A genetically engineered strain of high yield of triptolide ketone

Abstract

The application discloses a genetically engineered strain for high yield of triptolide ketone. The application belongs to the field of synthetic biology. The recombinant yeast strain is obtained by modifying a receptor yeast strain, and is as follows: a recombinant strain 1 for preparing triptolide ketone; or a recombinant strain 2 for preparing triptolide; or the recombinant yeast strain is a recombinant strain 3 for preparing triptolide ning B. By optimizing the expression of P450 enzymes in the downstream pathway, including screening homologous genes, introducing high-activity mutants and high-expression P450 enzymes, and by metabolic engineering strategies, the yeast metabolism and the heterologous expression environment of P450 enzymes are optimized, and the yield of triptolide ketone is further improved. The yield of triptolide ketone of the strain in a shake flask fermentation is 7.3 mg / L. Through scale-up in a fermenter, the yield of triptolide ketone of the strain THY230 in a 5L fermenter after 160h fermentation is 33.2 mg / L, and the strain has application prospect.

Description

Technical Field

[0001] This invention belongs to the field of synthetic biology, specifically relating to a genetically engineered strain that produces high levels of triptolide. Background Technology

[0002] Triptonide is an epoxy diterpenoid lactone extracted from Tripterygium wilfordii. It is an important monomeric component with anti-inflammatory, anti-reproductive, and immunosuppressive activities. It can be used to treat diseases such as rheumatoid arthritis (RA), nephropathy, and lupus erythematosus. A recent study showed that triptonide can be used as an effective and safe non-hormonal male contraceptive in mice and non-human primates, showing great promise for clinical application and market development.

[0003] Tripterygium lactones are found in very low concentrations in plants, and their complex and unstable chemical structures make plant extraction and chemical synthesis insufficient to meet the growing market demand. Heterologous production in microbial hosts is a sustainable and economically viable approach. The biosynthetic pathway of triptolide involves multiple CYP450 reactions, and the extremely low heterologous expression efficiency and activity of these CYP450s limit efficient heterologous production of triptolides, currently yielding only microgram-level production. Multiple optimizations of engineered strains are needed to improve yield, including increasing precursor yield, protein engineering and rational design, optimization of cofactors and electron transport chains, optimization of the CYP450 heterologous expression microenvironment, and regulation of key metabolic targets. Summary of the Invention

[0004] The technical problem to be solved by the present invention is how to prepare triptolide ketone using yeast and / or how to prepare at least one of the following intermediates: geraniol geraniol diphosphate, triptolide B and / or triptolide.

[0005] To address the above problems, this invention provides the following method for constructing recombinant yeast. In the method for constructing recombinant yeast provided by the present invention, the recombinant yeast is a recombinant strain obtained by modifying a recipient yeast. The recombinant yeast may be: recombinant strain 1 for preparing triptolide; recombinant strain 2 for preparing triptolide; or recombinant strain 3 for preparing triptolide B. The recipient yeast is a yeast with the following characteristics: S1. Does not contain the URA3 and Gal80 genes; the URA3 gene encodes whey glycoside 5-phosphate decarboxylase, and Gal80 is a key inhibitor of the galactose metabolism regulation system of Saccharomyces cerevisiae. S2 contains the Cas9 gene; The method for constructing the recombinant bacteria 1 includes steps 1) to 5). 1) Knock out the ERG9 gene of the recipient yeast; the ERG9 gene encodes squalene synthase. 2) Integration of the expression cassettes of the GGPPsa gene, BTS1 gene, ERG20 gene, IDI gene, and HMG2 gene into the genome of the recipient yeast. K6R Gene expression cassettes, tHMG1 gene expression cassettes, ERG10 gene expression cassettes, ERG13 gene expression cassettes, ERG8 gene expression cassettes, ERG13 gene expression cassettes, AnACLb and AnACLa gene expression cassettes, and MDH3, a gene encoding a truncated signal peptide. cyto Gene expression cassettes, RtME gene expression cassettes, CTP1 gene expression cassettes; 3) Integration of the tSmKSL-GGGS-CfTPS1 gene expression cassette into the genome of the recipient yeast, and integration of ThCYP82D274 into the genome of the recipient yeast. L234M Gene expression cassettes, AtCPR1 gene expression cassettes, integration of TwCYP71BE86 opt Gene expression cassettes, SOD1 gene expression cassettes, and CrCPR gene expression cassettes; 4) Integrating TrCYP71BE85 into the genome of the recipient yeast. WT Gene expression cassette, mutant TrCYP71BE85 T124G / G369S,F218N Gene expression cassettes, TwCPR5 gene expression cassettes, and TwCYB5 gene expression cassettes; 5) Integrating TwCYP82D213 into the genome of the recipient yeast opt Gene expression cassette, modified TwCYP82D213 opt The expression cassettes of the coding genes N77-274-t77TwCYP82D213opt (CP-6), MSBP3, INO2, HEM2, ZWF1, and URA3 were determined; and the OPI1 and HMX1 genes were knocked out. In one specific embodiment, the TwCYP82D213 opt The gene expression cassette integrates 6 copies; the N77-274-t77TwCYP82D213opt (CP-6) gene expression cassette integrates 6 copies.

[0006] The method for constructing the recombinant bacteria 2 includes steps 1) to 4). The method for constructing the recombinant bacteria 3 includes steps 1) to 3). In this article, the term "expression cassette" refers to a nucleic acid construct (DNA molecule) containing sufficient nucleic acid elements to express the target gene.

[0007] In one specific embodiment of the present invention, the expression cassette includes a promoter and a target gene; in another specific embodiment, the expression cassette includes a promoter, a target gene, and a terminator. The expression cassette may also include marker genes (such as TK, DHFR, CAT, and NEO genes), ribosome recognition and binding sites (SD), transcription factor binding sites (TFBS), enhancers, silencers, repressors, introns, poly(A) signal sequences, and / or mRNA splicing signal sequences. The elements within the expression cassette are operatively connected (either directly or indirectly via adapters).

[0008] The terms "operably linked," "operable connection," and "operable link" refer to the operative connection between segments of a nucleic acid sequence that are functionally related to each other. For example, operatively linked promoters, enhancers, open reading frames (OPFs), 5' and 3' UTRs, and terminator sequences result in the precise production of a nucleic acid molecule (e.g., RNA). In some embodiments, operatively linked nucleic acid elements lead to transcription of the OPF and ultimately to the production of a polypeptide (i.e., expression of the OPF).

[0009] In one specific embodiment, the method for constructing the recombinant bacterium 1 includes the following steps: M1), the gene encoding GGPPsa is integrated at site XI-3 in the recipient bacteria; M2) In the recipient bacteria, the coding genes for BTS1 and ERG20, driven by the promoter Gal10p, are integrated at the X-4 site. M3), knocking out the ERG9 encoding gene in the recipient bacteria; M4) Integrates the gene encoding IDI driven by the promoter Gal10p into the X-2 site of the recipient bacteria; M5) integrates HMG2, driven by the promoter PRS25Ai-eTDH3p, into the int14 site of the recipient bacteria. K6R The gene encoding tHMG1 and the gene encoding tHMG1 driven by the promoter PGK1p; M6) Integrates the following genes into the recipient bacteria at site XII-5: the gene encoding ERG10 driven by promoter PGK1p; the gene encoding ERG13 driven by promoter FBA1p; the gene encoding ERG8 driven by promoter TDH3p; and the gene encoding ERG12 driven by promoter TPI1p. M7) integrates the AnACLb and AnACLa coding genes driven by the promoter Gal1,10p into the XII-2 site of the recipient bacteria; and the MDH3 coding gene, whose signal peptide is truncated and driven by the promoter tHXT7p. cyto ; M8) integrates the RtME coding gene and the CTP1 coding gene into the recipient bacteria at the 22-site site. M9) integrates the tSmKSL-GGGS-CfTPS1 fusion gene at the int17 site in the recipient bacteria; M10), integrates ThCYP82D274 at the int4 site in the recipient bacteria. L234M The encoding gene; M11), integrates ThCYP82D274 at the XI-2 site in the recipient bacteria. L234M and AtCPR1; M12) integrates TwCYP71BE86 at site XII-3 in the recipient bacteria. opt The gene encoding SOD1; the gene encoding CrCPR; M13) integrates TwCYP71BE86 at site 17 in the recipient bacteria. opt The gene encoding SOD1; M14) integrates TrCYP71BE85, driven by the promoter SkGal2p, at the int19 site in the recipient bacteria. WT Encoding gene, mutant TrCYP71BE85 T124G,G369S,F218N The coding genes for TwCPR5 and TwCYB5; M15), and a copy of TrCYP71BE85 is re-integrated at site III-1 in the recipient bacteria. T124G,G369S,F218N The encoding gene; M16), integrating a total of 6 copies of TwCYP82D213 at sites V-1, XI-8, XII-4, XI-7, int16, and V-3 in the recipient bacteria. opt The encoding gene or 6 copies of the modified TwCYP82D213 opt The encoding gene N77-274-t77TwCYP82D213 opt (abbreviated as CP-6); M17) integrates N77-274-t77TwCYP82D213 at site int16 in the recipient bacteria. opt The gene encoding (CP-6); and a copy N77-274-t77TwCYP82D213 integrated at the V-3 site. optThe gene encoding (CP-6) and the gene encoding TwMSBP3 (membrane steroid-binding protein); M18) Overexpressed the coding genes for INO2 and ZWF1 in recipient bacteria; knocked out the coding gene for OPI1; knocked out the coding gene for HMX1; knocked out the coding gene for ROX1; integrated the coding gene for HEM2 at the XI-1 site; and integrated the coding gene for URA3 at the X-3 site. The present invention also provides recombinant bacteria obtained by the method described above.

