Saccharomyces cerevisiae genetically engineered bacterium for de novo synthesis of 7-DHC as well as construction method and application thereof

Abstract

The invention particularly relates to a saccharomyces cerevisiae genetically engineered bacterium for de novo synthesis of 7-DHC as well as a construction method and application of the saccharomyces cerevisiae genetically engineered bacterium and belongs to the technical field of biology. According to the invention, a saccharomyces cerevisiae engineering bacterium SC9 is taken as an original strain, multiple times of multiple copies of tHMG1, DHCR24 and ERG1 are carried out on TY transposon, multiple copies of ERG2, POS5, ERG7, ERG11, IDI1 and ERG3 are carried out on TY transposon, and POS5, ERG3 and DHCR24 are singly copied on a genome, so that the content of intracellular reducing coenzyme II is increased, and a main path for synthesizing 7-DHC is enhanced. The genome single copy ACL, ACS1, ERG10, ERG19 and the like are used for enhancing the utilization of saccharomyces cerevisiae ethanol and dynamically regulating and controlling the synthesis of 7-DHC. The saccharomyces cerevisiae gene engineering strain constructed by the invention has high product accumulation, and lays a foundation for replacing a traditional multi-step chemical synthesis route and realizing industrial production.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a genetically engineered strain of Saccharomyces cerevisiae for synthesizing 7-DHC, a key intermediate of active vitamin D3, and a construction method and application thereof. Background Art

[0002] Vitamin D3 (cholecalciferol, VD3) is a fat-soluble vitamin that helps maintain calcium phosphate homeostasis and plays a vital role in many biological processes, such as regulating calcium and phosphorus metabolism in the human body and promoting bone development. Vitamin D3 has an anti-rickets effect and is also called an anti-rickets vitamin. Vitamin D3 can also improve the body's immune ability and reduce the risk of cardiovascular disease. In addition, studies have shown that vitamin D is closely related to diseases such as hypertension, type 2 diabetes, immune regulation, cardiovascular disease, and various cancers.

[0003] As the physiological activity of vitamin D3 is gradually discovered and the aging population intensifies, the market demand for vitamin D3 is also growing. At present, the production of vitamin D and its derivatives has gradually failed to meet market needs. The existing vitamin D3 synthesis method is mainly chemical synthesis, which is further restricted by its expensive raw materials, multi-step chemical reactions, low purity, difficult extraction, and strong pollution. Synthetic biology has developed rapidly in the past decade, and has provided new ideas for the green production of high-value-added chemicals through the engineering design and construction of efficient cell factories. The microbial fermentation method mainly constructs metabolic synthesis pathways in microorganisms, uses simple carbon sources (such as glucose and ethanol) as substrates, biosynthesizes 7-DHC, and further obtains vitamin D3 through photoisomerization. Compared with the chemical synthesis method, the microbial fermentation reaction has the characteristics of less pressure on the environment, relatively moderate conditions, and sustainable production, and has a very broad application prospect. The safe, efficient and environmentally friendly de novo synthesis of 7-DHC using yeast as a cell factory provides new ideas and methods for the industrial production of 7-DHC.

[0004] 7-DHC is similar in structure to ergosterol in yeast. Saccharomyces cerevisiae can synthesize ergosterol from scratch. The de novo synthesis of 7-DHC in yeast can be achieved by modifying the ergosterol pathway in yeast. Therefore, yeast can be used as an excellent host for the production of drugs such as steroids. 7-DHC can be produced in Saccharomyces cerevisiae by knocking out ERG5 and ERG6 in the ergosterol synthesis pathway and heterologously expressing 24-sterol reductase DHCR24. This study improves 7-DHC production by knocking out related inhibitory genes, strengthening precursor supply, guiding the conversion of precursors to target products, increasing the supply of intracellular reducing power, using ethanol as a carbon source, and optimizing the utilization and tolerance of ethanol by Saccharomyces cerevisiae. This is of great significance for the subsequent development of steroid drugs. Summary of the invention

[0005] In view of the problems existing in the prior art, the purpose of the present invention is to design and provide a technical solution for de novo synthesis of 7-DHC high-yield brewer's yeast genetic engineering bacteria and its construction method and application.

[0006] The technical solution adopted by the present invention is:

[0007] A genetically engineered yeast for synthesizing 7-dehydrocholesterol from scratch is constructed by the following method: the genome of the bottom plate yeast Saccharomyces cerevisiae is enhanced to express truncated 3-hydroxy-3-methylglutaryl-CoA reductase tHMG1, squalene epoxidase ERG1, C-8 sterol isomerase ERG2, NADH kinase POS5, lanosterol demethylase ERG11, lanosterol synthase ERG7, isopentenyl pyrophosphate isomerase IDI1, acetyl-CoA synthetase ACS1, acetyl-CoA acetyltransferase ERG10, acetaldehyde dehydrogenase ALD6, acetyl-CoA hydrolase ACH1, mevalonate pyrophosphate decarboxylase ERG19, alcohol dehydrogenase ADH2, phospholipid biosynthesis regulator INO2, sterol transcription activator UPC2, GAL promoter activator protein GAL4, C-5 sterol desaturase ERG3, and heterologously express Gallus Gallus-derived 24-sterol reductase DHCR24, Salmonella enterica-derived acetyl-CoA synthetase ACS1, and Yarrowia lipolytica-derived citrate lyase ACL are used to knock out C-22 sterol desaturase ERG5, delta C-24 sterol methyltransferase ERG6, and ergosterol synthesis gene transcription repressor MOT3 required for ergosterol synthesis, thereby obtaining the cerevisiae strain that efficiently synthesizes 7-dehydrocholesterol.

[0008] Preferably, the chassis bacteria is Saccharomyces cerevisiae SC9, which can be constructed by the construction method disclosed in Chinese Patent 202310058515.6 (Genetically engineered Saccharomyces cerevisiae bacteria for synthesizing 7-DHC exocytosis and its construction and application).

[0009] Preferably, the enhanced expression refers to the enhanced expression of multiple copies of tHMG1, DHCR24, ERG1 (3-hydroxy-3-methylglutaryl-CoA reductase tHMG1, 24-sterol reductase DHCR24, squalene epoxidase ERG1 can be integrated into the repeated δ sequences at both ends of the Ty1Cons1 site of the transposon in the Saccharomyces cerevisiae genome), multiple copies of tHMG1, DHCR24, ERG2 (3-hydroxy-3-methylglutaryl-CoA reductase tHMG1, 24-sterol reductase DHCR24, C-8 sterol isothiocyanate can be expressed in the Saccharomyces cerevisiae genome). The repetitive sequences of the Ty2Cons site at both ends of the transposon in the Saccharomyces cerevisiae genome), multiple copies of tHMG1, DHCR24, ERG1 (the 3-hydroxy-3-methylglutaryl-CoA reductase tHMG1, 24-sterol reductase DHCR24, and squalene epoxidase ERG1 can be integrated into the repetitive δ sequences at both ends of the Ty1Cons2 site of the transposon in the Saccharomyces cerevisiae genome), multiple copies of tHMG1, DHCR24, and POS5 (the 3-hydroxy-3-methylglutaryl-CoA reductase tHMG1, 24-sterol reductase D HCR24, NADH kinase POS5 are integrated into the repetitive ω sequence at both ends of the Ty4Cons site of the transposon in the Saccharomyces cerevisiae genome), multiple copies of ERG7, ERG11, IDI1 (lanosterol synthase ERG7, lanosterol demethylase ERG11, isopentenyl pyrophosphate isomerase IDI1 can be integrated into the repetitive δ sequence at both ends of the Ty1Cons1 site of the transposon in the Saccharomyces cerevisiae genome), multiple copies of ERG2, ERG3, ERG1 (C-8 sterol isomerase ERG2, C-5 sterol desaturase ERG3, squalene epoxidase ERG 1 repetitive ω sequence integrated into the Ty3Cons site at both ends of the transposon in the Saccharomyces cerevisiae genome), as well as 1 copy of POS5, 1 copy of ERG3, 1 copy of DHCR24, 1 copy of ACL, 1 copy of ACS1, 1 copy of ERG10, 1 copy of ERG19, 1 copy of UPC2, 1 copy of ALD6, 1 copy of ACH1, 1 copy of IDI1, 1 copy of ADH2, 1 copy of INO2, and one copy of GAL4 that are enhanced in the genome.

[0010] Preferably, the Gene ID of tHMG1 is 42650, the Gene ID of IDI1 is 855986, the Gene ID of POS5 is 855913, the Gene ID of ERG2 is 855242, the Gene ID of ERG3 is 850745, the Gene ID of ERG1 is 853086, the Gene ID of ERG11 is 856398, the Gene ID of ERG7 is 856470, the Gene ID of ERG5 is 855029, the Gene ID of ERG6 is 855003, the Gene ID of MOT3 is 855092, the Gene ID of DHCR24 from Gallus Gallus is 424661, the Gene ID of ACH1 is 852266, the Gene ID of UPC2 is 851799, the Gene ID of ALD6 is 856044, the Gene ID of ACS1 is The Gene ID of the gene is 851245, the Gene ID of the ACS1 derived from Salmonella enterica is 1255801, the Gene ID of the ADH2 is 855349, the Gene ID of the ERG19 is 855779, the Gene ID of the INO2 is 851701, the Gene ID of the GAL4 is 855828, the Gene ID of the ACL derived from Yarrowia lipolytica is 2912112, the Gene ID of the OSH2 is 851543, the Gene ID of the YBR197C is 852496, the Gene ID of the YBL059W is 852221, the Gene ID of the YNR014W is 855748, and the Gene ID of the YMR206W is 855246.

[0011] Specifically, the present invention is through P TEF1 The promoter enhances the expression of DHCR24 through P GAP The promoter enhances the expression of ERG1 through P GAP The promoter enhances the expression of ERG2 through P PGK1 Promoter-enhanced expression of tHMG1, through P GAL7 The promoter enhances the expression of POS5 through P GAL1，10 The bidirectional promoter enhances the expression of ERG7 and ERG11 through P GAP The promoter enhances the expression of IDI1 through P GAL1，10 The bidirectional promoter enhances the expression of ERG3 and ERG2 through P GAL1，10 The bidirectional promoter enhanced the expression of ACL and ACS1, and the ethanol-induced promoter PFBA1 Enhanced expression of ACS1 from Salmonella enterica, via the ethanol-inducible promoter P HXT7 Enhanced expression of ERG10, through the promoter P ITR1 Enhanced expression of ERG19 by P GAL7 Promoter-enhanced expression of UPC2, through P GAL1，10 The bidirectional promoter enhances the expression of ALD6 and ACH1 through P TEF1 The promoter enhances the expression of ADH2, and the promoter P is induced by ethanol. FBA1 Enhanced expression of IDI1, through the promoter P YGP1 Enhanced expression of INO2 was achieved by inducing the promoter P HXT7 Enhanced expression of GAL4.

