Saccharomyces cerevisiae engineering bacteria with high yield of dammaradienol and construction method and application thereof

Abstract

The application discloses a saccharomyces cerevisiae engineering bacterium with high dammarendiol yield and a construction method and application thereof. In the present research, a dammarendiol engineering strain is constructed for the first time, efficient synthesis of dammarendiol is realized, the problem of insufficient natural source of rare ginsenoside is solved, the "green synthesis" and sustainable utilization of rare ginsenoside are promoted, a foundation is laid for subsequent construction of a rare ginsenoside cell factory. Moreover, the heterologous synthesis of dammarendiol, a rare ginsenoside skeleton with multiple double bonds, in saccharomyces cerevisiae is realized, which lays a solid foundation for subsequent realization of dammarendiol-type rare ginsenoside biosynthesis. In order to improve the yield of rare ginsenoside, realize "acquisition without planting", promote the sustainable development of traditional Chinese medicine resources, and break through the bottleneck of modernization of traditional Chinese medicine, a foundation is laid.

Description

Technical Field

[0001] This invention relates to the field of fermentation engineering, and in particular to a high-yield dammardienol-producing engineered Saccharomyces cerevisiae strain, its construction method, and its application. Background Technology

[0002] Saccharomyces cerevisiae, with its clear genetic information, simple operation, low cultivation requirements, and characteristics combining those of microorganisms and eukaryotes, exhibits significant advantages in the synthesis of terpenoids. Besides its inherent robustness, Saccharomyces cerevisiae is closer to plants, the main natural source of terpenoids, in terms of intracellular environment, organelles and other subcellular structures, and post-translational modification mechanisms compared to prokaryotes such as Escherichia coli. Furthermore, because Saccharomyces cerevisiae can produce large quantities of terpenoids, it is an excellent new natural product production system. Significant progress has been made in the synthesis of many terpenes in brewing yeasts. Other important terpenes, such as coumaric acid and limonene, can also be synthesized using Saccharomyces cerevisiae and have shown promising applications. Therefore, heterologous synthesis of these substances using Saccharomyces cerevisiae holds great promise. Currently, the understanding of its synthetic regulatory mechanisms is not yet complete, and these issues will be one of the key directions for future development. The MVA pathway provides a direct precursor source for terpenoids. Many important secondary metabolites, such as flavonoids and phenolic acids, can be obtained through this pathway. In recent years, terpenoids and some heterologous natural substances generated downstream of MVA, such as alkaloids, have increasingly been identified as being located on the endoplasmic reticulum (ER), and have shown high yields when combined with ER engineering. However, existing engineered bacteria still suffer from problems such as long fermentation cycles and low product yields.

[0003] Ginsenosides, as the main active ingredients in ginseng, have significant application value. However, their production and preparation currently face problems such as long production cycles, high costs, and difficulty in controlling quality. The use of synthetic biology methods to construct microbial cell factories has proven to be one of the most promising methods for the synthesis of natural products, and extensive research has preliminarily demonstrated the potential of this method for the large-scale production of ginsenosides. Current research has successfully constructed a biosynthetic pathway for protopanaxadiol in Saccharomyces cerevisiae for the first time, increasing the yield of protopanaxadiol by 262 times. Further optimization of the biphasic fermentation process ultimately increased the yield of protopanaxadiol to 1 g / L. Researchers first cloned and identified the key UGT and P450 reductase PgCPR1 required for the synthesis of rare ginsenosides CK, Rh2, Rg3, Rh1, and F1 from ginseng. Based on this, through the construction and optimization of cell factories, a process for producing rare ginsenosides CK, Rh2, Rg3, Rh1, and F1 using monosaccharide fermentation was realized, with current yields exceeding 2 g / L for each.

[0004] However, unlike conventional dammaradienol-type ginsenosides, the biosynthetic pathways of rare ginsenoside skeletons dammaradienol and ginsenoside Rk1 have not been elucidated, and no methods for constructing related engineered strains have been reported. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings and deficiencies of the prior art and provide a brewing yeast strain that produces high levels of dammardienol.

[0006] Another object of the present invention is to provide a method for constructing the above-mentioned engineered Saccharomyces cerevisiae that produces high levels of dammaradienol.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] A high-yield dammaradienol-producing engineered Saccharomyces cerevisiae strain has the following characteristics:

[0009] Starting with Saccharomyces cerevisiae, the tHMGR, ERG12, IDI, ERG13, ERG10, ERG19, and ERG8 genes were overexpressed, and then AtSQS2, AtSQE2, and SmFPS genes were introduced, followed by the introduction of the dammardienol synthase gene.

[0010] A high-squalene-producing engineered Saccharin yeast strain has the following characteristics:

[0011] Starting with Saccharomyces cerevisiae, the tHMGR, ERG12, IDI, ERG13, ERG10, ERG19, and ERG8 genes were overexpressed and then transformed into the AtSQS2, AtSQE2, and SmFPS genes to obtain the strain.

[0012] The overexpression of tHMGR, ERG12, IDI, ERG13, ERG10, ERG19, and ERG8 genes involves integrating these genes into the Gal7 and Gal10-Gal1 sites on the chromosome of Saccharomyces cerevisiae.

[0013] The tHMGR gene is a tHMGR gene expression cassette, which also contains the promoter P. PGK1 and Termination T ADH1 .

[0014] The ERG12 gene mentioned is an ERG12 gene expression cassette, which also contains the promoter P. PDC1 and Termination T ADH2 .

[0015] The IDI gene mentioned is an IDI gene expression cassette, which also contains the promoter P. ENO2and Termination T PDC1 .

[0016] The ERG13 gene mentioned is an ERG13 gene expression cassette, which also contains the promoter P. FBA1 and Termination T TDH2 .

[0017] The ERG10 gene mentioned is an ERG10 gene expression cassette, which also contains the promoter P. TEF1 and Termination T CYC1 .

[0018] The ERG19 gene mentioned is an ERG19 gene expression cassette, which also contains the promoter P. PYK1 and Termination T PGI1 .

[0019] The ERG8 gene mentioned is an ERG8 gene expression cassette, which also contains the promoter P. TDH3 and Termination T TPI1 .

[0020] The aforementioned transfer of the AtSQS2, AtSQE2, and SmFPS genes involves integrating these genes into the His3 locus of the Saccharomyces cerevisiae chromosome.

[0021] The AtSQS2 gene described is an AtSQS2 gene expression cassette, which also contains the promoter P. PGK1 and Termination T ADH1 .

[0022] The AtSQE2 gene described is an AtSQE2 gene expression cassette, which also contains the promoter P. TDH3 and Termination T TPI1 .

[0023] The SmFPS gene is a SmFPS gene expression cassette, which also contains the promoter P. TEF1 and Termination T CYC1 .

[0024] The dammardienol synthase gene is a gene of an enzyme capable of synthesizing dammardienol from squalene as a substrate; preferably at least one of OSC20433, SynOSC20433, and SynOSC20433 mutant.

[0025] The dammarenol synthase gene was introduced via shuttle vector or homologous recombination.

[0026] The shuttle vector is pESC-Ura; preferably, pESC-Ura is digested with BamHI and XohI restriction enzymes, ligated with the dammardienol synthase gene, and then transferred.

[0027] The aforementioned homologous recombination involves integrating the dammaradienol synthase gene into the chromosome of *Saccharomyces cerevisiae*; preferably, it is integrated into the *Saccharomyces cerevisiae* chromosome along with the AtSQE2 gene; more preferably, it involves integrating the gene with the promoter P... PGK1 and Termination T ADH1 The dammardienol synthase gene and the combination of promoter P TDH3 Termination of sub-T TPI1 It was integrated into the Saccharomyces cerevisiae chromosome along with the SynAtSQE2 gene expression cassette.

[0028] The engineered Saccharomyces cerevisiae strain that produces high levels of dammaradienol also replaces the natural promoter of the endogenous gene ERG7 with the weak promoter P. HXT1 .

[0029] The method described involves replacing the natural promoter of the endogenous gene ERG7 with the weak promoter P. HXT1 It was achieved through CRISPR / Cas9 gene editing technology.

[0030] The brewing yeast mentioned is brewing yeast CEN.PK2-1D.