[0010] The present invention also provides a method for preparing the following substance, wherein the substance may be Triptin B, Triptophenolide or Triptonide. 1) The Tripterygium wilfordii B is obtained by fermentation using the recombinant bacteria obtained in steps 1)-3) of the method described above; 2) The resorcinolone is obtained by fermentation using the recombinant bacteria obtained in steps 1)-4) of the method described above; 3) The triptolide is obtained by fermentation using the recombinant bacteria obtained in steps 1)-5) of the method described above.

[0011] The present invention also provides for the application of the methods described above in any of the following: 1) Application in the preparation of triptolide B; 2) Application in the preparation of raffinolactone; 3) Application in the preparation of triptolide.

[0012] The present invention also provides the application of the recombinant bacteria described above in any of the following: 1) Application in the preparation of triptolide B; 2) Application in the preparation of raffinolactone; 3) Application in the preparation of triptolide.

[0013] Compared with previous strains that produced triptolide, this study optimized the expression of downstream P450 enzymes, including screening for homologous genes, introducing highly active mutants and high-expression levels of P450 enzymes. In addition, through metabolic engineering strategies, the yeast metabolism and heterologous expression environment of P450 enzymes were optimized, further increasing the yield of triptolide. Attached Figure Description

[0014] Figure 1 This is a schematic diagram of the biosynthetic pathway of Tripterygium wilfordii lactone.

[0015] Figure 2The results are for the detection of geraniol (GGOH) in step (I) of Example 1.

[0016] Figure 3 The result is the detection result of Tripterygium wilfordii B in step (ii) of Example 1.

[0017] Figure 4 The results of the detection of cyclophosphamide in Example 2 are shown.

[0018] Figure 5 The results of the detection of triptolide in Example 3 are shown.

[0019] Figure 6 The results for triptolide in Example 3 are as follows: a. Tripterygium lactone production after INO2 overexpression and OPI1 knockout; b. Tripterygium lactone production after HEME2 overexpression and HMX1 and ROX1 knockout; c. Tripterygium lactone production after ZWF1 overexpression and URA3 gene integration.

[0020] Figure 7 The results are the detection results of the fed-feed fermentation scale-up production of Tripterygium wilfordii lactone in Example 4. Detailed Implementation

[0021] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0022] The following components used in this invention are all brewing yeast ( Saccharomyces cerevisiaeThe natural sequences of endogenous genes are available from public databases, including ERG20 (SGD:S000003703), ERG9 (SGD:S000001233), ERG12 (SGD:S000004821), ERG8 (SGD:S000004833), ERG10 (SGD:S000005949), ERG13 (SGD:S000004595), and BTS1. (SGD:S000005990), ERG20 (SGD:S000003703), CTP1 (SGD:S00000495), ZWF1 (SGD:S000005185), SOD1 (SGD:S000003865), OPI1 (SGD:S000001012), INO2 (SGD:S000002530), HMX1 (SGD:S000004195), HEM2 (SGD:S000003008), ROX1 (SGD:S000006269), URA3 (SGD:S000000747), TDH3 promoter (TDH3p, corresponding gene SGD:S000003424), PGK1 Promoters (PGK1p, corresponding gene SGD:S000000605), TEF1 promoter (TEF1p, corresponding gene SGD:S000006284), FBA1 promoter (FBA1p, corresponding gene SGD:S000001543), HXT7 promoter (tHXT7p, corresponding gene SGD:S000002750), TPI1 promoter (TPI1p, corresponding gene SGD:S000002457), GAL7 promoter (gal7p, corresponding gene SGD:S000000222), ENO2 terminator (ENO2t, corresponding gene SGD:SGD:S000001217), FBA1 Terminator (FBA1t, corresponding gene SGD:S000001543), TPS1 terminator (TPS1, corresponding gene SGD:S000000330), PDC1 terminator (PDC1, corresponding gene SGD:S000004034), CYC1 terminator (CYC1t, corresponding gene SGD:S000003809), TDH2 terminator (TDH2t, corresponding gene SGD:S000003769), ADH1 terminator (ADH1t, corresponding gene SGD:S000005446), ADH2 terminator (ADH2t, corresponding gene SGD:S000004918).

[0023] The tHMG1 gene element is obtained by removing the nucleotide sequence encoding the 530-amino acid membrane anchoring domain at the N-terminus of the wild-type HMG1 gene of Saccharomyces cerevisiae (SGD:S000004540), while retaining and expressing its C-terminal catalytic domain.

[0024] HMG2 K6R The gene element is based on the wild-type HMG2 gene of Saccharomyces cerevisiae (SGD:S000004442), with the sixth amino acid K mutated to R.

[0025] The MDH3 cyto The gene element is based on the wild-type Saccharomyces cerevisiae MDH3 gene (SGD:S000002236) with the removal of three amino acids at positions 341-343.

[0026] The complete base sequence information of all the above elements is publicly available through the Saccharomyces Genome Database (https: / / www.yeastgenome.org / ).

[0027] All homologous arm sequences used in this invention (including but not limited to X-2-us / -ds, ROX1-us / -ds, INO2-us / ds, etc.) are standard sequences designed based on the Saccharomyces cerevisiae S288C reference genome (https: / / www.yeastgenome.org) for specific gRNA targets (such as X-2, BTS1, MLS1p, etc.). Each homologous arm consists of a 500-600 bp genomic sequence upstream or downstream of the stated gRNA target. Their exact nucleotide sequences can be uniquely determined based on the coordinates of the gRNA target in the reference genome (e.g., via the Saccharomyces Genome Database, SGD), and therefore are not listed individually in this specification.

[0028] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0029] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0030] In the following examples, the GC-MS system was a ThermoFisher Scientific system, the column was a TG-5MS (ThermoFisher Scientific, USA, 0.25 mm × 30 m, 0.25 μm), and the injection program was as follows: initial temperature 100 ℃, hold for 1 min, increase to 190 ℃ at a rate of 30 ℃ / min, then increase to 280 ℃ at a rate of 10 ℃ / min, and finally increase to 300 ℃ at a rate of 30 ℃ / min, hold for 2 min, scan range m / z 50-500, and injection volume 1 μL.

[0031] In the following examples, the LC-MS system used was a ThermoFisher Scientific system, an OrbitrapExploris™ 240 mass spectrometer, and the column was a Waters T3 column (100 mm × 2.1 mm, 1.8 μm). Mobile phase A was 0.1% formic acid solution (solvent: water). Mobile phase B was 0.1% formic acid (solvent: acetonitrile). The flow rate was set to 0.4 mL / min. The mobile phase gradient was: 0–4 min, 30%B–34%B; 4–8 min, 34%B–52%B; 8–11.5 min, 52%B–52%B; 11.5–13 min, 52%B–90%B; 13–13.5 min, 90%B–30%B; 13.5–15 min, 30%B.

