A yeast with weakened acyl donor degradation, a modification method and application thereof in synthesis of simvastatin

Abstract

This invention discloses a yeast strain with weakened acyl donor degradation, a modification method, and its application in the synthesis of simvastatin, belonging to the field of genetic engineering. It describes a synthetic pathway for heterologous expression of simvastatin using Pichia pastoris as the host, with exogenously added α-dimethylbutyryl- S methyl mercaptopropionate (DMB-) S Simvastatin biosynthesis is achieved using MMP as an acyl donor. This invention significantly reduces the Pichia pastoris's reliance on DMB- by knocking out genes 2, 19, and 18 in the Pichia pastoris genome. S Degradation efficiency of simvastatin. Recombinant strains mlow-SV(Δ2Δ19) and mlow-SV(Δ2Δ19Δ18) produced 7.94 mg / L and 10.37 mg / L simvastatin, respectively, after 72 h of shake-flask fermentation, representing increases of 201.4% and 293.6% compared to recombinant strain mlow-SV(Δku70).

Description

Technical Field

[0001] This invention relates to a yeast strain for degrading acyl donors, a modification method thereof, and its application in the synthesis of simvastatin, belonging to the field of genetic engineering. Background Technology

[0002] Lovastatin, a polyketide compound, is a natural product isolated from Aspergillus terreus. It is effective in treating hypercholesterolemia, coronary heart disease, and preventing cardiovascular disease. It was approved by the U.S. Food and Drug Administration (FDA) in 1987, becoming the first clinically approved statin drug. Statins selectively inhibit HMG-CoA reductase, thereby significantly reducing cholesterol synthesis and blood lipid levels. Simvastatin is a semi-synthetic derivative of lovastatin, differing from lovastatin only in that it has an additional methyl group on the α-carbon atom of the butyrate ester side chain at the C-8 position, yet it possesses better efficacy and fewer side effects. Before generic versions were allowed on the market, simvastatin was the second best-selling drug in the United States, with annual sales exceeding $5 billion at one point. Therefore, optimizing the economic and technical indicators (cost and efficiency) of simvastatin synthesis has received widespread attention.

[0003] Currently, simvastatin is mainly produced industrially through chemical synthesis, as described in patents such as US4444784 and CN1019395B. These synthetic routes typically use lovastatin as a raw material, involving multiple steps such as group protection, group linkage, and deprotection. They also involve the use of organic reagents and demanding reaction conditions, and still require improvement in terms of cost-effectiveness and environmental compatibility. With the development of genetic engineering and synthetic biology, the synthesis of simvastatin using microbial factories is gradually attracting more attention.

[0004] Given the similarity in chemical structure between simvastatin and lovastatin, current research on the biosynthesis of simvastatin focuses on redirecting the lovastatin biosynthetic pathway to simvastatin by adding a specific acyl donor exogenously. Specifically, this involves the acyl donor providing an acyl group under the catalysis of the acyltransferase LovD, which then reacts with the metabolic intermediate monacolin J to generate simvastatin. Figure 1 To improve the efficiency of this conversion reaction, researchers screened for highly efficient acyl donors, such as DMB-. S-MMP (as described in CN101490271), and there are also reports of using protein engineering techniques to mutate the natural acyltransferase LovD to obtain the mutant LovD9 with higher catalytic efficiency (Jiménez-Osés G, Osuna S, Gao X, Sawaya MR, Gilson L, Collier SJ, Huisman GW, Yeates TO, Tang Y, Houk KN. The role of distant mutations and allosteric regulation on lovd active site dynamics. Nature Chemical Biology. 2014, 10(6): 431–436), providing a technical basis for the biosynthesis of simvastatin. While immobilized acyltransferases or hosts that heterologously express acyltransferases can drive the conversion of monacolin J and acyl donors to simvastatin (as described in CN103725726A and CN102695792A), de novo synthesis of simvastatin using high-performance microbial hosts can significantly reduce process complexity and cost.

[0005] Researchers have constructed the Monacoline J biosynthetic pathway in the eukaryotic model organism *Saccharomyces cerevisiae*, and demonstrated its effectiveness by recombinantly expressing LovD9 and adding exogenous DMB. S-MMP synthesized SVA at a titer of 5.9 mg / L in living cells, but production efficiency needs further improvement (Bond CM, Tang Y. Engineering Saccharomyces cerevisiae for production of simvastatin. Metabolic Engineering. 2019, 51: 1–8). Pichia pastoris, as one of the main eukaryotic expression systems, is generally recognized as safe (GRAS) by the FDA. It boasts simple culture, convenient genetic manipulation, high-density fermentation, and good post-translational modification capabilities. Currently, it has heterologously expressed over 5000 proteins and has the potential for efficient synthesis of simvastatin. Publicly available data reports the de novo synthesis of monacolin J using Pichia pastoris as a host, with a yield reaching 2.2 g / L (Liu Y, Bai C, Liu Q, Xu Q, Qian Z, Peng Q, Yu J, Xu M, Zhou X, Zhang Y, Cai M. Engineered ethanol-driven biosynthetic system for improving production of acetyl-CoA derived drugs in Crabtree-negative yeast. Metabolic Engineering. 2019, 54: 275–284). Therefore, in the recombinant Pichia pastoris strain that synthesizes monacolin J, further recombinant expression of the acyltransferase LovD9 and exogenous addition of DMB- S -MMPs have the potential to construct engineered strains that efficiently synthesize simvastatin. However, Pichia pastoris rapidly degrades DMB- S -MMP is α-dimethylbutyryl- S -propionic acid (DMB- S The characteristics of DMB-MPA in this engineered strain pose a significant challenge to its efficient production process. This was addressed by identifying and eliminating DMB-degrading molecules in Pichia pastoris. S The functional gene for -MMP can effectively enhance the biosynthetic efficiency of simvastatin in Pichia pastoris. Summary of the Invention

[0006] For recombinant Pichia pastoris strains that synthesize simvastatin, exogenously added DMB- SUsing MMP as an acyl donor, this invention weakens the degradation effect of Pichia pastoris on DMB-S-MMP by knocking out gene 2 and / or gene 19, or gene 2 and gene 19 and / or gene 18, thereby improving the synthesis efficiency of simvastatin.

