CN116179384A

CN116179384A - Saccharomyces cerevisiae genetically engineered bacterium synthesized by 7-DHC exocytosis and construction and application thereof

Info

Publication number: CN116179384A
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: 2023-05-30

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, and when 500mL shake flask biphasic fermentation is carried out by using the recombinant S.cerevisiae strain sc13, the total yield of 7-DHC reaches 28.189mg/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 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 synthesized by 7-DHC exocytosis and construction and application thereof

Field of the art

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

(II) background art

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. In operation Lisha Qia et al mention ΔGDH1, which 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 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 3g/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, and the Roger Schneiter et al study has 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.

(III) summary of the 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:

a saccharomyces cerevisiae strain synthesized by 7-DHC exocytosis is constructed and obtained by the following method: the genome of the Chaetoceros cerevisiae 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 diphosphate decarboxylase ERG19, and heterologously expressed a 24-sterol reductase DHCR24 from Gallus, a C-22 sterol desaturase ERG5, galactose/lactose metabolism regulator GAL80 which are required for synthesis of ergosterol, and a half acting as an inhibitor in the GAL4 systemCystine proteases GAL6 and MIG1, knocked out NADP ⁺ Specific glutamate dehydrogenase GDH1, knocked out diacylglycerol pyrophosphoric acid phosphatase DPP1, knocked out enzyme ADH3 capable of dehydrogenating alcohols, and heterologously expressed sterol transporters ST1 and PR-1 to obtain the Saccharomyces cerevisiae strain synthesized by 7-DHC exocytosis.

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 is realized by P _GAP Promoter enhanced expression of tHMG1 by P _GAL2 Enhanced expression of ERG1 by the promoter, through P _GAL1 Enhanced expression of ERG11 by the promoter through P _GAL1 Promoter enhanced expression of POS5, through P _GAL1，10 Bidirectional promoters are used for enhanced expression of ERG8 and ERG12, through P _GAL1，10 Bidirectional promoters for enhanced expression of ERG20 and ERG9 by P _GAL1，10 Bidirectional promoter enhanced expression of IDI1 and ERG19 by P _GAP Heterologous expression of DHCR24 from promoter by P _GAL1 The promoters express ST1 and PR-1 heterologous.

More specifically:

through P _GAL2 ERG1 expressed by promoter enhancement is integrated into GAL6 locus on saccharomyces cerevisiae genome, and Gene ID of GAL6 locus is 855482;

through P _GAL1 The ERG11 with enhanced expression of the promoter is integrated into GDH1 locus on the Saccharomyces cerevisiae genome, and the Gene ID of the GDH1 is 854557;

through P _GAL1 POS5 of promoter enhanced expression is integrated to MIG1 locus on Saccharomyces cerevisiae genome, and Gene ID of MIG1 is 852848;

through P _GAL1,10 The bidirectional promoter is used for enhancing the integration of ERG8 and ERG12 into GAL80 sites on the saccharomyces cerevisiae genome, and the Gene ID of GAL80 is 854954;

through P _GAL1,10 The bi-directional promoter enhances the integration of ERG9 and ERG20 into the DPP1 site on the Saccharomyces cerevisiae genome, the Gene ID of DPP1 is 851878;

through P _GAL1,10 The bidirectional promoter enhances the integration of IDI1 and ERG19 into the ADH3 site on the Saccharomyces cerevisiae genome, the Gene ID of ADH3 being 855107;

the Gallus is derived from and passes through P _GAP Heterologous expression of the promoter DHCR24 was integrated into the saccharomyces cerevisiae genome at the ERG5 site, which ERG5 Gene ID 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 the C-22 sterol desaturase ERG5 required for ergosterol synthesis, and carrying out P _GAP -DHCR24-T _CYC1 The fragment is integrated to an ERG5 enzyme site on a Saccharomyces cerevisiae CEN.PK2-1C genome, and the constructed Saccharomyces cerevisiae is named sc1 strain;

(2) Will P _GAL2 -ERG1-T _CYC1 The fragment was integrated into the sc1 strain genome, into the GAL6 site (Gene ID of GAL6 site is 855482), and the Saccharomyces cerevisiae mutant was constructedNamed sc2 strain;

(3) Will P _GAL1 -tHMG-T _CYC1 The fragment is integrated into delta sequences (the guide sequence is TGTTGGAATAGAAATCAACT) at two ends of Ty1 on the genome of the sc2 strain, and the saccharomyces cerevisiae strain named sc3 strain is constructed;

