CN116179384B

CN116179384B - A genetically engineered yeast strain for increasing 7-DHC production and its construction and application

Info

Publication number: CN116179384B
Application number: CN202310058515.6A
Authority: CN
Inventors: 柳志强; 柯霞; 潘子豪; 杜宏斐; 郑裕国
Original assignee: Zhejiang University of Technology ZJUT
Current assignee: Zhejiang University of Technology ZJUT
Priority date: 2023-01-18
Filing date: 2023-01-18
Publication date: 2025-08-22
Anticipated expiration: 2043-01-18
Also published as: CN116179384A

Abstract

The invention relates to a saccharomyces cerevisiae gene engineering strain synthesized by 7-DHC exocytosis, a construction method and application thereof. The invention constructs a recombinant S.cerevisiae strain sc13 capable of efficiently transporting 7-DHC to the outside of cells, when 500mL shake flask biphasic fermentation is carried out by using the recombinant S.cerevisiae strain sc13, the total 7-DHC yield reaches 28.189mg/g (secretion 11.701 mg/g), and compared with a control sc1 strain, the total 7-DHC yield is improved by 14.54 times, the total extracellular secretion yield is improved by 13.77 times, wherein the secretion yield of the extracellular 7-DHC accounts for 41.51 percent. The recombinant strain constructed by the invention has stronger exocrine capability and higher yield, provides a guiding thought for 7-DHC synthesis and simplified 7-DHC separation and extraction, and has wide application prospect.

Description

Saccharomyces cerevisiae genetically engineered bacterium for improving 7-DHC yield and construction and application thereof

Technical Field

The invention relates to a saccharomyces cerevisiae gene engineering strain synthesized by 7-DHC exocytosis, a construction method and application thereof.

Background

7-Dehydrocholesterol (7-dehydrocholesterol, 7-DHC) has multiple biological functions, can be directly converted into vitamin D3 under ultraviolet irradiation, and is important for maintaining bone calcium balance and protecting bone. In addition, 7-DHC is involved in the treatment of hyperlipidemia, and accumulation of 7-DHC at a high concentration contributes to the prevention of hyperlipidemia. 7-DHC has wide application in the industries of food, pharmacy and the like due to various biological activities. However, 7-DHC has a complex structure and limited sources, and is mainly obtained through lanolin extraction and semi-chemical synthesis, and the route has the disadvantages of high resource consumption and low extraction and synthesis efficiency.

Saccharomyces cerevisiae (Saccharomyces cerevisiae) is a natural synthetic pathway of ergosterol for edible fungi in a model, and the synthesis involves about 30 enzymes, and the whole pathway comprises three modules of mevalonate biosynthesis, farnesyl pyrophosphate biosynthesis and ergosterol biosynthesis. And the saccharomyces cerevisiae has short growth cycle and strong fermentation capacity, has a complete genetic operating system, and is an ideal chassis for de novo synthesis of sterol compounds. The current mainstream strategy is to utilize metabolic engineering strategies to achieve 7-DHC accumulation by knocking out the erg5/6 gene and heterologously expressing the C-24 reductase DHCR24 to reconstruct the ergosterol pathway. At present, through the strengthening of key limiting steps of 7-DHC on a synthesis path and combining strategies such as improving intracellular accumulation capacity based on modularized integration and organelle rational transformation strategies, in recent years, GUO and the like further screen the source of DHCR24, knock out endoplasmic reticulum membrane related gene PAH1 on the basis of enhancing the expression of the genes, and find that the genes are favorable for sterol synthesis. Lisha Qiu et al mention in work that ΔGDH1 not only increases the titer of 7-DHC, but also increases the growth rate of the strain. To optimize carbon metabolism, they used the CRISPRi technique to reduce ERG6 expression. WENQIAN WEI et al focused on the squalene postpathway and noted that there was an inhibitor of ERG2 expression, MOT3. After the inhibition of MOT3 is removed, CTT1 is integrated into a genome, cells are protected from being damaged by hydrogen peroxide, the synthesis of sterols is improved finally, the synthesis capacity of 7-DHC is improved remarkably, and the fermentation level of more than 3 g/L is realized by combining a high-density culture process of yeast, so that the method has an important alternative path depending on the traditional extraction process, and has a good market application prospect.

With the deep research of intracellular transport and storage mechanisms of ester substances, various endogenous lipid transport proteins are sequentially resolved, and can be combined with sterol targeted transport proteins through a non-vesicle transport way to realize the secretion. Sterols such as cholesterol are unevenly distributed in eukaryotic cells, and this phenomenon is most remarkable in Plasma Membranes (PM), which contain 60 to 80% of cell free cholesterol and 35 to 45% of lipids. This is a critical aspect of intracellular homeostasis, as changes in the concentration of sterols within the membrane may greatly alter the physical properties of the membrane (e.g., fluidity), affecting different processes such as signal transduction, membrane transport, or function of intact proteins. It is becoming increasingly clear that maintaining this intracellular sterol distribution is dependent on a tightly controlled synthesis, transport and storage system. Pathogen-associated yeast (PRY) proteins, a class of sterol-binding proteins, have three Pry family members in Saccharomyces cerevisiae, roger Schneiter et al have shown that Pry1 and Pry2 are involved in sterol transport and secretion, while Pry3 is a cell wall-bound protein. In addition, saccharomyces cerevisiae contains another endogenous sterol transport mechanism through NPC2 and NCR1 that are located in the late nucleus and on lysosomes. DHE-based live cell imaging experiments, NCR1 and NPC2 were found to be necessary to transport sterols to the vacuolar membrane, especially when the cells were starved. In addition, among the numerous transesterification proteins, cytoplasmic membrane sterol transporters such as Aus1p and Pdr11p are widely used for the extracellular transport of hydrophobic ester compounds such as carotene. In addition, in yeast cells, the lipid exchange family protein Lam1p-Lam6p anchored at the cell membrane contact site has a transesterification domain, and the water-transporting pocket can specifically recognize various sterol compounds and plays an important role in mediating the substance transport process of sterols independent of vesicles. Recently, sokolov et al reviewed the synthesis of ergosterol in s.cerevisiae cells and the conversion of the transport pathway, highlighting the important functions of Oshp and Lamp in promoting the non-vesicle transport pathway of sterols.

As a lipophilic compound, an excessive amount of 7-DHC in a limited yeast cell space is liable to cause intracellular accumulation and result in a product inhibitory effect, which becomes an important factor for limiting the ability of synthesizing unit cell sterols. The current research is focused on improving the accumulation of intracellular products, and the process of extraction needs to be performed through the processes of cell centrifugation, crushing, phase extraction, byproduct separation and the like, so that the process is complex, and the industrial production and application challenges are large. Transport engineering is considered one of the most promising strategies. Aiming at the transport proteins of various excavated ester compounds and complex transport paths thereof, if an effective extracellular secretion path can be constructed, the extracellular secretion synthesis of 7-DHC is realized, the metabolic pressure and the limited synthesis space limitation after intracellular accumulation of toxicity removal of the product can be reduced, the difficulty of downstream separation and purification can be simplified, the separation cost can be potentially reduced, and the method has important significance for promoting the industrial production of the de-novo synthesis path with a simple carbon source.