[0007] The first objective of this invention is to provide a method for improving the efficiency of yeast biosynthesis of simvastatin, the method being to knock out gene 2 and / or gene 19, or gene 2 and gene 19 and / or gene 18, on the Pichia pastoris genome, wherein genes 2, 19, and 18 are capable of degrading DMB- S - The function of MMP.

[0008] In one embodiment of the present invention, the nucleotide sequence of gene 2 (NCBI Gene ID: 8199732) is shown in SEQ ID NO.1.

[0009] In one embodiment of the present invention, the nucleotide sequence of gene 19 (NCBI Gene ID: 8196549) is shown in SEQ ID NO.2.

[0010] In one embodiment of the present invention, the nucleotide sequence of gene 18 (NCBI Gene ID: 8201321) is shown in SEQ ID NO.3.

[0011] In one embodiment of the present invention, the Pichia pastoris is a GS115Δku70 defective strain.

[0012] In one embodiment of the present invention, gene 2 is knocked out of the genome of the GS115Δku70 defective strain to obtain strain Δ2; gene 2 and gene 19 are knocked out to obtain strain Δ2Δ19; and gene 2, gene 19, and gene 18 are knocked out to obtain strain Δ2Δ19Δ18. DMB is degraded by strains Δ2, Δ2Δ19, and Δ2Δ19Δ18. S -Evaluate the efficiency of MMP.

[0013] A second objective of this invention is to provide genetically engineered bacteria obtained by constructing using any of the methods described above.

[0014] A third objective of this invention is to provide a yeast strain that synthesizes simvastatin, wherein the yeast strain uses GS115Δku70 as a host, knocks out genes 2 and 19, or genes 2, 19, and 18, and heterologously expresses the encoding genes for polyketide synthase LovB, acyl reductase LovC, phosphopanylthioethylamine transferase NpgA, thioesterase LovG, P450 monooxygenase LovA, P450 reductase CPR, and acyltransferase LovD9.

[0015] In one embodiment of the present invention, the protein amino acid sequence of the polyketide synthase LovB is shown in SEQ ID NO. 22 (GenBank No. AAD39830.1), the protein amino acid sequence of the enoyl reductase LovC is shown in SEQ ID NO. 23 (GenBank No. AAD34554.1), the protein amino acid sequence of the phosphate pantothenic thioethylamine transferase NpgA is shown in SEQ ID NO. 24 (GenBank No. USN24631.1), the protein amino acid sequence of the thioesterase LovG is shown in SEQ ID NO. 25 (GenBank No. XP_001209264.1), the protein amino acid sequence of the P450 monooxygenase LovA is shown in SEQ ID NO. 26 (GenBank No. AAD34552.1), and the protein amino acid sequence of the P450 reductase CPR is shown in SEQ ID NO. 22 (GenBank No. AAD39830.1). As shown in NO.27 (GenBank number XP_001214242.1), the protein amino acid sequence of acyltransferase LovD9 is shown in SEQ ID NO.28.

[0016] A third objective of this invention is to provide a method for synthesizing simvastatin, wherein the method utilizes an engineered yeast strain for simvastatin synthesis for fermentation culture, and exogenously adds α-dimethylbutyryl- during the fermentation process. S methyl mercaptopropionate (DMB- S Simvastatin was synthesized using MMP (-MMP).

[0017] In one embodiment of the present invention, the method includes the following steps: inoculating the yeast engineered strain into YPD medium and culturing it at 30°C and 200 rpm for 30 h; then inoculating the bacterial culture into YPD medium and culturing it at 30°C and 200 rpm for 12 h; then transferring the bacterial culture into fermentation medium YPLD and culturing it at 30°C and 200 rpm for 4 h, followed by induction with blue light, and adding DMB- at a final concentration of 2.4 mM at 58 h of culture. S-MMP, continue fermentation for 72 h.

[0018] A method for improving the efficiency of yeast biosynthesis of simvastatin, the method being to knock out gene 2 and / or gene 19 and / or gene 18 on the Pichia pastoris genome.

[0019] Preferably, the nucleotide sequence of gene 2 is shown in SEQ ID NO.1; the nucleotide sequence of gene 19 is shown in SEQ ID NO.2; and the nucleotide sequence of gene 18 is shown in SEQ ID NO.3.

[0020] Preferably, the yeast is Pichia pastoris.

[0021] Preferably, the Pichia pastoris is Pichia pastoris.

[0022] Preferably, the strain of Pichia pastoris is any one of NRRL-Y 11430 or its derivative, GS115 strain, GS115Δku70 deletion strain or its derivative.

[0023] A yeast strain that synthesizes simvastatin, in Pichia pastoris GS115Δku70, with gene 2 and / or gene 19 and / or gene 18 knocked out. Simultaneously, the encoding genes for polyketide synthase LovB, acyl reductase LovC, phosphopantoylthioethylamine transferase NpgA, thioesterase LovG, P450 monooxygenase LovA, P450 reductase CPR, and acyltransferase LovD9 were introduced.

[0024] Preferably, the nucleotide sequence of gene 2 is shown in SEQ ID NO.1; the nucleotide sequence of gene 19 is shown in SEQ ID NO.2; and the nucleotide sequence of gene 18 is shown in SEQ ID NO.3. The amino acid sequence of the polyketide synthase LovB is shown in SEQ ID NO. 22; the amino acid sequence of the acyl reductase LovC is shown in SEQ ID NO. 23; the amino acid sequence of the phosphate pantothenic thioethylamine transferase NpgA is shown in SEQ ID NO. 24; the amino acid sequence of the thioesterase LovG is shown in SEQ ID NO. 25; the amino acid sequence of the P450 monooxygenase LovA is shown in SEQ ID NO. 26; the amino acid sequence of the P450 reductase CPR is shown in SEQ ID NO. 27; and the amino acid sequence of the acyl transferase LovD9 is shown in SEQ ID NO. 28.