[0012] More specifically:

[0013] By P TEF1 , P TDH3 The promoter-enhanced expression of POS5 and ERG3 was integrated into the OSH2 site on the genome of the Saccharomyces cerevisiae strain, and the Gene ID of the OSH2 site is 851543;

[0014] By P GAL1 , P GAL10 The bidirectional promoter for enhanced expression of ERG7 and ERG2 was integrated into the ERG5 site on the Saccharomyces cerevisiae genome, and the Gene ID of the ERG5 site is 855029;

[0015] By P TEF1 , P GAP , P PGK1 Enhanced expression of DHCR24, ERG1, and tHMG1 was integrated into the Ty1Cons1 site on the yeast genome;

[0016] By P TEF1 The enhanced expression DHCR24 was integrated into the YPL062W site on the yeast genome, and the GeneID of YPL062W is 856043;

[0017] By P TEF1 , P GAP , P PGK1 Enhanced expression of DHCR24, ERG2, and tHMG1 integrated into the Ty2Cons site on the yeast genome;

[0018] By P TEF1 , P GAP , P PGK1 Enhanced expression of DHCR24, ERG1, and tHMG1 was integrated into the Ty1Cons2 site on the yeast genome;

[0019] By P TEF1 , P GAL7 , P PGK1 Enhanced expression of DHCR24, POS5, and tHMG1 integrated into the Ty4Cons site on the yeast genome;

[0020] By P GAL1 , P GAL10 The bidirectional promoter enhances the expression of ERG11 and ERG7 and simultaneously GAP The enhanced expression IDI was integrated into the Ty1Cons1 site on the Saccharomyces cerevisiae genome;

[0021] By P GAL1 , P GAL10 The bidirectional promoter enhances the expression of ERG2 and ERG3 and simultaneously GAP Enhanced expression of ERG1 was integrated into the Ty3Cons site on the Saccharomyces cerevisiae genome;

[0022] By P GAL1 , P GAL10 The bidirectional promoter enhances the expression of ACL and ACS1 and integrates them into the MOT3 site on the Saccharomyces cerevisiae genome, wherein the Gene ID of the MOT3 is 855092;

[0023] By P FBA1 , P HXT7 Ethanol-inducible promoter enhances expression of ACS1 L641P , ERG10 was integrated into the 208A site on the Saccharomyces cerevisiae genome;

[0024] By P ITR1 , P GAL7 Enhanced expression of ERG19 and UPC2 G888D Integrated into the YBR197C site on the Saccharomyces cerevisiae genome, the Gene ID of the YBR197C site is 852496;

[0025] By P GAL1 , P GAL10 The bidirectional promoter enhances the expression of ALD6 and ACH1 and integrates into the YBL059W site on the Saccharomyces cerevisiae genome, wherein the Gene ID of the YBL059W site is 852221;

[0026] By P FBA1 , P TEF1 The enhanced expression of IDI1 and ADH2 was integrated into the YNR014W site on the Saccharomyces cerevisiae genome, and the Gene ID of the YNR014W site is 855748;

[0027] By P YGP1 , PHXT7 To enhance the expression of INO2, GAL4 was integrated into the YMR206W site on the Saccharomyces cerevisiae genome, and the Gene ID of the YMR206W site is 855246;

[0028] The present invention also relates to a method for constructing the Saccharomyces cerevisiae strain, which comprises the following steps:

[0029] Using the engineered yeast SC9 of Saccharomyces cerevisiae as the chassis strain, T TEF -P TEF1 -POS5-T ADH1 -P TDH3 -ERG3-T CYC1 The fragment was integrated into the genome of the SC9 strain, integrated into the OSH2 locus (the Gene ID of the OSH2 locus is 851543), and the resulting Saccharomyces cerevisiae strain was named sd1;

[0030] T TEF -T ADH1 -ERG7-P GAL10 -P GAL1 -ERG2-T TPS1 The fragment was integrated into the genome of the sd1 strain, into the ERG5 locus (Gene ID of the ERG5 locus is 855029), and the resulting Saccharomyces cerevisiae strain was named the sd2 strain;

[0031] P TEF1 -DHCR24-T TPS1 -P GAP -ERG1-T TDH3 -P PGK1 -tHMG1-T CYC1 -HYGPMX6 fragment was integrated into the genome of sd2 strain, integrated into Ty1Cons1 site, and the resulting Saccharomyces cerevisiae strain was named sd3;

[0032] P TEF1 -DHCR24-T TPS1 The fragment was integrated into the genome of the sd3 strain, into the YPL062W site (Gene ID of YPL062W is 856043), and the constructed Saccharomyces cerevisiae strain was named sd4 strain;

[0033] P TEF1 -DHCR24-T TDH3 -P GAP -ERG2-T TPS1 -P PGK1 -tHMG1-T CYC1 -P HIS -HIS-T ADH1The fragment was integrated into the genome of the sd4 strain at the Ty2Cons site, and the resulting S. cerevisiae strain was named the sd5 strain;

[0034] P TEF1 -DHCR24-T TDH3 -P GAP -ERG1-T TPS1 -P PGK1 -tHMG1-T CYC1 -P TRP -TRP fragment was integrated into the genome of sd5 strain, integrated into Ty1Cons2 site, and the constructed Saccharomyces cerevisiae strain was named sd6;

[0035] P TEF1 -DHCR24-T TPS1 -P CAL7 -POS5-T TDH3 -P PGK1 -tHMG1-T CYC1 -NRSR fragment was integrated into the genome of sd6 strain, integrated into Ty4Cons site, and the constructed Saccharomyces cerevisiae strain was named sd7;

[0036] P GAL1 -ERG7-T TDH3 -P CAL7 -ERG11-T TPS1 -P GAP -IDI-T CYC1 -HYGPMX6 fragment was integrated into the genome of sd7 strain, integrated into Ty1Cons1 site, and the constructed Saccharomyces cerevisiae strain was named sd8;

[0037] T TEF- T ADH1 -ERG2-P CAL10 -P CAL1 -ERG3-T CYC1 -P CAP -ERG1-T TPS1 -P LEU The -LEU fragment was integrated into the genome of the sd8 strain and integrated into the Ty3Cons site to construct a Saccharomyces cerevisiae strain named sd9;

[0038] The C-24 sterol methyltransferase ERG6 required for ergosterol synthesis in the genome of the sd9 strain was knocked out, and the resulting Saccharomyces cerevisiae strain was named sd9-Δerg6;

[0039] T TEF -T ADH1 -ACL-P GAL10 -P GAL1-ACS1-T CYC1 The fragment was integrated into the genome of the sd9 strain, into the MOT3 locus (Gene ID of the MOT3 locus is 855092), and the resulting Saccharomyces cerevisiae strain was named se1;

[0040] T TPS1 -P FBA1 -ACS1 L641P -T ADH2 -P HXT7 -ERG10-T CYC1 The fragment was integrated into the genome of the se1 strain at the 208A site, and the resulting Saccharomyces cerevisiae strain was named se2;

[0041] T TPS1 -P ITR1 -ERG19-T ADH2 -P GAL7 -UPC2 G888D -T CYC1 The fragment was integrated into the genome of the se2 strain, into the YBR197C site (Gene ID of the YBR197C site is 852496), and the resulting Saccharomyces cerevisiae strain was named se3;

[0042] T TEF -T ADH1 -ALD6-P GAL10 -P GAL1 -ACH1-T CYC1 The fragment was integrated into the genome of the se3 strain, into the YBL059W site (Gene ID of the YBL059W site is 852221), and the resulting Saccharomyces cerevisiae strain was named se4;

[0043] T TPS1 -P FBA1 -IDI1-T ADH2 -P TEF1 -ADH2-T CYC1 The fragment was integrated into the genome of the se4 strain, integrated into the YNR014W site (Gene ID of the YNR014W site is 855748), and the resulting Saccharomyces cerevisiae strain was named se5;

[0044] T TEF -P YGP1 -INO2-T CYC1 -P HXT7 -GAL4-T TDH3 The fragment was integrated into the genome of the se5 strain, into the YMR206W site (Gene ID of the YMR206W site is 855246), and the resulting Saccharomyces cerevisiae strain was named se6;

[0045] The C-24 sterol methyltransferase ERG6 required for ergosterol synthesis in the se6 strain genome was knocked out, and the resulting Saccharomyces cerevisiae strain was named se6-Δerg6.

[0046] The invention also relates to the application of the saccharomyces cerevisiae strain in preparing 7-DHC by microbial fermentation.

[0047] Typically, the recombinant Saccharomyces cerevisiae is first inoculated into a seed culture medium to prepare a seed solution, and the prepared seed solution is then inoculated into a fermentation medium at an inoculation rate of 1 to 10% (v / v). The seed culture medium is usually a YPD culture medium. In the present invention, the YPD culture medium used is composed of the following: 20 g / L peptone, 10 g / L yeast extract, and 20 g / L anhydrous glucose. In one embodiment of the present invention, the YPD culture medium comprises per liter: 2% peptone, 1% yeast extract, and 2% anhydrous glucose.

[0048] Specifically, the application is: inoculating the saccharomyces cerevisiae strain into a shake flask fermentation medium, fermenting and culturing at 28-32° C. for 84-96 hours, and obtaining the 7-DHC by cell disruption and saponification extraction of the fermentation broth. The shake flask fermentation medium is composed of 20 g of tryptone, 40 g of anhydrous glucose, and 10 g of yeast extract per liter.

[0049] Specifically, the application is: the saccharomyces cerevisiae strain is inoculated into a 5L tank fermentation medium, and fermentation culture is carried out at 28-32°C for 100-130h. Feeding begins when the sugar content of the initial medium drops to 1g / L and the ethanol content drops to 10g / L (about 15h). In the early stage of feeding carbon source (15-50h), glucose is fed, and the sugar content of the fermentation medium is controlled below 1g / L and the ethanol content is controlled below 10g / L. Ethanol is fed in the later stage (50h to the end of fermentation), and the ethanol content of the fermentation medium is controlled at 15-25g / L. The fermentation broth is subjected to cell disruption and saponification extraction to obtain the 7-DHC. The 5L tank fermentation medium is composed of: 20g / L tryptone, 20g / L anhydrous glucose, 10g / L yeast extract, 5-10g / L potassium dihydrogen phosphate, 1-5g / L magnesium sulfate, 15-25g / L ammonium sulfate, 0.5-1.5g / L uracil, leucine, histidine and tryptophan.

