A de novo synthesis of 7-DHC Saccharomyces cerevisiae genetically engineered strain, its construction method and application

Abstract

This invention specifically relates to a de novo 7-DHC-synthesizing *Saccharomyces cerevisiae* genetically engineered strain, its construction method, and its applications, belonging to the field of biotechnology. Using *Saccharomyces cerevisiae* engineered strain SC9 as the starting strain, this invention utilizes multiple copies of tHMG1, DHCR24, and ERG1 via the TY transposon, multiple copies of ERG2, POS5, ERG7, ERG11, IDI1, and ERG3 via the TY transposon, and single copies of POS5, ERG3, and DHCR24 in the genome to increase the intracellular content of reducing coenzyme II and enhance the main pathway of 7-DHC synthesis. Single copies of ACL, ACS1, ERG10, and ERG19 in the genome enhance the utilization of ethanol in *Saccharomyces cerevisiae* and dynamically regulate the synthesis of 7-DHC. The *Saccharomyces cerevisiae* genetically engineered strain constructed by this invention exhibits high product accumulation, laying the foundation for replacing traditional multi-step chemical synthesis routes and realizing industrial production.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a genetically engineered strain of Saccharomyces cerevisiae that synthesizes 7-DHC, a key intermediate for the synthesis of active vitamin D3, as well as its construction method and application. Background Technology

[0002] Vitamin D3 (cholecalciferol, VD3) is a fat-soluble vitamin that helps maintain calcium phosphate homeostasis and plays a crucial role in numerous biological processes, such as regulating calcium and phosphorus metabolism and promoting bone development. Vitamin D3 has anti-rickets properties and is also known as the anti-rickets vitamin. It can also enhance the body's immunity and reduce the risk of cardiovascular disease. Furthermore, studies have shown a close correlation between vitamin D and diseases such as hypertension, type 2 diabetes, immune regulation, cardiovascular disease, and various cancers.

[0003] With the increasing discovery of the physiological activities of vitamin D3 and the aging population, the market demand for vitamin D3 is growing rapidly. Current production of vitamin D and its derivatives is gradually failing to meet market demand. Existing vitamin D3 synthesis methods are mainly chemical synthesis methods, which are limited by the high cost of raw materials, the need for multiple chemical reactions, low purity, difficult extraction, and high pollution. Synthetic biology has developed rapidly over the past decade, providing new ideas for the green production of high-value-added chemicals through the engineering design and construction of efficient cell factories. Microbial fermentation mainly involves constructing metabolic synthesis pathways within microorganisms, using simple carbon sources (such as glucose and ethanol) as substrates to biosynthesize 7-DHC, and further obtaining vitamin D3 through photoisomerization. Compared with chemical synthesis methods, microbial fermentation methods have the advantages of lower environmental stress, more moderate conditions, and sustainable production, making them very promising for applications. The safe, efficient, and environmentally friendly de novo synthesis of 7-DHC using yeast as a cell factory provides a new approach and method for the industrial production of 7-DHC.

[0004] 7-DHC is structurally similar to ergosterol in yeast, and *Saccharomyces cerevisiae* can synthesize ergosterol de novo. De novo synthesis of 7-DHC in yeast can be achieved by modifying the ergosterol pathway within yeast, thus making yeast an excellent host for the production of steroidal drugs. 7-DHC can be produced in *Saccharomyces cerevisiae* by knocking out ERG5 and ERG6 in the ergosterol synthesis pathway and heterologously expressing the 24-sterol reductase DHCR24. This study improved 7-DHC production by employing strategies such as knocking out relevant repressive genes, enhancing precursor supply, guiding the conversion of precursors to the target product, increasing intracellular reducing power supply, using ethanol as a carbon source, and optimizing ethanol utilization and tolerance in *Saccharomyces cerevisiae*. This has significant implications for the subsequent development of steroidal drugs. Summary of the Invention

[0005] To address the problems existing in the prior art, the purpose of this invention is to design and provide a technical solution for the de novo synthesis of 7-DHC high-yield Saccharomyces cerevisiae genetically engineered strains, its construction method, and its application.

[0006] The technical solution adopted in this invention is:

[0007] A de novo 7-dehydrocholesterol-synthesizing Saccharomyces cerevisiae genetically engineered strain was constructed using the following method: The Saccharomyces cerevisiae genome was enhanced with 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 synthase ACS1, acetyl-CoA acetyltransferase ERG10, aldehyde dehydrogenase ALD6, acetyl-CoA hydrolase ACH1, mevalonate pyrophosphate decarboxylase ERG19, alcohol dehydrogenase ADH2, phospholipid biosynthesis regulator INO2, sterol transcription activator UPC2, GAL promoter activator GAL4, and C-5 sterol desaturase ERG3, followed by heterologous expression of Gallus. The *Saccharomyces cerevisiae* strain that efficiently synthesizes 7-dehydrocholesterol was obtained by knocking out the following enzymes: 24-sterol reductase DHCR24 from *Gallus*, acetyl-CoA synthase ACS1 from *Salmonella enterica*, and citrate lyase ACL from *Yarrowia lipolytica*. This was achieved by also knocking out the C-22 sterol desaturase ERG5, δC-24 sterol methyltransferase ERG6, and the ergosterol synthesis gene transcription repressor MOT3.

[0008] Preferably, the substrate bacteria are Saccharomyces cerevisiae SC9, which can be constructed using the method disclosed in Chinese Patent 202310058515.6 (Saccharomyces cerevisiae genetically engineered strain synthesized by 7-DHC exocytosis and its construction and application).

[0009] Preferably, the enhanced expression refers to enhanced expression of multiple copies of tHMG1, DHCR24, and ERG1 (which can integrate 3-hydroxy-3-methylglutaryl-CoA reductase tHMG1, 24-sterol reductase DHCR24, and squalene epoxidase ERG1 into the repeating δ sequences at both ends of the Ty1Cons1 site of the Saccharomyces cerevisiae transposon) and multiple copies of tHMG1, DHCR24, and ERG2 (which can integrate 3-hydroxy-3-methylglutaryl-CoA reductase tHMG1, 24-sterol reductase DHCR24, and C-8 sterol isocyanate) into the Saccharomyces cerevisiae genome. The helicase ERG2 integrates into the repetitive sequences flanking the Ty2Cons site of the Saccharomyces cerevisiae transposon; multiple copies of tHMG1, DHCR24, and ERG1 (which can integrate 3-hydroxy-3-methylglutaryl-CoA reductase tHMG1, 24-sterol reductase DHCR24, and squalene epoxidase ERG1 into the repetitive δ sequences flanking the Ty1Cons2 site of the Saccharomyces cerevisiae transposon); and multiple copies of tHMG1, DHCR24, and POS5 (which can integrate 3-hydroxy-3-methylglutaryl-CoA reductase tHMG1, 24-sterol reductase D...) HCR24 and NADH kinase POS5 are integrated into the repeating ω sequences flanking the Ty4Cons site of the Saccharomyces cerevisiae transposon in the genome; multiple copies of ERG7, ERG11, and IDI1 (which can integrate lanosterol synthase ERG7, lanosterol demethylase ERG11, and isopentenyl pyrophosphate isomerase IDI1 into the repeating δ sequences flanking the Ty1Cons1 site of the Saccharomyces cerevisiae transposon in the genome); and multiple copies of ERG2, ERG3, and ERG1 (which can integrate C-8 sterol isomerase ERG2, C-5 sterol desaturase ERG3, and squalene epoxidase ERG1) are also present. 1. The repeat ω sequence integrated into both ends of the Ty3Cons site of the Saccharomyces cerevisiae transposon, and 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 1 copy of GAL4, which are enhancedly expressed on 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, and the Gene ID of ACS1 is... The gene IDs of the following gene types are as follows: ACS1 (from Salmonella enterica) is 1255801; ADH2 is 855349; ERG19 is 855779; INO2 is 851701; GAL4 is 855828; ACL (from Yarrowia lipolytica) is 2912112; OSH2 is 851543; YBR197C is 852496; YBL059W is 852221; YNR014W is 855748; and YMR206W is 855246.