[0025] Preferably, the yeast is Pichia pastoris.

[0026] Preferably, the use of the yeast engineered strains for synthesizing simvastatin as described in any of the foregoing descriptions in the preparation of simvastatin.

[0027] A method for synthesizing simvastatin, comprising fermenting and culturing a yeast engineered strain for synthesizing simvastatin as described above, wherein α-dimethylbutyryl-3- is exogenously added during fermentation. S methyl mercaptopropionate (DMB- S Simvastatin was synthesized using MMP (-MMP).

[0028] Compared with the prior art, the beneficial effects achieved by the present invention are: The recombinant Pichia pastoris strain mlow-SV(Δku70) can absorb exogenously added DMB- S -MMP is used as an acyl donor in the synthesis of simvastatin. However, Pichia pastoris degrades DMB- S The characteristics of MMPs resulted in a simvastatin production rate of only 2.64 mg / L in the recombinant strain mlow-SV (Δku70). Knockout of genes 2 and 19 in the Pichia pastoris genome, followed by further knockout of gene 18, significantly weakened the Pichia pastoris' resistance to DMB-. S The degradation effect of MMP by strains Δ2Δ19 and Δ2Δ19Δ18 on DMB was compared to that of strain GS115Δku70. S The efficiency of MMP decreased by 67.1% and 74.4%, respectively, laying an important foundation for the efficient synthesis of simvastatin by Pichia pastoris. Recombinant strains mlow-SV(Δ2Δ19) and mlow-SV(Δ2Δ19Δ18) produced 7.94 mg / L and 10.37 mg / L of simvastatin, respectively, after 72 h of shake-flask fermentation, representing increases of 201.4% and 293.6% compared to recombinant strain mlow-SV(Δku70). Attached Figure Description

[0029] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings: Figure 1 This is a schematic diagram of the biosynthetic pathway of simvastatin. Figure 2 Degradation of DMB by Pichia pastoris engineered strains S - Efficiency comparison chart of MMP; Figure 3 The graph shows the yield of simvastatin synthesized by engineered Pichia pastoris. Detailed Implementation

[0030] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; the reagents and biological materials mentioned are commercially available unless otherwise specified.

[0032] technical terms Pichia pastoris Komagataella phaffii The GS115Δku70 defective strain has been published in the 2019 paper "Liu Q, Shi X, Song L, Liu H, Zhou X, Wang Q, Zhang Y, Cai M. CRISPR–Cas9-mediated genomic multiloci integration in Pichia pastoris. Microbial Cell Factories. 2019, 18(1): 144" or in patent CN110079546A, with plasmid BB3pN_P. GAP -2Bbshandle_P LAT1 -Cas9 was published in the 2024 paper "Kang Y, Qian Z, Yu H, Lu J, Zhao Q, Qiao X, Ye M, Zhou X, Cai M. Programmable biosynthesis of plant-derived 4′-deoxyflavoneglycosides by an unconventional yeast consortium. Small Methods. 2024, 8(8):2301371". The seamless assembly kit ClonExpress Ultra one-step cloning kit V2 was purchased from Nanjing Novizan Biotechnology Co., Ltd., the restriction endonuclease T4 ligase was purchased from New England Biolabs, and the required DNA sequence was synthesized by Nanjing Genscript Biotech Co., Ltd.

[0033] Culture medium: The YPD medium used for culturing Pichia pastoris has the following formulation: 20 g / L peptone, 10 g / L yeast extract, and 20 g / L glucose. The YPLD medium used for culturing Pichia pastoris has the following formulation: 20 g / L peptone, 10 g / L yeast extract, and 10 g / L glucose. An additional 20 g / L of agar is added to the solid medium.

[0034] The HPLC analysis method was as follows: a C18 liquid chromatography column (Shimadzu; Shim-pack Scepeter C18-120 / 5μm) was used. The mobile phase B was acetonitrile, and the mobile phase A was a 1 mL / L ultrapure aqueous solution of acetic acid. The flow rate was 1 mL / min, and the injection volume was 20 μL. The proportion of phase A linearly decreased from 65% to 35% within 0–38 min to elute the sample. The UV detection wavelength was 238 nm.

[0035] Example 1: Knockout of genes 2, 19, and 18 in the genome of the GS115Δku70 defective strain

[0036] 1. The gene sequences involved in the gene knockout process are shown below: Gene 2 (SEQ ID NO.1), atggccaaaccaattcctgaaagcactctggaagacctcgggtattttcccaagggaactcccgttacctcagatccaatcataaacggcatacctttccgaaccaccttttggcccgttccagaaggtgttactgtgaaagggagaatactgtttgtacacggattggcagaacatgccctggtttacacagaaccgatggatttcttcagccaatctggatatgaatgtttcttctacgaccagagaggtgcaggtttgacggctaaatactataaaaatataggtgtcacgaacagcacgtatgtgtttgatgatttggaggctatcattgaattgaacttgaaagaggccgaacaataccaccataagctttttcttattggccattcaatgggcggagcaattgtcagtaactatgctataattggcaaacatagggacgagattagtggaattgtggcatgtgcccctctcatcgagactcaccctaagacatctccaaacatcatcttagaatacctggtgcgaggactcgtttatgtgatacccaaccacaagttcaacagtaagttaaatattgattttattacctccgataaaggttacactgagtttctgttgcaagacaggctttcagaccccattggatctctaattctgttccgtgatgcgttttaccgtggacgacgactattgactcccgagttctacaccaaattcaagaaggatttaccatatctggtcattcacggggccaaagattactgttgcagtggagactctgctaagaagtttgtagatcttatcaacaaaaacgagccaactgcccagcaaaccataacgttgtatgaagagggtaagcatagtcttctcttagaaaaagaagaacttaggtacaaagtgtacaatgatcttctaaagtttctggacgaccaggcatag;

[0037] 2. The target gene on the Pichia pastoris strain genome was knocked out using the CRISPR-Cas9 gene editing system. Relevant primers (Table 2) were designed for PCR amplification and the construction of related plasmids or donor DNA fragments, as detailed below: 1) The annealing products of primers 2sgRNAF / 2sgRNAR were ligated with plasmid BB3pN_P after digestion with restriction endonuclease BbsI using T4 ligase. GAP -2Bbshandle_P LAT1 - Cas9 ligation was performed to construct plasmid 2CRISPR.

