is mutated to a non-M amino acid, and the amino acid at position 418 is mutated to a non-W amino acid. Preferably, the 75th amino acid is mutated to A or S, the 118th amino acid is mutated to Q, the 394th amino acid is mutated to D or N, the 248th amino acid is mutated to R or K, the 469th amino acid is mutated to S or K, the 274th amino acid is mutated to F, M, L or I, the 173rd amino acid is mutated to C, the 107th amino acid is mutated to E, the 215th amino acid is mutated to A, the 369th amino acid is mutated to H, and the 418th amino acid is mutated to F.

3. A fusion protein, characterized in that, The fusion protein comprises the mutant of claim 1 and P450 reductase, wherein the mutant and P450 reductase are directly or indirectly linked, wherein the indirect link is through a linker sequence, such as GGGS, and preferably, the P450 reductase is derived from blue auricularia auricula, and the amino acid sequence of the P450 reductase is shown in SEQ ID NO.

3.

4. A recombinant expression vector, characterized in that, The vector contains a nucleic acid sequence encoding the fusion protein of claim 3, and preferably further contains a promoter and a terminator, preferably, the promoter is P. GAL The terminator is T FBA1 .

5. A method for preparing recombinant brewer's yeast, characterized in that, The method involves overexpressing genes encoding the ergosterol synthesis pathway in *Saccharomyces cerevisiae* as the starting strain, introducing exogenous genes 3β-HSD, StDWF5, GgDHCR24, and VcCYP90B27, and inserting a P gene at the ATF2 site. GAL1 For promoter, T FBA1 The nucleic acid sequence encoding the fusion protein of claim 3, regulated by the terminator, is preferably defined as follows: the starting strain is BY74742; preferably, the genes encoding the ergosterol synthesis pathway in *Saccharomyces cerevisiae* include ERG10, ERG13, tHMG1, ERG12, ERG8, ERG19, IDI1, ERG20, ERG9, and ERG1; the SG IDs of the amino acid sequences encoding the above genes are S000005949, S000004595, S000004540, S000004821, S000004833, S000005326, S000006038, S000003703, S000001233, and SGD:S000003407; the nucleic acid sequence encoding the 3β-HSD gene is shown in SEQ ID NO.1; and the amino acid sequence of the gene is uniprot. The amino acid sequence of the GgDHCR24 gene is M1BG0, the uniprot ID is A0A8V0ZY51, and the amino acid sequence of the VcCYP90B27 gene is shown in SEQ ID NO.

6. The insertion is to knock out the entire expression frame of the gene and insert the corresponding gene.

6. The method according to claim 5, characterized in that, Further, it includes integrating the nucleic acid sequence encoding the fusion protein of claim 3 at the GRE3, ura3, and DPP1 sites, and inserting the StDWF5 gene sequence at the GAL10 site, wherein the SG IDs of the amino acid sequences of the GRE3, ura3, and DPP1 genes are S000001146, S000000747, and S000002692.

7. The method according to claim 6, characterized in that, Insertion at the YPR1 gene site to the promoter P GAL10 and Termination T ADH1 The expressed tBtAdR gene sequence and promoter P GAL1 and Termination T ADH2 The expressed tBtAdx gene sequence; the CCC1 gene knocked out. The SG IDs of the amino acid sequences of the YPR1 and CCC1 genes are S000002776 and S000004210, respectively. The nucleotide sequence encoding tBtAdx is shown in SEQ ID NO.4, and the nucleotide sequence encoding tBtAdR is shown in SEQ ID NO.

5.

8. The recombinant brewer's yeast obtained using the method described in any one of claims 5-7.

9. The use of the mutant according to any one of claims 1-2, or the fusion protein according to claim 3, or the recombinant expression vector according to claim 4, or the recombinant Saccharomyces cerevisiae according to claim 8 in the production of progesterone, or in the preparation of progesterone products.

10. A method for producing progesterone, characterized in that, It includes the step of fermenting the recombinant brewer's yeast of claim 8 to produce progesterone, and optionally further includes the step of separating the produced progesterone.

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