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

[0012] More specifically:

[0013] Through P TEF1 P TDH3 Promoter enhancement 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 was 851543;

[0014] Through P GAL1 P GAL10 The bidirectional promoter enhances the expression of ERG7 and ERG2, which are integrated into the ERG5 site on the Saccharomyces cerevisiae genome. The Gene ID of the ERG5 site is 855029.

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

[0016] Through P TEF1 Enhanced expression of DHCR24 was integrated into the YPL062W site on the yeast genome, where the GeneID of YPL062W is 856043;

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

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

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

[0020] Through P GAL1 P GAL10 Bidirectional promoters enhance the expression of ERG11 and ERG7, and simultaneously via P GAP Enhanced expression of IDI integrated into the Ty1Cons1 site on the Saccharomyces cerevisiae genome;

[0021] Through P GAL1 P GAL10 Bidirectional promoters enhance the expression of ERG2 and ERG3, and simultaneously via P GAP Enhanced expression of ERG1 was integrated into the Ty3Cons site on the Saccharomyces cerevisiae genome;

[0022] Through P GAL1 P GAL10 The bidirectional promoter enhances the expression of ACL and ACS1, which are integrated into the MOT3 site on the Saccharomyces cerevisiae genome. The gene ID of MOT3 is 855092.

[0023] Through P FBA1 P HXT7 Ethanol-induced promoter enhances ACS1 expression L641P ERG10 is integrated into the 208A site on the Saccharomyces cerevisiae genome;

[0024] Through P ITR1 P GAL7 Enhanced expression of ERG19 and UPC2 G888D The YBR197C site, which is integrated into the genome of Saccharomyces cerevisiae, has a Gene ID of 852496.

[0025] Through P GAL1 P GAL10 The bidirectional promoter enhances the expression of ALD6 and ACH1, which are integrated into the YBL059W site on the Saccharomyces cerevisiae genome. The Gene ID of the YBL059W site is 852221.

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

[0027] Through P YGP1 PHXT7 Enhanced expression of INO2 and 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 aforementioned Saccharomyces cerevisiae strain, the method comprising the following steps:

[0029] Using the engineered Saccharomyces cerevisiae SC9 as the chassis strain, T TEF -P TEF1 -POS5-T ADH1 -P TDH3 -ERG3-T CYC1 The fragment was integrated into the genome of strain SC9 and into the OSH2 site (Gene ID of OSH2 site is 851543). 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 strain sd1 and into the ERG5 site (Gene ID of the ERG5 site is 855029). The resulting Saccharomyces cerevisiae strain was named sd2.

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

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

[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 strain sd4 and into the Ty2Cons site. The resulting Saccharomyces cerevisiae strain was named sd5.

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

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

[0036] P GAL1 -ERG7-T TDH3 -P CAL7 -ERG11-T TPS1 -P GAP -IDI-T CYC1 - The HYGPMX6 fragment was integrated into the genome of strain sd7 and into the Ty1Cons1 site. The resulting 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 strain sd8 and into the Ty3Cons site, resulting in the Saccharomyces cerevisiae strain named sd9.

[0038] The C-24 sterol methyltransferase ERG6, which is required for ergosterol synthesis, was knocked out in the genome of strain sd9, 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 strain sd9 and into the MOT3 site (Gene ID of MOT3 site 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 strain se1 and into site 208A, 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 strain se2 and into the YBR197C site (Gene ID of 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 strain se3 and into the YBL059W site (Gene ID of 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 strain se4 and into the YNR014W site (Gene ID of 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 strain se5 and into the YMR206W site (Gene ID of YMR206W site is 855246), and the resulting Saccharomyces cerevisiae strain was named se6.

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

[0046] The present invention also relates to the application of the aforementioned Saccharomyces cerevisiae strain in the microbial fermentation preparation of 7-DHC.

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

[0048] Specifically, the application involves inoculating the *Saccharomyces cerevisiae* strain into a shake-flask fermentation medium and fermenting it at 28–32°C for 84–96 hours. The fermentation broth is then subjected to cell disruption and saponification extraction to obtain the 7-DHC. The shake-flask fermentation medium consists of the following components: 20 g tryptone, 40 g anhydrous glucose, and 10 g yeast extract per liter.

[0049] Specifically, the application involves inoculating the *Saccharomyces cerevisiae* strain into a 5L fermentation medium and fermenting it at 28–32°C for 100–130 hours. Feeding begins when the initial sugar content of the medium drops to 1 g / L and the ethanol content drops to 10 g / L (approximately 15 hours). During the initial carbon source feeding phase (15–50 hours), glucose is added, maintaining the sugar content of the fermentation medium below 1 g / L and the ethanol content below 10 g / L. In the later phase (50 hours until the end of fermentation), ethanol is added, maintaining the ethanol content of the fermentation medium at 15–25 g / L. The fermentation broth is then subjected to cell disruption and saponification extraction to obtain the 7-DHC. The 5L fermentation medium consists of the following components: 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: This invention uses the engineered Saccharomyces cerevisiae SC9 as the starting strain, employing multiple copies of tHMG1, DHCR24, and ERG1 via TY transposons, multiple copies of ERG2, POS5, ERG7, ERG11, IDI1, and ERG3 via TY transposons, and single copies of POS5, ERG3, and DHCR24 in the genome to increase intracellular reductive coenzyme II content and enhance the main pathway of 7-DHC synthesis. Single copies of ACL, ACS1, ERG10, ERG19, UPC2, ALD6, ACH1, IDI1, ADH2, INO2, and GAL4 in the genome enhance the utilization of ethanol in Saccharomyces cerevisiae and dynamically regulate 7-DHC synthesis. The constructed engineered Saccharomyces cerevisiae se6-Δerg6 achieved a 7-DHC yield of 1400 mg / L when fermented in a 5L fermenter with fed-batch fermentation medium for 130 h. The Saccharomyces cerevisiae genetically engineered strain constructed by this invention has a high product accumulation, laying the foundation for replacing the traditional multi-step chemical synthesis route and realizing industrial production. Attached Figure Description

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

[0052] Figure 2 The graph shows the 7-DHC production of the engineered strain in shake-flask fermentation medium.

[0053] Figure 3 The graph shows the 7-DHC production of the engineered strain in shake-flask fermentation medium.

[0054] Figure 4 The graph shows the 7-DHC yield of the engineered strain sd9-Δerg6 in a 5L fermentation tank.

[0055] Figure 5 The graph shows the 7-DHC yield of the engineered strain se6-Δerg6 in a 5L fermentation tank. Detailed Implementation

[0056] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0057] The culture media involved in the examples are as follows:

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

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

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

[0061] 5L 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] Supplemented culture 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] Supplemental culture 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 20g tryptone, 0.5g NaCl, 5g yeast extract, 0.186g KCl, 1g 7MgCl2, and 20g agar powder.

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

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

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

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

[0071] Detection of 7-DHC content:

[0072] The fermented bacterial broth was centrifuged, and the bacterial cells were disrupted with 3M hydrochloric acid. Cell debris was collected by centrifugation, and intracellular sterols were extracted by saponification with 1.5 mol / L KOH methanol solution in a 60°C water bath. After the reaction was complete, the above liquid was extracted with n-hexane, and the obtained n-hexane was evaporated to dryness in a 75°C water bath. A certain amount of ethyl acetate was added to redissolve the residue, and the solution was filtered through a filter membrane into a liquid chromatography flask.