[0050] Beneficial effects: The invention uses the engineering bacteria SC9 of Saccharomyces cerevisiae as the starting strain, uses TY transposon multiple times, multiple copies of tHMG1, DHCR24, ERG1, uses TY transposon multiple copies of ERG2, POS5, ERG7, ERG11, IDI1, ERG3, and single copies of POS5, ERG3, DHCR24 in the genome to increase the content of intracellular reductive coenzyme II and strengthen the main pathway of 7-DHC synthesis. Single copies of ACL, ACS1, ERG10, ERG19, UPC2, ALD6, ACH1, IDI1, ADH2, INO2, GAL4 in the genome are used to enhance the utilization of ethanol in Saccharomyces cerevisiae and dynamically regulate the synthesis of 7-DHC. The 7-DHC production of the constructed brewer's yeast se6-Δerg6 reached 1400 mg / L during 130 h of batch feeding fermentation in a 5 L fermenter fermentation medium. The brewer's yeast genetic engineering strain constructed by the present invention has high product accumulation, laying a foundation for replacing the traditional multi-step chemical synthesis route and realizing industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 The mass spectrum and structural formula of 7-DHC of the engineered strain cultured in fermentation medium.

[0052] Figure 2 This is a graph showing the 7-DHC production of the engineered strain when cultured in shake flask fermentation medium.

[0053] Figure 3 This is a graph showing the 7-DHC production of the engineered strain when cultured in shake flask fermentation medium.

[0054] Figure 4 This is a graph showing the 7-DHC production of the engineered strain sd9-Δerg6 grown in 5L tank fermentation medium.

[0055] Figure 5 This is a graph showing the 7-DHC production of the engineered strain se6-Δerg6 when cultured in 5L tank fermentation medium. DETAILED DESCRIPTION

[0056] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0057] The culture medium involved in the embodiment is as follows:

[0058] SOB medium: contains 20g tryptone, 0.5g NaCl, 5g yeast extract, 0.186g KCl, and 1g MgCl2 per liter.

[0059] YPD medium: tryptone 20g / L, yeast extract 10g / L, anhydrous glucose 20g / L

[0060] Shake flask fermentation medium: tryptone 20 g / L, anhydrous glucose 40 g / L, yeast extract 10 g / L.

[0061] 5L tank fermentation medium: 20g / L tryptone, 20g / L anhydrous glucose, 10g / L yeast extract, 5-10g / L potassium dihydrogen phosphate, 1-5g / L magnesium sulfate, 15-25g / L ammonium sulfate, 0.5-1.5g / L uracil, leucine, histidine, and tryptophan.

[0062] Feed medium 1 (glucose): 400-600 g / L glucose, 80-100 g / L ammonium sulfate, 10-20 g / L potassium dihydrogen phosphate, 1-5 g / L magnesium sulfate, 0.5-1.5 g / L uracil, leucine, histidine, and tryptophan.

[0063] Feed medium 2 (ethanol): 80%-95% ethanol, 80-100 g / L ammonium sulfate, 5-10 g / L potassium dihydrogen phosphate, 1-5 g / L magnesium sulfate, 0.5-1.5 g / L uracil, leucine, histidine, and tryptophan.

[0064] SOB+Amp plates: Each liter contains 20 g tryptone, 0.5 g NaCl, 5 g yeast extract, 0.186 g KCl, 1 g7MgCl2, and 20 g agar powder.

[0065] YPD+G418 plate: 2% tryptone, 1% yeast extract, 2% anhydrous glucose, 2% agar powder, add 1 mL of 20 mg / mL G418 resistance stock solution per 100 mL.

[0066] YPD+G418+HYGPMX6 plate: 2% tryptone, 1% yeast extract, 2% anhydrous glucose, 2% agar powder, add 1 mL of 20 mg / mL G418 resistance stock solution per 100 mL, and add 1 mL of 20 mg / mL HYGPMX6 resistance stock solution per 100 mL.

[0067] YPD+G418+NRSR plate: 2% tryptone, 1% yeast extract, 2% anhydrous glucose, 2% agar powder, add 1 mL of 20 mg / mL G418 resistance stock solution per 100 mL, and add 1 mL of 20 mg / mL NRSR resistance stock solution per 100 mL.

[0068] SC-URA-HIS plate: For every 100 ml of culture medium, add 0.667 g of YNB inorganic nitrogen source medium, 2 g of glucose, 2 g of agar powder, and 20 mg of TRP and LEU amino acids.

[0069] SC-URA-TRP plate: For every 100 ml of culture medium, add 0.667 g of YNB inorganic nitrogen source medium, 2 g of glucose, 2 g of agar powder, and 20 mg of HIS and LEU amino acids.

[0070] SC-URA-LEU plate: For every 100 ml of culture medium, add 0.667 g YNB inorganic nitrogen source medium, 2 g glucose, 2 g agar powder, and 20 mg HIS and TRP amino acids.

[0071] Detection of 7-DHC content:

[0072] The fermented bacterial liquid was centrifuged, the bacterial cells were broken with 3M hydrochloric acid, and then centrifuged to collect the cell fragments, and the intracellular sterols were extracted by saponification with 1.5mol / L KOH methanol solution in a 60℃ water bath. After the reaction, the above liquid was extracted with n-hexane, and the obtained n-hexane was evaporated to dryness in a 75℃ water bath, and a certain amount of ethyl acetate was added to dissolve it, and filtered into the liquid phase bottle with a filter membrane.

[0073] The gas phase column used was Thermo Fisher's HP-5 (30m*0.25mm*0.25μm); the gas phase program was set as follows: inlet temperature: 300℃; program temperature: 190℃ (1min) 10℃ / min 300℃ (10min); injection volume: 1μL, split ratio 20:1; carrier gas: N2, flow rate 0.6mL / min; transfer rod temperature: 250℃.

[0074] Recombinant Saccharomyces cerevisiae OD 600 Detection method:

[0075] The yeast seed solution cultured for 16-24 hours was inoculated into a 500 ml shake flask containing 50 ml shake flask fermentation medium at a 2% inoculation rate and cultured at 30°C and 220 rpm. When sampling, the OD was measured using a UV spectrophotometer after dilution in an appropriate ratio. 600 .

[0076] The construction of the plasmids involved in the examples was carried out in E. coli Dh5α, and after the plasmid construction was completed, it was used as a template to amplify the expression cassette.

[0077] The plasmids involving PAM site mutations in the embodiments all need to be sequenced at the corresponding positions after construction to ensure that the pdc5 plasmid with the correct mutation at the PAM site is obtained.

[0078] The primer sequences involved in the examples are shown in Table 1:

[0079] Table 1: Primer list

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088]

[0089] Example 1: Cloning of genes required for 7-DHC biosynthesis

[0090] 1. Extraction of Saccharomyces cerevisiae Genomic DNA

[0091] The genomic DNA of Saccharomyces cerevisiae CEN.PK2-1C was extracted using a kit, and the specific steps are as follows:

[0092] (1) Take 1-3 mL of yeast culture medium that has been cultured for 18-24 hours and centrifuge at 12000 rpm for 1 minute at room temperature.

[0093] (2) Discard the supernatant, add 500 μL GenTLE Yeast Solution A to fully suspend the precipitate, and incubate at 37°C for 1 h.

[0094] (3) Add 100 μL of GenTLE Yeast Solution B, vortex gently to mix, and heat at 70°C for 10 min.

[0095] (4) Add 200 μl of GenTLE Yeast Solution C, gently shake to mix, cool on ice for 5 min, and centrifuge at 12,000 rpm and 4°C for 5 min.

[0096] (5) Transfer the supernatant to a new centrifuge tube. Add 1 / 2 volume of isopropanol (about 400 μL) of the supernatant, invert the tube to mix thoroughly, centrifuge at 12,000 rpm, 4°C for 5 min, and discard the supernatant.

[0097] (6) Add 500 μL of pre-cooled 70% ethanol to the precipitate, gently invert the precipitate to wash it, and centrifuge at 12,000 rpm and 4°C for 5 min.

[0098] (7) Discard the supernatant. Dry the DNA precipitate naturally at room temperature until there is no ethanol odor.

[0099] (8) Add 40-60 μL of TE Buffer or other dissolution buffer to dissolve the genomic DNA.

[0100] 2. Construction of tool plasmids

[0101] (1) Using the Saccharomyces cerevisiae CEN.PK2-1C genome as a template, TEF1t F and TEF1t R were used to obtain gene fragment T TEF1 , using ADH1t F and ADH1t R to obtain gene fragment T ADH1 , using GAL10p F and GAL1pR to obtain gene fragment P GAL1,10 , using CYC1t F and CYC1t R to obtain gene fragment T CYC1 ;

[0102] (2) The four segments T in step (1) are TEF1 、T ADH1 , P GAL1,10 、T CYC1 Fusion PCR was performed by PCR, and the correct band obtained by running the gel was cut and recovered to obtain the fusion gene fragment containing the bidirectional expression frame T TEF1 -T ADH1 -P GAL1,10 -T CYC1 ;

[0103] (3) Using pMD20 plasmid as template, primers LpMD20 F and LpMD20 R were used to obtain linearized pMD20 plasmid;

[0104] (4) Gene fragment T TEF1 -T ADH1 -P GAL1,10 -T CYC1 The plasmid was connected with the linearized pMD20 plasmid and introduced into the competent Dh5α. The primers V20F and V20R were used to verify the T TEF1 -T ADH1 -P GAL1,10 -T CYC1 Bidirectional expression frame T TEF1 -T ADH1 -P GAL1,10 -T CYC1 -pMD20 plasmid.

[0105] 3. Cloning of truncated 3-hydroxy-3-methylglutaryl-CoA reductase encoding gene (tHMG1), squalene epoxidase encoding gene (ERG1), lanosterol C-14 demethylase encoding gene (ERG11), C-8 sterol isomerase encoding gene (ERG2), NADH kinase encoding gene (POS5), lanosterol demethylase encoding gene (ERG11), lanosterol synthase encoding gene (ERG7), isopentenyl pyrophosphate isomerase encoding gene (IDI1), acetyl-CoA CoA synthetase (ACS1), acetyl-CoA acetyltransferase (ERG10), acetaldehyde dehydrogenase (ALD6), acetyl-CoA hydrolase (ACH1), mevalonate pyrophosphate decarboxylase (ERG19), alcohol dehydrogenase (ADH2), phospholipid biosynthesis regulator (INO2), transcription activator (UPC2), GAL promoter activator protein (GAL4), C-5 sterol desaturase (ERG3) were amplified by PCR using the Saccharomyces cerevisiae genome as a template and a high-fidelity enzyme (Phanta Max Super-Fidelity DNA Polymerase). 24-sterol reductase DHCR24 from Gallus Gallus and acetyl-CoA synthetase ACS1 from Salmonella enterica were synthesized by Qingke Bio after codon optimization. Citrate lyase ACL from Yarrowia lipolytica was amplified by PCR using the Yarrowia lipolytica genome as a template.