[0032] In the following examples, the plasmid p426-SNR52p-gRNA.CAN1.Y-SUP4t (also referred to as p426-URA3-gRNA) is a product from the AddGene website (https: / / www.addgene.org), catalog number: #43803. The plasmid p426-URA3-gRNA carries the SNR52 promoter and the gRNA backbone.

[0033] Information on the primers used in this invention is shown in Table 1, and information on the relevant strains is shown in Table 2.

[0034] Table 1. Primer Information

[0035] Table 2. Strains Information

[0036] The starting strain used in the following examples was *Saccharomyces cerevisiae* CEN.PK113, which expresses the Cas9 protein and has the arginine transporter CAN1 gene and the Gal80 gene knocked out. 5D * (Genome: Mataura3-52 HIS3 LEU2 TRPI MAL2-8CSUC2, Gal80Δ, CAN1Δ:TEFIp-CAS9-CYC1t), which enables efficient expression of pathway genes expressed after the promoters Gal7p and Gal1 / 10p under sugar-limited conditions. The originating strain is CEN.PK113. 5D * Specifically, it was obtained by knocking out the arginine transporter CAN1 gene and the Gal80 gene and integrating the Cas9 gene into the CEN.PK113-5D strain. CEN.PK113-5D (Mata ura3-52 HIS3 LEU2 TRPI MAL2-8C SUC2) was obtained from the BioVector plasmid vector bacterial strain cell protein antibody gene depository center - NTCC type culture depository center, website: http: / / www.biovector.net. CEN.PK113-5D and CEN.PK113... 5D * All are recorded in the literature: Jiang Meiling, Tian Zhenjiang, Tang Hao, et al. Creation of a high-efficiency Saccharomyces cerevisiae cell factory for the synthesis of ferrugin [J]. Chinese Journal of Traditional Chinese Medicine, 2025, 50(04):1031-1042. DOI:10.19540 / j.cnki.cjcmm.20241115.104. CEN.PK113 5D * Genotype (CEN.PK113-5D △CAN1:TEF1p-CAS9-CYC1t; Gal80△), CEN.PK113 5D * The strain can be obtained from the Laboratory of Synthetic Biology of Traditional Chinese Medicine, School of Traditional Chinese Medicine, Capital Medical University. This strain is only used to replicate the experiments of this invention and should not be used for other purposes.

[0037] The geraniol in the following examples is a compound with CAS: 24034-73-9, purchased from Shanghai Yuanye Biotechnology Co., Ltd., catalog number B28668; Tripterygium B is a compound with CAS: 189389-05-7, purchased from Shanghai Yuanye Biotechnology Co., Ltd., catalog number B33320; Triptophenolide is a compound with CAS: 74285-86-2, purchased from Shanghai Yuanye Biotechnology Co., Ltd., catalog number B20703; Tripterygium lactone is a compound with CAS: 38647-11-9, purchased from Shanghai Yuanye Biotechnology Co., Ltd., catalog number B20712.

[0038] The following examples used statistical software to process the data. The experimental results are expressed as mean ± standard deviation, and a one-way ANOVA test was used. P <0.05 (*) indicates statistical significance. P <0.01 (**) indicates high significance. P <0.001 (***) indicates a highly significant difference.

[0039] This study constructed a recombinant strain that produces triptolide through numerous experiments, and achieved de novo synthesis of triptolide using various strategies.

[0040] The biosynthetic pathway of triptolide is shown in the diagram. Figure 1 Specifically, it is as follows: 1) Geraniyl geraniyl pyrophosphate (GGPP), the precursor of triptolide in Saccharomyces cerevisiae, is synthesized via the mevalonate (MVA) pathway. Its first key intermediate, acetyl-coenzyme A (acetyl-CoA), is introduced and catalyzed by acetoacetyl-CoA thiolase (ERG10) and hydroxymethylglutaryl-CoA synthase (ERG13) to generate hydroxymethylglutaryl-CoA (HMG-CoA). 2) Subsequently, HMG-CoA is reduced to the key intermediate methanolate under the catalysis of hydroxymethylglutaryl-CoA reductase (HMGR; the isoenzyme in Saccharomyces cerevisiae is HMG1) or a truncated HMGR1, i.e. tHMG1; methanolate is then converted to isopentenyl pyrophosphate (IPP) and dimethylpropene pyrophosphate (DMAPP) by methanolate kinase ERG12, phosphate methanolate kinase ERG8, methanolate pyrophosphate decarboxylase ERG19, and isopentenyl pyrophosphate isomerase IDI1. 3) Subsequently, the two are catalyzed by farnesyl pyrophosphate synthase ERG20 to generate farnesyl diphosphate (FPP); 4) FPP is catalyzed by geraniol-geraniol diphosphate synthase (GGPPS) or BTS1 to generate geraniol-geraniol pyrophosphate (GGPP); GGPP is catalyzed by tanshinone (… Salvia miltiorrhiza Bunge The diterpenoid synthase SmKSL1 from the source and Coleus formosanus ( ) Coleus forskohlii The key intermediate, tanshinone diene, is generated under the catalysis of a fusion protein constructed from the diterpene synthase CfTPS1; tanshinone diene is then processed by Tripterygium wilfordii (Tripterygium wilfordii). Tripterygium wilfordii The four plant-derived pigment cells P450 enzymes, TwCYP82D274, TwCYP71BE86, TwCYP71BE85, and TwCYP82D213, catalyze the production of triptolide ketones. Specifically, TwCYP82D274 catalyzes the synthesis of 14-hydroxy-ascorbic acid triene from tanshinone diene, TwCYP71BE86 catalyzes the synthesis of triptolide B from 14-hydroxy-ascorbic acid triene, TwCYP71BE85 catalyzes the synthesis of triptolide B from triptolide, and TwCYP82D213 catalyzes the synthesis of triptolide ketones from triptolide.

[0041] Example 1: Method for constructing brewing yeast for producing Tripterygium wilfordii B (I) Obtaining THY04 and THY27 strains and their application in the production of geraniol (GGOH), a hydrolysis product of geraniol geraniol pyrophosphate (GGPP). One copy of GGPPsa (nucleotide sequence SEQ ID No:1) was integrated into the THY02 strain (CEN.PK113) using the CRISPR-Cas9 genome editing method. 5D *THY04 was obtained; the ERG9 gene in the THY04 strain was downregulated (upstream activation sequence of the ERG9 promoter was knocked out), and then a copy of the fusion protein BTS1-GGGGS3-ERG20, a copy of IDI, a copy of the truncated version of HMG1 tHMG1, and a copy of the HMG2 mutant HMG2 were integrated into it. K6R One copy of the ERG8 gene, one copy of the ERG10 gene, one copy of the ERG12 gene, one copy of the ERG13 gene, one copy of the AnACLa gene (nucleotide sequence SEQ ID No:2), one copy of the AnACLb gene (nucleotide sequence SEQ ID No:3), one copy of the RtME gene (nucleotide sequence SEQ ID No:4), one copy of the CTP1 gene, and one copy of the MDH3 signal peptide truncated MDH3. cyto Gene-acquired strain THY27.

[0042] 1. Construction of CRISPR-Cas9 vector and synthesis of repair fragments 1.1. Plasmid p426-URA3-XI-3-gRNA, p426-URA3-ERG9-gRNA, p426-URA3-X-4-gRNA, p426-URA3-X-2-gRNA, p Construction of 426-URA3-XII-5-gRNA, p426-URA3-int14-gRNA, p426-URA3-XII-2-gRNA, p426-URA3-22site-gRNA 1) Using plasmid p426-URA3-gRNA as a template, PaqCI (New England Biolabs, R0745S) Enzyme digestion yielded linearized plasmid p426-URA3-gRNA.

[0043] 2) Mix equal amounts of the gRNA sequence primers of XI-3, ERG9, X-2, X-4, XII-5, int14, XII-2, and 22site in Table 1, treat at 95℃ for 10 min, cool to room temperature, and obtain annealed short nucleic acid sequences, which include a 4bp sequence homologous to the SNR52 promoter, a 20bp gRNA sequence, and a 4bp sequence homologous to the gRNA backbone. The annealed short nucleic acid sequence and the linearized plasmid p426-URA3-gRNA were ligated using T4 ligase to obtain plasmids p426-URA3-XI-3-gRNA, p426-URA3-ERG9-gRNA, p426-URA3-X-4-gRNA, p426-URA3-X-2-gRNA, p426-URA3-XII-5-gRNA, p426-URA3-int14-gRNA, p426-URA3-XII-2-gRNA, and p426-URA3-22site-gRNA.