[0073] The gas chromatography column used was a Thermo Fisher Scientific HP-5 (30m*0.25mm*0.25μm); the gas chromatography program settings were as follows: inlet temperature: 300℃; programmed temperature rise: 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 brewer's yeast OD 600 Detection methods:

[0075] Yeast seed culture, after 16–24 hours of cultivation, was inoculated at a rate of 2% into 500 ml shake flasks containing 50 mL of shake-flask fermentation medium and incubated at 30°C and 220 rpm. Samples were taken and diluted appropriately before measuring the OD using a UV spectrophotometer. 600 .

[0076] The plasmids involved in the examples were constructed in E. coli Dh5α, and after the plasmids were constructed, they were used as templates to amplify expression cassettes.

[0077] In the examples, plasmids involving PAM site mutations must be sequenced at the corresponding positions after construction to ensure that the pdc5 plasmid with the correct PAM site mutation is obtained.

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

[0079] Table 1: Primer Table

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088]

[0089] Example 1: Cloning the gene required for 7-DHC biosynthesis

[0090] 1. Extraction of genomic DNA from Saccharomyces cerevisiae

[0091] Genomic DNA extraction from *Saccharomyces cerevisiae* CEN.PK2-1C was performed 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 h and centrifuge at 12000 rpm for 1 min at room temperature.

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

[0094] (3) Add 100 μL of GenTLE Yeast Solution B, gently shake 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) to the supernatant, invert the centrifuge tube to mix thoroughly, centrifuge at 12,000 rpm and 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. Allow the DNA precipitate to air dry at room temperature until there is no ethanol odor.

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

[0100] 2. Construction of tool plasmids

[0101] (1) Using the genome of Saccharomyces cerevisiae CEN.PK2-1C as a template, gene fragment T was obtained using TEF1t F and TEF1t R. TEF1 Gene fragment T was obtained using ADH1t F and ADH1t R. ADH1 Gene fragment P was obtained using GAL10pF and GAL1pR. GAL1,10 Gene fragment T was obtained using CYC1t F and CYC1t R. CYC1 ;

[0102] (2) Take the four segments T from step (1) TEF1 T ADH1 P GAL1,10 T CYC1 Fusion PCR was performed, and the correct bands obtained from gel chromatography were excised and recovered to obtain the fusion gene fragment T containing a bidirectional expression cassette. TEF1 -T ADH1 -P GAL1,10 -T CYC1 ;

[0103] (3) Using pMD20 plasmid as a template, linearized pMD20 plasmid was obtained using primers LpMD20 F and LpMD20 R;

[0104] (4) Gene fragment T TEF1 -T ADH1 -P GAL1,10 -T CYC1 The plasmid was ligated to a linearized pMD20 plasmid and introduced into competent cells Dh5α. The results were verified using primers V20F and V20R to confirm the presence of T... TEF1 -T ADH1 -P GAL1,10 -T CYC1 T in the two-way expression box TEF1 -T ADH1 -P GAL1,10 -T CYC1 -pMD20 plasmid.

[0105] 3. Cloning and truncating the following genes: 3-hydroxy-3-methylglutaryl-CoA reductase (tHMG1), squalene epoxidase (ERG1), lanosterol C-14 demethylase (ERG11), C-8 sterol isomerase (ERG2), NADH kinase (POS5), lanosterol demethylase (ERG11), lanosterol synthase (ERG7), isopentenyl pyrophosphate isomerase (IDI1), and acetyl coenzyme A reductase. Enzyme A synthase (ACS1), acetyl-CoA acetyltransferase (ERG10), aldehyde 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), and C-5 sterol desaturase (ERG3) were amplified by PCR using the *Saccharomyces cerevisiae* genome as a template and employing a high-fidelity DNA polymerase (Phanta Max). 24-sterol reductase DHCR24 from *Gallus gallus* and acetyl-CoA synthase ACS1 from *Salmonella enterica* were synthesized from the whole genome after codon optimization by Qingke Biotechnology. 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℃ for 5 min; (2) Denaturation at 95℃ for 30 s, annealing at 60℃ for 30 s, extension at 72℃ for 1 kb / min for 32 cycles; (3) Final extension at 72℃ for 5 min; (4) Storage at 16℃ for 1 min.

[0108] 4. Construction of sgRNA

[0109] Using plasmid pdc5 as a template, the primers listed in Table 1 were used to amplify the plasmid in reverse, such as using primers CERG6-F / CERG6-R, to obtain the linearized pdc5 plasmid. This plasmid was then introduced into competent cells Dh5α, plated on SOB+Amp plates, and cultured at 37°C to obtain the sgRNA-ERG6 plasmid.

[0110] Examples 2-11 were conducted with the aim of enhancing the de novo synthesis of 7-DHC along the main synthetic pathway.

[0111] Example 2: Effect of single-copy overexpression on enhancement of the coenzyme supply module and its effect on product accumulation

[0112] Based on the engineered Saccharomyces cerevisiae strain sc9, the endogenous Saccharomyces cerevisiae gene OSH2 was knocked out, and single-copy expression cassettes of the endogenous Saccharomyces cerevisiae genes ERG3 and POS5 were integrated into 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, with T TEF1 -T ADH1 -P GAL1,10 -T CYC1- Using pMD20 plasmid as a template, the expression cassette T was amplified using primers TEF1t F / CYC1t R. TEF1 -T ADH1 -P GAL1,10 -T CYC1 The upstream and downstream homologous arms of the OSH2 site were amplified using primers UOSH2-F / UOSH2-R and DOSH2-F / DOSH2-R, respectively. T TEF1 -T ADH1 -P GAL1,10 -T CYC1 The upstream and downstream homologous arms of the OSH2 site are connected to the linearized pMD20 plasmid.

[0115] (2) Using the above plasmids as templates, linearized plasmids containing upstream and downstream homologous arms and terminators of the OSH2 site were amplified using primers CYC1t F / TEF1t R. Gene ERG3 was amplified using primers ERG3-F / ERG3-R, gene POS5 was amplified using primers POS5-F / POS5-R, promoter TEF1 was amplified using primers P,TEF1-F / P,TEF1-R, and promoter TDH3 was amplified using primers P,TDH3-F / P,TDH3-R. The above fragments and plasmids were then ligated. The fragment donorΔOSH2::T was obtained from the recombinant plasmid using primer UOSH2-F / DOSH2-R. TEF -P TEF1 -POS5-T ADH1 -P TDH3 -ERG3-T CYC1 .

[0116] (3) Construction of sg RNA:

[0117] Using the sgRNA construction method described in Example 1, and with COSH2-F / COSH2-R as primers, the sgRNA-OSH2 plasmid was obtained.

[0118] (4) Combine the sgRNA-OSH2 and donorΔOSH2::T obtained in steps (2) and (3) TEF -P TEF1 -POS5-T ADH1 -P TDH3 -ERG3-T CYC1 The fusion gene fragment was transformed into competent cells of the constructed *Saccharomyces cerevisiae* strain SC9 and cultured on YPD+G418 plates at 30°C for 2-3 days. Single colonies with correct bands were selected to obtain the engineered strain, which was named *Saccharomyces cerevisiae* sd1.

[0119] Example 3: Effect of single-copy overexpression on the enhancement of the post-squalene module and its impact 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 cassettes of endogenous genes ERG7 and ERG2 of Saccharomyces cerevisiae were integrated into 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, with T TEF1 -T ADH1 -P GAL1,10 -T CYC1- Using pMD20 plasmid as a template, the expression cassette T was amplified using primers TEF1t F / CYC1t R. TEF1 -T ADH1 -P GAL1,10 -T CYC1 The upstream and downstream homologous arms of the ERG5 site were amplified using primers UERG5-F / UERG5-R and DERG5-F / DERG5-R, respectively. T TEF1 -T ADH1 -P GAL1,10 -T CYC1 The upstream and downstream homologous arms of the ERG5 site were connected to the linearized pMD20 plasmid.