[0106] The PCR reaction system (50 μL) is as follows: 2×Buffer: 25 μL, dNTPs (10 mM): 1 μL, Primer F: 1 μL, Primer R: 1 μL, Template: 1 μL, DNA polymerase: 0.5 μL, Water: 20.5 μL. The PCR program is as follows:

[0107] (1) Pre-denaturation at 95°C for 5 min; (2) Denaturation at 95°C for 30 s, annealing at 60°C for 30 s, extension at 72°C at 1 kb / min, 32 cycles; (3) Final extension at 72°C for 5 min; (4) Storage at 16°C for 1 min.

[0108] 4. Construction of sgRNA

[0109] Using plasmid pdc5 as a template, using primers described in Table 1, such as primers CERG6-F / CERG6-R, reversely amplify the plasmid to obtain a linearized pdc5 plasmid, and introduce it into competent Dh5α, spread it on a SOB+Amp plate, and culture it at 37°C to obtain a sgRNA-ERG6 plasmid.

[0110] Examples 2-11 were carried out with the goal of de novo synthesis of 7-DHC and strengthening the main synthesis pathway.

[0111] Example 2 Effect of single copy overexpression on the enhancement of the coenzyme supply module on product accumulation

[0112] Based on the engineered yeast sc9, the endogenous gene OSH2 of Saccharomyces cerevisiae was knocked out, and single-copy expression frames of the endogenous genes ERG3 and POS5 of Saccharomyces cerevisiae were integrated at this site to overexpress the rate-limiting step and promote the conversion of reduced coenzyme I to reduced coenzyme II.

[0113] The specific steps are as follows:

[0114] (1) Linearize pMD20 using primers LpMD20 F / LpMD20 R. TEF1 -T ADH1 -P GAL1,10 -T CYC1- The pMD20 plasmid was used as a template and the primers TEF1t F / CYC1t R were used to amplify the expression cassette T TEF1 -T ADH1 -P GAL1,10 -T CYC1 , primers UOSH2-F / UOSH2-R and DOSH2-F / DOSH2-R were used to amplify the upstream and downstream homology arms of the OSH2 locus. TEF1 -T ADH1 -P GAL1,10 -T CYC1 The upstream and downstream homology arms of the OSH2 site were ligated to the linearized pMD20 plasmid.

[0115] (2) Using the above plasmid as a template, primers CYC1t F / TEF1t R were used to amplify the linearized plasmid with the upstream and downstream homology arms and terminator of the OSH2 site, primers ERG3-F / ERG3-R were used to amplify the gene ERG3, primers POS5-F / POS5-R were used to amplify the gene POS5, primers P,TEF1-F / P,TEF1-R were used to amplify the promoter TEF1, and primers P,TDH3-F / P,TDH3-R were used to amplify the promoter TDH3, and the above fragments and plasmids were connected. Primers UOSH2-F / DOSH2-R were used to obtain the fragment donorΔOSH2::T from the obtained recombinant plasmid. TEF -P TEF1 -POS5-T ADH1 -P TDH3 -ERG3-T CYC1 .

[0116] (3) Construction of sg RNA:

[0117] The sgRNA construction method described in Example 1 was used, and COSH2-F / COSH2-R were used as primers to obtain the sgRNA-OSH2 plasmid.

[0118] (4) The sgRNA-OSH2 and donorΔOSH2::T constructed in steps (2) and (3) were added to TEF -P TEF1 -POS5-T ADH1 -P TDH3 -ERG3-T CYC1 The fusion gene fragment was transformed into the constructed Saccharomyces cerevisiae strain SC9 competent cell and cultured on a YPD+G418 plate at 30°C for 2-3 days. A single colony with the correct band was selected and the engineered strain was named Saccharomyces cerevisiae sd1.

[0119] Example 3 Effect of single copy overexpression on the enhancement of the post-squalene module on metabolic flux migration

[0120] Based on the aforementioned strain sd1, the endogenous gene ERG5 of Saccharomyces cerevisiae was knocked out, and single-copy expression frames of the endogenous genes ERG7 and ERG2 of Saccharomyces cerevisiae were integrated at this site to overexpress the rate-limiting gene of the 7-DHC main pathway synthesis.

[0121] The specific steps are as follows:

[0122] (1) Linearize pMD20 using primers LpMD20 F / LpMD20 R. TEF1 -T ADH1 -P GAL1,10 -T CYC1- The pMD20 plasmid was used as a template and the primers TEF1t F / CYC1t R were used to amplify the expression cassette T TEF1 -T ADH1 -P GAL1,10 -T CYC1 , primers UERG5-F / UERG5-R and DERG5-F / DERG5-R were used to amplify the upstream and downstream homology arms of the ERG5 locus. TEF1 -T ADH1 -P GAL1,10 -T CYC1 The upstream and downstream homology arms of the ERG5 site were ligated to the linearized pMD20 plasmid.

[0123] (2) Using the above plasmid as a template, primers CYC1t F / DERG5-R were used to amplify the linearized plasmid with the upstream and downstream homology arms and terminator of the ERG5 site, primers ERG7-F / ERG7-R were used to amplify the gene ERG7, primers ERG2-F / ERG2-R were used to amplify the gene ERG2, and primers GAL10p F / GAL1p R were used to obtain fragment PGAL1,10 , use primers T, TPS1-F / T, TPS1-R to amplify the terminator TPS1, and connect the above fragments and plasmid. Primers UERG5-F / DEG5-R obtain the fragment donorΔERG5::T from the obtained recombinant plasmid TEF -T ADH1 -ERG7-P GAL10 -P GAL1 -ERG2-T TPS1 .

[0124] (3) Construction of sg RNA:

[0125] The sgRNA construction method described in Example 1 was used to obtain the sgRNA-ERG5 plasmid using CERG5-F / CERG5-R as primers.

[0126] (4) The sgRNA-ERG5 and donorΔERG5::T constructed in steps (2) and (3) were added to TEF -T ADH1 -ERG7-P GAL10 -P GAL1 -ERG2-T TPS1 The fusion gene fragment was transformed into the constructed competent strain of Saccharomyces cerevisiae sd1 and cultured on a YPD+G418 plate at 30°C for 2-3 days. A single colony with the correct band was selected and the engineered strain was named Saccharomyces cerevisiae sd2.

[0127] Example 4 Effect of Ty1Cons1 multi-copy overexpression on the key rate-limiting step on 7-DHC accumulation

[0128] Based on the aforementioned strain sd2, multiple copies of the endogenous genes ERG1 and tHMG1 of Saccharomyces cerevisiae were integrated at the Ty1Cons1 site, DHCR24 was heterologously expressed, and the rate-limiting gene of the 7-DHC main pathway synthesis was overexpressed to strengthen the key rate-limiting step.

[0129] The specific steps are as follows:

[0130] (1) Primers UTY11-F / UTY11-R were used to amplify UTY11, DTY11-F / DTY11-R were used to amplify DTY11, P,TEF1-F / P,TEF1-R were used to amplify the promoter TEF1 (the same below), DHCR24-F / DHCR24-R were used to amplify the gene DHCR24 (the same below), T,TPS1-F / T,TPS1-R were used to amplify the terminator TPS1 (the same below), ERG1 gene was amplified with ERG1-F-20(GAP) / ERG1-R-20(TPS1O), and T,TDH3-F / T,TDH3 -R was used to amplify the terminator TDH3 (the same below), P,PGK1-F / PGK1-R was used to amplify the promoter PGK1, tHMG1-F-20(P,PGK1) / tHMG1-TCYC1-R was used to amplify the gene tHMG1, H-TCYC1-F / H-DTY11-R was used to amplify HYGPMX6, L-PMD20-F / L-PMD20-R was used to amplify the linearized plasmid PMD20 (the same below), multiple fragments were repeatedly fused PCR, multiple fragments and plasmids were connected, and the primers UTY11-F / DTY11-R were used to amplify donorΔTy1Cons1::P TEF1 -DHCR24-T TPS1 -P GAP -ERG1-T TDH3 -P PGK1 -tHMG1-T CYC1 -HYGPMX6.

[0131] (2) Construction of sg RNA:

[0132] The sgRNA construction method described in Example 1 was used, and CTY11-F / CTY11-R were used as primers to obtain the sgRNA-TY11 plasmid.

[0133] (3) The sgRNA-TY11 and donorΔTy1Cons1::P constructed in steps (1) and (2) were TEF1 -DHCR24-T TPS1 -P GAP -ERG1-T TDH3 -P PGK1 -tHMG1-T CYC1 -HYGPMX6 fusion gene fragment was transformed into the constructed Saccharomyces cerevisiae strain sd2 competent cell, and cultured on YPD+G418+HYGPMX6 plates at 30°C for 2-3 days. Single colonies with correct bands were selected to obtain the engineered strain named Saccharomyces cerevisiae sd3.

[0134] Example 5 Effect of knocking out YPL062W and overexpressing DHCR24 on promoting ethanol utilization and 7-DHC accumulation

[0135] Based on the aforementioned strain sd3, the endogenous gene YPL062W of Saccharomyces cerevisiae was knocked out to promote ethanol utilization, and a single copy expression cassette of DHCR24 was integrated at this site to overexpress the rate-limiting gene of the 7-DHC main pathway synthesis.

[0136] The specific steps are as follows:

[0137] (1) pMD20 was linearized using primers L-pMD20-F / L-pMD20-R, and the upstream and downstream homology arms of the YPL062W site were amplified using primers UYPL-F / UYPL-R and DYPL-F / DYPL-R, respectively, and fragments TEF1, TPS1, and gene DHCR24 (same as above) were fused by multi-fragment PCR and connected. Primers UYPL-F / DYPL-R were used to obtain the fragment donorΔYPL062W::P TEF1 -DHCR24-T TPS1 .

[0138] (2) Construction of sg RNA:

[0139] The sgRNA construction method described in Example 1 was used, and CYPL-F / CYPL-R were used as primers to obtain the sgRNA-YPL plasmid.

[0140] (3) The sgRNA-YPL and donorΔYPL062W::P constructed in steps (1) and (2) were added to TEF1 -DHCR24-T TPS1 The fusion gene fragment was transformed into the constructed competent yeast strain sd1 and cultured on a YPD+G418 plate at 30°C for 2-3 days. A single colony with the correct band was selected and the engineered strain was named Saccharomyces cerevisiae sd4.

[0141] Example 6 Effect of the key rate-limiting step of the squalene pathway on 7-DHC accumulation after Ty2Cons multi-copy overexpression

[0142] Based on the aforementioned strain sd4, the endogenous genes ERG2 and tHMG1 of Saccharomyces cerevisiae were integrated at the Ty2Cons site, DHCR24 was heterologously expressed, and the rate-limiting gene of the main pathway of 7-DHC synthesis was overexpressed to promote the conversion of yeast sterol to 7-DHC.