Disclosure of Invention

The invention aims to provide a saccharomyces cerevisiae gene engineering strain capable of transporting 7-DHC synthesized from the head into cells through metabolic engineering technology, and a construction method and application thereof.

The technical scheme adopted by the invention is as follows:

A7-DHC exocytosis synthesized saccharomyces cerevisiae strain is constructed by the following method that a chassis bacterium saccharomyces cerevisiae genome is subjected to enhanced expression of truncated 3-hydroxy-3 methylglutaryl coenzyme A reductase tHMG1, squalene epoxidase ERG1, NADH kinase POS5, lanosterol demethylase ERG11, squalene synthase ERG9, isopentenyl pyrophosphate isomerase IDI1, phosphovalerate kinase ERG8, mevalonate kinase ERG12, farnesyl pyrophosphate synthase ERG20, mevalonate decarboxylase ERG19, a Gallus Gallus-derived 24-sterol reductase DHCR24 is subjected to heterologous expression, C-22 sterol desaturase ERG5, galactose/lactose metabolism regulator GAL80 required by ergosterol synthesis and cysteine proteases GAL6 and MIG1 serving as inhibitors in a GAL4 system are subjected to knockdown, NADP ⁺ -specific glutamate dehydrogenase GDH1 is subjected to knockdown, DPP1 is subjected to knockdown, and 3-sterol phosphorylase ST can be expressed, and the yeast strain is subjected to exocytosis synthesized by the strain to the enzyme, and the strain is subjected to the variant expression of the alcohol-7-D1 is obtained.

Preferably, the Chaetomium is Saccharomyces cerevisiae CEN.PK2-1C.

The enhanced expression means enhanced expression of tHMG1 (3-hydroxy-3-methylglutaryl CoA reductase tHMG1 can be integrated into repeated delta sequences at two ends of the multi-copy site Ty 1) with multiple copy numbers on the saccharomyces cerevisiae genome, and enhanced expression of ERG1 with 1 copy number, ERG11 with 1 copy number, POS5 with 1 copy number, ERG9 with 1 copy number, ERG8 with 1 copy number, ERG12 with 1 copy number, IDI1 with 1 copy number, ERG20 with 1 copy number, ERG19 with 1 copy number on the genome. The heterologous expression refers to the expression of DHCR24 with multiple copy numbers on a Saccharomyces cerevisiae CEN.PK2-1C genome, and the multiple copy sites are repeated delta sequences at two ends of Ty 3.

The tHMG1 has a Gene ID of 42650, the IDI1 has a Gene ID of 855986, the ERG9 has a Gene ID of 856597, the POS5 has a Gene ID of 855913, the ERG8 has a Gene ID of 855260, the ER12 has a Gene ID of 855248, the ERG1 has a Gene ID of 853086, the ER11 has a Gene ID of 856398, the ERG20 has a Gene ID of 853272, the ERG19 has a Gene ID of 855779, the GAL80 has a Gene ID of 854954, the GAL6 has a Gene ID of 855482, the MIG1 has a Gene ID of 852848, the GDH1 has a Gene ID of 854557, the ERG5 has a Gene ID of 855029, the DPP1 has a Gene ID of 851878, and the ADH3 has a Gene ID of 855107.

Specifically, the invention enhances expression of tHMG1 by the P _GAP promoter, enhances expression of ERG1 by the P _GAL2 promoter, enhances expression of ERG11 by the P _GAL1 promoter, enhances expression of POS5 by the P _GAL1 promoter, enhances expression of ERG8 and ERG12 by the P _GAL1,10 bi-directional promoter, enhances expression of ERG20 and ERG9 by the P _GAL1,10 bi-directional promoter, enhances expression of IDI1 and ERG19 by the P _GAL1,10 bi-directional promoter, heterologous expression of DHCR24 by the P _GAP promoter, and heterologous expression of ST1 and PR-1 by the P _GAL1 promoter.

More specifically:

ERG1 expressed enhanced by the P _GAL2 promoter is integrated into the GAL6 site on the Saccharomyces cerevisiae genome, the Gene ID of the GAL6 site is 855482;

ERG11, which is expressed by the P _GAL1 promoter for enhanced integration into the GDH1 site on the Saccharomyces cerevisiae genome, the Gene ID of said GDH1 being 854557;

POS5, which is enhanced in expression by the P _GAL1 promoter, is integrated into the MIG1 locus on the Saccharomyces cerevisiae genome, and the Gene ID of the MIG1 is 852848;

Enhanced expression of ERG8 and ERG12 by a P _GAL1,10 bi-directional promoter integrated into the GAL80 locus on the saccharomyces cerevisiae genome, said GAL80 having a Gene ID of 854954;

Enhanced expression of ERG9 and ERG20 by a P _{GAL1 ,10} bi-directional promoter is integrated into the DPP1 site on the saccharomyces cerevisiae genome, the Gene ID of DPP1 is 851878;

The Gene ID of ADH3 is 855107, and IDI1 and ERG19 are expressed in a reinforced way through a P _{GAL1 ,10} bi-directional promoter and integrated into the ADH3 locus on the saccharomyces cerevisiae genome;

Gallus Gallus derived and heterologous expression of DHCR24 via the P _GAP promoter was integrated into the saccharomyces cerevisiae genome at the ERG5 site, which ERG5 Gene ID is 855029, with integration of the repeated delta sequences at both ends of the multicopy site Ty 3.

The invention also relates to a method for constructing the saccharomyces cerevisiae strain, which comprises the following steps:

(1) Knocking out C-22 sterol desaturase ERG5 required by ergosterol synthesis, integrating the P _GAP-DHCR24-T_CYC1 fragment into an ERG5 enzyme site on a Saccharomyces cerevisiae CEN.PK2-1C genome, and constructing the obtained Saccharomyces cerevisiae named sc1 strain;

(2) The P _GAL2-ERG1-T_CYC1 fragment is integrated on the genome of the sc1 strain and integrated on a GAL6 site (Gene ID of the GAL6 site is 855482), and a saccharomyces cerevisiae named sc2 strain is constructed;

(3) The P _GAL1-tHMG1-T_CYC1 fragment is integrated into delta sequences (the guide sequence is TGTTGGAATAGAAATCAACT) at two ends of Ty1 on the genome of the sc2 strain, and a saccharomyces cerevisiae strain named sc3 strain is constructed;

(4) Integrating the T _TEF-T_ADH1-ERG8-P_GAL10-P_GAL1-ERG12-T_CYC1 fragment into the GAL80 enzyme site (the Gene ID of GAL80 is 854954) on the genome of the sc3 strain, and constructing a saccharomyces cerevisiae strain named sc4 strain;