[0123] (2) Using the above plasmid as a template, a linearized plasmid containing upstream and downstream homologous arms and a terminator of the ERG5 site was amplified using primer CYC1t F / DERG5-R. The gene ERG7 was amplified using primer ERG7-F / ERG7-R, and the gene ERG2 was amplified using primer ERG2-F / ERG2-R. Fragment P was obtained using primer GAL10p F / GAL1p R.GAL1,10 The terminator TPS1 was amplified using primers T,TPS1-F / T,TPS1-R, and the above fragment and plasmid were ligated. The fragment donorΔERG5::T was obtained from the recombinant plasmid using primers UERG5-F / DEG5-R. TEF -T ADH1 -ERG7-P GAL10 -P GAL1 -ERG2-T TPS1 .

[0124] (3) Construction of sg RNA:

[0125] Using the sgRNA construction method described in Example 1, and with CERG5-F / CERG5-R as primers, the sgRNA-ERG5 plasmid was obtained.

[0126] (4) Combine the sgRNA-ERG5 and donorΔERG5::T obtained in steps (2) and (3) TEF -T ADH1 -ERG7-P GAL10 -P GAL1 -ERG2-T TPS1 The fusion gene fragment was transformed into competent cells of the constructed *Saccharomyces cerevisiae* strain sd1 and cultured on YPD+G418 plates at 30°C for 2-3 days. Single colonies with correct bands were selected to obtain the engineered strain, which was named *Saccharomyces cerevisiae* sd2.

[0127] Example 4: Effect of the key rate-limiting step of Ty1Cons1 multicopy overexpression 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 was overexpressed to strengthen the key rate-limiting step.

[0129] The specific steps are as follows:

[0130] (1) UTY11 was amplified using primers UTY11-F / UTY11-R, DTY11 was amplified using DTY11-F / DTY11-R, the promoter TEF1 was amplified using P,TEF1-F / P,TEF1-R (the same below), the gene DHCR24 was amplified using DHCR24-F / DHCR24-R (the same below), the terminator TPS1 was amplified using T,TPS1-F / T,TPS1-R (the same below), the gene ERG1 was amplified using ERG1-F-20(GAP) / ERG1-R-20(TPS1O), and the gene ERG1 was amplified using T,TDH3-F / T,TDH3 (the same below). -R amplification terminator TDH3 (the same below), P,PGK1-F / PGK1-R amplification of promoter PGK1, tHMG1 gene amplification with tHMG1-F-20(P,PGK1) / tHMG1-TCYC1-R amplification of HYGPMX6 with H-TCYC1-F / H-DTY11-R amplification of linearized plasmid PMD20 with L-PMD20-F / L-PMD20-R (the same below), multi-fragment repeat fusion PCR, multi-fragment and plasmid ligation, primer UTY11-F / DTY11-R amplification of donorΔTy1Cons1::P TEF1 -DHCR24-T TPS1 -P GAP -ERG1-T TDH3 -P PGK1 -tHMG1-T CYC1 -HYGPMX6.

[0131] (2) Construction of sg RNA:

[0132] Using the sgRNA construction method described in Example 1, with CTY11-F / CTY11-R as primers, the sgRNA-TY11 plasmid was obtained.

[0133] (3) Combine the sgRNA-TY11 and donorΔTy1Cons1::P obtained in steps (1) and (2) TEF1 -DHCR24-T TPS1 -P GAP -ERG1-T TDH3 -P PGK1 -tHMG1-T CYC1 The HYGPMX6 fusion gene fragment was transformed into competent cells of the constructed *Saccharomyces cerevisiae* strain sd2 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, which was named *Saccharomyces cerevisiae* sd3.

[0134] Example 5: Effect of YPL062W knockout overexpression of DHCR24 promoting ethanol utilization on 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. The upstream and downstream homologous arms of the YPL062W site were amplified using primers UYPL-F / UYPL-R and DYPL-F / DYPL-R, respectively. These were then fused with fragments TEF1, TPS1, and gene DHCR24 (as above) using multi-fragment fusion PCR, followed by ligation of the fragments. The fragment donorΔYPL062W::P was obtained from the recombinant plasmid using primers UYPL-F / DYPL-R. TEF1 -DHCR24-T TPS1 .

[0138] (2) Construction of sg RNA:

[0139] Using the sgRNA construction method described in Example 1, and with CYPL-F / CYPL-R as primers, the sgRNA-YPL plasmid was obtained.

[0140] (3) Combine the sgRNA-YPL and donorΔYPL062W::P obtained in steps (1) and (2). TEF1 -DHCR24-T TPS1 The fusion gene fragment was transformed into competent cells of the constructed *Saccharomyces cerevisiae* strain sd1 and cultured on YPD+G418 plates at 30°C for 2-3 days. Single colonies with correct bands were selected to obtain the engineered strain, which was named *Saccharomyces cerevisiae* sd4.

[0141] Example 6: Effect of Ty2Cons multicopy overexpression on key rate-limiting steps of the squalene pathway on 7-DHC accumulation

[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 7-DHC synthesis in the main pathway was overexpressed to promote the conversion of yeast sterols to 7-DHC.

[0143] The specific steps are as follows:

[0144] (1) UTY2 was amplified using primers UTY2-F / UTY2-R, DTY2 was amplified using DTY2-F / DTY2-R, DHCR24 with a TDH3 homologous arm was amplified using DHCR24-F / D24-R-TDH3 (the same below), the promoter GAP was amplified using P,GAP-F-TD / P,GAP-R, the gene ERG2 was amplified using ERG2-F-GAP / ERG2-R-TP, the promoter PGK1 was amplified using P,PGK1-F-TP / P,PGK1-R, the gene HIS was amplified using HIS-F / HIS-R, and the aforementioned fragments were amplified using multi-fragment repeat fusion PCR, multi-fragment and linear plasmid ligation, and the donorΔTy2Cons::P was amplified using primers UTY2-F / DTY2-R. 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] Using the sgRNA construction method described in Example 1, with CTY2-F / CTY2-R as primers, the sgRNA-TY2 plasmid was obtained.

[0147] (3) Combine the sgRNA-TY2 and donorΔTy2Cons::P obtained in steps (1) and (2). 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 competent cells of the constructed *Saccharomyces cerevisiae* strain sd4 and cultured on SC-URA-HIS plates at 30°C for 2-3 days. Single colonies with the correct bands were selected to obtain the engineered strain, which was named *Saccharomyces cerevisiae* sd5.

[0148] Example 7: Effect of key rate-limiting steps in the squalene pathway on 7-DHC accumulation after further multi-copy overexpression of Ty1Cons2

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

[0150] The specific steps are as follows:

[0151] (1) The plasmid P constructed in Implementation Case 4 TEF1 -DHCR24-T TPS1 -P GAP -ERG1-T TDH3 -P PGK1 -tHMG1-T CYC1 Using HYGPMX6 as a template, the linearized plasmid was amplified with LPMD20-R / P, TEF1-F, and then UTY12 was amplified with UTY12-F / UTY12-R and ligated to the linearized plasmid. The linearized plasmid was then amplified using LPMD20-F / T, CYC1-R-TRP as a template. The TRP fragment was amplified with PTRP-F-CYC1 / T, TEF-R-TY12D, and the DTY12 fragment was amplified with DTY12-F / DTY12-R, followed by multi-fragment ligation. 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] Using the sgRNA construction method described in Example 1, with CTY12-F / CTY12-R as primers, the sgRNA-TY12 plasmid was obtained.