[0143] The specific steps are as follows:

[0144] (1) Primers UTY2-F / UTY2-R were used to amplify UTY2, DTY2-F / DTY2-R were used to amplify DTY2, DHCR24-F / D24-R-TDH3 were used to amplify DHCR24 with TDH3 homology arms (the same below), P,GAP-F-TD / P,GAP-R were used to amplify the promoter GAP, ERG2-F-GAP / ERG2-R-TP were used to amplify the gene ERG2, P,PGK1-F-TP / P,PGK1-R were used to amplify the promoter PGK1, HIS-F / HIS-R were used to amplify the gene HIS, as well as the above-mentioned fragments, multiple fragments were repeatedly fused by PCR, multiple fragments and linear plasmids were connected, and primers UTY2-F / DTY2-R were used to amplify donorΔTy2Cons::P TEF1 -DHCR24-T TDH3 -P GAP -ERG2-T TPS1 -P PGK1 -tHMG1-T CYC1 -P HIS -HIS-T A DH1.

[0145] (2) Construction of sg RNA:

[0146] The sgRNA construction method described in Example 1 was used, and CTY2-F / CTY2-R were used as primers to obtain the sgRNA-TY2 plasmid.

[0147] (3) The sgRNA-TY2 and donorΔTy2Cons::P constructed in steps (1) and (2) were TEF1 -DHCR24-T TDH3 -P GAP -ERG2-T TPS1 -P PGK1 -tHMG1-T CYC1 -P HIS -HIS-T ADH1 The fusion gene fragment was transformed into the constructed competent yeast strain sd4 and cultured on SC-URA-HIS plates at 30°C for 2-3 days. The single colony with the correct band was selected and the engineered strain was named Saccharomyces cerevisiae sd5.

[0148] Example 7 Effect of the key rate-limiting step of the squalene pathway on 7-DHC accumulation after further overexpression of multiple copies of Ty1Cons2

[0149] Based on the aforementioned strain sd5, the endogenous genes ERG1 and tHMG1 of Saccharomyces cerevisiae were integrated at the Ty1Cons2 site, DHCR24 was heterologously expressed, and the rate-limiting gene was further overexpressed.

[0150] The specific steps are as follows:

[0151] (1) Plasmid P constructed by implementing Example 4 TEF1 -DHCR24-T TPS1 -P GAP -ERG1-T TDH3 -P PGK1 -tHMG1-T CYC1 -HYGPMX6 was used as a template to amplify the linearized plasmid using LPMD20-R / P, TEF1-F, and UTY12 was amplified using UTY12-F / UTY12-R and connected to the linearized plasmid. The above plasmid was used as a template to amplify the linearized plasmid using LPMD20-F / T, CYC1-R-TRP, the TRP fragment was amplified using PTRP-F-CYC1 / T, TEF-R-TY12D, and the DTY12 fragment was amplified using DTY12-F / DTY12-R, and multiple fragments were connected. The donorΔTy1Cons2::P was amplified using primers UTY12-F / DTY12-R TEF1 -DHCR24-T TDH3 -P GAP -ERG1-T TPS1 -P PGK1 -tHMG1-T CYC1 -P TRP -TRP.

[0152] (2) Construction of sg RNA:

[0153] The sgRNA construction method described in Example 1 was used, and CTY12-F / CTY12-R were used as primers to obtain the sgRNA-TY12 plasmid.

[0154] (3) The sgRNA-TY12 and donorΔTy1Cons2::P constructed in steps (1) and (2) were TEF1 -DHCR24-T TDH3 -P GAP -ERG1-T TPS1 -P PGK1 -tHMG1-T CYC1 -P TRP -TRP fusion gene fragment was transformed into the constructed Saccharomyces cerevisiae strain sd4 competent cell, and cultured on SC-URA-TRP plates at 30°C for 2-3 days. Single colonies with correct bands were selected to obtain the engineered strain named Saccharomyces cerevisiae sd6.

[0155] Example 8 Effect of strengthening the coenzyme supply module by overexpressing multiple copies of Ty4Cons on product accumulation

[0156] Based on the aforementioned strain sd6, the endogenous genes tHMG1 and POS5 of Saccharomyces cerevisiae were integrated at the Ty4Cons site, DHCR24 was heterologously expressed, and the rate-limiting gene was further overexpressed. Multi-copy overexpression of POS5 promoted the conversion of reduced coenzyme I to reduced coenzyme II.

[0157] The specific steps are as follows:

[0158] (1) Plasmid P constructed by implementing Example 4 TEF1 -DHCR24-T TPS1 -P GAP -ERG1-T TDH3 -P PGK1 -tHMG1-T CYC1 -HYGPMX6 was used as a template to amplify the linearized plasmid with LPMD20-R / P,TEF1-F, and UTY4 was amplified with UTY4-F / UTY4-R and connected to the linearized plasmid. The above plasmid was used as a template to amplify the linearized plasmid with LPMD20-F / T,CYC1-R-NAT, the fragment NAT was amplified with NAT-F / NAT-R, and the DTY4 fragment was amplified with DTY4-F / DTY4-R and connected to the linearized fragment. The above plasmid was reversely amplified again with primers T,TPS1-R-PGAL7 / T,TDH3-F-POS5, the promoter GAL7 was amplified with P,GAL7-F / P,GAL7-R, and the gene POS5 was amplified with POS5-F-GAL7 / POS5-R-TDH3, and multiple fragments were connected. The donorΔTy4Cons::P was amplified with primers UTY4-F / DTY4-R TEF1 -DHCR24-T TPS1 -P CAL7 -POS5-T TDH3 -P PGK1 -tHMG1-T CYC1 -NRSR.

[0159] (2) Construction of sg RNA:

[0160] The sgRNA construction method described in Example 1 was used to obtain the sgRNA-TY4 plasmid using CTY4-F / CTY4-R as primers.

[0161] (3) The sgRNA-TY4 and donorΔTy4Cons::P constructed in steps (1) and (2) were TEF1 -DHCR24-T TPS1 -P CAL7 -POS5-T TDH3 -P PGK1 -tHMG1-T CYC1The -NRSR fusion gene fragment was transformed into the constructed Saccharomyces cerevisiae strain sd6 competent cell, and cultured on YPD+G418+NRSR plates at 30°C for 2-3 days. A single colony with the correct band was selected to obtain an engineered strain named Saccharomyces cerevisiae sd7.

[0162] Example 9 Effect of Ty1Cons1 multi-copy overexpression on 7-DHC accumulation at the key rate-limiting step of the squalene pathway

[0163] Based on the aforementioned strain sd7, Ty1Cons1 was inserted again at this site because there were a large number of copies in the yeast genome. The endogenous genes ERG7, ERG11, and IDI1 of Saccharomyces cerevisiae were integrated at the Ty1Cons1 site to overexpress the rate-limiting gene.

[0164] The specific steps are as follows:

[0165] (1) Using the plasmid constructed in Example 4 as a template, amplifying the linear plasmid with T,CYC1-F / UTY11-R-PGAL1, amplifying the promoter GAL1 with P,GAL1-F / P,GAL1-R, amplifying the gene ERG7 with ERG7-F-GAL1 / ERG7-R-TDH3, amplifying the promoter GAL7 with P,CAL7-FT,TDH3 / P,GAL7-R, amplifying the gene ERG11 with ERG11-F-GAL7 / ERG11-R-TPS1, amplifying the promoter GAP with P,GAP-TPS1 / P,GAP-R, amplifying the gene IDI1 with IDI1-F-GAP / IDI1-R-CYC1, repeating fusion PCR for multiple fragments, and connecting multiple fragments and plasmids. Primers UTY11-F / DTY11-R amplified donorΔTy1Cons1::P GAL1 -ERG7-T TDH3 -P CAL7 -ERG11-T TPS1 -P GAP -IDI-T CYC1 -HYGPMX6.

[0166] (2) Construction of sg RNA:

[0167] The sgRNA construction method described in Example 1 was used to obtain the sgRNA-TY11 plasmid using CTY11-F / CTY11-R as primers.

[0168] (3) The sgRNA-TY11 and donorΔTy1Cons1::P constructed in steps (1) and (2) were GAL1 -ERG7-T TDH3 -P CAL7 -ERG11-T TPS1-P GAP -IDI-T CYC1 The -HYGPMX6 fusion gene fragment was transformed into the constructed Saccharomyces cerevisiae strain sd7 competent cell, and cultured on a YPD+G418+HYGPMX6 plate at 30°C for 2-3 days. A single colony with the correct band was selected to obtain an engineered strain named Saccharomyces cerevisiae sd8.

[0169] Example 10 Overexpression of Ty3Cons leads to significant accumulation of 7-DHC de novo synthesis Precursor reaction steps affect 7-DHC accumulation

[0170] Based on the strain sd8 constructed in Example 9, the endogenous genes ERG2, ERG3 and ERG1 of Saccharomyces cerevisiae were integrated at the Ty3Cons site to overexpress the rate-limiting gene, and further overexpress the key enzymes of the post-squalene pathway.

[0171] The specific steps are as follows:

[0172] (1) Linearize pMD20 using primers LpMD20 F / LpMD20 R. TEF1 -T ADH1 -P GAL1,10 -T CYC1- The pMD20 plasmid was used as a template and the primers TEF1t F / CYC1t R were used to amplify the expression cassette T TEF1 -T ADH1 -P GAL1,10 -T CYC1 The upstream and downstream homology arms of the TY3 locus were amplified using primers UTY3-F / UTY3-R and DTY3-F / DTY3-R, respectively, and the gene LEU was amplified using primers LEU-F-TPS1 / LEU-R. TEF1 -T ADH1 -P GAL1,10 -T CYC1 , the upstream and downstream homology arms of the TY3 site, and LEU were connected to the linearized pMD20 plasmid.

[0173] (2) Using the above plasmid as a template, primers CYC1t F / TEF1t R were used to amplify the linearized plasmid with the upstream and downstream homology arms and terminator of the TY3 site, primers ERG1-F-GAP / ERG1-R-TPS1 were used to amplify the gene ERG1, GAP-F-CYC1 / P, GAP-R were used to amplify the promoter GAP, primers ERG3-R-CYC1 / ERG3-F-GAL1 were used to amplify the gene ERG3, and primers ERG2-R-GAL10 / ERG2-F-ADH1 were used to amplify the gene ERG2. The above fragments and plasmids were combined with fragment P GAL1,10, multiple fragments repeated fusion PCR, multiple fragments connection. Primers UTY3-F / DTY3-R obtained fragment donorΔTy3Cons::T from the obtained recombinant plasmid TEF- T ADH1 -ERG2-P CAL10 -P CAL1 -ERG3-T CYC1 -P CAP -ERG1-T TPS1 -P LEU -LEU.