[0031] A method for constructing a high-yield dammaradienol-producing engineered Saccharomyces cerevisiae strain includes the following steps:

[0032] (1) Amplify Gal7-Up, Loxp-Ura-loxp, and P containing homologous fragments. PGK1 -tHMGR-T ADH1 P PDC1 -ERG12-T ADH2 P ENO2 -IDI-T PDC1 P FBA1 -ERG13-T TDH2 P TEF1 -ERG10-T CYC1 P PYK1 -ERG19-T PGI1 P TDH3 -ERG8-T TPI1 Gal7-Down was transferred together with Saccharomyces cerevisiae, and strain GQ01 was obtained through screening.

[0033] (2) Amplification of Gal10-Up, Loxp-Ura-loxp, and P containing homologous fragments PGK1 -tHMGR-T ADH1 P PDC1 -ERG12-T ADH2 P ENO2 -IDI-T PDC1 P FBA1 -ERG13-TTDH2 P TEF1 -ERG10-T CYC1 P PYK1 -ERG19-T PGI1 P TDH3 -ERG8-T TPI1 Gal1-Down was transferred together with strain GQ01, and strain GQ02 was obtained by screening.

[0034] (3) Amplify His3-Up, Loxp-Ura-loxp, and P containing homologous fragments. PGK1 -AtSQS2-T ADH1 P TDH3 -AtSQE2-T TPI1 P TEF1 -SmFPS-T CYC1 His3-Down was transferred into strain GQ02, and strain GQ03 was obtained by screening.

[0035] (4) The dammardienol synthase gene was amplified and linked to the vector backbone obtained by digesting the vector plasmid pESC-Ura with restriction fast digestion enzymes BamHI and XohI. The gene was then transformed into strain GQ03 and screened to obtain a high-dammardienol-producing Saccharomyces cerevisiae engineered strain.

[0036] Alternatively, amplify pcfb2988-Up and P containing homologous fragments. TDH3 -SynAtSQE2-T TPI1 The dammaradienol synthase gene and pcfb2988-Down were transferred into strain GQ03, and a high-yielding Saccharomyces cerevisiae engineered strain was obtained by screening.

[0037] The Gal7-Up and Gal7-Down segments are located at the Gal7 site in the Saccharomyces cerevisiae genome, wherein the nucleotide sequence of Gal7-Up is shown in SEQ ID NO.3 and the nucleotide sequence of Gal7-Down is shown in SEQ ID NO.4.

[0038] The Gal10-Up and Gal1-Down segments are located at the Gal10-Gal1 site in the genome of Saccharomyces cerevisiae, wherein the nucleotide sequence of Gal10-Up is shown in SEQ ID NO.5 and the nucleotide sequence of Gal1-Down is shown in SEQ ID NO.6.

[0039] The His3-Up and His3-Down segments are located at the His3 site in the Saccharomyces cerevisiae genome, wherein the nucleotide sequence of His3-Up is shown in SEQ ID NO.7 and the nucleotide sequence of His3-Down is shown in SEQ ID NO.8.

[0040] The pcfb2988-Up is an upstream fragment located at the Ty1cons2 site of the Saccharomyces cerevisiae genome, and the pcfb2988-Down is a downstream fragment carrying the selection marker gene Trp and located at the Ty1cons2 site of the Saccharomyces cerevisiae genome. The nucleotide sequence of pcfb2988-Up is shown in SEQ ID NO.9, and the nucleotide sequence of pcfb2988-Down is shown in SEQ ID NO.10.

[0041] The Loxp-Ura-loxp mentioned above is a fragment containing the selection marker gene Ura.

[0042] The P mentioned PGK1 -tHMGR-T ADH1 The tHMGR gene expression cassette also contains the promoter P PGK1 and Termination T ADH1 .

[0043] The P mentioned PDC1 -ERG12-T ADH2 The ERG12 gene expression cassette also contains the promoter P. PDC1 and Termination T ADH2 .

[0044] The P mentioned ENO2 -IDI-T PDC1 The IDI gene expression cassette also contains the promoter P. ENO2 and Termination T PDC1 .

[0045] The P mentioned FBA1 -ERG13-T TDH2 The ERG13 gene expression cassette also contains the promoter P. FBA1 and Termination T TDH2 .

[0046] The P mentioned TEF1 -ERG10-T CYC1 The ERG10 gene expression cassette also contains the promoter P. TEF1 and Termination T CYC1 .

[0047] The P mentioned PYK1 -ERG19-T PGI1 The ERG19 gene expression cassette also contains the promoter P.PYK1 and Termination T PGI1 .

[0048] The P mentioned TDH3 -ERG8-T TPI1 The ERG8 gene expression cassette also contains the promoter P. TDH3 and Termination T TPI1 .

[0049] The P mentioned PGK1 -AtSQS2-T ADH1 The AtSQS2 gene expression cassette also contains the promoter P. PGK1 and Termination T ADH1 .

[0050] The P mentioned TDH3 -AtSQE2-T TPI1 The AtSQE2 gene expression cassette also contains the promoter P. TDH3 and Termination T TPI1 .

[0051] The P mentioned TEF1 -SmFPS-T CYC1 The SmFPS gene expression cassette also contains the promoter P TEF1 and Termination T CYC1 .

[0052] The P mentioned TDH3 -SynAtSQE2-T TPI1 The SynAtSQE2 gene expression cassette also contains the promoter P. TDH3 and Termination T TPI1 .

[0053] The nucleotide sequence of the tHMGR gene is shown in the NCBI database sequence accession number NM_001182434.1.

[0054] The nucleotide sequence of the ERG12 gene is shown in the NCBI database sequence accession number NM_001182715.1.

[0055] The nucleotide sequence of the IDI gene is shown in the NCBI database sequence accession number NM_001183931.1.

[0056] The nucleotide sequence of the ERG13 gene is shown in the NCBI database sequence accession number NM_001182489.1.

[0057] The nucleotide sequence of the ERG10 gene is shown in the NCBI database sequence accession number NM_001183842.1.

[0058] The nucleotide sequence of the ERG19 gene is shown in the NCBI database sequence accession number NM_001183220.1.

[0059] The nucleotide sequence of the ERG8 gene is shown in the NCBI database sequence accession number NM_001182727.1.

[0060] The nucleotide sequence of the AtSQS2 gene is shown in the NCBI database sequence accession number NM_119631.3.

[0061] The nucleotide sequence of the AtSQE2 gene is shown in the NCBI database sequence accession number NM_127848.4.

[0062] The nucleotide sequence of the SmFPS gene is shown in the NCBI database sequence accession number HQ687768.1.

[0063] The nucleotide sequence of the SynAtSQE2 gene is shown in SEQ ID NO.11.

[0064] The nucleotide sequence of the screening marker gene Trp is shown in the NCBI database sequence accession number NP_010290.3.

[0065] The nucleotide sequence of the screening marker gene Ura is shown in the NCBI database sequence accession number 856692.

[0066] The promoter P PGK1 The nucleotide sequence is shown as positions 124102 to 124853 in the NCBI database sequence registry number CP135951.1.

[0067] The promoter P PDC1 The nucleotide sequence is shown as positions 216126 to 216925 in the NCBI database sequence registry number CP135955.1.

[0068] The promoter P ENO2 The nucleotide sequence is shown as positions 10603930 to 10604929 in the NCBI database sequence registry number CP029160.1.

[0069] The promoter P FBA1 The nucleotide sequence is shown as positions 5910688 to 5911509 in the NCBI database sequence registry number CP029160.1.

[0070] The promoter P TEF1The nucleotide sequence is shown as positions 707052 to 707481 in the NCBI database sequence registry number CP029160.1.

[0071] The promoter P PYK1 The nucleotide sequence is shown as positions 6308912 to 6309911 in the NCBI database sequence registry number CP029160.1.

[0072] The promoter P TDH3 The nucleotide sequence is shown as positions 9954286 to 9955085 in the NCBI database sequence registry number CP029160.1.

[0073] The promoter P HXT1 The nucleotide sequence is shown as positions 292634 to 293756 in the NCBI database sequence registry number CP036477.1.

[0074] The terminator T ADH1 The nucleotide sequence is shown as positions 4671969 to 4672126 in the NCBI database sequence registry number CP029160.1.

[0075] The terminator T ADH2 The nucleotide sequence is shown as positions 3398555 to 3398954 in the NCBI database sequence registry number CP029160.1.

[0076] The terminator T PDC1 The nucleotide sequence is shown as positions 3660519 to 3660915 in the NCBI database sequence registry number CP029160.1.

