A genetically engineered strain of Saccharomyces cerevisiae and its application

Abstract

This invention discloses a genetically engineered strain of *Saccharomyces cerevisiae* and its application in cholesterol synthesis. The strain was obtained through multiple genetic engineering steps, starting with *Saccharomyces cerevisiae* SquP1: DHCR24 gene was integrated at the YPL062W site to achieve 7-dehydrocholesterol synthesis; the ERG6 promoter was replaced with the ERG7 promoter and the DWF5 gene was integrated to achieve cholesterol synthesis; the TGL3, TGL4, and ROX1 genes were knocked out to enhance cholesterol accumulation; the FLX1 and ZWF1 genes were integrated to strengthen cofactor supply; the HXK1 promoter was replaced and the ERG11 gene was integrated to optimize the synthesis pathway; and auxotrophic genes were reintroduced to obtain the CC37N strain. This strain achieved a maximum yield of 6.23 g / L in a 5L bioreactor fermentation, providing an excellent strain for efficient cholesterol biosynthesis.

Description

Technical Field

[0001] This invention belongs to the field of microbial biosynthesis technology, specifically relating to a cholesterol-producing genetically engineered strain of Saccharomyces cerevisiae and its applications. Background Technology

[0002] Cholesterol is a key steroid compound widely distributed in animal cell membranes, playing an indispensable role in maintaining the dynamic fluidity and structural stability of cell membranes. Furthermore, as a precursor in biosynthesis, cholesterol plays a central role in the synthesis of key metabolic molecules such as steroid hormones, vitamin D, and bile acids. Given its multiple importance in organisms, cholesterol not only has wide applications in the medical field but is also widely used as an important raw material in various industries, including pharmaceutical excipients, cosmetics, food additives, and nutritional supplements.

[0003] Traditionally, cholesterol production has primarily relied on extraction from animal tissues or chemical synthesis. However, both methods have significant limitations: extracting cholesterol from animal tissues consumes vast amounts of animal resources, leading to high production costs and placing a heavy burden on the environment, while also being constrained by regulations and cultural customs; while chemical synthesis methods typically involve the use of toxic chemicals, posing potential safety hazards, and are complex and costly, making large-scale, efficient production difficult. Faced with these challenges, developing an efficient, economical, and environmentally friendly cholesterol production method has become a key research focus for both academia and industry.

[0004] In recent years, microbial fermentation has been widely used in the production of natural products due to its advantages such as low cost, high efficiency, and sustainability. Among these methods, *Saccharomyces cerevisiae* (Saccharomyces cerevisiae) is a particularly important microbial fermentation method. Saccharomyces cerevisiae As a highly safe and robust industrial microorganism, *Saccharomyces cerevisiae* has become an important industrial biomanufacturing platform due to its ease of cultivation, high transformation efficiency, and convenient genetic manipulation. This strain can synthesize various important metabolites such as ethanol, fatty acids, and steroidal compounds; its genome has been fully analyzed, and it possesses abundant resources for genetic engineering modification. Furthermore, the inherent ergosterol synthesis pathway of *Saccharomyces cerevisiae* further makes it an ideal microbial host for cholesterol synthesis.

[0005] Currently, most research focuses on optimizing the expression levels of key enzymes in the cholesterol synthesis pathway, such as HMG-CoA reductase (HMGR) and sterol reductase (ERG24), in order to increase cholesterol production. However, simply regulating the expression of these enzymes is insufficient for achieving ideal industrial application levels, as their synthesis process is still limited by insufficient energy supply and excessive metabolic diversion. Therefore, systematically optimizing each step of the cholesterol synthesis pathway and enhancing the metabolic flow in the Saccharomyces cerevisiae production process has become a core challenge in current research. Furthermore, Saccharomyces cerevisiae synthesizes and secretes large amounts of byproducts (such as ethanol and fatty acids) during metabolism, and their accumulation may not only inhibit cholesterol synthesis but also lead to carbon source waste and excessive energy consumption. Therefore, how to reduce byproduct generation and optimize carbon flow allocation to improve cholesterol synthesis efficiency is another research challenge that urgently needs to be overcome. In recent years, significant progress has been made in modifying Saccharomyces cerevisiae using genetic engineering techniques to address these challenges: on the one hand, the activity of key enzymes in the cholesterol synthesis pathway can be increased; on the other hand, cholesterol production has been further improved by optimizing metabolic flow, reducing byproduct generation, and enhancing cofactor supply. Nevertheless, achieving large-scale, cost-effective, and efficient cholesterol production still faces a series of technical bottlenecks. In the future, a more systematic and precise genetic engineering strategy is needed to continuously increase cholesterol production, laying the foundation for industrial applications. Summary of the Invention