(4) Will T _TEF -T _ADH1 -ERG8-P _GAL10 -P _GAL1 -ERG12-T _CYC1 The fragment is integrated into GAL80 enzyme site (Gene ID of GAL80 is 854954) on the genome of sc3 strain, and Saccharomyces cerevisiae strain named sc4 strain is constructed;

(5) Will T _TEF -P _GAL1 -POS5-T _CYC1 The fragment is integrated into the MIG1 locus (the Gene ID of the MIG1 is 852848) on the genome of the sc4 strain, and a saccharomyces cerevisiae strain named sc5 strain is constructed;

(6) Will T _TEF -T _ADH1 -ERG9-P _GAL10 -P _GAL1 -ERG20-T _CYC1 The fragment is integrated to a DPP1 site (the Gene ID of the DPP1 is 851878) on the genome of the sc5 strain, and a saccharomyces cerevisiae strain named sc6 strain is constructed;

(7) Will T _TEF -P _GAL1 -ERG11-T _CYC1 The fragment is integrated into a GDH1 locus (the Gene ID of the GDH1 is 854557) on the genome of the sc6 strain, and a saccharomyces cerevisiae strain named sc7 strain is constructed;

(8) Will T _TEF -T _ADH1 -IDI1-P _GAL10 -P _GAL1 -ERG19-T _CYC1 The fragment is integrated into the ADH3 locus (the Gene ID of the ADH3 is 855107) on the genome of the sc7 strain, and a saccharomyces cerevisiae strain named sc8 strain is constructed;

(9) Will P _GAL1 -DHCR24-T _CYC1 The 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 sc9 strain, and the Saccharomyces cerevisiae strain named sc13 strain is constructed, namely the 7-DHC exocytosis synthesized Saccharomyces cerevisiae strain. The Genebank sequence number of the sterol transporter sterol transporter (ST 1) is XP_717917.2 the Genebank sequence number of Fusarium odoratissimum NRRL 54006-derived PR-1 is XP_031058987.1.

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

Specifically, the application is as follows: inoculating the saccharomyces cerevisiae strain to a fermentation culture medium, adding 5-10% n-dodecane with the volume of the initial culture medium as an extractant, performing fermentation culture at 28-32 ℃ for 48-96 hours, and separating and purifying fermentation liquor to obtain the 7-DHC.

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

The beneficial effects of the invention are mainly as follows: the invention constructs a recombinant S.cerevisiae strain sc13 capable of efficiently transporting 7-DHC to the outside of cells, and when 500mL shake flask biphasic fermentation is carried out by using the recombinant S.cerevisiae strain sc13, the total yield of 7-DHC reaches 28.189mg/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 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.

(III) description of the drawings

FIG. 1 shows the difference in extracellular sterol distribution in a biphasic fermentation cell and quantitative analysis;

FIG. 2 is a graph showing the promotion of intracellular extracellular production of 7-DHC (mg/g) by overexpression of CAP family transporter (A) and by overexpression of NPC2 family transporter ST (B);

FIG. 3 shows intracellular and extracellular yields (mg/g) of 7-DHC of PR-1 after transformation of the s.cerevisiae sc9 over-expressed molecule;

FIG. 4 is a graph showing the promotion of extracellular production of 7-DHC by synergy of sterol transporters PR-1 and ST.

(IV) detailed description of the invention

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: each liter contains 20g tryptone, 0.5g NaCl, 5g yeast extract, 0.186g KCl, 1g MgCl ₂ 。

SD-Leu2 medium: 20mg uracil, 20mg tryptophan, 20mg histidine, 6.7g yeast nitrogen source without amino group and 20g glucose without water are contained in each liter.

SD-Ura3 Medium: leucine-containing 20mg, tryptophan 20mg, histidine 20mg, yeast nitrogen source without amino 6.7g and glucose without water 20g.

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

SOB+Amp plate: each liter contains 20g tryptone, 0.5g NaCl, 5g yeast extract, 0.186g KCl, 1g MgCl ₂ 20g of agar powder.

Ypd+nat plate: 2% tryptone, 1% yeast extract, 2% anhydrous dextrose, 2% agar powder, 1mL of 20mg/mL NAT-resistant mother liquor per 100 mL.

Ypd+g418 plates: 2% tryptone, 1% yeast extract, 2% anhydrous dextrose, 2% agar powder, 1mL of 20mg/mL of G418 resistant mother liquor per 100 mL.