[0038] 2) The annealing products of primers 19sgRNAF / 19sgRNAR were ligated with the plasmid BB3pN_P, which was then digested with restriction endonuclease BbsI, using T4 ligase. GAP -2Bbshandle_P LAT1 - Cas9 ligation was performed to construct plasmid 19CRISPR.

[0039] 3) The sequence between the PacI and BsaI restriction sites in plasmid 2CRISPR was ligated with the sequence between the XbaI and PacI restriction sites in plasmid 19CRISPR using T4 ligase to construct plasmid 2+19CRISPR.

[0040] 4) The annealing products of primers 18sgRNAF / 18sgRNAR were ligated with plasmid BB3pN_P, which was then digested with restriction endonuclease BbsI, using T4 ligase. GAP -2Bbshandle_P LAT1 - Cas9 ligation was used to construct the plasmid 18CRISPR.

[0041] 5) Using the genome of strain GS115Δku70 as a template, the sequence fragments at both ends of the coding region of gene 2 in the GS115Δku70 genome were amplified by PCR using primers 2donUPF / 2donUPR and 2donDOF / 2donDOR, respectively. The two sequence fragments were then ligated by overlap PCR to obtain the donor fragment 2donor.

[0042] 6) Using the genome of strain GS115Δku70 as a template, the sequence fragments at both ends of the coding region of gene 19 in the genome of GS115Δku70 were amplified by PCR using primers 19donUPF / 19donUPR and 19donDOF / 19donDOR, respectively. The two sequence fragments were then ligated by overlap PCR to obtain the donor fragment 19donor.

[0043] 7) Using the genome of strain GS115Δku70 as a template, the sequence fragments at both ends of the outer part of the coding region of gene 18 in the genome of GS115Δku70 were amplified by PCR using primers 18donUPF / 18donUPR and 18donDOF / 18donDOR, respectively. The two sequence fragments were then ligated by overlap PCR to obtain the donor fragment 18donor.

[0044] 8) Electroporate 100 ng of 2CRISPR and 1 μg of 2donor into competent cells of GS115Δku70 strain to knock out gene 2. After verifying the genotype and losing plasmid 2CRISPR, strain Δ2 was obtained.

[0045] 9) Electroporate 100 ng 2+19CRISPR, 1 μg 2donor and 1 μg 19donor into competent cells of GS115Δku70 strain to knock out genes 2 and 19. After verifying the genotype and losing plasmid 2+19CRISPR, strain Δ2Δ19 was obtained.

[0046] 10) Electroporate 100 ng of 18CRISPR and 1 μg of 18donor into competent cells of strain Δ2Δ19 to knock out gene 18. After verifying the genotype and losing plasmid 18CRISPR, strain Δ2Δ19Δ18 was obtained.

[0047] The names and sequences of the relevant primers are as follows: SEQ ID NO.4: 2sgRNAF,acactctagattctgtctgatgagtccgtgaggacgaaacgagtaagctcgtcacagaattagagatccaatg; SEQ ID NO.5: 2sgRNAR,aaaccattggatctctaattctgtgacgagcttactcgtttcgtcctcacggactcatcagacagaatctaga; SEQ ID NO.6: 19sgRNAF,acactctagaagaaccctgatgagtccgtgaggacgaaacgagtaagctcgtcggttctaaattatccagacg; SEQ ID NO.7: 19sgRNAR, aaaccgtctggataatttagaaccgacgagcttactcgtttcgtcctcacggactcatcagggttcttctaga; SEQ ID NO.8:18sgRNAF,acactctagacatttgctgatgagtccgtgaggacgaaacgagtaagctcgtccaaatgaccacctaaatctg; SEQ ID NO.9:18sgRNAR,aaaccagatttaggtggtcatttggacgagcttactcgtttcgtcctcacggactcatcagcaaatgtctaga; SEQ ID NO.10:2donUPF,gtgactggattaggcagaga; SEQ ID NO.11:2donUPR,tcgcatgagccattgacagttgcttaggat; SEQ ID NO.12:2donDOF,actgtcaatggctcatgcgaagtattctag; SEQ ID NO.13:2donDOR,ggtgcctattgtggaaatga; SEQ ID NO.14:19donUPF,ggtttgaagtaaggcatttg; SEQ ID NO.15:19donUPR,atgtggaagtagtgatggagagcgatagat; SEQ ID NO.16:19donDOF,ctccatcactacttccacataagcatcaag; SEQ ID NO.17:19donDOR,cttctagtttggtcttcatc; SEQ ID NO.18:18donUPF,ccttagccatatttcatttc; SEQ ID NO.19:18donUPR,tgctacctaccgtcgagtcaaactttatac; SEQ ID NO.20:18donDOF,tgactcgacggtaggtagcatgaatctgat; SEQ ID NO.21:18donDOR,cacacagaaccagaaatgtt。