(5) Integrating the T _TEF-P_GAL1-POS5-T_CYC1 fragment into the MIG1 locus (the Gene ID of the MIG1 is 852848) on the genome of the sc4 strain, and constructing a saccharomyces cerevisiae strain named sc5 strain;

(6) Integrating the T _TEF-T_ADH1-ERG9-P_GAL10-P_GAL1-ERG20-T_CYC1 fragment into the DPP1 locus (the Gene ID of the DPP1 is 851878) on the genome of the sc5 strain, and constructing and obtaining a saccharomyces cerevisiae strain named sc6 strain;

(7) Integrating the T _TEF-P_GAL1-ERG11-T_CYC1 fragment into the GDH1 locus on the genome of the sc6 strain (the Gene ID of the GDH1 is 854557), and constructing a Saccharomyces cerevisiae strain named sc7 strain;

(8) Integrating the T _TEF-T_ADH1-IDI1-P_GAL10-P_GAL1-ERG19-T_CYC1 fragment into the ADH3 locus (the Gene ID of the ADH3 is 855107) on the genome of the sc7 strain, and constructing a saccharomyces cerevisiae strain named sc8 strain;

(9) The P _GAL1-DHCR24-T_CYC1 fragment is integrated to a Ty3 locus (a guide sequence is ACGTTCATAAAACACATATG) on the genome of the sc8 strain, and a saccharomyces cerevisiae strain named sc9 strain is constructed;

(10) H1-P _GAL1-ST1- P_GAL1 -PR1-H2 is introduced into the genome of the sc9 strain, and the saccharomyces cerevisiae strain named sc13 strain is constructed, namely the saccharomyces cerevisiae strain synthesized by the 7-DHC exocytosis. The Genebank sequence number of the sterol transporter sterol transporter (ST 1) is XP_717917.2, and the Genebank sequence number of PR-1 of Fusarium odoratissimum NRRL 54006 origin is XP_031058987.1.

The invention also relates to application of the saccharomyces cerevisiae strain in preparation of 7-DHC by microbial fermentation.

The application is that the saccharomyces cerevisiae strain is inoculated to a fermentation culture medium, n-dodecane accounting for 5-10% of the volume of the initial culture medium is added as an extractant, fermentation culture is carried out for 48-96 hours at 28-32 ℃, and the 7-DHC is obtained through separation and purification of fermentation liquid.

Generally, recombinant saccharomyces cerevisiae is inoculated to a seed culture medium to prepare seed liquid, and the prepared seed liquid is inoculated to a fermentation culture medium in an inoculum size of 1-10% (v/v). The seed medium is typically YPD medium. In the invention, YPD medium is composed of 20g/L peptone, 10g/L yeast extract and 20g/L anhydrous glucose.

The invention has the main beneficial effects that a recombinant S.cerevisiae strain sc13 capable of efficiently transporting 7-DHC to the outside of cells is constructed, when 500mL shake flask biphasic fermentation is carried out by using the recombinant S.cerevisiae strain sc13, the total 7-DHC yield reaches 28.189mg/g (secretion 11.701 mg/g), and compared with a control sc1 strain, the total 7-DHC yield is improved by 14.54 times, the total extracellular secretion yield is improved by 13.77 times, wherein the extracellular 7-DHC secretion yield accounts for 41.51 percent. The recombinant strain constructed by the invention has stronger exocrine capability and higher yield, provides a guiding thought for 7-DHC synthesis and simplified 7-DHC separation and extraction, and has wide application prospect.

Drawings

FIG. 1 shows comparison of yields of starting strain sc1 and intracellular 7-DHC, ergot 5,8 dien 3 beta-ol, lanosterol from high yielding strain sc 9;

FIG. 2 shows the distribution difference and quantitative analysis of extracellular sterols in a double-phase fermentation cell, AB shows the detection result of gas chromatography, and CD shows the fermentation yield of an engineering strain;

FIG. 3 is a graph showing the promotion of intracellular extracellular production of 7-DHC by overexpression of CAP family transporter (A) and overexpression of NPC2 family transporter ST (B) (mg/g);

FIG. 4 shows intracellular and extracellular yields (mg/g) of 7-DHC from Saccharomyces cerevisiae sc9 over-expressed in the form of a modified PR-1.

Detailed Description

The invention will be further described with reference to the following specific examples, but the scope of the invention is not limited thereto:

the media referred to in the examples are as follows:

SOB medium containing 20g tryptone, 0.5g NaCl, 5g yeast extract, 0.186g KCl, 1g MgCl ₂ per liter.

SD-Leu2 medium containing 20mg uracil, 20mg tryptophan, 20mg histidine, 6.7g amino free yeast nitrogen source and 20g anhydrous glucose per liter.

SD-Ura3 medium containing leucine 20mg, tryptophan 20mg, histidine 20mg, amino free yeast nitrogen source 6.7g and anhydrous glucose 20g per liter.

Fermentation medium comprises 20g of tryptone, 40g of anhydrous glucose and 10g of yeast extract per liter.

SOB+Amp plate containing 20g tryptone, 0.5g NaCl, 5g yeast extract, 0.186g KCl, 1g MgCl ₂, 20g agar powder per liter.

YPD+NAT plate 1mL NAT-resistant mother liquor 20mg/mL was added per 100mL of 2% tryptone, 1% yeast extract, 2% anhydrous glucose, 2% agar powder.

YPD+G418 plates, 2% tryptone, 1% yeast extract, 2% anhydrous dextrose, 2% agar powder, 1mL of 20mg/mL G418 resistant mother liquor per 100 mL.

Detection of 7-DHC content:

centrifuging the fermented bacterial liquid, filtering the upper oil phase with a filter membrane, feeding into a liquid phase bottle, crushing the rest bacterial cells with 3N hydrochloric acid, centrifuging again to collect cell fragments, and saponifying with 1.5mol/L KOH methanol solution on a water bath at 60 ℃ to extract intracellular sterols. After the reaction is finished, extracting the liquid by using n-hexane, evaporating the obtained n-hexane on a water bath kettle at 75 ℃, adding a certain amount of ethyl acetate for re-dissolution, and filtering by using a filter membrane to enter a liquid phase bottle.

The gas phase column adopts the HP-5 (30 m*0.25 mm*0.25 mu m) of the Siemens, the gas phase program is set as follows, the temperature of a sample inlet is 300 ℃, the temperature program is 190 ℃ (1 min) 10 ℃ per minute 300 ℃ (10 min), the sample injection amount is 1 mu L, the split ratio is 20:1, the carrier gas is N ₂, the flow rate is 1.0 mL per minute, and the temperature of a transmission rod is 250 ℃.

The detection method of the recombinant saccharomyces cerevisiae OD ₆₀₀ comprises the following steps:

And (3) inoculating the yeast seed solution cultured for 16-24 hours into a 500m L shaking bottle filled with 100m L fermentation medium and 10m L dodecane according to the inoculum size of 1%, and culturing at 30 ℃ and 220 rpm. The OD ₆₀₀ was measured using an ultraviolet spectrophotometer after dilution in appropriate proportions at the time of sampling.