Detection of 7-DHC content:

centrifuging the fermented bacterial liquid, filtering the upper oil phase by using a filter membrane, and entering a liquid phase bottle; the residual thalli are crushed by 3N hydrochloric acid, and then are centrifuged to collect cell fragments, and 1.5mol/L KOH methanol solution is used for saponification extraction of intracellular sterol on a water bath kettle at 60 ℃. 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 is 0.25mm is 0.25 μm) of the Siemens fly; the gas phase procedure was set as follows: sample inlet temperature: 300 ℃; programming temperature: 190 ℃ (1 min) 10 ℃/min 300 ℃ (10 min); sample injection amount: 1 mu L, a split ratio of 20:1; carrier gas: n (N) ₂ The flow rate is 1.0mL/min; transfer bar temperature: 250 ℃.

Recombinant Saccharomyces cerevisiae OD ₆₀₀ Is characterized by comprising the following steps:

yeast seed solution for 16-24 h is inoculated into 500m L shake flask containing 100m L fermentation medium and 10m L dodecane according to an inoculum size of 1%, and placed at 30 ℃ for 220rpm culture. The OD is measured by an ultraviolet spectrophotometer after dilution according to proper proportion during sampling ₆₀₀ 。

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

/>

Example 1: construction of tool plasmid

The Saccharomyces cerevisiae CEN.PK2-1C genome is used as a template, and PG 2F, PG R is used to obtain a gene fragment P _GAL2 ；

Using pYES2 plasmid as template, using primer LpYES 2F, lpYES R to obtain linearization pYES2 plasmid;

fragment P of Gene _GAL2 Ligation with linearized pYES2 plasmid and introduction into competent Dh5α was verified with primer VG 2F, VG R to give a plasmid with P _GAL1 Substitution of promoter with P _GAL2 P of (2) _GAL2 -pYES2 plasmid.

The pYES2 plasmid is used as a template, and the primers emp F and emp R are used for obtaining linearization plasmid, and the linearization plasmid is transformed intoDh5α competence gives P alone _GAL1 -T _CYC1 Plasmid emp of empty expression cassette.

Example 2: 7-DHC-producing Saccharomyces cerevisiae chassis construction

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 rERG5-HOMO1F, rERG5-HOMO1R to obtain gene fragment ERG5-HOMO1;

obtaining a gene fragment ERG5-HOMO2 by using a primer rERG5-HOMO2F, rERG5-HOMO 2R;

the plasmid pcfb2312 is used as a template, and primers rNAT F and rNAT-G R are used for obtaining a fragment natMX;

p was obtained by using the primer fGAP-D F, rD24-ERG 5R _GAP -DHCR24-T _CYC1 A gene fragment.

(2) Four fragments ERG5-HOMO1, ERG5-HOMO2, natMX, P in step (1) _GAL1 -DHCR24-T _CYC1 Fusion PCR is carried out by adopting PCR, and correct bands obtained by gel running are cut and recovered to obtain fusion gene fragments delta ERG5-natMX-P containing ERG5 upstream and downstream homology arms _GAP -DHCR24-T _CYC1 。

(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 colony with correct band is selected to obtain strain CEN.PK2-1C delta ERG5-P _GAP -DHCR24-T _CYC1 Named Saccharomyces cerevisiae sc1.

Example 3: construction of high-yield 7-DHC Saccharomyces cerevisiae chassis

(1) Construction of sgRNA:

using the plasmid pdc5 as a template, the primers described in table 1 were used, and primer NCG 6R, NCG 6F was used; the linearized pdc5 plasmid was obtained, 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 was obtained with primer LP 2F, LP R _GAL2 -pYES2；

Combining the gene fragment ERG1 with linearization plasmid P _GAL2 The pYES2 is connected and is led into competent Dh5α, colony PCR verification is carried out by using a primer PG 2F, ERG R, and a gene fragment P is obtained by using primers LHP F and LPH 2R _GAL2 -ERG1-T _CYC1 ；

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

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

then the gene fragment P _GAL2 -ERG1-T _CYC1 Ligation with linearized plasmid pMD20-GAL6, PCR verification with primers HPP F, PH2-P R, and amplification with the same primers to give donor H1-P _GAL2 -ERG1-T _CYC1 -H2。