[0006] The purpose of this invention is to provide a method for constructing a high-cholesterol-producing engineered bacterium and its application, thereby solving the problem of low cholesterol production by brewer's yeast in the prior art.

[0007] To solve the above problems, the present invention adopts the following technical solution: In a first aspect, the present invention provides a genetically engineered strain of *Saccharomyces cerevisiae*, said strain being a *Saccharomyces cerevisiae* strain SquP1 as the starting strain, which integrates P at the YPL062W site. GAL1 For promoter and with T CYC1 The δ(24)-sterol reductase DHCR24 gene P, a terminator, is used for this purpose. GAL1 -DHCR24-T CYC1 The obtained Saccharomyces cerevisiae genetically engineered strain DC1; The NCBI Gene ID of the DHCR24 gene is 424661.

[0008] A second aspect of the present invention provides a genetically engineered strain of *Saccharomyces cerevisiae*, said strain being derived from strain DC1, with P... ERG6 Replace the promoter with P ERG7 The promoter-derived Saccharomyces cerevisiae strain DC4.

[0009] A third aspect of the present invention provides a genetically engineered strain of *Saccharomyces cerevisiae*, said strain being a starting strain DC4, which integrates P at the NEM1 site. GAL1 For promoter and with T CYC1 For the termination child Solanum lycopersium The source of δ(7)-cholesterol reductase DWF5 gene P GAL1 -DWF5-T CYC1 The obtained Saccharomyces cerevisiae genetically engineered strain CC1; The NCBI Gene ID of the DWF5 gene is 101256596.

[0010] In a fourth aspect, the present invention provides a genetically engineered strain of *Saccharomyces cerevisiae*, wherein the strain is *Saccharomyces cerevisiae* strain CC11 obtained by simultaneously knocking out the TGL3 and TGL4 genes, starting from strain CC1; The NCBI ID of the TGL3 gene is 855361; the NCBI Gene ID of the TGL4 gene is 853964.

[0011] In a fifth aspect, the present invention provides a genetically engineered strain of Saccharomyces cerevisiae, wherein the strain is Saccharomyces cerevisiae genetically engineered strain CC16 obtained by knocking out ROX1 from strain CC11 as the starting strain. The NCBI Gene ID of the ROX1 gene is 856178.

[0012] A sixth aspect of the present invention provides a genetically engineered strain of *Saccharomyces cerevisiae*, said strain being a starting strain CC16, which integrates P at the Int14 site. GAL1 For promoter and with T CYC1 The FLX1 gene P is a terminator. GAL1 -FLX1-T CYC1 And integrated at the Int20 site with P GAL1 For promoter and with T CYC1 The ZWF1 gene P is the terminator. GAL10 -ZWF1-T ADH1 The obtained Saccharomyces cerevisiae genetically engineered strain CC32; The NCBI Gene ID of the FLX1 gene is 854672; the NCBI Gene ID of the ZWF1 gene is 855480.

[0013] A seventh aspect of the present invention provides a genetically engineered strain of *Saccharomyces cerevisiae*, said strain being based on strain CC32 as the starting strain, with P... HXK1 Replace the promoter with P ADH2The promoter is integrated at the Int10 site with P GAL1 For promoter and with T CYC1 The ERG11 gene P is the terminator. GAL1 -ERG11-T CYC1 The obtained Saccharomyces cerevisiae genetically engineered strain CC37; The NCBI Gene ID of the ERG11 gene is 856398.