The construction of the plasmids involved in the examples was performed in E.coli Dh5α, and the plasmids were amplified as a template expression cassette after completion of the construction.

In the embodiment, the plasmid with the PAM locus mutation is subjected to sequencing at the corresponding position after construction is completed, so that the pdc5 plasmid with the PAM locus mutated correctly is obtained.

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

TABLE 1 primer list

Primer name	Primer sequences
		pY-G1F	AGTCCGATCCGGGGTTTTTTTTTGTTTGTTTATGTGTGTTTATTCGA
pY-G1R	GTGGGGATGATCCACTAGTATCATTATCAATACTGCCATTTCAAAGA
		rERG5-HOMO1F	CAATTACCAATCTCCGCATTGAC
rERG5-HOMO1R	GTATTCTGGGCCTCCATGTCTTTGTTAAAAGGTATTTATTGTCTATTGGAATAGC
		rERG5-HOMO2 F	CAGAACTTTGTCCAGACAATAAATCATATT
rERG5-HOMO2 R	ACTCTGAAGAGAATGAACCAAGG
		rNAT F	GACATGGAGGCCCAGAATAC
rNAT-G R	AATGGCAGTATTGATAATGACAGTATAGCGACCAGCATTCA
		fGAP-D F	TCATTATCAATACTGCCATTTCAAAGAAT
rD24-ERG5 R	ATTGTCTGGACAAAGTTCTGGCAAATTAAAGCCTTCGAGC
		VER H1 F	GTCTGCGAAGTCTCGTACCT
VER H2 R	TAGCAGATCATTAGCTGTAGCGTATG
		NCG6 F	TCAACATTTAAGTAAATCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAA
NCG6 R	GATTTACTTAAATGTTGACGATCATTTATCTTTCACTGCGGAG
		CE6 F	AGATTTCTATGAATATGGTTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAA
CE6 R	AACCATATTCATAGAAATCTGATCATTTATCTTTCACTGCGGAG
		P-G6 F	ATCTACTAGTCATATGGATTGTCCTCTTCCATCGATATCAGTAAGA
P-G6 R	TCGGTACCCGGGGATCCGATGGCCAAAGCACCCATTGG
		PG2 F	AGGTTGCAATTTCTTTTTCTATTAGTAGC
PG2 R	GCTTGGGGTTGCTGTGAAAC
		LpYES2 F	AAGCTGCGGCCCTGCATT
LpYES2 R	GGAGCTTCCAGGGGGAAAC
		emp F	TAACGTCAAGGAGTCTAGAGGGCCGCATCATGTAATTAGTTATGTCACGC
emp R	TGCGGCCCTCTAGACTCCTTGACGTTAAAGTATAG
		VG2 F	GCGTGGGGATGATCCACTAGTA
VG2 R	TGTAAGCGTGACATAACTAATTACATGA
		ERG1 F	ATGTCTGCTGTTAACGTTGCACC
ERG1 R	TTAACCAATCAACTCACCAAACAAA
		LP2 F	TCATGTAATTAGTTATGTCACGCTTACA
LP2 R	TACTAGTGGATCATCCCCACGC
		LHP F	CGGATTAGAAGCCGCCGA
LPH2 R	GCAAATTAAAGCCTTCGAGCG
		HPP R	GCTCGGCGGCTTCTAATCCGCAGTATAGCGACCAGCATTCACA
PH2-P F	GCTCGAAGGCTTTAATTTGCCTAAAGGACCCGTCTAACTCTAGTATCG
		HPP F	GTGGGGATGATCCACTAGTACGATGGCTAAGATTCCCGTCT
PH2-P R	TTAATGCAGGGCCGCAGCTTGGTGACAATAATAACGGGGTTGT
		VG1 F	CGGATTAGAAGCCGCCGAG
VG1 R	ACGCTCGAAGGCTTTAATTTGC
		T1t F	ATGGAATCCCAACAATTATCTCAACATTCACA
T1t R	CATTTAAACTATTAACTAACAAATGGATTCATTAG
		E8E20 R	ATGGACTACAACAAGAGATCTTCGGTC
E8E20 R	ACATTAAACGTTAGCAATATCTCGCATTATAG
		MP5 F	GCAAAGCCCATATCCAATGACACA
MP5 R	ACCAAGGGTGAATATGATTAATACTGC
		E9E20 F	CGTTTATTAAAACGCCTTTCAACATAGGG
E9E20 F	ATCAGAGGATCCCGGATGAGG
		E11G F	CATACAATTCAGAGTCACCTGGGA
E11G R	CACTAAGGACGGTAAGGTCTTGCCA
		IDE19 F	GCAATCCACAGCTGCAATCCCTA
IDE19 R	AGGTATTCTCTCATGTGGTGAAGTCC
		T3D24 F	GTCCTGTGTCCTGTGGTAGA
T3D24 R	CACAATACGACTGGCATCATTC
		125 F	GTTGAAGCTAGATATGGTTTCATTAAGTTGATTCATCA
125 R	CCATATCTAGCTTCAACTTCAACATAAGCACCTTCTT
		127 F	TTTGGCTGTTGCTAGAGCTTATACCAAAGATGATG
127 R	CTCTAGCAACAGCCAAATATTTACCAGGTGGAACTTCTT

Example 1 construction of tool plasmid

Taking Saccharomyces cerevisiae CEN.PK2-1C genome as a template, and obtaining a gene fragment P _GAL2 by using PG 2F, PG R;

using pYES2 plasmid as template and primer LpYES-F, lpYES R to obtain linearized pYES2 plasmid;

The gene fragment P _GAL2 was ligated with the linearized pYES2 plasmid and introduced into competent Dh5α, and verified with primer VG 2F, VG 2R to give a P _GAL2 -pYES2 plasmid with the P _GAL1 promoter replaced by P _GAL2.

The pYES2 plasmid is used as a template, and the primers emp F and emp R are used for obtaining linearized plasmids, and the linearized plasmids are transformed into Dh5α competence to obtain the plasmid emp with only a P _GAL1-T_CYC1 empty expression frame.

Example 2 construction of 7-DHC-producing Saccharomyces cerevisiae Chassis

The method comprises the following specific steps:

(1) Fragment synthesis:

The synthetic gene fragment DHCR24 (SEQ ID No. 1) was ligated into the pYES2 multiple cloning site.

The Saccharomyces cerevisiae CEN.PK2-1C genome was used as a template, primers described in Table 1 were used to obtain a gene fragment GAP using primers pY-G1F, pY-G1R, and it was used to replace the GAL1 promoter on plasmid pYES 2.

Using Saccharomyces cerevisiae CEN.PK2-1C genome as template, using the primers shown in Table 1, and using primer rERG-HOMO 1F, rERG5-HOMO1R to obtain gene fragment ERG5-HOMO1;

obtaining a gene fragment ERG5-HOMO2 by using a primer rERG-HOMO 2F, rERG5-HOMO 2R;

using plasmid pcfb2312 as template and primers rNAT F, rNAT-G R to obtain fragment natMX;

The P _GAP-DHCR24-T_CYC1 gene fragment is obtained by using the primers fGAP-D F and rD24-ERG 5R.