[0077] The terminator T TDH2 The nucleotide sequence is shown as positions 11545244 to 11545644 in the NCBI database sequence registry number CP029160.1.

[0078] The terminator T CYC1 The nucleotide sequence is shown as positions 1301 to 1607 in the NCBI database sequence accession number KC879308.1.

[0079] The terminator T PGI1 The nucleotide sequence is shown as positions 8108691 to 8109092 in the NCBI database sequence registry number CP029160.1.

[0080] The terminator T TPI1The nucleotide sequence is shown as positions 529841 to 530242 in the NCBI database sequence registry number CP134412.1.

[0081] The dammardienol synthase gene is a gene of an enzyme capable of synthesizing dammardienol from squalene as a substrate; preferably at least one of OSC20433, SynOSC20433, and SynOSC20433 mutant.

[0082] The nucleotide sequence of OSC20433 is shown in SEQ ID NO.1.

[0083] The nucleotide sequence of SynOSC20433 is shown in SEQ ID NO.2.

[0084] The mutant of SynOSC20433 is a nucleotide sequence obtained by mutation based on the nucleotide sequence shown in SEQ ID NO.4, and is one of the following: 7-9 positions mutated to AGG, 256-258 positions mutated to GAC, 271-273 positions mutated to ACT, 436-438 positions mutated to ACA, 502-504 positions mutated to TCT, 820-822 positions mutated to AGA, 862-864 positions mutated to GAA, 1045-1047 positions mutated to AGA, 1297-1299 positions mutated to GAT, and 1318-1320 positions mutated to AGA. The mutations at positions 1528-1530 are ACT, 1597-1599 are GTG, 1630-1632 are GAG, 1690-1692 are TGC, 1762-1764 are GAT, 1960-1962 are GAT, 2110-2112 are AGA, 2185-2187 are TGC, 2194-2196 are TCT, 2206-2208 are TAT, and 2212-2214 are ACA.

[0085] A method for producing dammardienol includes the following steps:

[0086] (1) Inoculate the engineered Saccharomyces cerevisiae with high dammardienol production into the culture medium for culture;

[0087] (2) After pyrolysis and extraction, the culture medium after cultivation yielded dammardienol.

[0088] The culture described in step (1) is carried out in defective SD-Trp liquid medium at 28-32°C for 2-3 days.

[0089] The extraction described in step (2) involves lysing the bacterial cells and then extracting with an organic solvent; preferably, hexane is used for extraction.

[0090] The dammardienol mentioned is Dammara-20,24-dien-3β-ol.

[0091] The present invention has the following advantages and effects compared with the prior art:

[0092] This study is the first to construct an engineered strain of dammaradienol, achieving efficient synthesis of dammaradienol and solving the problem of insufficient natural sources of rare ginsenosides. This promotes the "green synthesis" and sustainable utilization of rare ginsenosides, laying the foundation for the subsequent construction of rare ginsenoside cell factories. Furthermore, it achieved the heterologous synthesis of dammaradienol, a rare ginsenoside backbone with multiple double bonds, in *Saccharomyces cerevisiae*, laying a solid foundation for the subsequent biosynthesis of dammaradienol-type rare ginsenosides. This lays the foundation for increasing the yield of rare ginsenosides, achieving "uncultivated" production of rare ginsenosides, and promoting the sustainable development of traditional Chinese medicine resources. Attached Figure Description

[0093] Figure 1 This is a comparison diagram of wild strain CEN.PK2-1D and strain GQ01.

[0094] Figure 2 This is a comparison diagram of strains GQ01 and GQ02.

[0095] Figure 3 This is a comparison chart of strains GQ03 and GQ02.

[0096] Figure 4 This is a diagram of the pESC-Ura-OSC20433 expression vector and the results of gel electrophoresis verification.

[0097] Figure 5 This is a graph showing the GC-MS detection results of the pESC-Ura-OSC20433 product.

[0098] Figure 6 This is a GC-MS detection result of the target product peak of OSC20433 after preparation and purification.

[0099] Figure 7 This is a diagram showing the structural identification results of P1, the target product of OSC20433.

[0100] Figure 8 This is a comparison diagram of strains AE2001 and GQ03.

[0101] Figure 9 This is a comparison chart of strains AE2001 and AE2002.

[0102] Figure 10 This is a graph showing the relative yield results of the OSC20433 mutant and the wild-type control.

[0103] Figure 11 This is a comparison chart of strains AE2002 and AE2003.

[0104] Figure 12 This is a comparison chart of strains AE2003 and AE2004.

[0105] Figure 13 This is a graph showing the test results of strain AE2004 in a 2L fermenter. Detailed Implementation

[0106] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0107] Unless otherwise specified in the following implementation plan, the test conditions are generally as per standard test conditions or the test conditions recommended by the reagent company. Unless otherwise specified, all materials and reagents used are commercially available.

[0108] The plasmid pLB-P used in this invention PGK1 -tHMGR-T ADH1 pLB-P PDC1 -ERG12-T ADH2 pLB-P ENO2 -IDI-T PDC1 pLB-P FBA1 -ERG13-T TDH2 pLB-P TEF1 -ERG10-T CYC1 pUC19-P PYK1 -ERG19-T PGI1 pUC19-P TDH3 -ERG8-T TPI1pESC-Ura-AtSQS2, pESC-Ura-AtSQE2, and pESC-Ura-SmFPS were all stored in the laboratory of Guangzhou University of Chinese Medicine. Among them, pLB plasmid was obtained by constructing the promoter, gene, and terminator sequentially onto pLB plasmid using the pLB zero-background rapid cloning kit without multiple cloning sites. pcfb2988-Trp is a pcfb2988 plasmid including the Trp selection marker. The same applies to pUC19 and pESC-Ura plasmids. Unless otherwise specified, the expression cassettes are all located between the EcoRI and Hind III restriction sites. The plasmid construction methods are common existing techniques. For reference, please refer to literature such as "Lin Tingting, Wang Dong, Dai Zhubo, et al. Creation of a cell factory for the fermentation production of lupeol in Saccharomyces cerevisiae [J]. Chinese Journal of Traditional Chinese Medicine, 2016, 41(6):8. DOI:10.4268 / cjcmm20160606." etc.

[0109] Example 1: Construction of a high-squalene-producing Saccharin-producing Saccharin-producing Saccharin-producing chassis strain

[0110] 1.1 Construction of yeast strain GQ01

[0111] (1) Using plasmid pLB-P PGK1 -tHMGR-T ADH1 Using primers P with homologous arms from Table 1 as templates PGK1 -F and T ADH1 -R is used for cloning, amplifying fragment P. PGK1 -tHMGR-T ADH1 ; using plasmid pLB-P PDC1 -ERG12-T ADH2 Using primers P with homologous arms from Table 1 as templates PDC1 -F and T ADH2 -R is used for cloning, amplifying fragment P. PDC1 -ERG12-T ADH2 ; using plasmid pLB-P ENO2 -IDI-T PDC1 Using primers P with homologous arms from Table 1 as templates ENO2 -F and T PDC1 -R is used for cloning, amplifying fragment P. ENO2 -IDI-T PDC1 ; using plasmid pLB-P FBA1 -ERG13-T TDH2 Using primers P with homologous arms from Table 1 as templates FBA1 -F and T TDH2 -R is used for cloning, amplifying fragment P. FBA1 -ERG13-T TDH2 ; using plasmid pLB-P TEF1 -ERG10-TCYC1 Using primers P with homologous arms from Table 1 as templates TEF1 -F and T CYC1 -R is used for cloning, amplifying fragment P. TEF1 -ERG10-T CYC1 ; using plasmid pUC19-P PYK1 -ERG19-T PGI1 Using primers P with homologous arms from Table 1 as templates PYK1 -F and T PGI1 -R is used for cloning, amplifying fragment P. PYK1 -ERG19-T PGI1 ; using plasmid pUC19-P TDH3 -ERG8-T TPI1 Using primers P with homologous arms from Table 1 as templates TDH3 -F and T TPI1 -R is used for cloning, amplifying fragment P. TDH3 -ERG8-T TPI1 Using the genome of wild-type strain CEN.PK2-1D as a template, the Gal7-Up fragment was cloned using primers Gal7-up-F and Gal7-up-R (Table 1), and the Gal7-Down fragment was cloned using primers Gal7-down-F and Gal7-down-R. Using plasmid pESC-Ura-loxp as a template, the Loxp-Ura-loxp fragment was cloned using primers Loxp-Ura-loxp-F and Loxp-Ura-loxp-R (Table 1). After PCR amplification, the 10 cloned fragments were detected by gel electrophoresis. If a correct positive band appeared, the fragments were purified using a kit. After purification, the 10 fragments were concentrated to 30 μL.