[0154] (3) Combine the sgRNA-TY12 and donorΔTy1Cons2::P obtained in steps (1) and (2) TEF1 -DHCR24-T TDH3 -P GAP -ERG1-T TPS1 -P PGK1 -tHMG1-T CYC1 -P TRP The TRP fusion gene fragment was transformed into competent cells of the constructed *Saccharomyces cerevisiae* strain sd4 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, which was named *Saccharomyces cerevisiae* sd6.

[0155] Example 8: Effect of Ty4Cons multicopy overexpression coenzyme supply module enhancement 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. Multiple copies of POS5 were overexpressed to promote the conversion of reduced coenzyme I to reduced coenzyme II.

[0157] The specific steps are as follows:

[0158] (1) The plasmid P constructed in Implementation Case 4 TEF1 -DHCR24-T TPS1 -P GAP -ERG1-T TDH3 -P PGK1 -tHMG1-T CYC1 Using HYGPMX6 as a template, the linearized plasmid was amplified with LPMD20-R / P, TEF1-F, and UTY4 was amplified with UTY4-F / UTY4-R and ligated to the linearized plasmid. The linearized plasmid was then amplified using LPMD20-F / T, CYC1-R-NAT as a template, the NAT fragment was amplified with NAT-F / NAT-R, and the DTY4 fragment was amplified with DTY4-F / DTY4-R and ligated to the linearized fragment. The plasmid was then amplified again in reverse 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 for multi-fragment ligation. 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] Using the sgRNA construction method described in Example 1, with CTY4-F / CTY4-R as primers, the sgRNA-TY4 plasmid was obtained.

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

[0162] Example 9: Effect of Ty1Cons1 multicopy overexpression on key rate-limiting steps of the squalene pathway on 7-DHC accumulation

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

[0164] The specific steps are as follows:

[0165] (1) Using the plasmid constructed in Example 4 as a template, the linear plasmid was amplified with T,CYC1-F / UTY11-R-PGAL1, the promoter GAL1 was amplified with P,GAL1-F / P,GAL1-R, the gene ERG7 was amplified with ERG7-F-GAL1 / ERG7-R-TDH3, the promoter GAL7 was amplified with P,CAL7-FT,TDH3 / P,GAL7-R, the gene ERG11 was amplified with ERG11-F-GAL7 / ERG11-R-TPS1, the promoter GAP was amplified with P,GAP-TPS1 / P,GAP-R, and the gene IDI1 was amplified with IDI1-F-GAP / IDI1-R-CYC1. Multiple fragment repeat fusion PCR was performed, and multiple fragments and plasmids were ligated. Primers UTY11-F / DTY11-R were used to amplify donorΔTy1Cons1::P. GAL1 -ERG7-T TDH3 -P CAL7 -ERG11-T TPS1 -P GAP -IDI-T CYC1 -HYGPMX6.

[0166] (2) Construction of sg RNA:

[0167] Using the sgRNA construction method described in Example 1, with CTY11-F / CTY11-R as primers, the sgRNA-TY11 plasmid was obtained.

[0168] (3) Combine the sgRNA-TY11 and donorΔTy1Cons1::P obtained in steps (1) and (2) GAL1 -ERG7-T TDH3 -P CAL7 -ERG11-T TPS1-P GAP -IDI-T CYC1 The HYGPMX6 fusion gene fragment was transformed into competent cells of the constructed *Saccharomyces cerevisiae* strain sd7 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, which was named *Saccharomyces cerevisiae* sd8.

[0169] Example 10: Effect of Ty3Cons overexpression of 7-DHC on the de novo synthesis of a significantly accumulating precursor reaction step on 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, the rate-limiting gene was overexpressed, and the key enzyme of the squalene pathway was further overexpressed.

[0171] The specific steps are as follows:

[0172] (1) Linearize pMD20 using primers LpMD20 F / LpMD20 R, with T TEF1 -T ADH1 -P GAL1,10 -T CYC1- Using pMD20 plasmid as a template, the expression cassette T was amplified using primers TEF1t F / CYC1t R. TEF1 -T ADH1 -P GAL1,10 -T CYC1 The upstream and downstream homologous arms of the TY3 site 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. The T... TEF1 -T ADH1 -P GAL1,10 -T CYC1 The upstream and downstream homologous arms of the TY3 site, the LEU, and the linearized pMD20 plasmid were connected.

[0173] (2) Using the above plasmid as a template, amplify the linearized plasmid with upstream and downstream homologous arms and terminator containing the TY3 site using primer CYC1t F / TEF1t R. Amplify the gene ERG1 using primer ERG1-F-GAP / ERG1-R-TPS1. Amplify the promoter GAP using GAP-F-CYC1 / P,GAP-R. Amplify the gene ERG3 using primer ERG3-R-CYC1 / ERG3-F-GAL1. Amplify the gene ERG2 using primer ERG2-R-GAL10 / ERG2-F-ADH1. Combine the above fragments and plasmid with fragment P. GAL1,10Multi-fragment repeat fusion PCR, multi-fragment ligation. Primers UTY3-F / DTY3-R were used to obtain the fragment donorΔTy3Cons::T from the acquired 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] Using the sgRNA construction method described in Example 1, with CTY3-F / CTY3-R as primers, the sgRNA-TY3 plasmid was obtained.

[0176] (4) Combine the sgRNA-TY3 and donorΔTy3Cons::T obtained in steps (2) and (3) 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 competent cells of the constructed *Saccharomyces cerevisiae* strain sd8 and cultured on SC-URA-LEU plates at 30°C for 2-3 days. Single colonies with the correct bands were selected to obtain the engineered strain, which was named *Saccharomyces cerevisiae* sd9.

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

[0178] The specific steps are as follows:

[0179] (1) Construction of donor fragments:

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

[0181] (2) Construction of sg RNA:

[0182] Using the sgRNA construction method described in Example 1, and with CERG6-F / CERG6-R as primers, the sgRNA-ERG6 plasmid was obtained.

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

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

[0185] Examples 12-18 were conducted with the aim of enhancing ethanol utilization and regulating the dynamic synthesis of 7-DHC.

[0186] Example 12 Effect of increased supply of sterol synthesis precursor acetyl-CoA on 7-DHC accumulation

[0187] Based on 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, thereby increasing the supply of cytoplasmic acetyl-CoA.

[0188] The specific steps are as follows:

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

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

[0191] (3) Construction of sg RNA:

[0192] Using the sgRNA construction method described in Example 1, and with CMOT3-F / CMOT3-R as primers, the sgRNA-MOT3 plasmid was obtained.

[0193] (4) Combine the sgRNA-MOT3 and donorΔMOT3::T obtained in steps (2) and (3) TEF -T ADH1 -ACL-P GAL10 -P GAL1 -ACS1-T CYC1 The fusion gene fragment was transformed into competent cells of the constructed *Saccharomyces cerevisiae* strain sd9 and cultured on YPD+G418 plates at 30°C for 2-3 days. Single colonies with correct bands were selected to obtain the engineered strain, which was named *Saccharomyces cerevisiae* se1.

[0194] Example 13: Dynamic Regulation of 7-DHC Synthesis Using an Ethanol-Inducible Promoter

[0195] Based on the strain se1 constructed in Example 12, the endogenous gene ERG10 of Saccharomyces cerevisiae was integrated at site 208A, and the acetyl-CoA synthase ACS1, derived from Salmonella enterica with a 641-amino acid mutation to proline, was heterologously expressed. The promoter overexpression of ACS was induced by ethanol. L641P ERG10 is used to dynamically regulate the synthesis of 7-DHC.