[0174] (3) Construction of sg RNA:

[0175] The sgRNA construction method described in Example 1 was used to obtain the sgRNA-TY3 plasmid using CTY3-F / CTY3-R as primers.

[0176] (4) The sgRNA-TY3 and donorΔTy3Cons::T constructed in steps (2) and (3) were TEF- T ADH1 -ERG2-P CAL10 -P CAL1 -ERG3-T CYC1 -P CAP -ERG1-T TPS1 -P LEU The -LEU fusion gene fragment was transformed into the constructed Saccharomyces cerevisiae strain sd8 competent cell and cultured on SC-URA-LEU plates at 30°C for 2-3 days. A single colony with the correct band was selected to obtain an engineered strain named Saccharomyces cerevisiae sd9.

[0177] Example 11 Effect of blocking the endogenous ergot synthesis pathway of sd9 on 7-DHC accumulation

[0178] The specific steps are as follows:

[0179] (1) Construction of donor fragment:

[0180] Using the genome of Saccharomyces cerevisiae CEN.PK2-1C as a template, the upstream and downstream homology arms of the ERG6 locus were amplified using primers UERG6-F / UERG6-R-DE6 and DERG6-F-UE6 / DERG6-R, respectively, and the homology arms were connected into donorΔERG6 using the fusion PCR method.

[0181] (2) Construction of sg RNA:

[0182] The sgRNA construction method described in Example 1 was used to obtain the sgRNA-ERG6 plasmid using CERG6-F / CERG6-R as primers.

[0183] (3) The sgRNA-CERG6 and donorΔERG6 fusion gene fragments constructed in steps (1) and (2) were transformed into the competent strain of Saccharomyces cerevisiae sd9 and cultured on YPD+G418 plates at 30°C for 2-3 days. Single colonies with correct bands were selected and the obtained strain was named Saccharomyces cerevisiae sd9-Δerg6.

[0184] The engineered bacteria sd9-Δerg6 obtained in Example 2-11 was fermented in a shake flask fermentation medium at 30°C and 220 rpm for 96 hours, and the cells were collected and 7-DHC in the cells was detected. The results showed ( Figure 2 (as shown) The 7-DHC production of Saccharomyces cerevisiae sd9-Δerg6 after 96 h of fermentation was 215 mg / L.

[0185] In order to enhance the utilization of ethanol and the dynamic synthesis and regulation of 7-DHC, Examples 12-18 were carried out.

[0186] Example 12 Effect of increasing the supply of acetyl-CoA, a precursor for sterol synthesis, on 7-DHC accumulation

[0187] On the basis of the strain sd9 constructed in Example 10, the endogenous gene MOT3 of Saccharomyces cerevisiae was knocked out, and the endogenous gene ACS1 of Saccharomyces cerevisiae was integrated at this site to heterologously express the citrate lyase ACL from Yarrowia lipolytica to increase the supply of cytoplasmic acetyl-CoA.

[0188] The specific steps are as follows:

[0189] (1) Linearize pMD20 using primers LpMD20 F / LpMD20 R. TEF1 -T ADH1 -P GAL1,10 -T CYC1- The pMD20 plasmid was used as a template and the primers TEF1t F / CYC1t R were used to amplify the expression cassette T TEF1 -T ADH1 -P GAL1,10 -T CYC1 , primers UMOT3-F / UMOT3-R and DMOT3-F / DMOT3-R were used to amplify the upstream and downstream homology arms of the MOT3 locus. TEF1 -T ADH1 -P GAL1,10 -T CYC1 The upstream and downstream homology arms of the MOT3 site were ligated to the linearized pMD20 plasmid.

[0190] (2) Using the above plasmid as a template, primers CYC1t F / TEF1t R were used to amplify the linearized plasmid with the upstream and downstream homology arms of the MOT3 site and the terminator, primers ACL-F / ACL-R were used to amplify the gene ACL, and primers ACS1-F / ACS1-R were used to amplify the gene ACS1. The above fragments and plasmids were combined with P GAL1,10 Primers UMOT3-F / DMOT3-R were used to obtain the fragment donorΔMOT3::T from the obtained recombinant plasmid. TEF -T ADH1 -ACL-P GAL10 -P GAL1 -ACS1-T CYC1 .

[0191] (3) Construction of sg RNA:

[0192] The sgRNA construction method described in Example 1 was used to obtain the sgRNA-MOT3 plasmid using CMOT3-F / CMOT3-R as primers.

[0193] (4) The sgRNA-MOT3 and donorΔMOT3::T constructed in steps (2) and (3) were added to TEF -T ADH1 -ACL-P GAL10 -P GAL1 -ACS1-T CYC1 The fusion gene fragment was transformed into the constructed competent yeast strain sd9 and cultured on a YPD+G418 plate at 30°C for 2-3 days. A single colony with the correct band was selected and the engineered strain was named Saccharomyces cerevisiae se1.

[0194] Example 13 Dynamic regulation of 7-DHC synthesis by ethanol-inducible promoter

[0195] Based on the strain se1 constructed in Example 12, the endogenous gene ERG10 of Saccharomyces cerevisiae was integrated at the 208A site, and the acetyl-CoA synthetase ACS1 from Salmonella enterica with the amino acid 641 mutated to proline was heterologously expressed, and ACS was overexpressed with an ethanol-inducible promoter. L641P , ERG10 dynamically regulates 7-DHC synthesis.

[0196] The specific steps are as follows:

[0197] (1) Plasmid PET was linearized using primers LPET-F / LPET-R. The upstream and downstream homology arms of 208A were amplified using primers U208A-F / U208A-R and D208A-F / D208A-R, respectively. The promoter FBA1 was amplified using primers P,FBA1-F-TPS1 / P,FBA1-R. The acetyl-CoA synthetase ACS1 from Salmonella enterica with amino acid 641 mutated to proline was amplified using primers SACS1-F / SACS1-R. The terminator ADH2 was amplified using primers T,ADH2-F / T,ADH2-R (the same below). The promoter HXT7 was amplified using primers P,HXT7-F-ADH2 / P,HXT7-R. The gene ERG10 was amplified using primers ERG10-F-HXT7 / ERG10-R-CYC1. Multiple fragments were repeatedly fused with the terminator TPS1 by PCR, and the multiple fragments were connected to the plasmid. Primers U208A-F / D208A-R were used to obtain the fragment donorΔ208A::T from the obtained recombinant plasmid. TPS1 -P FBA1 -ACS1 L641P -T ADH2 -P HXT7 -ERG10-T CYC1 .

[0198] (2) Construction of sg RNA:

[0199] The sgRNA construction method described in Example 1 was used to obtain the sgRNA-208A plasmid using C208A-F / C208A-R as primers.

[0200] (3) The sgRNA-208A and donorΔ208A::T constructed in steps (1) and (2) were TPS1 -P FBA1 -ACS1 L641P -T ADH2 -P HXT7 -ERG10-T CYC1 The fusion gene fragment was transformed into the constructed Saccharomyces cerevisiae strain se1 competent cell and cultured on a YPD+G418 plate at 30°C for 2-3 days. A single colony with the correct band was selected to obtain an engineered strain named Saccharomyces cerevisiae se2.

[0201] Example 14 Effect of overexpression of sterol synthesis regulatory factors and rate-limiting steps on 7-DHC accumulation

[0202] Based on the strain se2 constructed in Example 13, the endogenous gene ERG19 of Saccharomyces cerevisiae was integrated at the YBR197C site, the sterol transcription activator UPC2 with the 888 amino acid mutated to aspartic acid was overexpressed, the rate-limiting enzyme ERG19 of the mevalonate pathway was overexpressed with the constitutive promoter ITR1, and the sterol synthesis transcription regulator UPC2 was overexpressed with the inducible promoter GAL7 to promote the extension of cytoplasmic acetyl-CoA to sterol synthesis.

[0203] The specific steps are as follows:

[0204] (1) Primers LPET-F / LPET-R were used to linearize plasmid PET, and UYBR-F / UYBR-R and DYBR-F / DYBR-R were used to amplify the upstream and downstream homology arms of YBR197C, respectively. The promoter ITR1 was amplified with P,ITR1-F-TPS1 / P,ITR1-R, the gene ERG19 was amplified with ERG19-F-ITR1 / ERG19-R-ADH2, the promoter GAL7 was amplified with P,GAL7-F-ADH2 / P,GAL7-R, and the sterol transcription activator UPC2 with amino acid mutated from 888 to aspartic acid, as well as the terminators TPS1, ADH2, and CYC1 were amplified with UPC2-F-GAL7 / UPC2-R-CYC1. Multi-fragment fusion PCR was performed and the multi-fragment was connected with the linearized plasmid. Primers UYBR-F / DYBR-R were used to obtain the fragment donorΔYBR197C::T from the obtained recombinant plasmid. TPS1 -P ITR1 -ERG19-T ADH2 -P GAL7 -UPC2 G888D -T CYC1 .

[0205] (2) Construction of sg RNA:

[0206] The sgRNA construction method described in Example 1 was used, and CYBR-F / CYBR-R were used as primers to obtain the sgRNA-YBR197C plasmid.

[0207] (3) The sgRNA-YBR197C and donorΔYBR197C::T constructed in steps (1) and (2) were added to TPS1 -P ITR1 -ERG19-T ADH2 -P GAL7 -UPC2 G888D -T CYC1 The fusion gene fragment was transformed into the constructed Saccharomyces cerevisiae strain se2 competent cell, and cultured on a YPD+G418 plate at 30°C for 2-3 days. A single colony with the correct band was selected to obtain an engineered strain named Saccharomyces cerevisiae se3.

[0208] Example 15 Overexpression of the ethanol utilization pathway increases the supply of sterol synthesis precursors

[0209] Based on the strain se3 constructed in Example 14, the endogenous genes ALD6 and ACH1 of Saccharomyces cerevisiae were integrated at the YBL059W site to promote the utilization of ethanol and balance the content of cytosolic acetyl-CoA and mitochondrial acetyl-CoA.

[0210] The specific steps are as follows:

[0211] (1) Linearize pMD20 using primers LpMD20 F / LpMD20 R. TEF1 -T ADH1 -P GAL1,10 -T CYC1- The pMD20 plasmid was used as a template and the primers TEF1t F / CYC1t R were used to amplify the expression cassette T TEF1 -T ADH1 -P GAL1,10 -T CYC1 , primers UYBL-F / UYBL-R and DYBL-F / DYBL-R were used to amplify the upstream and downstream homology arms of the YBL059W site, respectively. TEF1 -T ADH1 -P GAL1,10 -T CYC1 The upstream and downstream homology arms of the YBL059W site were ligated to the linearized pMD20 plasmid.