[0014] In an eighth aspect of the present invention, a genetically engineered strain of Saccharomyces cerevisiae is provided, wherein the strain is obtained by adding a auxotrophic gene at the Int9 site to strain CC37 as the starting strain. The auxotrophic gene expression cassette includes: His3 gene expression cassette, specifically P His3 -His3-T ADH1 ; Leu2 gene expression cassette, specifically P Leu2 -Leu2-T CYC1 ; Ura3 gene expression cassette, specifically P Ura3 -Ura3-T PGK1 ; Trp1 gene expression cassette, specifically P Ura3 -Ura3-T PGK1 .

[0015] According to a preferred embodiment of the present invention, the connection sequence of the His3 gene expression cassette and the Leu2 gene expression cassette is T. ADH1 -His3-P His3 -P Leu2 -Leu2-T CYC1 ; The connection sequence of the Ura3 gene expression cassette and the Trp1 gene expression cassette is T. PGK1 -Ura3-P Ura3 -P Trp1 -Trp1-T TPS1 .

[0016] A ninth aspect of the present invention provides the application of the genetically engineered strain of Saccharomyces cerevisiae as described above in the synthesis of cholesterol.

[0017] The beneficial effects of this invention are as follows: (1) This invention uses Saccharomyces cerevisiae SquP1 as the starting strain to obtain expression Gallus gallus δ(24)-sterols from Recombinant Saccharomyces cerevisiae containing the reductase gene DHCR24 synthesized 7-dehydrocholesterol. This was achieved by weakening the expression of the competing pathway gene ERG6. Solanum lycopersium The recombinant Saccharomyces cerevisiae obtained from the δ(7)-cholesterol reductase DWF5 gene achieved cholesterol synthesis.

[0018] (2) This invention further enhances the accumulation of cholesterol in Saccharomyces cerevisiae by knocking out the degradation genes TGL3 and TGL4, which are important components of lipid droplets, and on this basis, knocking out the non-pathway gene ROX1.

[0019] (3) This invention enhances the supply of cofactors in the strain by integrating single copies of FLX1 (increasing FAD supply) and ZWF1 (increasing NADPH supply), thereby increasing cholesterol accumulation again.

[0020] (4) This invention dynamically regulates the rate of ethanol synthesis by replacing the HXK1 (hexokinase) promoter and increases the expression of the rate-limiting gene ERG11 by single-copy integration, thereby improving the ability of Saccharomyces cerevisiae to synthesize cholesterol de novo in vivo.

[0021] (5) Among the high cholesterol-producing strains of Saccharomyces cerevisiae obtained in this invention, strain CC37N has the most outstanding results. When fermented in YPD medium for 120 h in shake flasks, the cholesterol yield was 978.1 mg / L. When fermented in a 5-liter bioreactor, the cholesterol yield reached a maximum of 6.23 g / L. Attached Figure Description

[0022] Figure 1 A schematic diagram of the metabolism of cholesterol biosynthesis in Saccharomyces cerevisiae; Figure 2 The image information of the expression plasmid pESC-GAL1-DHCR24 in Example 1; Figure 3 The results show the detection of cholesterol production by the genetically engineered Saccharomyces cerevisiae CC37N in a 5L fermenter in Example 9. Detailed Implementation

[0023] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the techniques used in the embodiments are conventional practices in the art, or experimental methods recommended by the reagent kit and instrument manufacturers. Unless otherwise specified, the reagents and materials used in the embodiments are commercially available.

[0024] The following description of the sources of experimental materials used in the examples: Saccharomyces cerevisiae CEN.PK2-1C is a commercial strain that can be obtained through regular commercial channels.

[0025] Saccharomyces cerevisiae SquP1: Its construction method is referenced in "De novo biosynthesis of betulinic acid in engineered yeast". Saccharomyces cerevisiae ” (Bioorganic Chemistry, 2024, 152: 107737. 2024-08-22.) PTCL (CAS9) plasmid: This plasmid is preserved in our laboratory, and its nucleotide sequence is shown in SEQ ID NO.1.

[0026] The primers used in the following examples have sequences shown in Tables 1 and 2.