(2) And (3) performing fusion PCR on the four fragments ERG5-HOMO1, ERG5-HOMO2, natMX and P _GAP-DHCR24-T_CYC1 obtained in the step (1) by adopting PCR, and performing gel cutting recovery on the correct band obtained by gel running to obtain a fusion gene fragment delta ERG5-natMX-P _GAP-DHCR24-T_CYC1 containing the upstream and downstream homology arms of ERG 5.

(3) The fusion gene fragment in step (2) was transformed into Saccharomyces cerevisiae CEN.PK2-1C strain competence, cultured on YPD+NAT plates at 30℃for 2-3 days, and single colony PCR verification was performed using primers VER H1F and VER H2R described in Table 1. Single colonies with correct bands were selected to give strain CEN.PK2-1 C.DELTA.ERG5-P _GAP-DHCR24-T_CYC1, designated Saccharomyces cerevisiae sc1.

Example 3 construction of high yield 7-DHC Saccharomyces cerevisiae Chassis

(1) Construction of sgRNA:

The plasmid pdc5 was used as a template, the primers shown in Table 1 were used, and the primer NCG 6R, NCG F was used to obtain a linearized pdc5 plasmid, which was introduced into competent Dh5α, spread on SOB+Amp plates, and cultured at 37℃to obtain pdc5-NCG6 plasmid.

(2) Construction of the donor fragment

Taking Saccharomyces cerevisiae CEN.PK2-1C as a template, and using a primer ERG 1F, ERG R to obtain a gene fragment ERG1;

Linearized plasmid P _GAL2 -pYES2 is obtained by using primer LP 2F, LP R;

Connecting the gene segment ERG1 with linearization plasmid P _GAL2 -pYES2, introducing into competent Dh5α, performing colony PCR verification by using primer PG 2F, ERG R, and obtaining gene segment P _GAL2-ERG1-T_CYC1 by using primers LHP F and LPH 2R;

obtaining a target gene segment GAL6 by using primers P-G6F, P-G6R;

Connecting the plasmid with a linearization plasmid pMD20, directly verifying by using primers HPP R and PH2-P F, and linearizing the newly obtained pMD20-GAL6 plasmid by using the same primers;

And then connecting the gene fragment P _GAL2-ERG1-T_CYC1 with a linearization plasmid pMD20-GAL6, carrying out PCR verification by using primers HPP F and PH2-P R, and amplifying by using the same primers to obtain the donor H1-P _GAL2-ERG1-T_CYC1 -H2.

(3) The sgRNA dc5-NCG6 and the donor H1-P _GAL2-ERG1-T_CYC1 -H2 constructed in (1) and (2) are simultaneously introduced into the competence of Saccharomyces cerevisiae sc1, cultured on YPD+G418 plates at 30 ℃ for 2-3 days, and subjected to PCR verification by using P-G6F, VG 1R, thereby obtaining Saccharomyces cerevisiae chassis sc2.

The subsequent chassis construction method is the same as the above method, namely, the pam site of pdc5 plasmid is mutated by using a primer to obtain sgRNA, then the gene to be over-expressed and the gene to be knocked out are cloned from Saccharomyces cerevisiae by using the primer, then the gene to be over-expressed is inserted between a promoter and a terminator on plasmid pYES2, the target knocked-out gene is connected to plasmid pMD20, and then the complete expression frame on pYES2 is cloned by using the primer and is inserted into pMD20 containing the target knocked-out gene to obtain a knocked-out substitution fragment. Finally, the two fragments obtained above are introduced into saccharomyces cerevisiae competence, and are cultured for 2-3 days at 30 ℃ on a YPD+G418 flat plate, so that a subsequent saccharomyces cerevisiae chassis is obtained, and finally, a high-yield 7-DHC saccharomyces cerevisiae chassis sc9 is constructed, which is specifically as follows:

(1) The P _GAL1-tHMG1-T_CYC1 fragment is integrated into delta sequences (the guide sequence is TGTTGGAATAGAAATCAACT) at two ends of Ty1 on the genome of the sc2 strain by using a primer T1T F/R, and a saccharomyces cerevisiae strain named sc3 strain is constructed;

(2) Integrating the T _TEF-T_ADH1-ERG8-P_GAL10-P_GAL1-ERG12-T_CYC1 fragment into the GAL80 enzyme site (the Gene ID of GAL80 is 854954) on the genome of the sc3 strain by using a primer E8E 20F/R, and constructing a saccharomyces cerevisiae strain named sc4 strain;

(3) The T _TEF-P_GAL1-POS5-T_CYC1 fragment is integrated into the MIG1 locus (the Gene ID of the MIG1 is 852848) on the genome of the sc4 strain by using a primer MP 5F/R, and a saccharomyces cerevisiae strain named sc5 strain is constructed;

(4) The T _TEF-T_ADH1-ERG9-P_GAL10-P_GAL1-ERG20-T_CYC1 fragment is integrated into the DPP1 locus (the Gene ID of the DPP1 is 851878) on the genome of the sc5 strain by using a primer E9E 20F/R, and a saccharomyces cerevisiae strain named sc6 strain is constructed;

(5) The T _TEF-P_GAL1-ERG11-T_CYC1 fragment is integrated into the GDH1 locus on the genome of the sc6 strain by using a primer E11G F/R (the Gene ID of the GDH1 is 854557), and a saccharomyces cerevisiae strain named sc7 strain is constructed;

(6) The T _TEF-T_ADH1-IDI1-P_GAL10-P_GAL1-ERG19-T_CYC1 fragment is integrated into the ADH3 locus (the Gene ID of the ADH3 is 855107) on the genome of the sc7 strain by using a primer IDE 19F/R, and a saccharomyces cerevisiae strain named sc8 strain is constructed;

(7) The P _GAL1-DHCR24-T_CYC1 fragment was integrated into the Ty3 site (guide sequence ACGTTCATAAAACACATATG) on the genome of the sc8 strain using primer T3D 24F/R, and the construction yielded a Saccharomyces cerevisiae strain designated sc9 strain.

Example 4 analysis of Saccharomyces cerevisiae sterol cell out product in oil-water two-phase fermentation System

For further enrichment of 7-DHC, an oil-water biphasic fermentation process is used here, the specific operating method being as follows:

And (3) respectively culturing the recombinant saccharomyces cerevisiae strains sc1 and sc9 for 16-24 hours to prepare seed liquid, inoculating the prepared seed liquid into a 500mL conical flask filled with 100mL fermentation medium and 10mL dodecane according to the inoculum size of 2% (v/v), and culturing for 96 hours at 30 ℃ and 220rpm to prepare fermentation liquor.