[0196] The specific steps are as follows:

[0197] (1) The plasmid PET was linearized using primers LPET-F / LPET-R. Homologous arms of 208A were amplified using U208A-F / U208A-R and D208A-F / D208A-R, respectively. The promoter FBA1 was amplified using P,FBA1-F-TPS1 / P,FBA1-R. The acetyl-CoA synthase ACS1, derived from Salmonella enterica with a 641 amino acid mutation to proline, was amplified using SACS1-F / SACS1-R. The terminator ADH2 was amplified using T,ADH2-F / T,ADH2-R. The promoter HXT7 was amplified using P,HXT7-F-ADH2 / P,HXT7-R. The gene ERG10 was amplified using ERG10-F-HXT7 / ERG10-R-CYC1. The gene was then fused with the terminator TPS1 using multiple fragment repeat PCR, and the multiple fragments were ligated to the plasmid. Primers U208A-F / D208A-R were used to obtain the fragment donorΔ208A::T from the recombinant plasmid. TPS1 -P FBA1 -ACS1 L641P -T ADH2 -P HXT7 -ERG10-T CYC1 .

[0198] (2) Construction of sg RNA:

[0199] Using the sgRNA construction method described in Example 1, with C208A-F / C208A-R as primers, the sgRNA-208A plasmid was obtained.

[0200] (3) Combine the sgRNA-208A and donorΔ208A::T obtained in steps (1) and (2) TPS1 -P FBA1 -ACS1 L641P -T ADH2 -P HXT7 -ERG10-T CYC1 The fusion gene fragment was transformed into competent cells of the constructed *Saccharomyces cerevisiae* strain se1 and cultured on YPD+G418 plates at 30°C for 2-3 days. Single colonies with correct bands were selected to obtain the engineered strain, which was named *Saccharomyces cerevisiae* se2.

[0201] Example 14 Effects of overexpression of sterol synthesis regulators and rate-limiting steps on 7-DHC accumulation

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

[0203] The specific steps are as follows:

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

[0205] (2) Construction of sg RNA:

[0206] Using the sgRNA construction method described in Example 1, and with CYBR-F / CYBR-R as primers, the sgRNA-YBR197C plasmid was obtained.

[0207] (3) Combine the sgRNA-YBR197C and donorΔYBR197C::T obtained in steps (1) and (2) TPS1 -P ITR1 -ERG19-T ADH2 -P GAL7 -UPC2 G888D -T CYC1 The fusion gene fragment was transformed into competent cells of the constructed *Saccharomyces cerevisiae* strain se2 and cultured on YPD+G418 plates at 30°C for 2-3 days. Single colonies with correct bands were selected to obtain the engineered strain, which was 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 ethanol utilization and balance the content of cytoplasmic acetyl-CoA and mitochondrial acetyl-CoA.

[0210] The specific steps are as follows:

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

[0212] (2) Using the above plasmid as a template, a linearized plasmid containing the upstream and downstream homologous arms of the YBL059W site and a terminator was amplified using primers CYC1t F / TEF1t R. The gene ALD6 was amplified using primers ALD6-F / ALD6-R, and the gene ACH1 was amplified using primers ACH1-F / ACH1-R. The above fragments and plasmid were then compared with P... GAL110 Ligation was performed. Primers UYBL-F / DYBL-R were used to obtain the fragment donorΔYBL059W::T from the acquired recombinant plasmid. TEF -T ADH1 -ALD6-P GAL10 -P GAL1 -ACH1-T CYC1 .

[0213] (3) Construction of sg RNA:

[0214] Using the sgRNA construction method described in Example 2, and with CYBL-F / CYBL-R as primers, the sgRNA-YBL059W plasmid was obtained.

[0215] (4) Combine the sgRNA-YBL059W and donorΔYBL059W::T obtained in steps (2) and (3). TEF -T ADH1 -ALD6-P GAL10 -P GAL1 -ACH1-T CYC1 The fusion gene fragment was transformed into competent cells of the constructed *Saccharomyces cerevisiae* strain se3 and cultured on YPD+G418 plates at 30°C for 2-3 days. Single colonies with correct bands were selected to obtain the engineered strain, which was 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 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 by inducing the promoter with ethanol.

[0218] The specific steps are as follows:

[0219] (1) Using primers LPET-F / LPET-R, plasmid PET was linearized. Homologous arms of YNR014W were amplified using UYNR-F / UYNR-R and DYNR-F / DYNR-R, respectively. Promoter FBA1 was amplified using P,FBA1-F-TPS1 / P,FBA1-R, gene IDI1 was amplified using IDI1-F-FBA1 / IDI1-R-ADH2, promoter TEF1 was amplified using P,TEF1-F-ADH2 / P,TEF1-R, and gene ADH2 was amplified using ADH2-F-TEF1 / ADH2-R-CYC1. Multiple fragments were then fused with terminators TPS1, ADH2, and CYC1, and ligated to the linearized plasmid. The fragment donorΔYNR014W::T was obtained from the recombinant plasmid using primers UYNR-F / DYNR-R. TPS1 -P FBA1 -IDI1-T ADH2 -P TEF1 -ADH2-T CYC1 .

[0220] (2) Construction of sg RNA:

[0221] Using the sgRNA construction method described in Example 1, and with CYNR-F / CYNR-R as primers, the sgRNA-YNR014W plasmid was obtained.

[0222] (3) Combine the sgRNA-YNR014W and donorΔYNR014W::T obtained in steps (1) and (2)TPS1 -P FBA1 -IDI1-T ADH2 -P TEF1 -ADH2-T CYC1 The fusion gene fragment was transformed into competent cells of the constructed *Saccharomyces cerevisiae* strain se4 and cultured on YPD+G418 plates at 30°C for 2-3 days. Single colonies with the correct bands were selected, and the resulting engineered strain was named *Saccharomyces cerevisiae* se5.

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

[0224] Based on the strain se5 constructed in Example 16, the endogenous gene INO2 from *Saccharomyces cerevisiae* was integrated at the YMR206W site, expanding the endoplasmic reticulum membrane and increasing the site for 7-DHC synthesis. This was achieved through P... HXT7 GAL4 expression was enhanced by feeding with ethanol to activate high levels of GAL4 expression, which in turn further activated the rate-limiting gene overexpressed by the gal promoter.

[0225] The specific steps are as follows:

[0226] (1) Using primers LPET-F / LPET-R, plasmid PET was linearized. Homologous arms of YMR206W were amplified using UYMR-F / UYMR-R and DYMR-F / DYMR-R, respectively. Promoter YGP1 was amplified using P,YGP1-F-TEF / P,YGP1-R, gene INO2 was amplified using INO2-F-YGP1 / INO2-R-CYC1, promoter HXT7 was amplified using P,HXT7-R / P,HXT7-F-CYC1, gene GAL4 was amplified using GAL4-F / GAL4-R, and terminators TEF, CYC1, and TDH3 were also amplified. Multi-fragment fusion PCR and ligation of multiple fragments to the linearized plasmid were performed. The fragment donorΔYMR206W::T was obtained from the recombinant plasmid using primers UYMR-F / DYMR-R. TEF -P YGP1 -INO2-T CYC1 -P HXT7 -GAL4-T TDH3 .

[0227] (2) Construction of sg RNA:

[0228] Using the sgRNA construction method described in Example 1, and with CYMR-F / CYMR-R as primers, the sgRNA-YMR206W plasmid was obtained.

[0229] (3) Combine the sgRNA-YMR206W and donorΔYMR206W::T obtained in steps (1) and (2).TEF -P YGP1 -INO2-T CYC1 -P HXT7 -GAL4-T TDH3 The fusion gene fragment was transformed into competent cells of the constructed *Saccharomyces cerevisiae* strain se5 and cultured on YPD+G418 plates at 30°C for 2-3 days. Single colonies with the correct bands were selected to obtain the engineered strain, which was named *Saccharomyces cerevisiae* se6.