[0112] Table 1 Primers for constructing the upstream expression module of MVA

[0113]

[0114] (2) The 10 purified fragments Gal7-Up, Loxp-Ura-loxp, P PGK1 -tHMGR-T ADH1 P PDC1 -ERG12-T ADH2 P ENO2 -IDI-T PDC1 P FBA1 -ERG13-T TDH2 P TEF1 -ERG10-T CYC1 P PYK1 -ERG19-T PGI1 PTDH3 -ERG8-T TPI1 Gal7-Down was transformed into wild-type strain CEN.PK2-1D using the lithium acetate yeast transformation method and plated on SD-Ura solid medium, waiting 2-3 days. After single colonies grew, positive single colonies were picked for propagation and induced expression (deficient SD-Ura medium, glucose-induced expression, 30℃). After 2-3 days of expression, the bacterial cells were extracted, lysed, and extracted with n-hexane. The squalene yield was detected by GC-MS. GC-MS analysis showed that the squalene yield of strain GQ01 was significantly increased, reaching 277.14±16.42 mg / L, nearly 34 times higher than that of the wild type. Figure 1 As shown in B.

[0115] 1.2 Construction of yeast strain GQ02

[0116] (1) Using the genome of wild-type strain CEN.PK2-1D as a template, the Gal10-Up fragment was cloned using primers Gal10-up-F and Gal10-up-R in Table 2, and the Gal1-Down fragment was cloned using primers Gal1-down-F and Gal1-down-R. Using plasmid pESC-Ura-loxp as a template, the Loxp-Ura-loxp fragment was cloned using primers Gal10-Loxp-Ura-F in Table 2 and primers Loxp-Ura-loxp-R in Table 1. The remaining MVA upstream pathway genes were amplified according to the method in 1.1. After PCR amplification, the 10 fragments were detected, purified, and recovered, and then concentrated to 30 μL.

[0117] Table 2 Primers for constructing the upstream expression module of the MVA pathway overexpression at Gal1-Gal10 sites.

[0118]

[0119] (2) Combine the 10 fragments Gal10-Up, Loxp-Ura-loxp, P PGK1 -tHMGR-T ADH1 P PDC1 -ERG12-T ADH2 P ENO2 -IDI-T PDC1 P FBA1 -ERG13-T TDH2 P TEF1 -ERG10-T CYC1 P PYK1 -ERG19-T PGI1 P TDH3 -ERG8-T TPI1Gal1-Down was transformed into strain GQ01 using the lithium acetate yeast transformation method and plated on SD-Ura solid medium, waiting 2-3 days. After single colonies grew, it was propagated and induced to express using the method described in 1.1. After 2-3 days of expression, extraction and detection were performed to determine the squalene content in the bacterial cells. GC-MS analysis showed that the squalene yield of strain GQ02 was slightly increased, reaching 334.20±14.40 mg / L. Figure 2 As shown in B.

[0120] 1.3 Construction of yeast strain GQ03

[0121] (1) Using plasmid pLB-P PGK1 -tHMGR-T ADH1 Using the primers FXPCR-P in Table 3 as templates PGK1 -R and FXPCR-T ADH1 -F was used for cloning, amplifying the vector fragment pLB-P. PGK1 -T ADH1 Using plasmid pESC-Ura-AtSQS2 as a template, the gene fragment AtSQS2 was amplified by cloning with primers AtSQS2-F and AtSQS2-R (containing homologous arms) listed in Table 3. The vector fragment pLB-P PGK1 -T ADH1 Homologous recombination was performed with the AtSQS2 gene fragment. The homologous product was then used to transform *E. coli* DH5α competent cells. After platelets formed, *E. coli* colony PCR was performed, and positive single clones were selected for sequencing. After successful sequencing, plasmid was extracted to obtain plasmid pLB-P. PGK1 -AtSQS2-T ADH1 .

[0122] (2) Using plasmid pUC19-P TDH3 -ERG8-T TPI1 Using the primers FXPCR-P in Table 3 as templates TDH3 -R and FXPCR-T TPI1 Cloning was performed using the -F method, amplifying the vector fragment pUC19-P. TDH3 -T TPI1 Using plasmid pESC-Ura-AtSQE2 as a template, the gene fragment AtSQE2 was amplified by cloning with primers AtSQE2-F and AtSQE2-R (containing homologous arms) listed in Table 3. The vector fragment pUC19-P... TDH3 -T TPI1 After homologous recombination, transformation, screening, and sequencing verification of the AtSQE2 gene fragment, plasmid was extracted to obtain plasmid pUC19-P. TDH3 -AtSQE2-T TPI1 .

[0123] (3) Using plasmid pLB-P TEF1 -ERG10-T CYC1 Using the primers FXPCR-P in Table 3 as templates TEF1 -R and FXPCR-T CYC1 -F was used for cloning, amplifying the vector fragment pLB-P. TEF1 -T CYC1 Using plasmid pESC-Ura-SmFPS as a template, the gene fragment SmFPS was amplified by cloning with primers SmFPS-F and SmFPS-R (containing homologous arms) listed in Table 3. The vector fragment pLB-P was then... TEF1 -T CYC1 After homologous recombination, transformation, screening, and sequencing verification of the gene fragment SmFPS, plasmid was extracted to obtain plasmid pLB-P. TEF1 -SmFPS-T CYC1 .

[0124] (4) Using the plasmid pLB-P obtained in step (1) PGK1 -AtSQS2-T ADH1 Using primer P with homologous arms from Table 3 as a template PGK1 -F and T ADH1 -R1 is used for cloning, amplifying fragment P. PGK1 -AtSQS2-T ADH1 The plasmid pUC19-P obtained in step (2) TDH3 -AtSQE2-T TPI1 Using primer P with homologous arms from Table 3 as a template TDH3 -F1 and T ADH1 -R is used for cloning, amplifying fragment P. TDH3 -AtSQE2-T TPI1 The plasmid pLB-P obtained in step (3) TEF1 -SmFPS-T CYC1 Using primer P with homologous arms from Table 3 as a template TEF1 -F and T CYC1 -R1 is used for cloning, amplifying fragment P. TEF1 -SmFPS-T CYC1Using the wild-type CEN.PK2-1D genome as a template, the His3-Up fragment was cloned using primers His3-up-F and His3-up-R (Table 3), and the His3-Down fragment was cloned using primers His3-down-F and His3-down-R. Using the plasmid Loxp-Ura-loxp as a template, the Loxp-Ura-loxp fragment was cloned using primers Loxp-Ura-loxp-F1 and Loxp-Ura-loxp-R (Table 1). After PCR amplification, the six fragments were detected, purified, recovered, and concentrated to 30 μL.

[0125] Table 3 Primers for constructing the downstream expression module of the MVA pathway overexpression at His3 site.

[0126]

[0127]

[0128] (5) Combine the 6 fragments His3-Up, Loxp-Ura-loxp, P PGK1 -AtSQS2-T ADH1 P TDH3 -AtSQE2-T TPI1 P TEF1 -SmFPS-T CYC1 His3-Down was transformed into strain GQ02 using the lithium acetate yeast transformation method and plated on SD-Ura solid medium, waiting 2-3 days. After single colonies grew, propagation and expression induction were performed according to the method in 1.1. After 2-3 days of expression, extraction and detection were performed to determine the squalene production in the cells. GC-MS analysis showed that the squalene production of strain GQ03 increased to 421.58±33.89 mg / L, a 50-fold increase compared to the wild-type strain. Figure 3 As shown in B.