[0048] 3. Strains GS115Δku70 and yeast genetically engineered strains Δ2, Δ2Δ19, and Δ2Δ19Δ18 were inoculated into 24-well plates containing 2 mL of YPD medium and cultured at 30°C and 200 rpm for 30 h in a shaker. The bacterial culture was then inoculated at a ratio of 1% (v / v) into 250 mL Erlenmeyer flasks containing 50 mL of YPD medium and cultured at 30°C and 200 rpm for 12 h in a shaker. Finally, the bacterial culture was transferred to 24-well plates containing 2 mL of YPD medium at a final inoculation volume of ≈1, and cultured at 30°C and 200 rpm for 6 h in a shaker. Then, 1.2 mM DMB was added to achieve a final concentration of 1. S -MMP (DMB- S -MMP was dissolved in DMSO solution at a 1:1 volume ratio. After 6 h of further culture, 1 mL of the fermentation broth was thoroughly mixed with 4 mL of ethyl acetate, extracted using a vacuum rotary evaporator, and then dissolved in 1 mL of methanol. The sample solution was filtered through a 0.22 μm filter membrane and analyzed by HPLC for DMB- S -MMP degradation product DMB- S -MPA content.

[0049] like Figure 2 As shown, DMB- in the fermentation broth of strain GS115Δku70 S -MMP degradation product DMB- S -MPA peak area reached 3458, DMB- in fermentation broth of yeast genetically engineered strains Δ2, Δ2Δ19 and Δ2Δ19Δ18 S The peak areas of -MPA were 1516, 1137, and 887, respectively, compared to the peak areas of DMB degradation by strain GS115Δku70. S The efficiency of MMP was reduced by 56.2%, 67.1%, and 74.4%, respectively.

[0050] Example 2: Construction of Simvastatin Synthetic Strains 1. The sequences or numbers of the enzymes and expression regulatory elements involved in the simvastatin synthesis pathway involved in the strain construction process are as follows: SEQ ID NO.23:LovC,AAD34554.1,MGDQPFIPPPQQTALTVNDHDEVTVWNAAPCPMLPRDQVYVRVEAVAINPSDTKMRGQFATPWAFLGTDYAGTVVAVGSDVTHIQVGDRVYGAQNEMCPRTPDQGAFSQYTVTRGRVWAKIPKGLSFEQAAALPAGISTAGLAMKLLGLPLPSPSADQPPTHSKPVYVLVYGGSTATATVTMQMLRLSGYIPIATCSPHNFDLAKSRGAEEVFDYRAPNLAQTIRTYTKNNLRYALDCITNVESTTFCFAAIGRAGGHYVSLNPFPEHAATRKMVTTDWTLGPTIFGEGSTWPAPYGRPGSEEERQFGEDLWRIAGQLVEDGRLVHHPLRVVQGGFDHIKQGMELVRKGELSGEKLVVRLEGP; SEQ ID NO.24:NpgA,USN24631.1,MVQDTSSASTSPILTRWYIDTRPLTASTAALPLLETLQPADQISVQKYYHLKDKHMSLASNLLKYLFVHRNCRIPWSSIVISRTPDPHRRPCYIPPSGSQEDSFKDGYTGINVEFNVSHQASMVAIAGTAFTPNSGGDSKLKPEVGIDITCVNERQGRNGEERSLESLRQYIDIFSEVFSTAEMANIRRLDGVSSSSLSADRLVDYGYRLFYTYWALKEAYIKMTGEALLAPWLRELEFSNVVAPAAVAESGDSAGDFGEPYTGVRTTLYKNLVEDVRIEVAALGGDYLFATAARGGGIGASSRPGGGPDGSGIRSQDPWRPFKKLDIERDIQPCATGVCNCLS; SEQ ID NO.25:LovG,XP_001209264.1,MRYQASPALVKAPRALLCIHGAGCSPAIFRVQLSKLRAALRENFEFVYVTAPFPSSAGPGILPVFADLGPYYSWFESSSDNNHNGPSVSERLAAVHDPIRRTIVDWQTQHPHIPIVGAIGFSEGALVTTLLLWQQQMGHLPWLPRMSVALLICPWYQDEASQYMRNEVMKNHDDDNDSKDTEWQEELVIRIPTLHLQGRDDFALAGSKMLVARHFSPREAQVLEFAGQHQFPNRPRDVLEVINRFRKLCVTAQTLE; SEQ ID NO.26:LovA,AAD34552.1,MTVDALTQPHHLLSLAWNDTQQHGSWFAPLVTTSAGLLCLLLYLCSSGRRSDLPVFNPKTWWELTTMRAKRDFDANAPSWIESWFSQNDKPIRFIVDSGYCTILPSSMADEFRKMKELCMYKFLGTDFHSHLPGFDGFKEVTRDAHLITKVVMNQFQTQAPKYVKPLANEASGIITDIFGDSNEWHTVPVYNQCLDLVTRTVTFIMVGSKLAHNEEWLDIAKHHAVTMAIQARQLRLWPVILRPLVHWLEPQGAKLRAQVRRARQLLDPIIQERRAERDACRAKGIEPPRYVDSIQWFEDTAKGKWYDAAGAQLAMDFAGIYGTSDLLIGGLVDIVRHPHLLEPLRDEIRTVIGQGGWTPASLYKLKLLDSCLKESQRVKPVECATMRSYALQDVTFSNGTFIPKGELVAVAADRMSNPEVWPEPAKYDPYRYMRLREDPAKAFSAQLENTNGDHIGFGWHPRACPGRFFASKEIKMMLAYLLIRYDWKVVPDEPLQYYRHSFSVRIHPTTKLMMRRRDEDIRLPGSL; SEQ ID NO.27:CPR,XP_001214242.1,; LovD9(SEQ ID NO.28),MVMGSNIDAAVAADPVVLMETAFRKAVESSQIPGAVLMARDASGRLNYTRCFGARTVRRDENQLPPLQVDTPCRLASATKLLTTIMALQCMERGLVR LDETVDRLLPDLCAMPVLEGFDDAGNPRLRERRGKITLRHLLTHTSGLSYVFLHPLLREYVAQGHLQGAEKFGIQNRFAPPLVNDPGAEWIYGAGIDWAGKLVERA TGLDLEQYLQENICAPLGITDMTFKLQQRPDMLARRADMTHRNSSDGKLRYDDTVYFRHDGEECFGGQGVFSSPGSYMKVLHSLLKRDGLLLQPGTVDLMFQPALE PRLEEQMNQHMDASPHINYGGPMPMVMRRSFGLGGIALEDLDGENWRRKGSMTFGGGPNIIWQIDPKAGLCTLVFFQLEPWSDPVCRDLTRTFEKAIYAQYQQG; SEQ ID NO.29:lexO- cP AOX1 ,tgctgtatataaaccagtggttatatgtacagtactgctgtataaaccagtggttatatgtacagtacggcgcgctagcggggctataaactagtggggccctaaccctacttgacagcaatatataaacagaa ggaagctgccctgtcttaaacctttttttatcatcattattagctctttcatattgcgactggttccaattgacaagcttttgatttaacgacttttaacgacaacttgaagatcaaaaaacaactaattattcgaa; SEQ ID NO.30:P mlow