[0212] (2) Using the above plasmid as a template, primers CYC1t F / TEF1t R were used to amplify the linearized plasmid with the upstream and downstream homology arms and terminator of the YBL059W site, primers ALD6-F / ALD6-R were used to amplify the gene ALD6, and primers ACH1-F / ACH1-R were used to amplify the gene ACH1. The above fragments and plasmids were combined with P GAL110 Primers UYBL-F / DYBL-R were used to obtain the fragment donorΔYBL059W::T from the obtained recombinant plasmid. TEF -T ADH1 -ALD6-P GAL10 -P GAL1 -ACH1-T CYC1 .

[0213] (3) Construction of sg RNA:

[0214] The sgRNA construction method described in Example 2 was used, and CYBL-F / CYBL-R were used as primers to obtain the sgRNA-YBL059W plasmid.

[0215] (4) The sgRNA-YBL059W and donorΔYBL059W::T constructed in steps (2) and (3) were added to TEF -T ADH1 -ALD6-P GAL10 -P GAL1 -ACH1-T CYC1 The fusion gene fragment was transformed into the constructed Saccharomyces cerevisiae strain se3 competent cell, and cultured on a YPD+G418 plate at 30°C for 2-3 days. A single colony with the correct band was selected to obtain an engineered strain named Saccharomyces cerevisiae se4.

[0216] Example 16 Effect of Overexpression of Ethanol Utilization Pathway to Enhance Key Rate-Limiting Steps on 7-DHC Accumulation

[0217] Based on the strain se4 constructed in Example 15, the endogenous genes IDI1 and ADH2 of Saccharomyces cerevisiae were integrated at the YNR014W site to enhance ethanol utilization and strengthen the main pathway with an ethanol-inducible promoter.

[0218] The specific steps are as follows:

[0219] (1) Plasmid PET was linearized using primers LPET-F / LPET-R, and the upstream and downstream homology arms of YNR014W were amplified using primers UYNR-F / UYNR-R and DYNR-F / DYNR-R, respectively. The promoter FBA1 was amplified using primers P,FBA1-F-TPS1 / P,FBA1-R, the gene IDI1 was amplified using primers IDI1-F-FBA1 / IDI1-R-ADH2, the promoter TEF1 was amplified using primers P,TEF1-F-ADH2 / P,TEF1-R, and the gene ADH2 was amplified using primers ADH2-F-TEF1 / ADH2-R-CYC1. The fragments were fused by PCR and connected to the linearized plasmid using primers UYNR-F / DYNR-R. The fragment donorΔYNR014W::T was obtained from the obtained recombinant plasmid. TPS1 -P FBA1 -IDI1-T ADH2 -P TEF1 -ADH2-T CYC1 .

[0220] (2) Construction of sg RNA:

[0221] The sgRNA construction method described in Example 1 was used, and CYNR-F / CYNR-R were used as primers to obtain the sgRNA-YNR014W plasmid.

[0222] (3) The sgRNA-YNR014W and donorΔYNR014W::T constructed in steps (1) and (2) were added toTPS1 -P FBA1 -IDI1-T ADH2 -P TEF1 -ADH2-T CYC1 The fusion gene fragment was transformed into the constructed Saccharomyces cerevisiae strain se4 competent cell and cultured on a YPD+G418 plate at 30°C for 2-3 days. A single colony with the correct band was selected to obtain an engineered strain named Saccharomyces cerevisiae se5.

[0223] Example 17 Effect of cascade amplification of regulatory factors by inducible promoter overexpression on 7-DHC accumulation

[0224] Based on the strain se5 constructed in Example 16, the endogenous gene INO2 of Saccharomyces cerevisiae was integrated at the YMR206W site to expand the endoplasmic reticulum membrane and increase the site for 7-DHC synthesis. HXT7 The expression of GAL4 was enhanced, and ethanol feeding was used to activate the expression of GAL4 in large quantities, and GAL4 was used to further activate the rate-limiting gene overexpressed by the gal promoter.

[0225] The specific steps are as follows:

[0226] (1) Plasmid PET was linearized using primers LPET-F / LPET-R, and the upstream and downstream homology arms of YMR206W were amplified using UYMR-F / UYMR-R and DYMR-F / DYMR-R, respectively. The promoter YGP1 was amplified using P,YGP1-F-TEF / P,YGP1-R, the gene INO2 was amplified using INO2-F-YGP1 / INO2-R-CYC1, the promoter HXT7 was amplified using P,HXT7-R / P,HXT7-F-CYC1, the gene GAL4 was amplified using GAL4-F / GAL4-R, and the terminators TEF, CYC1, and TDH3 were amplified. Multi-fragment fusion PCR was performed and the multi-fragment was connected to the linearized plasmid. Primers UYMR-F / DYMR-R were used to obtain the fragment donorΔYMR206W::T from the obtained recombinant plasmid. TEF -P YGP1 -INO2-T CYC1 -P HXT7 -GAL4-T TDH3 .

[0227] (2) Construction of sg RNA:

[0228] The sgRNA construction method described in Example 1 was used, and CYMR-F / CYMR-R were used as primers to obtain the sgRNA-YMR206W plasmid.

[0229] (3) The sgRNA-YMR206W and donorΔYMR206W::T constructed in steps (1) and (2) were added toTEF -P YGP1 -INO2-T CYC1 -P HXT7 -GAL4-T TDH3 The fusion gene fragment was transformed into the constructed Saccharomyces cerevisiae strain se5 competent cell and cultured on a YPD+G418 plate at 30°C for 2-3 days. A single colony with the correct band was selected to obtain an engineered strain named Saccharomyces cerevisiae se6.

[0230] (4) Using the above recombinant plasmid ΔYMR206W::T TEF -P YGP1 -INO2-T CYC1 -P HXT7 -GAL4-T TDH3 As a template, the plasmid was reversely amplified with primers T, TEF-R-PTEF1 / INO2-F-PTEF1, and connected with promoter TEF1. The donorΔYMR206W::T was obtained in the same way as step (1). TEF -P TEF1 -INO2-T CYC1 -P HXT7 -GAL4-T TDH3 The above recombinant plasmid ΔYMR206W::T TEF -P YGP1 -INO2-T CYC1 -P HXT7 -GAL4-T TDH3 As a template, the plasmid was reversely amplified with primers T, TEF-R-GAL1 / INO2-F-GAL1, and ligated with promoter GAL1. The donorΔYMR206W::T was obtained in the same manner as step (1). TEF -P GAL1 -INO2-T CYC1 -P HXT7 -GAL4-T TDH3 The above recombinant plasmid ΔYMR206W::T TEF -P YGP1 -INO2-T CYC1 -P HXT7 -GAL4-T TDH3 As a template, the plasmid was reversely amplified with primers T, TEF-R-FBA1 / INO2-F-FBA1, and the promoter FBA1 was amplified with primers P, FBA1-F / P, FBA1-R, and connected with the plasmid, and the donorΔYMR206W::T was obtained in the same way as step (1). TEF -P FBA1 -INO2-T CYC1 -P HXT7 -GAL4-T TDH3The donor was introduced into se5 in the same manner as step (3) to obtain engineered strains se5-tef1-ino2, se5-gal1-ino2, and se5-fba1-ino2. The shake flask level validation analysis was performed in the same manner as in Example 11. The results were as follows: Figure 3 As shown, overexpression of the phospholipid biosynthesis regulator INO2 with a constitutive promoter had a significant inhibitory effect on bacterial growth, while the acetic acid and ethanol inducible promoter had little effect on bacterial growth. The 7-DHC production of the se6 strain increased by 15%, and the engineered strain se6 overexpressing ino2 with the acetic acid inducible promoter YGP1 was subjected to the next metabolic transformation.

[0231] Example 18 Effect of Blocking the Endogenous Ergosterol Synthesis Pathway of SE6 on 7-DHC Accumulation

[0232] The specific steps are as follows:

[0233] (1) Construction of donor fragment:

[0234] Using the genome of Saccharomyces cerevisiae CEN.PK2-1C as a template, the upstream and downstream homology arms of the ERG6 locus were amplified using primers UERG6-F / UERG6-R-DE6 and DERG6-F-UE6 / DERG6-R, respectively, and the homology arms were connected into donorΔERG6 using the fusion PCR method.

[0235] (2) Construction of sg RNA:

[0236] The sgRNA construction method described in Example 1 was used to obtain the sgRNA-ERG6 plasmid using CERG6-F / CERG6-R as primers.

[0237] (3) The sgRNA-CERG6 and donorΔERG6 fusion gene fragments constructed in steps (1) and (2) were transformed into the competent cells of the Saccharomyces cerevisiae se6 strain and cultured on a YPD+G418 plate at 30°C for 2-3 days. A single colony with the correct band was selected and the obtained strain was named Saccharomyces cerevisiae se6-Δerg6.

[0238] Example 19 Fermentation of sd9-Δerg6 and se6-Δerg6 engineered bacteria in 5L tanks

[0239] (1) Under sterile conditions, single colonies of strains sd9-Δerg6 and se6-Δerg6 were inoculated into YPD medium and cultured at 30°C and 220 rpm for 24 h to prepare activated bacterial solution for scale-up culture;

[0240] (2) Under sterile conditions, the activated bacterial liquid of sd9-Δerg6 and se6-Δerg6 was inoculated into a 500 ml YPD shake flask containing 50 ml YPD medium at a 2% inoculation rate, 0.5 g / L to 1 g / L uracil was added exogenously, and the culture was shaken at 30°C and 220 rpm for 24 to 36 h to prepare the fermentation seed liquid;

[0241] (3) Batch fed fermentation was carried out in a 5L fermenter with a liquid volume of 3L. The initial ventilation volume was 3L / min, the initial stirring was 300rpm, the culture temperature was 30°C, and the pH was controlled at 5.5 with 50% ammonia water. After the initial dissolved oxygen dropped to 30%, the dissolved oxygen was controlled at about 30% by controlling the stirring speed and air flow. When the glucose content dropped to 1g / L and the ethanol content dropped to 10g / L, feeding (about 15h) was started. In the early stage of fermentation (15-50h), feed medium 1 was used to feed the carbon source glucose, and the sugar content of the fermentation medium was controlled to be below 1g / L and the ethanol content was controlled to be below 10g / L. In the late stage of fermentation (50h to the end of fermentation), feed medium 2 was used to feed the carbon source ethanol, and the ethanol content of the fermentation medium was controlled to be 15-25g / L. The fermentation ended after 100h-130h. The fermentation broth was subjected to cell disruption and saponification extraction to obtain the 7-DHC, such as Figure 4 , Figure 5 As shown, the final production of strains sd9-Δerg6 and se6-Δerg6 reached 800 mg / L and 1400 mg / L, respectively.