[0129] Example 2: Identification of OSC20433 metabolites and construction of engineered yeast strains

[0130] 2.1 Biochemical Functional Identification of OSC20433

[0131] 2.1.1 Construction of the pESC-Ura-OSC20433 expression vector

[0132] The inventors discovered a protein involved in the biosynthesis of dammaradienol from Artemisia argyi H.Lév. & Vaniot (Asteraceae family), named OSC20433, with the corresponding nucleotide sequence shown in SEQ ID NO.2. A commercial company was commissioned to synthesize a gene-containing vector based on the OSC20433 gene sequence. Cloning was performed using primers OSC20433-F and OSC20433-R (Table 4), amplifying the target gene fragment OSC20433. The vector plasmid pESC-Ura was digested using the restriction enzymes BamHI and XohI. Homologous ligation of the target gene fragment and the digested vector plasmid was performed, followed by transformation into competent E. coli cells. Colony PCR verification was performed using primers F1-F and F1-R (Table 4), and positive single-clone transformants were sent for sequencing. After successful sequencing, plasmid extraction was performed to obtain plasmid pESC-Ura-OSC20433, as shown in the plasmid map. Figure 4 As shown in Figure A.

[0133] Table 4 Primers for constructing the OSC20433 expression vector plasmid

[0134]

[0135] 2.1.2 Heterologous functional characterization of OSC20433 yeast

[0136] The successfully constructed yeast expression vector pESC-Ura-OSC20433 was transformed into the high-squalene-producing chassis strain GQ03 using the lithium acetate conversion method and plated on SD-Ura solid medium. After single colonies grew on the plate, yeast colony PCR was performed for verification, and positive single colonies were selected. High-density fermentation was carried out in a 5L fermenter using YNB-deficient medium, and galactose was added to induce expression. Fermentation was completed after 5-7 days. The fermentation cells and fermentation supernatant were extracted with n-hexane, followed by separation and purification by reverse-phase chromatography using a methanol-water gradient elution. The 52-62 fraction was collected and purified by normal-phase silica gel column chromatography using a 35:1 petroleum ether-ethyl acetate mobile phase. The 21 fraction was purified using 78% isopropanol-water as the mobile phase, and evaporated to dryness to obtain the target product sample P1.

[0137] The purified sample P1 was subjected to... 1 H-NMR, 13 The C-NMR spectrum was analyzed, and the measured data were imported into a database for querying. The retrieved data was compared and analyzed with the processed data. The product peak P1 was identified as Dammara-20,24-dien-3β-ol, confirming that the OSC20433 gene is dammaradienol synthase. The NMR data are as follows: Figure 7 As shown.

[0138] 2.1.3 Construction of yeast strain AE2001

[0139] (1) Using plasmid pESC-Ura-OSC20433 as a template, primers P with homologous arms as shown in Table 5 were used. PGK1 -OSC20433-F and OSC20433-T ADH1 Cloning was performed using -R, amplifying the target gene fragment OSC20433; plasmid pLB-P was used. PGK1 -tHMGR-T ADH1 Using the primers FXPCR-P in Table 5 as templates PGK1 -R and FXPCR-T ADH1 -F was used for cloning, amplifying the vector fragment pLB-P. PGK1 -T ADH1 The vector fragment pLB-P PGK1 -T ADH1 After homologous recombination, transformation, screening, and sequencing verification of the gene fragment OSC20433, plasmid was extracted to obtain plasmid pLB-P. PGK1 -OSC20433-T ADH1 .

[0140] (2) Using plasmid pUC19-P TDH3 -AtSQE2-T TPI1 Using the primer pcfb2988-P with homologous arms from Table 5 as a template... TDH3 -F and pcfb2988-P PGK1 -R is used for cloning, amplifying fragment P. TDH3 -AtSQE2-T TPI1 plasmid pLB-P prepared in step (1) PGK1 -OSC20433-T ADH1 Using the primer pcfb2988-P with homologous arms from Table 5 as a template... PGK1 -F and pcfb2988-T ADH1 -R is used for cloning, amplifying fragment P. PGK1 -OSC20433-T ADH1 Using plasmid pcfb2988-Trp as a template, primers pcfb2988-up-P with homologous arms (listed in Table 5) were used. TDH3 The fragment pcfb2988-Up was cloned using primers -F and pcfb2988-up-R, and then cloned using primers pcfb2988-T with homologous arms as shown in Table 5. ADH1 The Trp-tagged fragment pcfb2988-Down was cloned from -down-F and pcfb2988-down-R. After PCR amplification, the four fragments were detected, purified, recovered, and concentrated to 30 μL.

[0141] Table 5 Primers for OSC20433 overexpression gene cloning module

[0142]

[0143] (3) Combine the four fragments pcfb2988-Up, P TDH3 -AtSQE2-T TPI1 P PGK1 -OSC20433-T ADH1 pcfb2988-Down was transformed into strain GQ03 using the lithium acetate yeast transformation method, plated on SD-Trp solid medium, and left for 2-3 days. After single colonies grew, yeast colony PCR was performed for verification, and the positive single colony was named strain AE2001.

[0144] (4) Select positive monoclonal colonies and culture them in 3 ml of SD-Trp-deficient medium for 8-12 h. Then, transfer them at a ratio of 2% to 50 ml of shake flasks for glucose-induced expression for 3 days. Take samples every 12 h to detect their colony density (OD). 600 After expression, the bacterial cells were lysed, and the yields of squalene and dammaradienol in the cells were detected. The GC-MS results are shown below. Figure 8 As shown, a large amount of precursor squalene remained, and the catalytic efficiency of the OSC20433 cyclase was low, resulting in a low conversion rate and a dammaradienol yield of only 31.58 ± 1.65 mg / L. To further improve the dammaradienol yield in strain AE2001, we sent the two genes AtSQE2 and OSC20433 integrated in this step to General Biotechnology (Anhui) Co., Ltd. for codon optimization to improve their catalytic efficiency and obtain a higher yield of dammaradienol.

[0145] 2.1.4 Construction of yeast strain AE2002

[0146] (1) Using the codon-optimized plasmid pUC57-SynOSC20433 as a template, the target gene fragment SynOSC20433 (SEQ ID NO.4) was amplified by cloning with primers SynOSC20433-F and SynOSC20433-R containing homologous arms as shown in Table 6; plasmid pLB-P PGK1 -tHMGR-T ADH1 Using the primers FXPCR-P in Table 6 as templates PGK1 -R and FXPCR-T ADH1 -F was used for cloning, amplifying the vector fragment pLB-P. PGK1 -T ADH1 The vector fragment pLB-P PGK1 -TADH1 After homologous recombination, transformation, screening, and sequencing verification of the gene fragment SynOSC20433, plasmid was extracted to obtain plasmid pLB-P. PGK1 -SynOSC20433-T ADH1 .

[0147] (2) Using the codon-optimized plasmid pUC57-SynAtSQE2 as a template, the target gene fragment SynAtSQE2 was amplified by cloning with primers SynAtSQE2-F and SynAtSQE2-R containing homologous arms as shown in Table 6; the primer FXPCR-P in Table 6 was used. TDH3 -R and FXPCR-T TPI1 Cloning was performed using the -F method, amplifying the vector fragment pUC19-P. TDH3 -T TPI1 ; the vector fragment pUC19-P TDH3 -T TPI1 After homologous recombination, transformation, screening, and sequencing verification of the gene fragment SynAtSQE2, plasmid was extracted to obtain plasmid pUC19-P. TDH3 -SynAtSQE2-T TPI1 .

[0148] (3) Using the plasmid pUC19-P obtained in step (2) TDH3 -SynAtSQE2-T TPI1 Using the primer pcfb2988-P with homologous arms from Table 5 as a template... TDH3 -F and pcfb2988-P PGK1 -R is used for cloning, amplifying fragment P. TDH3 -SynAtSQE2-T TPI1 The plasmid pLB-P obtained in step (1) PGK1 -SynOSC20433-T ADH1 Using the primer pcfb2988-P with homologous arms from Table 5 as a template... PGK1 -F and pcfb2988-T ADH1 -R is used for cloning, amplifying fragment P. PGK1 -SynOSC20433-T ADH1 Using plasmid pcfb2988-Trp as a template, primers pcfb2988-up-P with homologous arms (listed in Table 5) were used. TDH3 The fragment pcfb2988-Up was cloned using primers -F and pcfb2988-up-R, and then cloned using primers pcfb2988-T with homologous arms as shown in Table 5. ADH1The Trp-tagged fragment pcfb2988-Down was cloned from -down-F and pcfb2988-down-R. After PCR amplification, the four fragments were detected, purified, recovered, and concentrated to 30 μL.