The gas phase analysis method is as described above. Upon analysis of the intracellular products, it was found that in addition to 7-DHC, there were also fucosterol, ergot 5,7 dien 3 beta-ol, ergot 5,8 dien 3 beta-ol and lanosterol. Upon analysis of the extracellular product, it was found that only 7-DHC and fucosterol were present, and this combination analyzed for structural differences between fucosterol and the other three by-products, and it was possible that only sterols of the cholesterol class could be transported out of the cell (see fig. 1, 2).

Example 5 construction and characterization of the Saccharomyces cerevisiae transport and cell-out pathway

Plasmid containing ST1 (SEQ ID No. 2), ST2 (SEQ ID No. 3), ST3 (SEQ ID No. 4), ST4 (SEQ ID No. 5), pYES2 (SEQ ID No. 6) of ST5 was introduced into Saccharomyces cerevisiae SC9 competent, cultured on SC-URA plate at 30℃for 4-5 days, and pcr validation was performed using VG 1F, VG R in Table 1 to obtain Saccharomyces cerevisiae chassis SC9-ST1, SC9-ST2, SC9-ST3, SC9-ST4, SC9-ST5.

The Genebank sequence number of the sterol transporter sterol transporter (ST 1) is XP_717917.2, the Genebank sequence number of the ST2 from CANDIDA VISWANATHII is RCK66207.1, the Genebank sequence number of the ST3 from Candida parapsilosis is XP_036664935.1, the Genebank sequence number of the ST4 from Scheffersomyces stipitis is KAG2730877.1, and the Genebank sequence number of the ST5 from Pachysolen tannophilus NRRL Y-2460 is ODV98224.1.

The fermentation specifically comprises the following steps:

(1) The recombinant saccharomyces cerevisiae strains sc1, emp, sc9-ST1, sc9-ST2, sc9-ST3, sc9-ST4 and sc9-ST5 are respectively cultured for 16-24 hours at 30 ℃ and 220rpm to prepare seed liquid, the prepared seed liquid is inoculated into a 500mL conical flask filled with 100mL of fermentation medium and 10mL of dodecane according to the inoculum size of 2% (v/v), and the seed liquid is cultured for 96 hours at 30 ℃ and 220rpm to prepare the fermentation liquid.

(2) Quantitative analysis of extracellular 7-DHC:

and centrifuging the fermentation liquor, sucking the upper dodecane, filtering the fermentation liquor in a liquid phase sample injection bottle through a filter membrane, performing gas chromatography detection, and converting the fermentation liquor with the peak area of a 7-DHC standard product to obtain the fermentation yield of the engineering strain. The remaining broth was resuspended and the OD ₆₀₀ was measured using an ultraviolet spectrophotometer after 10-fold dilution of the broth was aspirated.

As shown in Table 2 and FIG. 3B, the sc9-ST1 strain overexpressing ST1 protein had a 7-DHC content of 6.486mg/g and an OD ₆₀₀ of 10.11 in extracellular dodecane.

(3) Yield of intracellular 7-DHC was calculated:

8mL of the resuspended broth was aspirated, washed with deionized water and resuspended, and then broken in boiling water for 5min in a 3mL 3N hydrochloric acid 10mL ep tube. After centrifugation, the supernatant was removed and washed with deionized water. The intracellular sterols were extracted by saponification with 1.5mol/L KOH in methanol at 60℃in a water bath. After the reaction is finished, extracting the liquid by using n-hexane, evaporating the obtained n-hexane on a water bath kettle at 75 ℃, adding a certain amount of ethyl acetate for re-dissolution, and filtering by using a filter membrane to enter a liquid phase bottle. The gas chromatography detection was carried out, and the engineering strain fermentation yield was obtained by conversion with the peak area of the 7-DHC standard, and the results are shown in Table 2 and FIG. 3B.

TABLE 2 overexpression of NPC2 Transporter family intracellular extracellular 7-DHC yield of Saccharomyces cerevisiae and OD after fermentation ₆₀₀

Strain	Extracellular 7-DHC (mg/g)	Intracellular 7-DHC (mg/g)	OD₆₀₀
				sc1	0.861	0.488	7.11
sc9	3.376	7.186	8.17
				emp	3.255	6.412	9.07
sc9-ST1	6.486	6.729	10.11
				sc9-ST2	4.675	6.057	10.54
sc9-ST3	5.273	2.642	9.89
				sc9-ST4	4.966	2.668	9.37
sc9-ST5	4.365	4.415	10.32

Among them, recombinant S.cerevisiae strain sc9-ST1 was the highest in yield, and when 500mL shake flask biphasic fermentation was performed using this, the total yield of 7-DHC reached 13.215mg/g (secretion 6.486 mg/g). In this strain, the total secretion and total yield of 7-DHC were increased to 6.81 and 7.63 fold, respectively, of the control sc1 strain (1.94 mg/g and 0.85 mg/g) by overexpressing ST transporter.

(II) plasmid pYES2 containing PRY1, bacterial Pry (Bac Pry gene sequence see SEQ ID No. 10), vertebrate CRISP protein (Vcp gene sequence see SEQ ID No. 9), apolipoprotein E isoform b precursor (AEp gene sequence see SEQ ID No. 8), plant-PR-1 (Pr-1 gene sequence see SEQ ID No. 7), NPC intracellular cholesterol transporter 1 homolog 1b isoform X4 (NPCX 4 gene sequence see SEQ ID No. 11) was introduced into competent Saccharomyces cerevisiae SC9, cultured on SC-URA plate at 30℃for 4-5 days, and pc R-verified using VG 1F, VG R in Table 1 to obtain Saccharomyces cerevisiae chassis SC9-PRY1, SC9-Bac Pry, SC9-VCp, SC9-AEp, SC9-PR1, SC 9-NPCX.

The Genebank sequence number of PR-1 of Fusarium odoratissimum NRRL 54006 is XP_031058987.1, the Genebank sequence number of Bacterial Pry of Yarrowia lipolytica is KAB8284425.1, the Genebank sequence number of Vertebrate CRISP protein of homosapiens is AAI07708.1, the Genebank sequence number of Apolipoprotein E isoform b precursor of homosapiens is NP_001289620.1, and the NPC intracellular cholesterol transporter 1 homolog 1b isoform X4 sequence number of Rosa chinensis is NC_037093.1.

The recombinant saccharomyces cerevisiae strains sc1, emp, sc9-PRY1, sc9-Bac Pry, sc9-VCp, sc9-AEp, sc9-PR1 and sc9-NPCX4 are respectively cultured for 16-24 hours at 30 ℃ and 220rpm to prepare seed liquid, the prepared seed liquid is inoculated into a 500mL conical flask filled with 100mL of fermentation medium and 10mL of dodecane according to the inoculum size of 2% (v/v), and the seed liquid is cultured for 96 hours at 30 ℃ and 220rpm to prepare the fermentation liquid.

Yield of extracellular 7-DHC was calculated:

The results are shown in Table 3 and FIG. 3A.