Claims

1. A genetically engineered yeast for synthesizing 7-DHC from scratch, characterized in that: The method is constructed by the following method: the genome of the bottom fungus Saccharomyces cerevisiae is used to enhance the expression of truncated 3-hydroxy-3-methylglutaryl-CoA reductase tHMG1, squalene epoxidase ERG1, C-8 sterol isomerase ERG2, NADH kinase POS5, lanosterol demethylase ERG11, lanosterol synthase ERG7, isopentenyl pyrophosphate isomerase IDI1, acetyl-CoA synthetase ACS1, acetyl-CoA acetyltransferase ERG10, acetaldehyde dehydrogenase ALD6, acetyl-CoA hydrolase ACH1, mevalonate pyrophosphate decarboxylase ERG19, alcohol dehydrogenase ADH2, phospholipid biosynthesis regulator INO2, sterol transcription activator UPC2, GAL promoter activator protein GAL4, C-5 sterol desaturase ERG3, and heterologously express 24-sterol reductase DHCR24 from Gallus Gallus, acetyl-CoA synthetase ACS1 from Salmonella enterica, Yarrowia lipolytica, knocked out the C-22 sterol desaturase ERG5, δC-24 sterol methyltransferase ERG6 and ergosterol synthesis gene transcription repressor MOT3 required for ergosterol synthesis, and obtained the cerevisiae strain that efficiently synthesizes 7-dehydrocholesterol.

2. A genetically engineered yeast for synthesizing 7-DHC from scratch as claimed in claim 1, characterized in that: The base bacteria is Saccharomyces cerevisiae SC9.

3. A genetically engineered yeast for synthesizing 7-DHC from scratch as claimed in claim 1, characterized in that: The amino acid at position 641 of the acetyl-CoA synthetase ACS1 derived from Salmonella enterica is mutated from leucine to proline, and the amino acid at position 888 of the sterol transcription activator UPC2 is mutated from glycine to aspartic acid.

4. A genetically engineered yeast for synthesizing 7-DHC from scratch as claimed in claim 1, characterized in that: The enhanced expression specifically refers to the enhanced expression of multiple copies of tHMG1 and DHCR24, multiple copies of ERG1 and POS5, multiple copies of ERG11, IDI1 and ERG7, and multiple copies of ERG2 and ERG3 on the Saccharomyces cerevisiae genome.

5. A genetically engineered yeast for synthesizing 7-DHC from scratch as claimed in claim 1, characterized in that: By P TEF1 The promoter enhances the expression of DHCR24 through P GAP The promoter enhances the expression of ERG1 through P GAP The promoter enhances the expression of ERG2 through P PGK1 Promoter-enhanced expression of tHMG1, through P GAL7 The promoter enhances the expression of POS5 through P GAL1，10 The bidirectional promoter enhances the expression of ERG7 and ERG11 through P GAP The promoter enhances the expression of IDI1 through P GAL1，10 The bidirectional promoter enhances the expression of ERG3 and ERG2 through P GAL1，10 The bidirectional promoter enhanced the expression of ACL and ACS1, and the ethanol-induced promoter P FBA1 Enhanced expression of ACS1 from Salmonella enterica, via the ethanol-inducible promoter P HXT7 Enhanced expression of ERG10, through the promoter P ITR1 Enhanced expression of ERG19 by P GAL7 Promoter-enhanced expression of UPC2, through P GAL1，10 The bidirectional promoter enhances the expression of ALD6 and ACH1 through P TEF1 The promoter enhances the expression of ADH2, and the promoter P is induced by ethanol. FBA1 Enhanced expression of IDI1, through the promoter P YGP1 Enhanced expression of INO2 was achieved by inducing the promoter P HXT7 Overexpression of GAL4.

6. A genetically engineered yeast for synthesizing 7-DHC from scratch as claimed in claim 1, characterized in that: Phospholipid biosynthesis regulator INO2, sterol transcription activator UPC2, and GAL promoter activator protein GAL4 were overexpressed using an inducible promoter.

7. The method for constructing a genetically engineered yeast for synthesizing 7-DHC from scratch according to any one of claims 1 to 6, characterized in that: The method comprises the following steps: enhancing the expression of truncated 3-hydroxy-3-methylglutaryl-CoA reductase tHMG1, squalene epoxidase ERG1, C-8 sterol isomerase ERG2, NADH kinase POS5, lanosterol demethylase ERG11, lanosterol synthase ERG7, isopentenyl pyrophosphate isomerase IDI1, acetyl-CoA synthetase ACS1, acetyl-CoA acetyltransferase ERG10, acetaldehyde dehydrogenase ALD6, acetyl-CoA hydrolase ACH1, mevalonate pyrophosphate decarboxylase ERG19, alcohol dehydrogenase ADH2, phospholipid biosynthesis regulatory factor INO2, sterol transcription activator UPC2, GAL promoter activator protein GAL4, C-5 sterol desaturase ERG3 in the genome of the bottom fungus Saccharomyces cerevisiae, heterologously expressing 24-sterol reductase DHCR24 from Gallus Gallus, acetyl-CoA synthetase ACS1 from Salmonella enterica, Yarrowia lipolytica, knocked out the C-22 sterol desaturase ERG5, δC-24 sterol methyltransferase ERG6 and ergosterol synthesis gene transcription repressor MOT3 required for ergosterol synthesis, and obtained the cerevisiae strain that efficiently synthesizes 7-dehydrocholesterol.

8. The method according to claim 1, characterized in that The specific steps include: (1) Using strain SC9 as the base strain, T TEF -P TEF1 -POS5-T ADH1 -P TDH3 -ERG3-T CYC1 The fragment was integrated into the genome of the SC9 strain at the OSH2 site, and the resulting S. cerevisiae strain was named the sd1 strain; (2) T TEF -T ADH1 -ERG7-P GAL10 -P GAL1 -ERG2-T TPS1 The fragment was integrated into the genome of the sd1 strain at the ERG5 site, and the resulting S. cerevisiae strain was named the sd2 strain; (3) P TEF1 -DHCR24-T TPS1 -P GAP -ERG1-T TDH3 -P PGK1 -tHMG1-T CYC1 -HYGPMX6 fragment was integrated into the genome of sd2 strain, integrated into Ty1Cons1 site, and the resulting Saccharomyces cerevisiae strain was named sd3; (4) P TEF1 -DHCR24-T TPS1 The fragment was integrated into the genome of the sd3 strain at the YPL062W site, and the resulting S. cerevisiae strain was named the sd4 strain; (5)P TEF1 -DHCR24-T TDH3 -P GAP -ERG2-T TPS1 -P PGK1 -tHMG1-T CYC1 -P HIS -HIS-T ADH1 The fragment was integrated into the genome of the sd4 strain at the Ty2Cons site, and the resulting S. cerevisiae strain was named the sd5 strain; (6) P TEF1 -DHCR24-T TDH3 -P GAP -ERG1-T TPS1 -P PGK1 -tHMG1-T CYC1 -P TRP -TRP fragment was integrated into the genome of sd5 strain, integrated into Ty1Cons2 site, and the constructed Saccharomyces cerevisiae strain was named sd6 strain; (7)P TEF1 -DHCR24-T TPS1 -P CAL7 -POS5-T TDH3 -P PGK1 -tHMG1-T CYC1 -NRSR fragment was integrated into the genome of sd6 strain, integrated into Ty4Cons site, and the constructed Saccharomyces cerevisiae strain was named sd7; (8)P GAL1 -ERG7-T TDH3 -P CAL7 -ERG11-T TPS1 -P GAP -IDI-T CYC1 -HYGPMX6 fragment was integrated into the genome of sd7 strain, integrated into Ty1Cons1 site, and the constructed Saccharomyces cerevisiae strain was named sd8; (9) T TEF- T ADH1 -ERG2-P CAL10 -P CAL1 -ERG3-T CYC1 -P CAP -ERG1-T TPS1 -P LEU The -LEU fragment was integrated into the genome of the sd8 strain and integrated into the Ty3Cons site to construct a Saccharomyces cerevisiae strain named sd9; (10) Knockout of the C-24 sterol methyltransferase ERG6 required for ergosterol synthesis in the genome of the sd9 strain, and the resulting Saccharomyces cerevisiae strain was named sd9-Δerg6; (11) T TEF -T ADH1 -ACL-P GAL10 -P GAL1 -ACS1-T CYC1 The fragment was integrated into the genome of the sd9 strain at the MOT3 site, and the resulting Saccharomyces cerevisiae strain was named se1; (12) T TPS1 -P FBA1 -ACS1 L641P -T ADH2 -P HXT7 -ERG10-T CYC1 The fragment was integrated into the genome of the se1 strain at the 208A site, and the resulting Saccharomyces cerevisiae strain was named se2; (13) T TPS1 -P ITR1 -ERG19-T ADH2 -P GAL7 -UPC2 G888D -T CYC1 The fragment was integrated into the genome of the se2 strain at the YBR197C site, and the resulting Saccharomyces cerevisiae strain was named se3; (14) T TEF -T ADH1 -ALD6-P GAL10 -P GAL1 -ACH1-T CYC1 The fragment was integrated into the genome of the se3 strain at the YBL059W site, and the resulting Saccharomyces cerevisiae strain was named se4; (15) T TPS1 -P FBA1 -IDI1-T ADH2 -P TEF1 -ADH2-T CYC1 The fragment was integrated into the genome of the se4 strain at the YNR014W site, and the resulting Saccharomyces cerevisiae strain was named se5; (16) T TEF -P YGP1 -INO2-T CYC1 -P HXT7 -GAL4-T TDH3 The fragment was integrated into the genome of the se5 strain at the YMR206W site, and the resulting Saccharomyces cerevisiae strain was named se6; (17) The C-24 sterol methyltransferase ERG6 required for ergosterol biosynthesis was knocked out in the se6 strain genome, and the resulting Saccharomyces cerevisiae strain was named se6-Δerg6.

9. Use of the genetically engineered yeast Saccharomyces cerevisiae that synthesizes 7-DHC de novo as claimed in any one of claims 1 to 6 in the preparation of 7-DHC by microbial fermentation.

10. The use according to claim 9, characterized in that The brewer's yeast strain is inoculated into a fermentation medium, and fermentation culture is carried out at 28-32° C. for 84-96 hours. The fermentation liquid is subjected to cell disruption and saponification extraction to obtain the 7-DHC.

11. The use according to claim 9, characterized in that The brewer's yeast strain is inoculated into a fermentation medium in a 5L fermenter, and fermentation culture is carried out at 28-32°C for 100-130h. Feeding is started when the sugar content of the initial culture medium drops to 1g / L and the ethanol content drops to 10g / L. Glucose is fed in the early stage of feeding carbon source, and the sugar content of the fermentation medium is controlled to be below 1g / L and the ethanol content is controlled to be below 10g / L. Ethanol is fed in the later stage, and the ethanol content of the fermentation medium is controlled to be 15-25g / L. The fermentation broth is subjected to cell disruption and saponification extraction to obtain the 7-DHC.

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