[0149] Table 6 Primers for the SynOSC20433 gene overexpression cloning module

[0150]

[0151] (4) Combine the four fragments pcfb2988-Up, P TDH3 -SynAtSQE2-T TPI1 P PGK1 -SynOSC20433-T ADH1 pcfb2988-Down was transformed into strain GQ03 using the lithium acetate yeast transformation method, plated on SD-Trp solid medium, and allowed to grow for 2-3 days. After single colonies emerged, yeast colony PCR was performed for verification, and the results are as follows. Figure 9 As shown, the positive monoclonal colony was named strain AE2002.

[0152] (5) Cultured according to the method in 2.1.3, with colony density measured every 12 hours. After expression, the bacterial cells were lysed to detect the dammaradienol yield. GC-MS results are as follows: Figure 9 As shown in the figure, the experimental results indicate that the catalytic efficiency of the OSC20433 gene was improved after codon optimization, and the dammardienol yield was 59.21±3.36 mg / L.

[0153] Example 3: Site-directed mutagenesis study of the OSC20433 gene

[0154] 3.1 Construction of mutants

[0155] (1) Select the mutation sites closest to the front or rear end for full plasmid mutation, such as K3R, E86D, N91T, F168S, M729C, C732S, F736Y, and E738T. These eight mutant plasmids were then cloned. Using plasmid pESC-Ura-OSC20433 as a template, the corresponding primers in Table 7 were used for full plasmid cloning. After amplification, gel electrophoresis was performed for verification. If a correct positive band appeared, 5 μL of the PCR reaction sample was taken, digested with digestive enzyme DnpI for 40-50 min, and inactivated at 80℃ for 5 min. After the reaction, the inactivated sample was transformed into E. coli DH5α competent cells. After platelets grew, E. coli colony PCR was performed, and positive single clones were selected for sequencing. After correct sequencing, plasmid extraction was performed to obtain the mutant plasmid pESC-Ura-OSC20433. K3RpESC-Ura-OSC20433 E86D pESC-Ura-OSC20433 N91T pESC-Ura-OSC20433 F168S pESC-Ura-OSC20433 M729C pESC-Ura-OSC20433 C732S pESC-Ura-OSC20433 F736Y pESC-Ura-OSC20433 E738T .

[0156] (2) For other mutant plasmids located in the middle, overlapping PCR cloning can be used for construction. Using plasmid pESC-Ura-OSC20433 as a template, the 5′ end of the mutant plasmid is amplified using primers OSC20433-F and related mutant primers R in Table 7; the 3′ end of the mutant plasmid is amplified using related mutant primers F and OSC20433-R in Table 7. After amplification, gel electrophoresis is used to verify whether the bands are correct. If the amplified bands are correct, the next round of overlapping PCR cloning can be performed. Using the gene fragments at the 5′ and 3′ ends of the amplified mutant plasmid as templates, amplification is performed using primers OSC20433-F and OSC20433-R in Table 7 to amplify the full-length fragment with the mutation site OSC20433. After amplification, homologous recombination was performed with the enzyme-digested vector pESC-Ura. The homologous product was then transformed into E. coli DH5α competent cells. After platelet growth, E. coli colony PCR was performed, and positive single clones were selected for sequencing. After successful sequencing, plasmid was extracted to obtain the mutant plasmid pESC-Ura-OSC20433. Q288E pESC-Ura-OSC20433 E433D pESC-Ura-OSC20433 Q544E pESC-Ura-OSC20433 K349R pESC-Ura-OSC20433 N510T pESC-Ura-OSC20433 K440R pESC-Ura-OSC20433 K274R pESC-Ura-OSC20433 E588D pESC-Ura-OSC20433 E654D pESC-Ura-OSC20433 K146T pESC-Ura-OSC20433 K704R pESC-Ura-OSC20433 I533V pESC-Ura-OSC20433 F168S .

[0157] Table 7 OSC20433 Mutant Primers

[0158]

[0159]

[0160] 3.2 Analysis of Mutation Results

[0161] We transformed the successfully mutated plasmid into the high-squalene-producing Saccharomyces cerevisiae strain GQ03 using lithium acetate conversion for induced expression and verified its mutant function. Following the method in 2.1.2, we transformed the mutant plasmid prepared in 3.1 into the high-squalene-producing chassis strain GQ03 for verification. After induction, we extracted and detected changes in dammaradienol production in the bacterial cells. Using the peak area of ​​dammaradienol in the wild-type control pESC-Ura-OSC20433 as the standard, we quantitatively analyzed the production of the control and the mutant. The results are as follows: Figure 10 As shown, the production of F168S, P564C, and C732S decreased significantly, while the production of E86D, K146T, E433D, and E654D increased significantly, with E654D showing the most significant increase.

[0162] Example 4: Construction of strain AE2003 overexpressing the SynOSC20433 mutant E654D

[0163] 4.1 Construction of yeast strain AE2003

[0164] (1) Using plasmid pLB-P PGK1 -SynOSC20433-T ADH1 Using the primer SynOSC20433 from Table 5-1 as a template E654D -F and SynOSC20433 E654D -R was used for full plasmid cloning, amplifying the full-length fragment of SynOSC20433 with the E654D mutation site. After transformation, screening, and sequencing verification, the plasmid was extracted to obtain plasmid pLB-P. PGK1 -SynOSC20433 E654D -T ADH1 .

[0165] (2) Using the plasmid pLB-P obtained in step (1) PGK1 -SynOSC20433 E654D -T ADH1 Using the primer pcfb2798-P with homologous arms from Table 8 as a template... PGK1 -F and T ADH1 -R2 is used for cloning, amplifying fragment P. PGK1-SynOSC20433 E654D -T ADH1 ; using plasmid pUC19-P TDH3 -SynAtSQE2-T TPI1 Using primer P with homologous arms from Table 8 as a template TDH3 -F2 and T TPI1 -down-R is used for cloning, amplifying fragment P. TDH3 -SynAtSQE2-T TPI1 Using plasmid pcfb2798-Leu as a template, primers pcfb2798-up-F and pcfb2798-up-P with homologous arms as shown in Table 8 were used. PGK1 The fragment pcfb2798-Up was cloned using -R, and the primer pcfb2798-T with homologous arms from Table 8 was used. TPI1 The Leu-tagged fragment pcfb2798-Down was cloned from -down-F and pcfb2798-down-R. After PCR amplification, the four fragments were detected, purified, recovered, and concentrated to 30 μL.

[0166] Table 8. Overexpression of SynOSC20433 E654D Gene cloning module primers

[0167]

[0168] (3) Combine the above 4 fragments pcfb2798-Up, P TDH3 -SynAtSQE2-T TPI1 P PGK1 -SynOSC20433 E654D -T ADH1 pcfb2798-Down was transformed into strain AE2002 using the lithium acetate yeast transformation method, and then plated on SD-Trp-Leu solid medium. After 2-3 days, once a single colony had grown, the positive single colony was named strain AE2003.

[0169] 4.2 Shake-flask fermentation and detection of yeast strain AE2003

[0170] Cultured according to the method in 2.1.3, and samples were taken every 12 hours to detect the colony density (OD). 600 After expression, the bacterial cells were lysed to detect the dammaradienol yield in the cells. GC-MS results are shown below. Figure 11 As shown, the dammaradienol production of strain AE2003, which was transformed with the SynOSC20433 mutant E654D, increased to 101.85±5.73 mg / L.

[0171] Example 5 utilizes PHXT1 Construction of strain AE2004 with downregulated ERG7 gene expression

[0172] 5.1 Construction of yeast strain AE2004

[0173] (1) To further increase yield, the natural promoter of the endogenous gene ERG7 was replaced with the weak promoter P. HXT1 Using the genome of wild-type strain CEN.PK2-1D as a template, the primers in Table 9 were used for cloning, and the target fragments ERG7-Up, ERG7-Down, and ERG7-P were amplified, respectively. HXT1 After amplification, the three cloned fragments are detected by gel electrophoresis. If the correct positive band appears, the fragments can be purified by column chromatography using the kit. After purification, the three fragments can be concentrated to 30 μL.