[0044] 2. Preparation of repair fragments 2.1 The repair fragment XI-3-us-Gal10p-GGPPsa-ADH1t-XI-3-ds, from 5' to 3', consists of the upstream homologous arm XI-3-us, the promoter Gal10p, the gene GGPPsa, the terminator ADH1t, and the downstream homologous arm XI-3-ds. These fragments are overlapped. The overlap method is as follows.

[0045] 1) Preparation of overlap system.

[0046] The overlap system consisted of 25 µL of 2×PrimeStar STAR Max premix 12.5 µL, upstream homologous arm XI-3-us, promoter Gal10p, gene GGPPsa, terminator ADH1t, downstream homologous arm XI-3-ds, and ddH2O.

[0047] 2) Take the above overlap system, react it, and obtain the product.

[0048] The reaction conditions were: denaturation at 98℃ for 2 min; denaturation at 98℃ for 10 s, annealing at 55℃ for 15 s, extension at 72℃ for 30 s, for 15 cycles; and a final extension at 72℃ for 10 min.

[0049] Take 2 μL of the overlap system product obtained in step 2) and use it as a template. Use primers composed of XI-3-us-F and XI-3-ds-R to amplify it by PCR to obtain the repair fragment XI-3-us-Gal10p-GGPPsa-ADH1t-XI-3-ds.

[0050] 2.2 Construct the following repair fragments according to the method in step 2: X-4-us-Gal10p-BTS1-GGGGS3-ERG20-ADH1t-X-4-ds, ERG9-us-ERG9-ds, and int14-us-FBA1t-HMG2. K6R -PRS25Ai-eTDH3p, repair fragment PGK1p-tHMG1-PGI1t-int14-ds, repair fragment XII-5-us-PGK1p-ERG10-TEF1t, repair fragment FBA1p-ERG13-ADH2t, repair fragment TDH3p-ERG8-CYC1t, repair fragment TPI1p-ERG12-ENO2t-XII-5-ds, XII-2-us-ADH1t-AnACLb-Gal1,10p-AnACLa-TEF1t, repair fragment tHXT7p-MDH3 cyto -HIS3t-XII-2-ds, Repair Fragment 22site-us-FBA1t-RtME-TDH3p-PGK1p, CTP1-PGI1t-22site-ds.

[0051] 3. Construction of THY04 and THY27 strains 3.1 Obtaining the THY04 strain 1) CEN.PK113-5D * Methods for preparing yeast competent cells From CEN.PK113-5D * Single colonies were picked from the plate and inoculated into 2 mL of YPD medium. The culture was activated overnight for 12 h. The activated bacterial culture was then inoculated into 20 mL of YPD medium and cultured for 4-5 h. The cells were collected at 4000 rpm, the medium was removed, and the cells were resuspended in sterile water. The cells were collected again at 4000 rpm, the supernatant was removed, and the washing was repeated twice. The cells were then transferred to 1.5 mL EP tubes, 0.1 M LiAc was added, and the cells were resuspended. The cells were aliquoted into 50 µL tubes and collected at 4000 rpm. This bacterial cell is CEN.PK113-5D. * Yeast competent cells. 2) YPD culture medium preparation method: 1% yeast extract, 2% peptone, then add distilled water to a final volume of 1L, sterilize at 121 ℃ for 15 min; finally add glucose filtered through a 0.22 µm filter membrane to a final concentration of 2%.

[0052] 3) Yeast transformation steps, positive transformant verification methods, and gRNA plasmid removal methods. The plasmid p426-URA3-XI-3-gRNA obtained in step 1.1 and the repair fragment XI-3-us-Gal10p-GGPPsa-ADH1t-XI-3-ds obtained in step 2.1 were added to CEN.PK113-5D according to Table 3. * Yeast competent cells.

[0053] Table 3. Yeast competent cell transformation system

[0054] Place a drop of the resuspended solution in the center of an SD-Ura plate and spread it evenly with a spreader until all the bacterial solution is absorbed. Incubate the plate upside down in a 30°C incubator for 2-3 days.

[0055] SC-Complete plates: Add distilled water to 8 g of SC-Complete (a product of Pankino, catalog number YGM003A series) and 20 g of agar powder, then bring the volume to 1 L with distilled water and sterilize at 121 °C for 15 min; finally, add glucose filtered through a 0.22 µm filter membrane to a final concentration of 2%, pour into sterile plates and cool.

[0056] Single colonies were picked from SD-Ura plates and subjected to colony PCR. Specifically, the picked colonies were placed in 20 mmol / L NaOH solution and lysed at 99°C for 20 min, followed by centrifugation for 2 min. The supernatant was collected and used as a template for PCR amplification with specific primers to obtain the PCR product. The reaction conditions were: 98°C pre-denaturation for 3 min; 95°C denaturation for 15 s, 55°C annealing for 15 s, 68°C extension for 30-60 s, for 30 cycles; and a final extension at 68°C for 10 min.

[0057] The PCR amplification products were subjected to agarose gel electrophoresis, and then the following judgment was made: if the PCR amplification product obtained by a certain single clone meets the expected DNA fragment size, then the single clone is a positive clone.

[0058] Four positive clones were randomly selected and streaked on 5-FOA plates. They were then incubated at 30°C for 2-3 days. The resulting colonies were then streaked on both SD-Ura and SC-plates for verification. The colonies were incubated at 30°C for 2-3 days. If the transformant could not grow on SD-Ura medium, it was considered that the transformant had successfully eliminated the URA3 selection marker. The yeast strain obtained on the corresponding SC-plate was named THY04.

[0059] 5-FOA plates: Add distilled water to 8g SC medium and 20g agar powder, then bring the volume to 1L with distilled water and sterilize at 121℃ for 20min; finally, when the temperature of the medium drops to about 40℃, add 5-FOA stock solution (obtained by dissolving 5-fluoroorotic acid (5-FOA) powder in dimethyl sulfoxide (DMSO)) in a sterile laminar flow hood and make the final concentration of 5-FOA 0.1%, then pour into sterile plates and cool.

[0060] 3.2 Obtaining the THY27 strain Following step 3.1, plasmid p426-URA3-X-4-gRNA and repair fragment X-4-us-Gal10p-BTS1-GGGGS3-ERG20-ADH1t-X-4-ds were added sequentially to THY04 yeast competent cells according to Table 3. Positive clones were selected and streaked onto 5-FOA plates. The clones that grew were strains that had lost the gRNA plasmid, resulting in strain THY13. Following step 3.1, plasmid p426-URA3-ERG9-gRNA and repair fragment ERG9-us-ERG9-ds were added sequentially to THY13 yeast competent cells according to Table 3. Positive clones were selected and streaked onto 5-FOA plates. The clones that grew were strains that had lost the gRNA plasmid, resulting in strain THY15. Following step 3.1, plasmid p426-URA3-X-2-gRNA and repair fragment X-2-us-Gal10-IDI-TPS1t-X-2-ds were added to THY15 yeast competent cells according to Table 3. Positive clones were selected and streaked onto 5-FOA plates. The clones that grew were the strain that had lost the gRNA plasmid, resulting in strain THY19. Following step 3.1, plasmid p426-URA3-int14-gRNA and repair fragment int14-us-FBA1t-HMG2 were added... K6RPRS25Ai-eTDH3p and PGK1p-tHMG1-PGI1t-int14-ds were added sequentially to THY19 yeast competent cells according to Table 3. Positive clones were selected and streaked onto 5-FOA plates. The clones that grew were strains that had lost the gRNA plasmid, resulting in strain THY25. Following step 3.1, plasmid p426-URA3-XII-5-gRNA and repair fragments XII-5-us-PGK1p-ERG10-TEF1t, FBA1p-ERG13-ADH2t, TDH3p-ERG8-CYC1t, and TPI1p-ERG12-ENO2t-XII-5-ds were added sequentially to THY25 yeast competent cells according to Table 3. Positive clones were selected and streaked onto 5-FOA plates. The clones that grew were strains that had lost the gRNA plasmid, resulting in strain THY26. Following step 3.1, plasmids p426-URA3-XII-2-gRNA, p426-URA3-22site-gRNA, and repair fragments XII-2-us-ADH1t-AnACLb-Gal1, 10p-AnACLa-TEF1t, tHXT7p-tMDH3-HIS3t-XII-2-ds, 22site-us-FBA1t-RtME-TDH3p-PGK1p, and CTP1-PGI1t-22site-ds were added sequentially to THY26 yeast competent cells according to Table 3. Positive clones were selected and streaked onto 5-FOA plates. The clones that grew were strains that had lost the gRNA plasmids, resulting in strain THY27.