Yield of intracellular 7-DHC was calculated:

8mL of the resuspended broth was aspirated, washed with deionized water and resuspended, and then broken in boiling water for 5min in a 10mL ep tube with 3mL3N hydrochloric acid. After centrifugation, the supernatant was removed and washed with deionized water. The intracellular sterols were extracted by saponification with 1.5mol/L KOH in methanol at 60℃in a water bath. After the reaction is finished, extracting the liquid by using n-hexane, evaporating the obtained n-hexane on a water bath kettle at 75 ℃, adding a certain amount of ethyl acetate for re-dissolution, and filtering by using a filter membrane to enter a liquid phase bottle. The gas chromatography detection was carried out, and the engineering strain fermentation yield was obtained by conversion with the peak area of the 7-DHC standard, and the results are shown in Table 3 and FIG. 3A.

TABLE 3 overexpression of CAP Transporter family intracellular and extracellular 7-DHC yields from Saccharomyces cerevisiae and OD after completion of the reaction ₆₀₀

Strain	Extracellular 7-DHC (mg/g)	Intracellular 7-DHC (mg/g)	OD₆₀₀
				sc1	0.850	1.086	7.11
sc9	3.376	7.186	8.17
				emp	3.255	6.412	9.07
sc9-PRY1	3.739	5.944	10.65
				sc9-Bac Pry	2.338	4.912	10.84
sc9-VCp	3.064	5.466	11.03
				sc9-AEp	2.378	5.121	9.77
sc9-PR1	4.306	6.539	9.81
				sc9-NPCX4	3.118	5.682	10.16

Among them, recombinant S.cerevisiae strain sc9-PR1 was the highest in yield, and when 500mL shake flask biphasic fermentation was performed using this, the total yield of 7-DHC reached 10.845mg/g (secretion 4.306 mg/g). In this strain, the total secretion and total yield of 7-DHC were increased to 5.59 and 5.07 times, respectively, that of the control sc1 strain by overexpressing PR-1 transporter.

Example 6 modification of Transporter PR-1 molecule and characterization of overexpression of Saccharomyces cerevisiae fermentation

(1) The plasmid pYES2-PR1 is used as a template, the primers (95F/R, 112F/R, 125F/R and 127F/R) in the table 1 are used for obtaining linearized pYES2-PR1 plasmids which are mutated into alanine at the corresponding sites (95, 112, 125 and 127 sites of Pr-1 protein), the linearized pYES2-PR1 plasmids are transferred into Dh5α competence, coated on SOB+Amp plates and cultured at 37 ℃ to obtain site-mutated plasmids, and the 95PR-1, 112PR-1, 125PR-1 and 127PR-1 plasmids.

(2) The plasmid constructed in (1) was introduced into Saccharomyces cerevisiae sc9 competent, cultured on SD-Leu2 plates at 30℃for 4-5 days, and pcr verification was performed using VG 1F, VG R in Table 1 to obtain Saccharomyces cerevisiae strains sc9-95PR-1, sc9-112PR-1, sc9-125PR-1, sc9-127PR-1.

The fermentation specifically comprises the following steps:

(1) The recombinant Saccharomyces cerevisiae strains emp, sc9-PR-1, sc9-95PR-1, sc9-112PR-1, sc9-125PR-1 and sc9-127PR-1 are respectively prepared. Culturing for 16-24 h at 30 ℃ and 220rpm to prepare seed liquid, inoculating the prepared seed liquid into a 500mL conical flask filled with 100mL fermentation medium and 10mL dodecane according to the inoculum size of 2% (v/v), and culturing for 96h at 30 ℃ and 220rpm to prepare fermentation liquor.

(2) Yield of extracellular 7-DHC was calculated:

The results are shown in Table 4 and FIG. 4.

(3) Yield of intracellular 7-DHC was calculated:

8mL of the resuspended broth was aspirated, washed with deionized water and resuspended, and then broken in boiling water for 5min in a 10mL ep tube with 3mL3N hydrochloric acid. After centrifugation, the supernatant was removed and washed with deionized water. The intracellular sterols were extracted by saponification with 1.5mol/L KOH in methanol at 60℃in a water bath. After the reaction is finished, extracting the liquid by using n-hexane, evaporating the obtained n-hexane on a water bath kettle at 75 ℃, adding a certain amount of ethyl acetate for re-dissolution, and filtering by using a filter membrane to enter a liquid phase bottle. The gas chromatography test was carried out, and the fermentation yield of the engineering strain was obtained by conversion with the peak area of the 7-DHC standard, and the results are shown in Table 4 and FIG. 4.

TABLE 4 intracellular and extracellular 7-DHC yields of recombinant Saccharomyces cerevisiae modified with different PR-1 and OD after fermentation ₆₀₀

Strain	Extracellular 7-DHC (mg/g)	Intracellular 7-DHC (mg/g)	OD₆₀₀
				sc9	3.376	7.039	8.17
emp	3.614	7.079	9.07
				sc9-PR-1	5.170	7.572	9.81
sc9-95 PR-1	4.642	7.408	10.69
				sc9-112 PR-1	5.951	9.239	11.03
sc9-125 PR-1	5.746	9.848	10.87
				sc9-127 PR-1	5.605	7.638	10.46

Example 7 enhancement and characterization of the extracellular secretory pathway of 7-DHC

(1) The plasmid pdc is used as a template, the primers CE 6F and CE 6R are used for obtaining linearization plasmids, the linearization plasmids are transferred into Dh5α competence, coated on SOB+Amp plates, and cultured at 37 ℃ to obtain the PAM locus mutation plasmids of gRNA, pdc5-CE6 plasmids.

(2) The Saccharomyces cerevisiae CEN.PK2-1C is used as a template, and a primer E6F, E R is used to obtain a gene fragment ERG6, and a primer VG 1F, VG R is used to obtain an expression frame P _GAL1 -ST1 and P _GAL1 -PR1;

connecting the gene segment ERG6 with a linearization plasmid pMD20, and introducing the gene segment ERG6 into competent Dh5α to obtain a plasmid pMD20-ERG6;

The expression frames P _GAL1 -ST1 and P _GAL1 -PR1 are respectively connected with the linearization plasmid pMD20-ERG6, so that a single knockout over-expression fragment H1-P _GAL1-ST1-H2,H1-P_GAL1 -PR1-H2 can be obtained, and a simultaneous over-expression knockout fragment H1-P _GAL1-ST1- P_GAL1 -PR1-H2 can also be obtained.

(3) The sgrnas pdc5-CE6 and H1-P _GAL1-ST1-H2,H1-P_GAL1-PR1-H2,H1-P_GAL1-ST1- P_GAL1 -PR1-H2 constructed in (1) and (2) were simultaneously introduced into s.cerevisiae sc9 competence, respectively, and cultured on ypd+g418 plates at 30 ℃ for 2-3 days, and pcr validation was performed using E6F, E R, to obtain s.cerevisiae sc10, sc11, sc12, sc13.