(3) The sgRNA dc5-NCG6 and the donor H1-P constructed in (1) and (2) were subjected to _GAL2 -ERG1-T _CYC1 While introducing H2 into Saccharomyces cerevisiae sc1 competence, culturing on YPD+G418 plate at 30deg.C for 2-3 days, and performing PCR verification by using P-G6F, VG R to obtain 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 on YPD+G418 flat plates at 30 ℃ for 2-3 days, 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) Will P _GAL1 -tHMG-T _CYC1 The fragment is integrated into delta sequences (TGTTGGAATAGAAATCAACT of a guide sequence) 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) Will T _TEF -T _ADH1 -ERG8-P _GAL10 -P _GAL1 -ERG12-T _CYC1 The fragment was integrated into the GAL80 enzyme site on the sc3 strain genome using primer E8E 20F/R (the Gene ID of GAL80 is 854954), and a Saccharomyces cerevisiae strain named sc4 strain was constructed;

(3) Will T _TEF -P _GAL1 -POS5-T _CYC1 The 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) Will T _TEF -T _ADH1 -ERG9-P _GAL10 -P _GAL1 -ERG20-T _CYC1 The fragment is integrated into a DPP1 site (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) Will T _TEF -P _GAL1 -ERG11-T _CYC1 The fragment was integrated into the GDH1 site on the genome of the sc6 strain using primer E11G F/R (the Gene ID of said GDH1 is 854557), and the construction yielded a Saccharomyces cerevisiae strain designated sc7 strain;

(6) Will T _TEF -T _ADH1 -IDI1-P _GAL10 -P _GAL1 -ERG19-T _CYC1 The fragment was integrated into the ADH3 site on the genome of the sc7 strain using primer IDE 19F/R (the Gene ID of the ADH3 is 855107), and a Saccharomyces cerevisiae strain named sc8 strain was constructed;

(7) Will P _GAL1 -DHCR24-T _CYC1 The fragment was integrated into the sc8 strain genome at the Ty3 site (guide sequence ACGTTCATAAAACACATATG) using the primer T3D 24F/R,the constructed Saccharomyces cerevisiae strain was designated sc9 strain.

Example 4: analysis of Saccharomyces cerevisiae sterol cell-out product in oil-water double-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:

the recombinant saccharomyces cerevisiae strains sc1 and sc9 are respectively cultured for 16-24 hours under the condition of being prepared to obtain 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 under the condition of being at 30 ℃ and 220rpm to obtain 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).

Example 5: construction and characterization of saccharomyces cerevisiae transport 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 ST2 from Candida viswanathii is RCK66207.1, the Genebank sequence number of ST3 from Cand ida parapsilosis is XP_036664935.1, the Genebank sequence number of ST4 from Schefferso myces stipitis is KAG2730877.1, and the Genebank sequence number of ST5 from Pachysolen ta nnophilus 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. Resuspension the remaining fermentation broth, sucking the fermentation broth, diluting 10 times, and measuring OD with ultraviolet spectrophotometer ₆₀₀ 。

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

(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 3mL3N hydrochloric acid 10mL ep tube. After centrifugation, the supernatant was removed and washed with deionized water. The intracellular sterol is saponified and extracted by 1.5mol/L KOH methanol solution on a water bath kettle at 60 ℃. 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 Saccharomyces cerevisiae intracellular extracellular 7-DHC yield and OD after fermentation ₆₀₀

Strain	Extracellular 7-DHC (mg/g)	Intracellular 7-DHC (mg/g)	OD ₆₀₀
				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-ST 2	4.675	6.057	10.54
sc9-ST 3	5.273	2.642	9.89
				sc9-ST 4	4.966	2.668	9.37
sc9-ST 5	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), ve rtebrate CRISP protein (Vcp gene sequence see SEQ ID No. 9), apolipoprotein E isofor m 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 1homolog 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 was verified using VG 1F, VG 1R in Table 1 to obtain Saccharomyces cerevisiae chassis SC9-PRY1, SC9-Bac Pry, SC9-VCp, SC9-AEp, SC9-PR1, SC9-NPCX4.

The Genebank sequence number of PR-1 from Fusarium odoratissimum NRRL 54006 is XP_031058987.1, the Genebank sequence number of Bacterial Pry from Yarrowia lipolytica is KAB8284425.1, the Genebank sequence number of Vertebrate CRISP2 protein from homosapiens is AAI07708.1, the Genebank sequence number of Apolipoprotein E isoform b precur sor from homosapiens is NP_001289620.1, and the Genebank sequence number of NPC intracel lular cholesterol transporter 1homolog 1b isoform X4 from 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 an 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:

As shown in Table 3 and FIG. 3, the content of 7-DHC in extracellular dodecane of sc9-PR1 strain overexpressing PR-1 was 17.60mg/L, OD ₆₀₀ Reaching 9.81.