[0174] Table 9P HXT1 downregulation of ERG7 gene expression module primers

[0175]

[0176] The above three segments are ERG7-Up, ERG7-Down, and ERG7-P. HXT1 and CRISPR / Cas9 plasmid pCRCT-GTR-URA2-△P ERG7 (The preparation method of this vector can be found in the literature Zhang, Y., Wang, J., Wang, Z. et al. A gRNA-tRNA array for CRISPR-Cas9 based rapid multiplexed genome editing in Saccharomyces cerevisiae. Nat Commun 10, 1053 (2019). https: / / doi.org / 10.1038 / s41467-019-09005-3, and the gRNA sequence used is gtgggcgacgattattggta.) The vector was transformed into strain AE2003 using the lithium acetate yeast transformation method and plated on SD-Trp-Leu-Ura solid medium, waiting 2-3 days. After single colonies grew, yeast colony PCR verification was performed, and the positive single colony was named strain AE2004.

[0177] 5.2 Shake-flask fermentation and detection of yeast strain AE2004

[0178] Cultured according to the method in 2.1.3, and samples were taken every 12 hours to detect the colony density (OD). 600After expression, the bacterial cells were lysed to detect the dammaradienol yield. GC-MS results are shown below. Figure 12 As shown, the dammaradienol yield of strain AE2004 was 145.67±8.06 mg / L.

[0179] 5.3 Fermentation of yeast strain AE2004 in a 2L fermenter

[0180] Positive single colonies of strain AE2004 were picked from plates and cultured overnight (12 h) in 3 mL of SD-Trp-Leu-Ura deficient liquid medium at 30°C and 200 rpm in a yeast shaker. A 5% transfer was made to 30 mL of deficient liquid medium in a new 250 mL Erlenmeyer flask and cultured for 12 h at 30°C and 200 rpm. This process was repeated three times: a 5% transfer to 30 mL of deficient liquid medium in a new 250 mL Erlenmeyer flask and cultured for 12 h at 30°C and 200 rpm; finally, a 5% transfer was made to 100 mL of deficient liquid medium in a new 500 mL Erlenmeyer flask and cultured for 12 h at 30°C and 200 rpm to obtain the fermentation seed culture. The seed culture was then inoculated into 800 mL of YPD liquid medium in a sterilized 2 L fermenter, with the appropriate initial sugars, trace elements, and vitamins added. During fermentation, the temperature is maintained at 30℃, and ammonia is automatically added to maintain the pH at around 5.5. Dissolved oxygen is linked to the fermentation speed; the initial speed is 300 rpm, maintaining dissolved oxygen at around 40% fluctuation, with an air flow rate of 2 L / min. Defoaming is also linked to the fermentation tank. When dissolved oxygen drops from 100% to around 40%, the feeding switch is activated, linked to dissolved oxygen levels. Feeding is initiated when dissolved oxygen suddenly increases to above 55%. (Appropriate trace elements and vitamins can be added to the feed sugar.) Cell density (OD) is measured every 24 hours. 600 Dammaradienol yield was measured. After 7 days of fermentation, the cell weight was measured to be 144g, and the final dammaradienol yield was 1.037g / L. The test results are as follows. Figure 13 As shown.

[0181] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A high-yield dammaradienol-producing engineered Saccharomyces cerevisiae strain, characterized in that: Starting with Saccharomyces cerevisiae, the tHMGR, ERG12, IDI, ERG13, ERG10, ERG19, and ERG8 genes were overexpressed, and then AtSQS2, AtSQE2, and SmFPS genes were introduced, followed by the introduction of the dammardienol synthase gene. The overexpression of tHMGR, ERG12, IDI, ERG13, ERG10, ERG19, and ERG8 genes involves integrating these genes into the Gal7 and Gal10-Gal1 sites on the chromosome of Saccharomyces cerevisiae. The aforementioned transfer of AtSQS2, AtSQE2, and SmFPS genes involves integrating the AtSQS2, AtSQE2, and SmFPS genes into the His3 site on the chromosome of Saccharomyces cerevisiae. The dammaradienol synthase gene is a gene for an enzyme that can synthesize dammaradienol using squalene as a substrate. The dammardienol synthase gene is one of OSC20433, SynOSC20433, OSC20433 mutant, and SynOSC20433 mutant; The dammarenol synthase gene was introduced via shuttle vector or homologous recombination. The shuttle carrier is pESC-Ura; The aforementioned homologous recombination involves combining promoter P... PGK1 and Termination T ADH1 The dammardienol synthase gene expression cassette and the combined promoter P TDH3 and Termination T TPI1 The SynAtSQE2 gene expression cassette was integrated into the Ty1cons2 site on the Saccharomyces cerevisiae chromosome; The nucleotide sequence of OSC20433 is shown in SEQ ID NO.1; The nucleotide sequence of SynOSC20433 is shown in SEQ ID NO.2; The mutant of OSC20433 is a nucleotide sequence obtained by mutation based on the nucleotide sequence shown in SEQ ID NO.1, and is one of the following: 7-9 positions mutated to AGG, 256-258 positions mutated to GAC, 436-438 positions mutated to ACA, 1045-1047 positions mutated to AGA, 1297-1299 positions mutated to GAT, 1528-1530 positions mutated to ACT, 1762-1764 positions mutated to GAT, 1960-1962 positions mutated to GAT, 2110-2112 positions mutated to AGA, and 2212-2214 positions mutated to ACA; The mutant of SynOSC20433 is a nucleotide sequence obtained by mutating GAT at positions 1960-1962 based on the nucleotide sequence shown in SEQ ID NO.

2. The nucleotide sequence of the SynAtSQE2 gene is shown in SEQ ID NO.

11.

2. The engineered brewer's yeast according to claim 1, characterized in that: The tHMGR gene is a tHMGR gene expression cassette, which also contains the promoter P. PGK1 and Termination T ADH1 ; The ERG12 gene mentioned is an ERG12 gene expression cassette, which also contains the promoter P. PDC1 and Termination T ADH2 ; The IDI gene mentioned is an IDI gene expression cassette, which also contains the promoter P. ENO2 and Termination T PDC1 ; The ERG13 gene mentioned is an ERG13 gene expression cassette, which also contains the promoter P. FBA1 and Termination T TDH2 ; The ERG10 gene mentioned is an ERG10 gene expression cassette, which also contains the promoter P. TEF1 and Termination T CYC1 ; The ERG19 gene mentioned is an ERG19 gene expression cassette, which also contains the promoter P. PYK1 and Termination T PGI1 ; The ERG8 gene mentioned is an ERG8 gene expression cassette, which also contains the promoter P. TDH3 and Termination T TPI1 ; The AtSQS2 gene described is an AtSQS2 gene expression cassette, which also contains the promoter P. PGK1 and Termination T ADH1 ; The AtSQE2 gene described is an AtSQE2 gene expression cassette, which also contains the promoter P. TDH3 and Termination T TPI1 ; The SmFPS gene is a SmFPS gene expression cassette, which also contains the promoter P. TEF1 and Termination T CYC1 .

3. The engineered brewer's yeast strain according to claim 1, characterized in that: The engineered Saccharomyces cerevisiae strain that produces high levels of dammaradienol will also contain endogenous genes. ERG7 The natural promoter is replaced with the weak promoter P. HXT1 ; The aforementioned endogenous genes ERG7 The natural promoter is replaced with the weak promoter P. HXT1 It was achieved through CRISPR / Cas9 gene editing technology; The promoter P HXT1 The nucleotide sequence is shown as positions 292634 to 293756 in the NCBI database sequence registry number CP036477.

1.