[0061] 4. Application of THY04 and THY27 strains in the production of geraniol geraniol diphosphate (GGOH).

[0062] 4.1 Preparation of Fermentation Culture Medium 1) Preparation of trace metal element mother liquor: 4.5 g / L calcium chloride dihydrate (CaCl2) 4.5 g / L zinc sulfate heptahydrate (ZnSO4) 7H2O), 3 g / L ferrous sulfate heptahydrate (FeSO4) 7H2O), 1 g / L boric acid (H3BO3), 1 g / L manganese chloride tetrahydrate (MnCl2) 4H2O), 0.4 g / L sodium molybdate dihydrate (Na2MoO4) 2H2O), 0.3 g / L cobalt chloride hexahydrate (CoCl2) 6H2O), 0.1 g / L anhydrous copper sulfate (CuSO4) The following ingredients were added: 5H₂O, 0.1 g / L potassium iodide (KI), and 15 g / L ethylenediaminetetraacetic acid (EDTA). All components except EDTA were dissolved in 900 mL of ultrapure water, and the pH was adjusted to 6 to prepare a trace metal solution. The solution will turn deep orange or brown at this point. The solution was then gently heated, and EDTA was added. Finally, the pH was adjusted to 4, the solution volume was adjusted to 1 L, and the solution was sterilized in an autoclave at 121 °C for 20 min. After sterilization, the culture medium was allowed to cool at room temperature. Upon cooling, the color of the culture medium changed from clear green to clear purple. The prepared metal element stock solution was stored at 4 °C.

[0063] 2) Preparation of Vitamin Stock Solution 50 mg / L Biotin, 200 mg / L p-aminobenzoic acid, 1 g / L nicotinic acid, 1 g / L calcium pantothenate, 1 g / L pyridoxine-HCl, 1 g / L thiamine-HCl, and 25 g / L myo-inositol. First, dissolve biotin in 20 mL of 0.1 M sodium hydroxide solution and add 900 mL of ultrapure water. Adjust the pH to 6.5 with hydrochloric acid, then add the vitamins except for myo-inositol, and finally add myo-inositol. Adjust the pH of the culture medium to 6.5 again, and finally adjust the volume to 1 L. After filtration sterilization in a clean bench, store at 4°C.

[0064] Table 4. 1 L of minimal medium (inorganic salts)

[0065] 4.2. Four single clones of THY04 and THY27 were selected for parallel experiments. The experiments performed on each single clone are as follows: 1) Add 2 ml of minimal medium containing 2% glucose to a 12 mL shaker tube (see Table 4 for medium formulation and preparation method), then inoculate a single colony and incubate at 30°C and 450 rpm for 18-24 h on a shaker to obtain a bacterial culture. Inoculate the bacterial culture into a 100 mL shake flask containing 20 ml of minimal medium, and simultaneously add 10% n-dodecane, a two-phase organic reagent, to the shake flask for two-phase fermentation. Incubate at 30°C and 220 rpm on a shaker for 96 h.

[0066] 2) Determine the OD of the fermentation broth 600nm And take the average of 4 parallel experiments.

[0067] 3) Take 10 μL of the two-phase reagent n-dodecane from the culture medium after fermentation in step 3.1.1, dilute it with 90 μL of chromatographically pure ethyl acetate, and perform GC-MS detection (to detect the metabolic components of the organic phase). Take 2 mL of the fermentation broth obtained in step 3.1.1, add 2 mL of chromatographically pure ethyl acetate, sonicate for 30 min, and vortex thoroughly for 20 min; then centrifuge at 13000 rpm for 12 min, and collect the supernatant; finally, dilute the collected supernatant with 1 volume of ethyl acetate and perform GC-MS detection (to detect intracellular metabolic components).

[0068] 4.3 Sample Testing and Results The results are as follows Figure 2 As shown, strain THY27 produced the highest yield of geraniol (GGOH), the hydrolysis product of the precursor geraniol-geraniol pyrophosphate (GGPP), with a post-fermentation GGOH yield of approximately 106.5 mg / L. This represents an approximately 11-fold increase compared to the initial strain THY04. This demonstrates that the constructed strain for producing the precursor geraniol-geraniol pyrophosphate (GGPP) can provide sufficient precursors for the subsequent construction of the strain producing tripterygine B.

[0069] (II) Obtaining THY70, THY75, and THY76 strains and their application in the production of Triptin B 1. Construction of CRISPR-Cas9 vector and synthesis of repair fragments Cut off the salvia miltiorrhiza ( Salvia miltiorrhiza The tSmKSL1 gene and *Cyclocarya pubescens* (from which the gene originates) Coleus forskohlii The CfTPS1 gene from the source was optimized for codons preferred by Saccharomyces cerevisiae, and then the fusion protein tSmKSL1-GGGS-CfTPS1 (nucleotide sequence is SEQ ID No:5) was constructed. It was then integrated into the int17 site of strain THY27 using the CRISPR-Cas9 genome editing method to obtain strain THY38.

[0070] Two copies of the Kunming crabapple ( Tripterygium hypoglaucum )Source: ThCYP82D274 L234M (The ThCYP82D274 gene underwent codon optimization followed by a mutation at amino acid position 234) (nucleotide sequence is SEQ ID No:6) and a copy of Arabidopsis thaliana ( Arabidopsis thaliana The CPR1 gene AtCPR1 (nucleotide sequence SEQ ID No:7) from the source was integrated into the int4 site of the THY38 strain to obtain strain THY56.

[0071] Integrating a copy of the codon-optimized TwCYP71BE86 gene from Tripterygium wilfordii (nucleotide sequence SEQ ID No:8) and a copy of the periwinkle gene (… Catharanthus roseus The CPR gene CrCPR (nucleotide sequence SEQ ID No: 9) from strain THY56 was incorporated into the XI-6 site of strain THY70; a copy of the expression element SkGal10p-TwCYP71BE86-GGGGS3-SOD1 (nucleotide sequence of SkGal10p is SEQ ID No: 10) and a copy of CrCPR were incorporated into the XI-6 site of strain THY56 to obtain strain THY75; another copy of the expression element SkGal10p-TwCYP71BE86-GGGGS3-SOD1 was incorporated into the 17site site of strain THY75 to obtain strain THY76. 1. Construction of CRISPR-Cas9 vector and synthesis of repair fragments 1.1 Following step (I) 1.1, replace primers gRNA-XI-3-F and gRNA-XI-3-R with gRNA sequences at sites int17, int4, XI-2, XII-3, and 17site, respectively (primer sequences are shown in Table 1; the gRNA sequences are located within the primers at each site), to obtain the plasmids p426-URA3-in17-gRNA, p426-URA3-int4-gRNA, p426-URA3-XI-2-gRNA, p426-URA3-XII-3-gRNA, and p426-URA3-17site-gRNA. 1.2 Preparation of Repair Fragments Construct the repair fragment int17-us-GAL10p-tSmKSL-GGGS-CfTPS1-TPS1t-int17-ds and the repair fragment int4-us-GAL10p-ThCYP82D274 according to the method in step (I) 2.1. L234M -TDH2t-int4-ds, XI-2-us-TDH2t-ThCYP82D274 L234M -GAL10p, Gal7p-AtCPR1-TPS1t-XI-2-ds, XII-3-us-PDC1t-TwCYP71BE86 opt -GAL10p、XII-3-us-PDC1t-SOD1 - GGGGS3-TwCYP71BE86 opt -SkGal2p, Gal7p-CrCPR-TPS1t-XII-3-ds, 17site-us-SkGal2p-TwCYP71BE86opt - GGGGS3-SOD1-PDC1t-17site-ds 2. Obtaining strains THY70, THY75, and THY76 2.1 Obtaining THY70 and THY75 strains Following the method in step (I) 3.1, the plasmid p426-URA3-int17-gRNA obtained in step (II) 1.1 and the repair fragment int17-us-GAL10p-tSmKSL-GGGS-CfTPS1-CYC1t-int17-ds obtained in step (II) 1.2 were added to THY27 yeast competent cells according to Table 3. Positive clones were selected and streaked onto 5-FOA plates. The clones that grew were strains that had lost the gRNA plasmid, and strain THY38 was obtained.