[0230] (4) Using the above recombinant plasmid ΔYMR206W::T TEF -P YGP1 -INO2-T CYC1 -P HXT7 -GAL4-T TDH3 Using the template, the plasmid was reverse-amplified with primers T,TEF-R-PTEF1 / INO2-F-PTEF1 and ligated to the promoter TEF1. The donorΔYMR206W::T was obtained in step (1). TEF -P TEF1 -INO2-T CYC1 -P HXT7 -GAL4-T TDH3 The above recombinant plasmid ΔYMR206W::T was used. TEF -P YGP1 -INO2-T CYC1 -P HXT7 -GAL4-T TDH3 Using the template, the plasmid was reverse-amplified with primers T,TEF-R-GAL1 / INO2-F-GAL1 and ligated to the promoter GAL1. The donorΔYMR206W::T was obtained in step (1). TEF -P GAL1 -INO2-T CYC1 -P HXT7 -GAL4-T TDH3 The above recombinant plasmid ΔYMR206W::T was used. TEF -P YGP1 -INO2-T CYC1 -P HXT7 -GAL4-T TDH3 Using a template, the plasmid was reverse-amplified with primers T,TEF-R-FBA1 / INO2-F-FBA1, and the promoter FBA1 was amplified with primers P,FBA1-F / P,FBA1-R. The plasmid was then ligated to the plasmid, and the donorΔYMR206W::T was obtained in the same step (1). TEF -P FBA1 -INO2-T CYC1 -P HXT7 -GAL4-T TDH3In step (3), the donors were introduced into SE5, and the resulting engineered strains se5-tef1-ino2, se5-gal1-ino2, and se5-fba1-ino2 were subjected to shake-flask level verification analysis as in Example 11. The results are as follows: Figure 3 As shown, overexpression of the phospholipid biosynthesis regulator INO2 via the constituent promoter significantly inhibited bacterial growth, while the acetic acid-ethanol-induced promoter had little effect on bacterial growth. The se6 strain showed a 15% increase in 7-DHC production. Further metabolic modification of the engineered strain se6, which overexpresses ino2 via the acetic acid-induced promoter YGP1, is planned.

[0231] Example 18 Effect of blocking the SE6 endogenous ergosterol synthesis pathway on 7-DHC accumulation

[0232] The specific steps are as follows:

[0233] (1) Construction of donor fragments:

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

[0235] (2) Construction of sg RNA:

[0236] Using the sgRNA construction method described in Example 1, and with CERG6-F / CERG6-R as primers, the sgRNA-ERG6 plasmid was obtained.

[0237] (3) The sgRNA-CERG6 and donorΔERG6 fusion gene fragments obtained in steps (1) and (2) were transformed into competent cells of Saccharomyces cerevisiae se6 strain and cultured on YPD+G418 plates at 30℃ for 2-3 days. Single colonies with correct bands were selected and the strain was named Saccharomyces cerevisiae se6-Δerg6.

[0238] Example 19: Fermentation culture of engineered bacteria sd9-Δerg6 and se6-Δerg6 in a 5L tank.

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

[0240] (2) Under aseptic conditions, the activated bacterial solutions of sd9-Δerg6 and se6-Δerg6 were inoculated into a 500ml YPD shake flask containing 50ml YPD medium at a 2% inoculation rate. 0.5g / L to 1g / L of uracil was added exogenously. The culture was carried out at 30℃ and 220rpm for 24 to 36 hours to prepare the fermentation seed liquid.

[0241] (3) Batch feeding fermentation was carried out in a 5L fermenter with a liquid volume of 3L. The initial aeration rate was 3L / min, the initial stirring speed was 300rpm, the culture temperature was 30℃, 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 around 30% by controlling the stirring speed and air flow. Feeding was started when the glucose content dropped to 1g / L and the ethanol content dropped to 10g / L (about 15h). In the early stage of fermentation (15-50h), fed medium 1 was used to feed glucose as the carbon source, and the sugar content of the fermentation medium was controlled below 1g / L and the ethanol content was controlled below 10g / L. In the later stage of fermentation (50h to the end of fermentation), fed medium 2 was used to feed ethanol as the carbon source, and the ethanol content of the fermentation medium was controlled at 15-25g / L. 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 yields of strains sd9-Δerg6 and se6-Δerg6 reached 800 mg / L and 1400 mg / L, respectively.

Claims

1. A de novo synthetic 7-DHC Saccharomyces cerevisiae genetically engineered strain, characterized in that, It was constructed by the following method: enhancing the expression of truncated 3-hydroxy-3-methylglutaryl-CoA reductase in the genome of *Saccharomyces cerevisiae*. tHMG1 squalene epoxygenase ERG1 C-8 sterol isomerase ERG2 NADH kinase POS5 Lanosterol demethylase ERG11 Lanosterol synthase ERG7 Isopentenyl pyrophosphate isomerase IDI1 Acetyl-CoA synthase ACS1 Acetyl-CoA acetyltransferase ERG10 acetaldehyde dehydrogenase ALD6 Acetyl-CoA hydrolase ACH1 Mevalonate pyrophosphate decarboxylase ERG19 Ethanol dehydrogenase ADH2 Phospholipid biosynthesis regulators INO2 Sterol transcription activator UPC2 GAL promoter activator protein GAL4 C-5 sterol desaturase ERG3 Heterologous expression Gallus Gallus 24-sterol reductase from [source] DHCR24 , Salmonella enterica Acetyl-CoA synthase from [source] ACS1 , Yarrowia lipolytica citrate lyase from [source] ACL Knock out the C-22 sterol desaturase required for ergosterol synthesis ERG5 δC-24 sterol methyltransferase ERG6 Ergosterol synthesis gene transcription repressor MOT3 The *Saccharomyces cerevisiae* strain that efficiently synthesizes 7-dehydrocholesterol was obtained. The Salmonella enterica Acetyl-CoA synthase from [source] ACS1 The 641st amino acid is mutated from leucine to proline, a sterol transcription activator. UPC2 The amino acid at position 888 is mutated from glycine to aspartic acid; Specifically, the following steps are included: (1) Using strain SC9 as the chassis strain, T TEF -P TEF1 -POS5-T ADH1 -P TDH3 -ERG3-T CYC1 The fragment was integrated into the genome of strain SC9, and integrated into... OSH2 The site, the OSH2 The gene ID of the site is 851543, and the constructed Saccharomyces cerevisiae strain is named sd1. (2) T TEF -T ADH1 -ERG7-P GAL10 -P GAL1 -ERG2-T TPS1 Fragments were integrated into the genome of strain sd1 and into... ERG5 The site, the ERG5 The Gene ID of the site is 855029, and the constructed Saccharomyces cerevisiae strain is named sd2. (3) P TEF1 -DHCR24-T TPS1 -P GAP -ERG1-T TDH3 -P PGK1 -tHMG1-T CYC1 -HYGPMX6 The fragment was integrated into the genome of strain sd2 and integrated into... Ty1Cons1 The site was determined, and the resulting Saccharomyces cerevisiae strain was named sd3. (4) P TEF1 -DHCR24-T TPS1 Fragments were integrated into the genome of strain sd3 and integrated into... YPL062W The site, the YPL062W The Gene ID is 856043, and the constructed Saccharomyces cerevisiae strain is named sd4. (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 strain sd4 and integrated into... Ty2Cons The constructed Saccharomyces cerevisiae strain was named sd5. (6) P TEF1 -DHCR24-T TDH3 -P GAP -ERG1-T TPS1 -P PGK1 -tHMG1-T CYC1 -P TRP -TRP The fragment was integrated into the genome of strain sd5 and integrated into... Ty1Cons2 The site was determined, and the resulting Saccharomyces cerevisiae strain was named sd6. (7) P TEF1 -DHCR24-T TPS1 -P CAL7 -POS5-T TDH3 -P PGK1 -tHMG1-T CYC1 -NRSR The fragment was integrated into the genome of strain sd6, and integrated into... Ty4Cons The site was determined, and the resulting Saccharomyces cerevisiae strain was named sd7. (8) P GAL1 -ERG7-T TDH3 -P CAL7 -ERG11-T TPS1 -P GAP -IDI-T CYC1 -HYGPMX6 The fragment was integrated into the genome of strain sd7 and integrated into... Ty1Cons1 The site was determined, and the resulting 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 -LEU The fragment was integrated into the genome of strain sd8 and integrated into... Ty3Cons The site was determined, and the resulting Saccharomyces cerevisiae strain was named sd9. (10) Knock out the C-24 sterol methyltransferase required for ergosterol synthesis in the genome of strain sd9 ERG6 The constructed brewing yeast was named sd9- Δerg6 strain; (11) will T TEF -T ADH1 -ACL-P GAL10 -P GAL1 -ACS1-T CYC1 The fragment was integrated into the genome of strain sd9 and integrated into... MOT3 The site, the MOT3 The Gene ID is 855092, and the constructed Saccharomyces cerevisiae strain is named se1. (12) will T TPS1 -P FBA1 -ACS1 L641P -T ADH2 -P HXT7 -ERG10-T CYC1 Fragments were integrated into the genome of strain se1 and into... 208A The site was determined, and the resulting Saccharomyces cerevisiae strain was named SE2. (13) will T TPS1 -P ITR1 -ERG19-T ADH2 -P GAL7 -UPC2 G888D -T CYC1 The fragment was integrated into the genome of strain se2 and integrated into... YBR197C The site, the YBR197C The Gene ID of the site is 852496, and the resulting Saccharomyces cerevisiae strain is named se3. (14) will T TEF -T ADH1 -ALD6-P GAL10 -P GAL1 -ACH1-T CYC1 Fragments were integrated into the genome of strain SE3, and integrated into... YBL059W The site, the YBL059W The Gene ID of the site is 852221, and the resulting Saccharomyces cerevisiae strain is named se4. (15) will T TPS1 -P FBA1 -IDI1-T ADH2 -P TEF1 -ADH2-T CYC1 The fragment was integrated into the genome of strain SE4 and integrated into... YNR014W The site, the YNR014W The Gene ID of the site is 855748, and the resulting Saccharomyces cerevisiae strain is named SE5. (16) will T TEF -P YGP1 -INO2-T CYC1 -P HXT7 -GAL4-T TDH3 The fragment was integrated into the genome of strain SE5, and integrated into... YMR206W The site, the YMR206W The Gene ID of the site is 855246, and the resulting Saccharomyces cerevisiae strain is named SE6. (17) Knock out the C-24 sterol methyltransferase required for ergosterol synthesis in the genome of the SE6 strain. ERG6 The constructed brewing yeast was named se6- Δerg6 strains.