4. A method for constructing a high-yield dammaradienol-producing engineered Saccharomyces cerevisiae strain, characterized in that... Includes the following steps: (1) Amplification of Gal7-Up, Loxp-Ura-loxp, and P containing homologous fragments PGK1 -tHMGR-T ADH1 P PDC1 -ERG12-T ADH2 P ENO2 -IDI-T PDC1 P FBA1 -ERG13-T TDH2 P TEF1 -ERG10-T CYC1 P PYK1 -ERG19-T PGI1 P TDH3 -ERG8-T TPI1 Gal7-Down was transferred together with Saccharomyces cerevisiae, and strain GQ01 was obtained through screening. (2) Amplification of Gal10-Up, Loxp-Ura-loxp, and P containing homologous fragments PGK1 -tHMGR-T ADH1 P PDC1 -ERG12-T ADH2 P ENO2 -IDI-T PDC1 P FBA1 -ERG13-T TDH2 P TEF1 -ERG10-T CYC1 P PYK1 -ERG19-T PGI1 P TDH3 -ERG8-T TPI1 Gal1-Down was transferred together with strain GQ01, and strain GQ02 was obtained by screening. (3) Amplify His3-Up, Loxp-Ura-loxp, and P containing homologous fragments. PGK1 -AtSQS2-T ADH1 P TDH3 -AtSQE2-T TPI1 P TEF1 -SmFPS-T CYC1 His3-Down was transferred into strain GQ02, and strain GQ03 was obtained by screening. (4) Amplify the dammardienol synthase gene and use a restriction fast cutase. Bam HI and Xoh I. The vector backbone obtained by digesting the vector plasmid pESC-Ura was ligated and transformed into strain GQ03. The Saccharomyces cerevisiae engineered strain with high dammardienol production was screened to obtain the strain. Alternatively, amplify pcfb2988-Up and P containing homologous fragments. TDH3 -SynAtSQE2-T TPI1 The dammaradienol synthase gene and pcfb2988-Down were transferred into strain GQ03, and a high-dammaradienol-producing Saccharomyces cerevisiae engineered strain was obtained by screening. The dammaradienol synthase gene is a gene for an enzyme that can synthesize dammaradienol using squalene as a substrate. The dammardienol synthase gene is one of OSC20433, SynOSC20433, OSC20433 mutant, and SynOSC20433 mutant; The nucleotide sequence of OSC20433 is shown in SEQ ID NO.1; The nucleotide sequence of SynOSC20433 is shown in SEQ ID NO.2; The mutant of OSC20433 is a nucleotide sequence obtained by mutation based on the nucleotide sequence shown in SEQ ID NO.1, and is one of the following: 7-9 positions mutated to AGG, 256-258 positions mutated to GAC, 436-438 positions mutated to ACA, 1045-1047 positions mutated to AGA, 1297-1299 positions mutated to GAT, 1528-1530 positions mutated to ACT, 1762-1764 positions mutated to GAT, 1960-1962 positions mutated to GAT, 2110-2112 positions mutated to AGA, and 2212-2214 positions mutated to ACA; The mutant of SynOSC20433 is a nucleotide sequence obtained by mutating GAT at positions 1960-1962 based on the nucleotide sequence shown in SEQ ID NO.

2. The nucleotide sequence of the SynAtSQE2 gene is shown in SEQ ID NO.

11.

5. The construction method according to claim 4, characterized in that: The Gal7-Up and Gal7-Down segments are located at the Gal7 site in the Saccharomyces cerevisiae genome, wherein the nucleotide sequence of Gal7-Up is shown in SEQ ID NO.3 and the nucleotide sequence of Gal7-Down is shown in SEQ ID NO.4; The Gal10-Up and Gal1-Down segments are located at the Gal10-Gal1 site in the genome of Saccharomyces cerevisiae, wherein the nucleotide sequence of Gal10-Up is shown in SEQ ID NO.5 and the nucleotide sequence of Gal1-Down is shown in SEQ ID NO.6; The His3-Up and His3-Down segments are located at the His3 site in the Saccharomyces cerevisiae genome, wherein the nucleotide sequence of His3-Up is shown in SEQ ID NO.7 and the nucleotide sequence of His3-Down is shown in SEQ ID NO.

8. The pcfb2988-Up is an upstream fragment located at the Ty1cons2 site of the Saccharomyces cerevisiae genome, and the pcfb2988-Down is a downstream fragment carrying the selectable marker gene Trp and located at the Ty1cons2 site of the Saccharomyces cerevisiae genome. The nucleotide sequence of pcfb2988-Up is shown in SEQ ID NO.9, and the nucleotide sequence of pcfb2988-Down is shown in SEQ ID NO.

10. The Loxp-Ura-loxp mentioned above is a fragment containing the selection marker gene Ura; The P mentioned PGK1 -tHMGR-T ADH1 The tHMGR gene expression cassette also contains the promoter P PGK1 and Termination T ADH1 ; The P mentioned PDC1 -ERG12-T ADH2 The ERG12 gene expression cassette also contains the promoter P. PDC1 and Termination T ADH2 ; The P mentioned ENO2 -IDI-T PDC1 The IDI gene expression cassette also contains the promoter P. ENO2 and Termination T PDC1 ; The P mentioned FBA1 -ERG13-T TDH2 The ERG13 gene expression cassette also contains the promoter P. FBA1 and Termination T TDH2 ; The P mentioned TEF1 -ERG10-T CYC1 The ERG10 gene expression cassette also contains the promoter P. TEF1 and Termination T CYC1 ; The P mentioned PYK1 -ERG19-T PGI1 The ERG19 gene expression cassette also contains the promoter P. PYK1 and Termination T PGI1 ; The P mentioned TDH3 -ERG8-T TPI1 The ERG8 gene expression cassette also contains the promoter P. TDH3 and Termination T TPI1 ; The P mentioned PGK1 -AtSQS2-T ADH1 The AtSQS2 gene expression cassette also contains the promoter P. PGK1 and Termination T ADH1 ; The P mentioned TDH3 -AtSQE2-T TPI1 The AtSQE2 gene expression cassette also contains the promoter P. TDH3 and Termination T TPI1 ; The P mentioned TEF1 -SmFPS-T CYC1 The SmFPS gene expression cassette also contains the promoter P TEF1 and Termination T CYC1 ; The P mentioned TDH3 -SynAtSQE2-T TPI1 The SynAtSQE2 gene expression cassette also contains the promoter P. TDH3 and Termination T TPI1 .

6. The engineered Saccharomyces cerevisiae according to claim 1 or the construction method according to claim 5, characterized in that: The nucleotide sequence of the tHMGR gene is shown in the NCBI database sequence accession number NM_001182434.1; The nucleotide sequence of the ERG12 gene is shown in the NCBI database sequence accession number NM_001182715.1; The nucleotide sequence of the IDI gene is shown in the NCBI database sequence accession number NM_001183931.1; The nucleotide sequence of the ERG13 gene is shown in the NCBI database sequence accession number NM_001182489.1; The nucleotide sequence of the ERG10 gene is shown in the NCBI database sequence accession number NM_001183842.1; The nucleotide sequence of the ERG19 gene is shown in the NCBI database sequence accession number NM_001183220.1; The nucleotide sequence of the ERG8 gene is shown in the NCBI database sequence accession number NM_001182727.1; The nucleotide sequence of the AtSQS2 gene is shown in the NCBI database sequence accession number NM_119631.3; The nucleotide sequence of the AtSQE2 gene is shown in the NCBI database sequence accession number NM_127848.4; The nucleotide sequence of the SmFPS gene is shown in the NCBI database sequence accession number HQ687768.1; The promoter P PGK1 The nucleotide sequence is shown as positions 124102 to 124853 in the NCBI database sequence accession number CP135951.1; The promoter P TDH3 The nucleotide sequence is shown as positions 9954286 to 9955085 in the NCBI database sequence accession number CP029160.1; The terminator T ADH1 The nucleotide sequence is shown as positions 4671969 to 4672126 in the NCBI database sequence registry number CP029160.1; The terminator T TPI1 The nucleotide sequence is shown as positions 529841 to 530242 in the NCBI database sequence registry number CP134412.

1.

7. The construction method according to claim 5, characterized in that: The promoter P PDC1 The nucleotide sequence is shown as positions 216126 to 216925 in the NCBI database sequence accession number CP135955.1; The promoter P ENO2 The nucleotide sequence is shown as positions 10603930 to 10604929 in the NCBI database sequence accession number CP029160.1; The promoter P FBA1 The nucleotide sequence is shown as positions 5910688 to 5911509 in the NCBI database sequence registry number CP029160.1; The promoter P TEF1 The nucleotide sequence is shown as positions 707052 to 707481 in the NCBI database sequence registry number CP029160.1; The promoter P PYK1 The nucleotide sequence is shown as positions 6308912 to 6309911 in the NCBI database sequence registry number CP029160.1; The terminator T ADH2 The nucleotide sequence is shown as positions 3398555 to 3398954 in the NCBI database sequence registry number CP029160.1; The terminator T PDC1 The nucleotide sequence is shown as positions 3660519 to 3660915 in the NCBI database sequence registry number CP029160.1; The terminator T TDH2 The nucleotide sequence is shown as positions 11545244 to 11545644 in the NCBI database sequence registry number CP029160.1; The terminator T CYC1 The nucleotide sequence is shown as positions 1301 to 1607 in the NCBI database sequence accession number KC879308.1; The terminator T PGI1 The nucleotide sequence is shown as positions 8108691 to 8109092 in the NCBI database sequence registry number CP029160.1.

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