[0051] 2. Based on the pathway enzyme or expression regulatory element sequences shown in Table 3, design relevant primers (Table 4), perform PCR amplification and construct relevant plasmids, as detailed below: 1) With P mlow Using -LVAD as a template, the gene fragment was amplified using primers mlow-LVAD-F / mlow-LVAD-R. The gene fragment was then integrated into the expression vector plasmid pPICZB between the BglII and SalI sites using a seamless assembly kit, resulting in plasmid pZB-P. mlow -LVAD.

[0052] 2) Using lexO-cPAOX1 as a template, the gene fragment was amplified using primers lexO-cAOX1-F / cAOX1-R. Using LovB as a template, the gene fragment was amplified using primers LovB-F / LovB-R. The two gene fragments were then integrated into the expression vector plasmid pPICZB between the BglII and SalI sites using a seamless assembly kit, resulting in plasmid pZB-lexO-cP. AOX1 -LovB.

[0053] 3) Using LexO-cP AOX1 Using lexO-cAOX1-F / cAOX1-R as a template, the gene fragment was amplified. Using LovC as a template, the gene fragment was amplified using LovC-F / LovC-R. The two gene fragments were then integrated into the expression vector plasmid pPICZB between the BglII and SalI sites using a seamless assembly kit, resulting in the plasmid pZB-lexO-cP. AOX1 -LovC.

[0054] 4) Using LexO-cP AOX1 Using lexO-cAOX1-F / cAOX1-R as a template, the gene fragment was amplified. Using NpgA as a template, the gene fragment was amplified using primers NpgA-F / NpgA-R. The two gene fragments were then integrated into the expression vector plasmid pPICZB between the BglII and SalI sites using a seamless assembly kit, resulting in plasmid pZB-lexO-cP. AOX1 -NpgA.

[0055] 5) Using LexO-cP AOX1Using lexO-cAOX1-F / cAOX1-R as a template, the gene fragment was amplified. Using LovG as a template, the gene fragment was amplified using LovG-F / LovG-R. The two gene fragments were then integrated into the expression vector plasmid pPICZB between the BglII and SalI sites using a seamless assembly kit, resulting in plasmid pZB-lexO-cP. AOX1 -LovG.

[0056] 6) Using LexO-CP AOX1 Using lexO-cAOX1-F / cAOX1-R as a template, the gene fragment was amplified. Using LovG as a template, the gene fragment was amplified using LovA-F / LovA-R. The two gene fragments were then integrated into the expression vector plasmid pPICZB between the BglII and SalI sites using a seamless assembly kit, resulting in plasmid pZB-lexO-cP. AOX1 -LovA.

[0057] 7) Using LexO-CP AOX1 Using lexO-cAOX1-F / cAOX1-R as a template, the gene fragment was amplified. Using CPR as a template, the gene fragment was amplified using primers CPR-F / CPR-R. The two gene fragments were then integrated into the expression vector plasmid pPICZB between the BglII and SalI sites using a seamless assembly kit, resulting in plasmid pZB-lexO-cP. AOX1 -CPR.

[0058] 8) Using LexO-cP AOX1 Using lexO-cAOX1-F / cAOX1-R as a template, the gene fragment was amplified. Using LovD9 as a template, the gene fragment was amplified using LovD9-F / LovD9-R. The two gene fragments were then integrated into the expression vector plasmid pPICZB between the BglII and SalI sites using a seamless assembly kit, resulting in plasmid pZB-lexO-cP. AOX1 -LovD9.

[0059] 9) Using plasmid pZB-lexO-cP AOX1 Using -NpgA as a template, lexO-cP was amplified using primers TTB-lexOF / TT-BR. AOX1 The -NpgA gene fragment was inserted into plasmid pZB-lexO-cP using a seamless assembly kit. AOX1 The plasmid pZ-lexO1cA-CN was constructed by using the BamHI site of -LovC.

[0060] 10) Using plasmid pZB-lexO-cP AOX1 Using -LovG as a template, lexO-cP was amplified using primers TTB-lexOF / TT-BR. AOX1 The -LovG gene fragment was inserted into the BamHI site of plasmid pZ-lexO1cA-CN using a seamless assembly kit to construct plasmid pZ-lexO1cA-CNG.

[0061] 11) Using plasmid pZB-P mlow Using LVAD as a template, primers TTB-GAPF / TT-BR are used to amplify P mlow The -LVAD gene fragment was inserted into the BamHI site of plasmid pZ-lexO1cA-CNG using a seamless assembly kit to construct plasmid pZ-lexO1cA-CNG_P. mlow -LVAD.