[0072] Following the method in step (I) 3.1, combine the plasmids p426-URA3-int4-gRNA and p426-URA3-XI-2-gRNA obtained in step (II) 1.1 with the repair fragment int4-us-GAL10p-ThCYP82D274 obtained in step (II) 1.2. L234M -TDH2t-int4-ds, XI-2-us-TDH2t-ThCYP82D274 L234M -GAL10p and Gal7p-AtCPR1-TPS1t-XI-2-ds were added to THY38 yeast competent cells according to Table 3. Positive clones were selected and streaked onto 5-FOA plates. The clones that grew were strains that had lost the gRNA plasmid, and strain THY56 was obtained.

[0073] Following the method in step (I) 3.1, combine the plasmid p426-URA3-XII-3-gRNA obtained in step (II) 1.1 with the repair fragment XII-3-us-PDC1t-TwCYP71BE86 obtained in step (II) 1.2. opt -GAL10p or XII-3-us-PDC1t-SOD1 - GGGGS3-TwCYP71BE86 opt -SkGal2p, and then the repair fragment Gal7p-CrCPR-TPS1t-XII-3-ds were added to THY56 yeast competent cells in sequence according to Table 3. Positive clones were selected and streaked onto 5-FOA plates. The clones that grew were strains that had lost the gRNA plasmid. The resulting strains were named THY70 and THY75, respectively.

[0074] Following the method in step (I) 3.1, combine the plasmid p426-URA3-17site-gRNA obtained in step (II) 1.1 with the repair fragment 17site-us-SkGal2p-TwCYP71BE86 obtained in step (II) 1.2. opt - GGGGS3-SOD1-PDC1t-17site-ds were added to THY75 yeast competent cells in accordance with Table 3. Positive clones were selected and streaked onto 5-FOA plates. The clones that grew were strains that had lost the gRNA plasmid. The resulting strain was named THY76.

[0075] 3. Application of THY70, THY75, and THY76 strains in the production of Triptin B 3.1 Fermentation of Strains and Sample Preparation 1) Four single clones of strains THY70, THY75, and THY76 were selected for parallel experiments. The experiments performed on each single clone are as follows: 2) Add 2 ml of minimal medium containing 2% glucose to a 12 mL shaker tube (see Table 4 for medium formulation and preparation method), then inoculate with a single colony and incubate at 30°C and 450 rpm for 18-24 h on a shaker to obtain a bacterial suspension. Inoculate the bacterial suspension into a 100 mL shake flask containing 20 ml of minimal medium and incubate at 30°C and 220 rpm on a shaker for 96 h.

[0076] 3) Determine the OD of the fermentation broth 600nm And take the average of 4 parallel experiments.

[0077] 4) Take 3 mL of the fermentation broth obtained in step 3.1, add 3 mL of chromatographic grade ethyl acetate, sonicate for 30 min, and vortex thoroughly for 20 min; then centrifuge at 13000 rpm for 12 min and collect the supernatant; take 2 mL of ethyl acetate for rotary concentration, evaporate to dryness, and redissolve in 100 µL of chromatographic grade methanol, then centrifuge at 13000 rpm for 12 min and collect the supernatant. Take 90 µL for LC-MS detection (the purpose is to detect the metabolic components of the strain).

[0078] 3.2 Sample Testing and Results The results are as follows Figure 3 As shown, strain THY76 produced the highest yield of the substrate triptolide B, with a post-fermentation yield of approximately 6.1 mg / L, a 5.5-fold increase compared to the initial strain THY70. A biosynthetic pathway for downstream triptolide was constructed using THY76 as the substrate strain. Example 2: Obtaining THY175, THY178, and THY179 strains and their application in the production of raffinolactone.

[0079] Northeast Tripterygium wilfordii ( Tripterygium regeli ) source TrCYP71BE85 WT The gene (nucleotide sequence SEQ ID No:11) or the mutant gene TrCYP71BE85 T124G / G369S / F218N The nucleotide sequence of TwCPR5 (SEQ ID No:12), the P450 reductase derived from Tripterygium wilfordii (SEQ ID No:13), and TwCYB5 (SEQ ID No:14) were integrated into strain THY76 to obtain strains THY175 and THY178, respectively. An additional copy of TrCYP71BE85 was also integrated. T124G / G369S / F218N Strain THY179 was obtained from strain THY178.

[0080] 1. Construction of CRISPR-Cas9 vector and synthesis of repair fragments 1.1. Following step (I) 1.1 of Example 1, replace primers gRNA-XI-3-F and gRNA-XI-3-R with gRNA sequences at sites int19 and III-1, respectively (primer sequences are shown in Table 1) to obtain plasmids p426-URA3-int19-gRNA and p426-URA3-III-1-gRNA.

[0081] 1.2. Construct the repair fragment int19-us-FBA1t-TrCYP71BE85 according to the method in step (I) 2.1 of Example 1. WT - SkGal2p-Gal7p, int19-us-FBA1t-TrCYP71BE85 T124G / G369S / F218N - SkGal2p-Gal7p, TwCPR5-TPS1t-Gal1p-TwCYB5-ADH1t-int19-ds, III-1-us-FBA1t-TrCYP71BE85 T124G / G369S / F218N - SkGal2p-III-1-ds.

[0082] 2. Obtaining strains THY175, THY178, and THY179 2.1 Obtaining strains THY175 and THY178 The plasmid p426-URA3-int19-gRNA obtained in step 1.1 and the repair fragment int19-us-FBA1t-TrCYP71BE85 obtained in step 1.2 were combined. WT - SkGal2p-Gal7 or int19-us-FBA1t-TrCYP71BE85 T124G / G369S / F218N - SkGal2p-Gal7p and TwCPR5-TPS1t-Gal1p-TwCYB5-ADH1t-int19-ds were added to THY76 yeast competent cells in accordance with Table 3. Positive clones were selected and streaked onto 5-FOA plates. The clones that grew were strains that had lost the gRNA plasmid. The resulting strains were named THY175 and THY178, respectively.

[0083] 2.2 Obtaining the THY179 strain The plasmid p426-URA3-III-1-gRNA obtained in step 1.1 and the repair fragment III-1-us-FBA1t-TrCYP71BE85 obtained in step 1.2 were combined. T124G / G369S / F218N - SkGal2p-III-1-ds were added to THY178 yeast competent cells in accordance with Table 3. Positive clones were selected and streaked onto 5-FOA plates. The clones that grew were strains that had lost the gRNA plasmid. The resulting strains were named THY179.

[0084] 3. Application of THY175 and THY179 strains in the production of triptophenolide.

[0085] 3.1 Fermentation of the strain and sample preparation are described in Example 1 (II) 3.1.

[0086] 3.2 Sample Testing and Results Test results are shown Figure 4 The results showed that the resveratrol content of strain THY179 increased to 5.1 mg / L, while the resveratrol content of strain THY175 was approximately 85 µg / L. The resveratrol content of strain THY179 increased by approximately 65 times. Using strain THY179 as the substrate, the biosynthetic pathway of triptolide was further constructed.