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 sterol is saponified and extracted by 1.5mol/L KOH methanol solution on a water bath kettle at 60 ℃. 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 Saccharomyces cerevisiae intracellular extracellular 7-DHC production and OD after reaction termination ₆₀₀

Strain	Extracellular 7-DHC (mg/g)	Intracellular 7-DHC (mg/g)	OD ₆₀₀
				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 of 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 transport protein PR-1 molecule and fermentation characterization of overexpression saccharomyces cerevisiae

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

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

The fermentation specifically comprises the following steps:

(1) The recombinant Saccharomyces cerevisiae strains emp, sc9-PR-1, sc9-95 PR-1, sc9-112 PR-1, sc9-125 PR-1 and sc9-127 PR-1 are respectively prepared. Culturing at 30 ℃ and 220rpm for 16-24 h 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 at 30 ℃ and 220rpm for 96h to prepare the fermentation liquid.

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

As shown in Table 4 and FIG. 3, the 7-DHC content in extracellular dodecane after mutation of amino acid 112 of PR1 to alanine was 21.92mg/L, OD ₆₀₀ Reaching 11.03.

(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 sterol is saponified and extracted by 1.5mol/L KOH methanol solution on a water bath kettle at 60 ℃. 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. 3.

Table 4: different PR-1 modified recombinant Saccharomyces cerevisiae intracellular and extracellular 7-DHC yield and OD after fermentation ₆₀₀

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

(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) Taking Saccharomyces cerevisiae CEN.PK2-1C as a template, and using a primer E6F, E6R to obtain a gene fragment ERG6; obtaining expression frame P by using primer VG 1F, VG 1R _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;

will express frame P _GAL1 -ST1 and P _GAL1 PR1 is respectively connected with linearization plasmid pMD20-ERG6 to obtain single knockout over-expression fragment H1-P _GAL1 -ST1-H2，H1-P _GAL1 PR1-H2, it is also possible to obtain a simultaneous over-expression of the knockout fragment H1-P _GAL1 -ST1-P _GAL1 -PR1-H2。

(3) The sgRNAs pdc5-CE6 and H1-P constructed in (1) and (2) were combined _GAL1 -ST1-H2，H1-P _GAL1 -PR1-H2，H1-P _GAL1 -ST1-P _GAL1 PR1-H2 was simultaneously introduced into the competence of Saccharomyces cerevisiae sc9, and cultured on YPD+G418 plates at 30℃for 2-3 days, and pcr was performed using E6F, E6R to obtain Saccharomyces 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 to 24 hours at the temperature of 30 ℃ and the speed of 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 the temperature of 30 ℃ and the speed of 220rpm to prepare fermentation liquid.

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

separating the fermentation brothAnd sucking the upper dodecane after the heart, filtering the upper dodecane in a liquid phase sample injection bottle through a filter membrane, performing gas chromatography detection, and converting the peak area of the 7-DHC standard product to obtain the fermentation yield of the engineering strain. Resuspension the remaining fermentation broth, sucking the fermentation broth, diluting 10 times, and measuring OD with ultraviolet spectrophotometer ₆₀₀ 。

As shown in Table 5 and FIG. 4, the sc13 strain with ERG6 knocked out and ST1 and PR-1 transporter overexpressed had a 7-DHC content of 11.701mg/g and OD in extracellular dodecane ₆₀₀ Reaching 7.78. (3) calculating the yield of intracellular 7-DHC:

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 sterol is saponified and extracted by 1.5mol/L KOH methanol solution on a water bath kettle at 60 ℃. 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 5 and FIG. 4.

Table 5: PR-1 and ST1 substitution ERG6 strain intracellular extracellular 7-DHC yield and OD after fermentation ₆₀₀

Strain	Extracellular 7-DHC (mg/g)	Intracellular 7-DHC (mg/g)	OD ₆₀₀
				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 28.189mg/g (secretion 11.701 mg/g), and compared with the 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 saccharomyces cerevisiae gene engineering bacterium for 7-DHC exocytosis synthesis is constructed and obtained by the following method: 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 by genome of Saccharomyces cerevisiaePhosphovalerate kinase ERG8, mevalonate kinase ERG12, farnesyl pyrophosphate synthase ERG20, mevalonate diphosphate decarboxylase ERG19, and heterologously express the galus Gallus-derived 24-sterol reductase DHCR24, knock out the C-22 sterol desaturase ERG5, galactose/lactose metabolism regulating protein GAL80, and cysteine proteases GAL6 and MIG1 acting as inhibitors in GAL4 system, knock out NADP ⁺ Specific glutamate dehydrogenase GDH1, knocked out diacylglycerol pyrophosphoric acid phosphatase DPP1, knocked out enzyme ADH3 capable of dehydrogenating alcohols, and heterologously expressed sterol transport proteins ST1 and PR-1 to obtain the Saccharomyces cerevisiae genetically engineered bacterium for 7-DHC exocytosis synthesis.