[0062] 12) Using plasmid pZ-lexO1cA-CNG_P mlow Using LVAD as a template, lexO1cA-CNG_P was amplified using primers TTB-boxF / TT-BR. mlow -LVAD gene fragment, which was inserted into plasmid pZB-lexO-cP using a seamless assembly kit. AOX1 The plasmid pZ-lexO1cA-BCNG_P was constructed by using the BamHI site of -LovB. mlow -LVAD.

[0063] 13) Using plasmid pZB-lexO-cP AOX1 - Using CPR as a template, amplify lexO-cP using primers TTB-lexOF / TT-BR. AOX1 - The CPR gene fragment was inserted into plasmid pZB-lexO-cP using a seamless assembly kit. AOX1 The plasmid pZ-lexO1cA-AC was constructed by using the BamHI site of -LovA.

[0064] 14) Using plasmid pZB-lexO-cP AOX1 -LovD9 was used as a template to amplify lexO-cP using primers TTB-lexOF / TT-BR. AOX1 Using the -LovD9 gene fragment and pZ-lexO1cA-AC as a template, the entire DNA fragment of the plasmid was amplified using primers TT-BF / TTAOX1-BR. The two fragments were then ligated using a seamless assembly kit to construct the plasmid pZ-lexO1cA-ACD9.

[0065] 15) Using plasmid pZ-lexO1cA-ACD9 as a template, the lexO1cA-ACD9 gene fragment was amplified using primers pPSCD9F / pPSCD9R. The plasmid backbone was amplified using the expression vector plasmid pPIC3.5K as a template and primers pPF / pPR. The two DNA fragments were ligated using a seamless assembly kit to construct plasmid pP-lexO1cA-ACD9.

[0066] The names and sequences (5' to 3') of the relevant primers are as follows: SEQ ID NO.31: mlow-LVAD-F,tttggtcatgcatgagatcagatcttttttgtagaaatgtcttggtgtc; SEQ ID NO.32: mlow-LVAD-R, caatgatgatgatgatgatggtctggatcttcgacttttcttttctt; SEQ ID NO.33: lexO-cAOX1-F, ggattttggtcatgagatcagatcttgctgtatataaaaccagtgg; SEQ ID NO.34: cAOX1-R,ttcgaataattagttgttttttgatcttc; SEQ ID NO.35: LovB-F,aaacaactaattattcgaaggtaccatggctcaatctatgtatcct; SEQ ID NO.36: LovB-R,caatgatgatgatgatgatggtctgccagcttcagggcgggattcat; SEQ ID NO.37: LovC-F, aacaactaattattcgaaatgggcgaccagccattcat; SEQ ID NO.38: LovC-R, caatgatgatgatgatgatggtccggcccctcgagccgaaccacg; SEQ ID NO.39: NpgA-F, aacaactaattattcgaaatggtgcaagacacatcaagc; SEQ ID NO.40: NpgA-R, caatgatgatgatgatgatggtcggataggcaattacacaccc; SEQ ID NO.41:LovG-F,aacaactaattattcgaaatgcgttaccaagcatctcc; SEQ ID NO.42:LovG-R,caatgatgatgatgatgatggtcctccaatgtctgggccgtcacaca; SEQ ID NO.43:LovA-F,aacaactaattattcgaaatgactgttgacgctttg; SEQ ID NO.44:LovA-R,caatgatgatgatgatgatggtccaaagaacctggcaatctaatg; SEQ ID NO.45:CPR-F,aacaactaattattcgaaatggctcaactcgacactc; SEQ ID NO.46:CPR-R,caatgatgatgatgatgatggtctgaccacacgtcctcctggtag; SEQ ID NO.47:LovD9-F,aaaacaactaattattcgaaatggttatgggtagtaacatcg; SEQ ID NO.48:LovD9-R,caatgatgatgatgatgatggtctccttgttggtactgggcgtag; SEQ ID NO.49:TTB-lexOF,gtgagaccttcgtttgtgcagatcttgctgtatataaaaccagtgg; SEQ ID NO.50:TT-BR,gaagctatggtgtgtgggggatcc; SEQ ID NO.51:TTB-GAPF,gtgagaccttcgtttgtgcagatcttttttgtagaaatgtcttggtg; SEQ ID NO.52:TTB-boxF,gtgagaccttcgtttgtgcgggattttggtcatgagatcag; SEQ ID NO.53:TT-BF,ccccacacaccatagcttcaaaatg; SEQ ID NO.54: TTAOX1-BR, cgcacaaacgaaggtctcac; SEQ ID NO.55: pPF,gttcgtttgtgcaagcttatc; SEQ ID NO.56: pPR, ggtacctactagtggatcatc; SEQ ID NO.57: pPSCD9F,gatgatccactagtaggtaccggattttggtcatgagatcag; SEQ ID NO. 58: pPSCD9R, gataagcttgcacaaacgaacgaagctatggtgtgtgggggatcc.

[0067] 3. Construct a Pichia pastoris genetically engineered strain capable of synthesizing simvastatin using relevant plasmids. The specific steps are as follows: 1) The plasmid pZ-lexO1cA-BCNG_P mlow -LVAD was linearized with BlnI restriction enzyme and electroporated into competent cells of GS115Δku70 strain. Transformants were picked from plates with the corresponding resistance for verification. The strain with the correct expression cassette and a single copy was named mlow-MJ(Δku70).

[0068] 2) After linearizing the plasmid pP-lexO1cA-ACD9 with SalI restriction enzyme, it was electroporated into mlow-MJ competent cells. Transformants were picked from plates with the corresponding resistance for verification. The strain that was verified to be a single copy of the expression cassette was named mlow-SV(Δku70).

[0069] Example 3: Following the method described in Example 1, genes 2 and 19, or genes 2, 19, and 18, were knocked out in the Pichia pastoris genetically engineered strain that synthesized simvastatin. The specific steps are as follows: 1) 100 ng 2+19CRISPR, 1 μg 2donor and 1 μg 19donor were electroporated into mlow-SV(Δku70) competent cells to knock out genes 2 and 19. After verifying the genotype and losing plasmid 2+19CRISPR, strain mlow-SV(Δ2Δ19) was obtained.