The fermentation specifically comprises the following steps:

(1) The recombinant saccharomyces cerevisiae strains sc9, sc10, sc11, sc12 and sc13 are respectively cultured for 16-24 hours at the temperature of 30 ℃ and the rpm of 220 to prepare seed liquid, the prepared seed liquid is inoculated into a 500mL conical flask filled with 100mL of fermentation medium and 10mL of dodecane according to the inoculum size of 2% (v/v), and the seed liquid is cultured for 96 hours at the temperature of 30 ℃ and the rpm of 220 to prepare fermentation liquid.

(2) Yield of extracellular 7-DHC was calculated:

As a result, as shown in Table 5, the sc13 strain, which knocked out ERG6 and overexpressed ST1 and PR-1 transporter, had a 7-DHC content of 11.701mg/g and an OD ₆₀₀ of 7.78. (3) Yield of intracellular 7-DHC was calculated:

8mL of the resuspended broth was aspirated, washed with deionized water and resuspended, and then broken in boiling water for 5min in a 10mL ep tube with 3mL3N hydrochloric acid. After centrifugation, the supernatant was removed and washed with deionized water. The intracellular sterols were extracted by saponification with 1.5mol/L KOH in methanol at 60℃in a water bath. After the reaction is finished, extracting the liquid by using n-hexane, evaporating the obtained n-hexane on a water bath kettle at 75 ℃, adding a certain amount of ethyl acetate for re-dissolution, and filtering by using a filter membrane to enter a liquid phase bottle. The gas chromatography detection was carried out, and the fermentation yield of the engineering strain was obtained by conversion with the peak area of the 7-DHC standard, and the results are shown in Table 5.

TABLE 5 production of extracellular 7-DHC by PR-1 and ST1 substitution of ERG6 Strain and OD after fermentation ₆₀₀

Strain	Extracellular 7-DHC (mg/g)	Intracellular 7-DHC (mg/g)	OD₆₀₀
				sc9	3.870	11.531	5.94
Sc10	5.241	18.784	7.82
				Sc11	9.832	18.214	7.67
Sc12	11.124	16.392	7.89
				Sc13	11.701	16.497	7.78

Wherein, the highest yield is recombinant S.cerevisiae strain sc13, when 500mL shake flask biphasic fermentation is carried out by using the recombinant S.cerevisiae strain sc13, the total yield of 7-DHC reaches 28.198mg/g (secretion 11.701 mg/g), and compared with a control sc1 strain, the total yield of 7-DHC is improved by 14.54 times, the total extracellular secretion is improved by 13.77 times, wherein, the secretion yield of extracellular 7-DHC accounts for 41.51 percent.

Claims

1. A method for constructing a genetically engineered yeast strain for increasing the extracellular secretion yield of 7-DHC, characterized in that the method comprises the following steps:

(1) The C-22 sterol desaturase ERG5 required for ergosterol synthesis was knocked out, and the P _GAP - DHCR24 -T _CYC1 fragment was integrated into the ERG5 site on the genome of Saccharomyces cerevisiae CEN.PK2-1C. The resulting Saccharomyces cerevisiae strain was named sc1; wherein the DHCR24 sequence is shown in SEQ ID No. 1;

(2) The P _GAL2 - ERG1 - T _CYC1 fragment was integrated into the genome of the sc1 strain at the GAL6 site, and the expression of ERG1 was enhanced through the P _GAL2 promoter to construct a Saccharomyces cerevisiae strain named sc2;

(3) The P _GAL1 - tHMG1 - T _CYC1 fragment was integrated into the δ sequence at both ends of Ty1 in the genome of the sc2 strain, and the expression of tHMG1 was enhanced by the P _GAL1 promoter to construct a Saccharomyces cerevisiae strain named sc3 strain; the Gene ID of the tHMG1 is 42650;

(4) The _TEF - _TADH1 - ERG8 - _PGAL10 - _PGAL1 - ERG12 - _TCYC1 fragment was integrated into the GAL80 site on the sc3 strain genome, and the expression of ERG8 and ERG12 was enhanced by the _PGAL1,10 bidirectional promoter to construct a Saccharomyces cerevisiae strain named sc4 strain;

(5) The T _TEF - _PGAL1 - POS5 - T _CYC1 fragment was integrated into the MIG1 site on the sc4 strain genome, and the expression of POS5 was enhanced by the _PGAL1 promoter to construct a Saccharomyces cerevisiae strain named sc5 strain;

(6) The _TEF - _TADH1 - ERG9 - _PGAL10 - _PGAL1 - ERG20 - _TCYC1 fragment was integrated into the DPP1 site on the sc5 strain genome, and the expression of ERG20 and ERG9 was enhanced by the _PGAL1,10 bidirectional promoter to construct a Saccharomyces cerevisiae strain named sc6;

(7) The _TEF - _PGAL1 - ERG11 - _TCYC1 fragment was integrated into the GDH1 site of the sc6 strain genome, and the expression of ERG11 was enhanced by the _PGAL1 promoter to construct a Saccharomyces cerevisiae strain named sc7;

(8) The _TTEF - _TADH1 - IDI1 - _PGAL10 - _PGAL1 - ERG19 - _TCYC1 fragment was integrated into the ADH3 site on the sc7 strain genome, and the expression of IDI1 and ERG19 was enhanced by the _PGAL1,10 bidirectional promoter to construct a Saccharomyces cerevisiae strain named sc8;

(9) The P _GAL1 - DHCR24 - T _CYC1 fragment was integrated into the Ty3 site of the sc8 strain genome, and DHCR24 was heterologously expressed through the P _GAL1 promoter to construct a Saccharomyces cerevisiae strain named sc9;

(10) The expression frames _PGAL1 - ST1 and _PGAL1 - PR1 were connected to the linearized plasmid pMD20- ERG6 to obtain the simultaneously overexpressed knockout fragment H1- _PGAL1 - ST1 - _PGAL1 -PR1-H2, and H1- _PGAL1 - ST1 - _PGAL1 - PR1 - H2 was introduced into the sc9 strain genome to construct a Saccharomyces cerevisiae strain that knocked out ERG6 and overexpressed ST1 and PR1 transporters, which was named sc13 strain, i.e., the genetically engineered Saccharomyces cerevisiae strain that increases the extracellular secretion yield of 7-DHC; the Genebank sequence number of the ST1 is XP_717917.2, and the Genebank sequence number of the PR1 is XP_031058987.1.

2. A genetically engineered yeast strain of Saccharomyces cerevisiae that increases the extracellular secretion yield of 7-DHC, constructed by the method of claim 1.

3. Use of the genetically engineered yeast Saccharomyces cerevisiae according to claim 2 in the production of 7-DHC by microbial fermentation.

4. The use according to claim 3, characterized in that the use comprises: inoculating the genetically engineered yeast Saccharomyces cerevisiae into a fermentation medium, adding n-dodecane at 5-10% of the volume of the initial medium, fermenting at 28-32°C for 48-96 hours, and separating and purifying the fermentation broth to obtain the 7-DHC.