2. The de novo synthetic 7-DHC Saccharomyces cerevisiae genetically engineered strain as described in claim 1, characterized in that, The substrate bacteria are Saccharomyces cerevisiae SC9.

3. The de novo synthetic 7-DHC Saccharomyces cerevisiae genetically engineered strain as described in claim 1, characterized in that, The enhanced expression specifically refers to enhancing the expression of multiple copies in the Saccharomyces cerevisiae genome. tHMG1, DHCR24 Multiple copies ERG1 , POS5 Multiple copies ERG11 , IDI1 , ERG7 Multiple copies ERG2 , ERG3 .

4. The de novo synthetic 7-DHC Saccharomyces cerevisiae genetically engineered strain as described in claim 1, characterized in that, Through P TEF1 Promoter enhancement expression DHCR24 Through P GAP Promoter enhancement expression ERG1 Through P GAP Promoter enhancement expression ERG2 Through P PGK1 Promoter enhancement expression tHMG1 Through P GAL7 Promoter enhancement expression POS5 Through P GAL1，10 Bidirectional promoter enhancement expression ERG7 and ERG11 Through P GAP Promoter enhancement expression IDI1 Through P GAL1，10 Bidirectional promoter enhancement expression ERG3 and ERG2 Through P GAL1，10 Bidirectional promoter enhancement expression ACL and ACS1 By inducing the promoter P through ethanol FBA1 Strengthen expression Salmonella enterica Source ACS1 By inducing the promoter P through ethanol HXT7 Strengthen expression ERG10 via the promoter P ITR1 Strengthen expression ERG19, Through P GAL7 Promoter enhancement expression UPC2, Through P GAL1，10 Bidirectional promoter enhancement expression ALD6 and ACH1, Through P TEF1 Promoter enhancement expression ADH2 By inducing the promoter P through ethanol FBA1 Strengthen expression IDI1 via the promoter P YGP1 Strengthen expression INO2 By inducing the promoter P through ethanol HXT7 overexpression GAL4.

5. The de novo synthetic 7-DHC Saccharomyces cerevisiae genetically engineered strain as described in claim 1, characterized in that, Regulatory factors of phospholipid biosynthesis by inducible promoters INO2 Sterol transcription activator UPC2 GAL promoter activator protein GAL4 It has been expressed.

6. The de novo synthesized 7-DHC Saccharomyces cerevisiae genetically engineered strain according to any one of claims 1-5, characterized in that, Includes the following steps: Enhanced expression of 3-hydroxy-3-methylglutaryl-CoA reductase in the genome of *Saccharomyces cerevisiae* tHMG1 squalene epoxygenase ERG1 C-8 sterol isomerase ERG2 NADH kinase POS5 Lanosterol demethylase ERG11 Lanosterol synthase ERG7 Isopentenyl pyrophosphate isomerase IDI1 Acetyl-CoA synthase ACS1 Acetyl-CoA acetyltransferase ERG10 acetaldehyde dehydrogenase ALD6 Acetyl-CoA hydrolase ACH1 Mevalonate pyrophosphate decarboxylase ERG19 Ethanol dehydrogenase ADH2 Phospholipid biosynthesis regulators INO2 Sterol transcription activator UPC2 GAL promoter activator protein GAL4 C-5 sterol desaturase ERG3 Heterologous expression Gallus Gallus 24-sterol reductase from [source] DHCR24 , Salmonella enterica Acetyl-CoA synthase from [source] ACS1 , Yarrowia lipolytica citrate lyase from [source] ACL Knock out the C-22 sterol desaturase required for ergosterol synthesis ERG5 δC-24 sterol methyltransferase ERG6 Ergosterol synthesis gene transcription repressor MOT3 The Saccharomyces cerevisiae strain that efficiently synthesizes 7-dehydrocholesterol was obtained.

7. The application of the de novo 7-DHC synthesized Saccharomyces cerevisiae genetically engineered strain as described in any one of claims 1-5 in the microbial fermentation preparation of 7-DHC.

8. The application as described in claim 7, characterized in that, The *Saccharomyces cerevisiae* strain was inoculated into a fermentation medium and fermented at 28–32°C for 84–96 h. The fermentation broth was then subjected to cell disruption and saponification extraction to obtain the 7-DHC.

9. The application as described in claim 7, characterized in that, The *Saccharomyces cerevisiae* strain was inoculated into a 5L fermenter and fermented at 28–32°C for 100–130 h. Feeding was initiated when the sugar content of the initial culture medium decreased to 1 g / L and the ethanol content decreased to 10 g / L. Initially, glucose was added as the carbon source, and the sugar content and ethanol content of the fermentation medium were controlled below 1 g / L and below 10 g / L, respectively. Later, ethanol was added to maintain the ethanol content of the fermentation medium at 15–25 g / L. The fermentation broth was then subjected to cell disruption and saponification extraction to obtain the 7-DHC.

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