[0087] Example 3: Obtaining strains THY189, THY191, THY192, THY195, THY202, THY207, THY211, THY221, and THY230 and their application in the production of triptolide. （ Applications of Triptonide Integrating 6 copies of TwCYP82D213 opt (nucleotide sequence is SEQ ID No:15) The gene was inserted into strain THY179 to obtain strain THY189; four copies of the gene N77-274-t77TwCYP82D213 were integrated. opt (CP-6) (nucleotide sequence SEQ ID No:16) was incorporated into strain THY179 to obtain strain THY191; two additional copies of the CP-6 gene were incorporated into strain THY191 to obtain strain THY192; two additional copies of the CP-6 gene and one copy of the membrane steroid-binding protein (MSBP) gene TwMSBP3 (nucleotide sequence SEQ ID No:16) derived from Tripterygium wilfordii were incorporated into strain THY192. No:17) Strawberry THY195 was obtained by integrating THY191 into strain THY191; strain THY202 was obtained by replacing the promoter of the endogenous transcription factor INO2 in strain THY195 with the strong promoter TDH3p and knocking out the gene OPI1; strain THY207 was obtained by integrating one copy of the 5-aminolevulinic acid dehydratase HEM2 at the XI-1 site of strain THY202 and knocking out the heme oxygenase HMX1; strain THY211 was obtained by knocking out the ROX1 gene in strain THY207; strain THY221 was obtained by replacing the promoter of the glucose-6-phosphate dehydrogenase gene ZWF1 in strain THY211 with the strong promoter GAL10p; strain THY230 was obtained by integrating one copy of the URA3 gene at the X-3 site of strain THY221.

[0088] 1. Construction of CRISPR-Cas9 vector and synthesis of repair fragments 1.1 Following step (I) 1.1 of Example 1, replace primers gRNA-XI-3-F and gRNA-XI-3-R with gRNA sequences at sites V-1, XI-8, XI-7, V-3, int16, XII-4, IN02, OPI1, HMX1, XI-1, ZWF1, X-3, and ROX1 respectively (primer sequences are shown in Table 1), obtaining plasmids p426-URA3-V-1-gRNA, p426-URA3-XI-8-gRNA, p426-URA3-XI-7-gRNA, p426-URA3-V-3-gRNA, p426-URA3-int16-gRNA, p426-URA3-XII-4-gRNA, p426-URA3-IN02-gRNA, p426-URA3-OPI1-gRNA, p426-URA3- HMX1-gRNA, p426-URA3-XI-1-gRNA, p426-URA3-ZWF1-gRNA, p426-URA3-X-3-gRNA, p426-URA3-ROX1-gRNA.

[0089] 1.2 Preparation of Repair Fragments 1) Construct the repair fragment V-1-us-SkGal2p-CP-6-ENO2t-V-1-ds according to the method in step (I) 2.1 of Example 1. Replace the CP-6 gene with the unmodified gene TwCYP82D213. opt The repaired fragment V-1-us-SkGal2p-TwCYP82D213 was obtained. opt -ENO2t-V-1-ds.

[0090] 2) Following the method in step (I) 2.1 of Example 1, replace the homologous arms of the V-1-us-SkGal2p-CP-6-ENO2t-V-1-ds repair fragment with the homologous arms of the XI-8, XI-7, XII-4, V-3, and int16 sites respectively to obtain the repair fragments XI-8-us-SkGal2p-CP-6-ENO2t-XI-8-ds, XI-7-us-SkGal2p-CP-6-ENO2t-XI-7-ds, XII-4-us-SkGal2p-CP-6-ENO2t-XII-4-ds, V-3-us-SkGal2p-CP-6-ENO2t-V-3-ds, and in16-us-SkGal2p-CP-6-ENO2t-in16-ds.

[0091] 3) Replace the gene in 2) above with TwCYP82D213 optThe repaired fragment XI-8-us-SkGal2p-TwCYP82D213 was obtained. opt -ENO2t-XI-8-ds,XI-7-us-SkGal2p-TwCYP82D213 opt- ENO2t-XI-7-ds, XII-4-us-SkGal2p-TwCYP82D213 opt -ENO2t-XII-4-ds, in16-us-SkGal2p-TwCYP82D213 opt -ENO2t-in16-ds, V-3-us-SkGal2p-TwCYP82D213 opt -ENO2t-V-3-ds.

[0092] 4) Construct the repair fragments V-3-us-Gal10p-TwMSBP3-ADH2t-SkGal2p, CP-6-ENO2t-V-3-ds, OPI1-us-OPI1-ds, INO2-us-TDH3p-INO2-ds, HMX1-us-HMX1-ds, XI-1-us-TDH3p-HEM2-HEM1t-XI-1-ds, ROX1-us-ROX1-ds, ZWF1-us-Gal10p-ZWF1-ds, and X-3-us-URA3-X-3-ds according to the method in step (I) 2.1.

[0093] 2. Obtaining strains THY189, THY191, THY192, THY195, THY202, THY207, THY211, THY221, and THY230 2.1 Obtaining the THY189 strain 1) Combine the plasmids p426-URA3-V-1-gRNA and p426-URA3-XI-8-gRNA obtained in step 1.1 with the repair fragment XI-8-us-SkGal2p-TwCYP82D213 obtained in step 1.2. opt -ENO2t-XI-8-ds,V-1-us-SkGal2p-TwCYP82D213 opt -ENO2t-V-1-ds were added to THY179 yeast competent cells in accordance with Table 3. Positive clones were selected and streaked onto 5-FOA plates. The clones that grew were strains that had lost the gRNA plasmid. The resulting strains were named THY187.

[0094] 2) Combine the plasmids p426-URA3-XI-7-gRNA and p426-URA3-XII-4-gRNA obtained in step 1.1 with the repair fragment XI-7-us-SkGal2p-TwCYP82D213 obtained in step 1.2. opt- ENO2t-XI-7-ds, XII-4-us-SkGal2p-TwCYP82D213 opt -ENO2t-XII-4-ds were added to THY187 yeast competent cells in accordance with Table 3. Positive clones were selected and streaked onto 5-FOA plates. The clones that grew were strains that had lost the gRNA plasmid. The resulting strains were named THY188.

[0095] 3) Combine the plasmids p426-URA3-V-3-gRNA and p426-URA3-int16-gRNA obtained in step 1.1 with the repair fragment in16-us-SkGal2p-TwCYP82D213 obtained in step 1.2. opt -ENO2t-in16-ds, V-3-us-SkGal2p-TwCYP82D213 opt -ENO2t-V-3-ds were added to THY188 yeast competent cells in accordance with Table 3. Positive clones were selected and streaked onto 5-FOA plates. The clones that grew were strains that had lost the gRNA plasmid. The resulting strains were named THY189.

[0096] 2.2 Obtaining strains THY190, THY191, THY192, and THY195 1) Replace the repair fragment in step 1) of 2.1 above with XI-8-us-SkGal2p-CP-6-ENO2t-XI-8-ds, V-1-us-SkGal2p-CP-6-ENO2t-V-1-ds, and name the resulting strain THY190.

[0097] 2) Replace the repair fragment in step 2) of 2.1 above with XI-7-us-SkGal2p-CP-6-ENO2t-XI-7-ds and XII-4-us-SkGal2p-CP-6-ENO2t-XII-4-ds, and name the resulting strains THY191 respectively.

[0098] 3) Replace the repair fragment in step 3) of 2.1 above with in16-us-SkGal2p-CP-6-ENO2t-in16-ds and V-3-us-SkGal2p-CP-6-ENO2t-V-3-ds, and name the resulting strains THY192 respectively.

[0099] 4) The strains obtained from the repair fragments V-3-us-Gal10p-TwMSBP3-ADH2t-SkGal2p, CP-6-ENO2t-V-3-ds, and in16-us-SkGal2p-CP-6-ENO2t-in16-ds in step 3) of 2.1 above are named THY195.

[0100] 2.3 Obtaining strains THY202, THY207, THY211, THY221, and THY230 1) The plasmids p426-URA3-OPI1-gRNA and p426-URA3-INO2-gRNA obtained in step 1.1 and the repair fragments OPI1-us-OPI1-ds and INO2-us-TDH3p-INO2-ds obtained in step 1.2 were added to THY195 yeast competent cells in accordance with Table 3. Positive clones were selected and streaked onto 5-FOA plates. The clones that grew were the strains that had lost the gRNA plasmids. The resulting strains were named THY202.

[0101] 2) The plasmids p426-URA3-XI-1-gRNA and p426-URA3-HMX1-gRNA obtained in step 1.1, and the repair fragments HMX1-us-HMX1-ds and XI-1-us-TDH3p-HEM2-HEM1t-XI-1-ds obtained in step 1.2, were added to THY202 yeast competent cells in accordance with Table 3. Positive clones were selected and streaked onto 5-FOA plates. The clones that grew were the strains that had lost the gRNA plasmids. The resulting strains were named THY207.