2. The saccharomyces cerevisiae genetically engineered bacterium of claim 1 wherein the chassis bacterium is saccharomyces cerevisiae cen.pk2-1C.

3. The saccharomyces cerevisiae genetically engineered bacterium of claim 1 wherein: through P _GAP Promoter enhanced expression of tHMG1 by P _GAL2 Enhanced expression of ERG1 by the promoter, through P _GAL1 Enhanced expression of ERG11 by the promoter through P _GAL1 Promoter enhanced expression of POS5, through P _GAL1，10 Bidirectional promoters are used for enhanced expression of ERG8 and ERG12, through P _GAL1，10 Bidirectional promoters for enhanced expression of ERG20 and ERG9 by P _GAL1，10 Bidirectional promoter enhanced expression of IDI1 and ERG19 by P _GAP Heterologous expression of DHCR24 from promoter by P _GAL1 The promoters express ST1 and PR-1 heterologous.

4. The method for constructing saccharomyces cerevisiae genetically engineered bacteria of claim 1, wherein the method comprises the following steps:

(2) Will P _GAL2 -ERG1-T _CYC1 Integration of fragments intoOn the genome of the sc1 strain, integrating the strain into GAL6 locus to construct and obtain a saccharomyces cerevisiae named sc2 strain;

(3) Will P _GAL1 -tHMG-T _CYC1 The fragment is integrated into delta sequences at two ends of Ty1 on the genome of the sc2 strain, and the saccharomyces cerevisiae strain named sc3 strain is constructed;

(4) Will T _TEF -T _ADH1 -ERG8-P _GAL10 -P _GAL1 -ERG12-T _CYC1 The fragment is integrated to GAL80 enzyme locus on the genome of sc3 strain, and the saccharomyces cerevisiae strain is constructed and obtained and named sc4 strain;

(5) Will T _TEF -P _GAL1 -POS5-T _CYC1 The fragment is integrated to the MIG1 locus on the genome of the sc4 strain, and the saccharomyces cerevisiae strain named sc5 strain is constructed;

(6) Will T _TEF -T _ADH1 -ERG9-P _GAL10 -P _GAL1 -ERG20-T _CYC1 The fragment is integrated to DPP1 locus on the genome of sc5 strain, and Saccharomyces cerevisiae strain named sc6 strain is constructed;

(7) Will T _TEF -P _GAL1 -ERG11-T _CYC1 The fragment is integrated to GDH1 locus on the genome of sc6 strain, and the saccharomyces cerevisiae strain is constructed and obtained and named sc7 strain;

(8) Will T _TEF -T _ADH1 -IDI1-P _GAL10 -P _GAL1 -ERG19-T _CYC1 The fragment is integrated to ADH3 locus on the genome of sc7 strain, and the saccharomyces cerevisiae strain named sc8 strain is constructed;

(9) Will P _GAL1 -DHCR24-T _CYC1 The fragment is integrated to a Ty3 locus on the genome of the sc8 strain, and a saccharomyces cerevisiae strain named sc9 strain is constructed;

(10) H1-P _GAL1 -ST1-P _GAL1 And introducing PR1-H2 into the genome of the sc9 strain, and constructing and obtaining a saccharomyces cerevisiae strain named sc13 strain, namely the saccharomyces cerevisiae gene engineering strain synthesized by the 7-DHC exocytosis.

5. The use of the Saccharomyces cerevisiae genetically engineered bacterium of claim 1 in microbial fermentation to prepare 7-DHC.

6. The application according to claim 5, characterized in that the application is: inoculating the saccharomyces cerevisiae genetic engineering bacteria to a fermentation culture medium, adding n-dodecane accounting for 5-10% of the volume of the initial culture medium as an extracting agent, performing fermentation culture at 28-32 ℃ for 48-96 hours, and separating and purifying fermentation liquor to obtain the 7-DHC.