[0070] 2) Electroporate 100 ng of 18CRISPR and 1 μg of 18donor into the competent cells of mlow-SV(Δ2Δ19) to knock out gene 18. After verifying the genotype and losing plasmid 18CRISPR, strain mlow-SV(Δ2Δ19Δ18) was obtained.

[0071] Example 4: Shake-flask fermentation of the Pichia pastoris genetically engineered strain for synthesizing simvastatin was performed, as detailed below: Yeast strains *mlow-SV* (Δku70), *mlow-SV* (Δ2Δ19), and *mlow-SV* (Δ2Δ19Δ18) were inoculated into 24-well plates containing 3 mL of YPD medium and cultured in a shaker at 30°C and 200 rpm for 30 h. The bacterial culture was then inoculated at a ratio of 1% (v / v) into 250 mL Erlenmeyer flasks containing 50 mL of YPD medium and cultured in a shaker at 30°C and 200 rpm for 12 h. The bacterial culture was then transferred to 250 mL Erlenmeyer flasks containing 50 mL of YPLD fermentation medium at a final inoculation rate of OD600≈0.5, with glucose added at a final concentration of 10 g / L every 24 h. After culturing in a shaker at 30°C and 200 rpm for 4 h, blue light induction was initiated. At 58 h, DMB-100 solution was added at a final concentration of 2.4 mM. S -MMP (DMB- S -MMP of DMSO solution was dissolved at a 1:1 volume ratio. Fermentation continued for 72 h, then 1 mL of the fermentation broth was thoroughly mixed with 4 mL of ethyl acetate. After extraction using a vacuum rotary evaporator, 1 mL of 0.05 M sodium hydroxide methanol solution was added to dissolve the sample. The sample solution was filtered through a 0.22 μm filter membrane, and the compound concentration was quantitatively analyzed by HPLC.

[0072] like Figure 3 As shown, the exogenous addition of the acyl donor DMB- S Under the condition of -MMP, the recombinant strain mlow-SV(Δku70) was able to synthesize simvastatin, but the yield of simvastatin only reached 2.64 mg / L after fermentation in shake flasks for 72 h.

[0073] like Figure 3 As shown, the recombinant strains mlow-SV(Δ2Δ19) and mlow-SV(Δ2Δ19Δ18) produced 7.94 mg / L and 10.37 mg / L of simvastatin, respectively, after 72 h of shake-flask fermentation, representing increases of 201.4% and 293.6% compared to the recombinant strain mlow-SV(Δku70). This indicates that knockout of genes 2 and 19, or genes 2, 19, and 18, can significantly reduce the susceptibility of Pichia pastoris to DMB- S -MMP degradation effect, thereby improving the efficiency of Pichia pastoris in synthesizing simvastatin.

[0074] The above embodiments are not intended to limit the present invention. Any modifications and alterations made by those skilled in the art without departing from the spirit and principle of the present invention shall also fall within the protection scope of the present invention. The protection scope of the present invention shall be defined by the claims.

Claims

1. A method for improving the efficiency of yeast biosynthesis of simvastatin, characterized in that, The method involves knocking out gene 2 and / or gene 19 and / or gene 18 on the Pichia pastoris genome.

2. The method as described in claim 1, characterized in that, The nucleotide sequence of gene 2 is shown in SEQ ID NO.1; the nucleotide sequence of gene 19 is shown in SEQ ID NO.2; and the nucleotide sequence of gene 18 is shown in SEQ ID NO.

3.

3. The method as described in claim 1, characterized in that, The yeast in question is Pichia pastoris.

4. The method as described in claim 3, characterized in that, The Pichia pastoris is Pichia pastoris.

5. The method as described in claim 4, characterized in that, The strain of *Pichia pastoris* is any one of NRRL-Y 11430 or its derivative, GS115 strain, GS115Δku70 deletion strain or its derivative.

6. A yeast strain for synthesizing simvastatin, characterized in that, Gene 2 and / or gene 19 and / or gene 18 were knocked out in Pichia pastoris GS115Δku70; Simultaneously, the encoding genes for polyketide synthase LovB, acyl reductase LovC, phosphopantoylthioethylamine transferase NpgA, thioesterase LovG, P450 monooxygenase LovA, P450 reductase CPR, and acyltransferase LovD9 were introduced.

7. The yeast strain for synthesizing simvastatin as described in claim 6, characterized in that, The nucleotide sequence of gene 2 is shown in SEQ ID NO.1; the nucleotide sequence of gene 19 is shown in SEQ ID NO.2; and the nucleotide sequence of gene 18 is shown in SEQ ID NO.

3. The protein amino acid sequence of the polyketide synthase LovB is shown in SEQ ID NO.22; the protein amino acid sequence of the enoyl reductase LovC is shown in SEQ ID NO.

23. The amino acid sequence of the protein of phosphopantoylthioethylamine transferase NpgA is shown in SEQ ID NO.24; the amino acid sequence of the protein of thioesterase LovG is shown in SEQ ID NO.25; the amino acid sequence of the protein of P450 monooxygenase LovA is shown in SEQ ID NO.26; the amino acid sequence of the protein of P450 reductase CPR is shown in SEQ ID NO.27; and the amino acid sequence of the protein of acyltransferase LovD9 is shown in SEQ ID NO.

28.

8. The yeast strain for synthesizing simvastatin as described in claim 7, characterized in that, The yeast in question is Pichia pastoris.

9. The use of the yeast engineered strain for synthesizing simvastatin as described in any one of claims 6 to 8 in the preparation of simvastatin.

10. A method for synthesizing simvastatin, characterized in that: Fermentation culture of yeast engineered strains that synthesize simvastatin according to any one of claims 6 to 8, wherein α-dimethylbutyryl-3 ... S 1-Methyl mercaptopropionate, used to synthesize simvastatin.

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