[0102] 3) The plasmid p426-URA3-ROX1-gRNA obtained in step 1.1 and the repair fragment ROX1-us-ROX1-ds obtained in step 1.2 were added to THY207 yeast competent cells in accordance with Table 3. Positive clones were selected and streaked onto 5-FOA plates. The clones that grew were the strains that had lost the gRNA plasmid. The resulting strains were named THY211.

[0103] 4) The plasmid p426-URA3-ZWF1-gRNA obtained in step 1.1 and the repair fragment ZWF1-us-Gal10p-ZWF1-ds obtained in step 1.2 were added to THY211 yeast competent cells in accordance with Table 3. Positive clones were selected and streaked onto 5-FOA plates. The clones that grew were the strains that had lost the gRNA plasmid. The resulting strains were named THY221.

[0104] 5) The plasmid p426-URA3-X-3-gRNA obtained in step 1.1 and the repair fragment X-3-us-URA3-X-3-ds obtained in step 1.2 were added to THY221 yeast competent cells in accordance with Table 3. Positive clones were selected and streaked onto 5-FOA plates. The clones that grew were the strains that had lost the gRNA plasmid. The resulting strains were named THY230.

[0105] 3. Application of THY189, THY191, THY192, THY195, THY202, THY207, THY211, THY221, and THY230 in the production of Tripterygium wilfordii lactone.

[0106] 3.1 Fermentation of the strain and sample preparation are described in Example 1 (II) 3.1.

[0107] 3.2 Sample Testing and Results Test results are shown Figure 5 Six copies of TwCYP82D213 were integrated into strain THY179. opt The yield of triptolide in strain THY189 was only 6.0 µg / L; strain THY191, which integrated 4 copies of the CP-6 gene into strain THY179, increased the yield to 193.9 µg / L; strain THY192, which further integrated 2 copies of the CP-6 gene into strain THY191, increased the yield to 335.5 µg / L; and strain THY195, which integrated 2 copies of the CP-6 gene and 1 copy of TwMSBP3 into strain THY191, significantly increased the yield to 1.85 mg / L.

[0108] Test results are shown Figure 6 In strain THY195, overexpression of INO2 and ZWF1, knockout of OPI1 and HMX1, and integration of one copy of HEM2 and URA3 resulted in strain THY230, which significantly increased the yield of triptolide to 7.3 mg / L, a 2394-fold increase compared to the initial strain THY189.

[0109] Example 4: Scale-up production of triptonide using strain THY230 in a fermenter. 1. Preparation of Fermentation Seed Liquid Add 2 ml of minimal medium containing 2% glucose to a 12 mL shaker tube (see Table 3 for medium formulation and preparation method), then inoculate with a single colony and incubate at 30°C and 450 rpm for 18-24 h on a shaker to obtain the bacterial culture. Add 100 mL of minimal medium containing 2% glucose to a 500 mL shaker flask, then add 2 mL of the bacterial culture and incubate at 30°C and 220 rpm for 18-24 h on a shaker to obtain the seed culture for fermenter cultivation. Detect the bacterial culture density (OD). 600 .

[0110] 2. Preparation of fermentation tank culture medium The initial working volume was 2L, and the 2L culture medium consisted of the following components: 5g / L (NH4)2SO4, 3g / L KH2PO4, 0.5g / L MgSO4·7H2O, 20g / L glucose, 4mL of metal elements, 2mL of trace elements, and 40g glucose. The formulation of the metal elements and trace elements is shown in step 4.1 of Example 1.

[0111] The supplemental culture medium was 1L (5X): 500g / L glucose, 25g / L (NH4)2SO4, 15g / L KH2PO4, 2.5g / L MgSO4·7H2O, 10mL of metal elements, and 5mL of trace elements. The formulation of metal elements and trace elements is shown in step 4.1 of Example 1.

[0112] 3. Fermentation conditions and feed control Fed-batch fermentation of triptolide was carried out in a 5 L bioreactor (instrument model BLBIO-1GC-4, Shanghai). The initial working volume was 2 L. After adding the initial culture medium from step 2, it was autoclaved together with the fed culture medium. After preparing the fermenter, the fermentation seed liquid obtained in step 1 was added to the fermenter to adjust the initial bacterial density (OD). 600 The pH was set to 0.5. The temperature was set to 30°C, and the stirring speed was set to 200 rpm / min. The pH was automatically controlled using 4M KOH and 2M HCl. The feed rate was adjusted by measuring the levels of glucose and ethanol (SBA-40E++ biosensor analyzer). During the fed-batch culture, samples were taken every 24 hours to determine product yield and OD. 600 value.

[0113] 4. Detection of fermentation results Fermentation broth was collected at each time point (diluted according to the OD value of the bacterial broth before extraction), and the detection method is described in step 3.1 of Example 1. Results are shown below. Figure 7The yield of triptolide from strain THY230 was 33.2 mg / L after fermentation in a 5L fermenter for 160 h.

[0114] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A method for constructing recombinant yeast, characterized in that, The recombinant yeast is a recombinant strain obtained by modifying a recipient yeast. The recombinant yeast is: Recombinant strain 1 used for the preparation of triptolide; Recombinant bacteria 2 used to prepare raffinolactone; The recombinant yeast strain is recombinant strain 3 used to prepare Tripterygium wilfordii B; The recipient yeast is a yeast with the following characteristics: S1. Does not contain the URA3 and Gal80 genes; the URA3 gene encodes whey glycoside 5-phosphate decarboxylase, and Gal80 is a key inhibitor of the galactose metabolism regulation system of Saccharomyces cerevisiae. S2 contains the Cas9 gene; The method for constructing the recombinant bacteria 1 includes steps 1) to 4). 1) Knock out the upstream activation sequence of the ERG9 gene promoter in the recipient yeast; the ERG9 gene encodes squalene synthase. 2) The expression cassettes of the GGPPsa gene, BTS1 gene, and ERG20 gene, and HMG2 gene are integrated into the genome of the recipient yeast. K6R Gene expression cassettes, tHMG1 gene expression cassettes, ERG10 gene expression cassettes, ERG13 gene expression cassettes, ERG8 gene expression cassettes, ERG13 gene expression cassettes, AnACLb and AnACLa gene expression cassettes, and MDH3, a gene encoding a truncated signal peptide. cyto Gene expression cassettes, RtME gene expression cassettes, CTP1 gene expression cassettes; tSmKSL and CfTPS1 gene expression cassettes; ThCYP82D274 L234M Gene expression cassettes, AtCPR1 gene expression cassettes; TwCYP71BE86 opt Gene expression cassette; CrCPR gene expression cassette 3) Integrating TrCYP71BE85 into the genome of the recipient yeast. WT Gene expression cassette, mutant TrCYP71BE85 T124G / G369S / F218N Gene expression cassettes, TwCPR5 gene expression cassettes, and TwCYB5 gene expression cassettes; 4) Integrating TwCYP82D213 into the genome of the recipient yeast opt Gene expression cassette, modified TwCYP82D213 opt The expression cassettes of the coding genes N77-274-t77TwCYP82D213opt (CP-6), MSBP3, INO2, HEM2, ZWF1, and URA3 were determined; and the OPI1 and HMX1 genes were knocked out. The method for constructing the recombinant bacteria 2 includes steps 1) to 3). The method for constructing the recombinant bacteria 3 includes steps 1) to 2).

2. The recombinant bacteria obtained by the method of claim 1.

3. A method for preparing the following substance, characterized in that: The substance is triptolide B, triptolide, or triptolide ketone; 1) The recombinant bacteria obtained by the method described in claim 1)-2) of Tripterygium wilfordii B are fermented; 2) The recombinant bacteria obtained by fermentation of the resorcinolone lactone (in steps 1-3 of the method according to claim 1); 3) The triptolide is obtained by fermentation with the recombinant bacteria obtained in steps 1)-4) of the method according to claim 1.

4. The application of the method of claim 1 in any of the following: 1) Application in the preparation of triptolide B; 2) Application in the preparation of raffinolactone; 3) Application in the preparation of triptolide.

5. The use of the recombinant bacteria according to claim 2 in any of the following: 1) Application in the preparation of triptolide B; 2) Application in the preparation of raffinolactone; 3) Application in the preparation of triptolide.

6. A method for producing triptolide, comprising the following steps: fermenting the recombinant bacteria of claim 2, collecting the fermentation product, and obtaining triptolide therefrom.

7. The use of the method of claim 6 in the preparation of triptolide.

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