[0027] Table 1. Primer sequences for plasmid construction

[0028] Table 2. Primer sequences for engineered bacteria construction

[0029] Unless otherwise specified, the gene engineering operations involved in the following embodiments are all conventional operations in the art or operations performed in accordance with the relevant product instructions.

[0030] Example 1: Construction of expression cassette plasmid 1.1 Construction of expression plasmid pESC-GAL1-DHCR24 S1: First, design primers for amplifying the target gene DHCR24: P GAL1 -DHCR24-F / T CYC1 -DHCR24-R was amplified using the synthetic gene fragment DHCR24, which was optimized according to the codons of Saccharomyces cerevisiae, as a template. The nucleotide sequence of the codon-optimized DHCR24 is shown in SEQ ID NO.2. The target fragment was recovered by gel electrophoresis.

[0031] S2: Using plasmid pESC-LEU (GenBank: AF063849.1) as a template, the empty vector plasmid backbone fragment was amplified by cas-pGal10-pGal1-R / cas-tCYC1-F loop p, and the plasmid backbone fragment was recovered by gel electrophoresis.

[0032] S3: The target fragment obtained in S1 and the plasmid backbone fragment obtained in S2 were ligated using seamless cloning. The circular ligation system was transformed into *E. coli* DH5α competent cells. The cells were screened for Amp resistance on plates and verified by colony PCR and sequencing to obtain the successfully constructed positive recombinant plasmid pESC-GAL1-DHCR24. Its plasmid map is shown below. Figure 2 As shown.

[0033] 1.2 Construction of guide RNA plasmids The method for constructing guide RNA plasmids, taking PSCM-YPL062W as an example, is as follows: Using primer gRNA-YPL062W-F / R, a PSCM (gRNA) plasmid already present in the laboratory was used as a template. The nucleotide sequence of the PSCM (gRNA) plasmid is shown in SEQ ID NO.3. The linearized gRNA plasmid fragment was amplified, digested with DPNⅠ enzyme, and recombined through seamless cloning. The ligation system was transformed into E. coli DH5α competent cells. After screening with Amp-resistant plates, two single colonies were randomly selected for sequencing verification, and the successfully constructed positive recombinant plasmid PSCM-YPL062W was obtained.

[0034] Example 2: Construction of the genetically engineered Saccharomyces cerevisiae DC1 In this embodiment, a strain of Saccharomyces cerevisiae DC1 capable of synthesizing 7-dehydrocholesterol was constructed.

[0035] 2.1 Construction of engineered bacteria DC1 was obtained by integrating DHCR24 into the YPL062W site using SquP1 (which had been transformed into the PTCL (CAS9) plasmid) as the starting strain.

[0036] The specific operating steps are as follows: (1) The artificial gene fragment δ(24)-sterol reductase gene DHCR24 (NCBI Gene ID 424661) was synthesized. After codon preference optimization using *Saccharomyces cerevisiae* as the expression host, its sequence is shown in SEQ ID NO.2. Primer P was used. GAL1 -DHCR24-F / T CYC1 The gene fragment DHCR24 was obtained by amplification with DHCR24-R. The expression plasmid pESC-GAL1-DHCR24 was constructed as described in section 1.1 of Example 1. The plasmid pESC-GAL1-DHCR24 was then amplified using primers cas-TADH1-F / cas-Tcyc1-R to obtain the target fragment T. ADH1 -P GAL10 -P GAL1 -D HCR24 -T CYC1; (2) Design guide RNA plasmid primers for knocking out the YPL062W site, namely: gRNA-YPL062W-F / R to amplify the linearized PSCM (gRNA)-YPL062W DNA fragment and transform it into E. coli DH5α competent cells; (3) Regarding the knockout of the YPL062W site, the specific steps are as follows: primers YPL062W-UF / UR and YPL062W-DF / DR were designed to amplify the upstream and downstream homologous arms YPL062W-UP / DOWN using genomic CEN.PK2-1C, and the DNA fragments were recovered by gel electrophoresis; YPL062W-UP, YPL062W-DOWN and T were then fused using fusion PCR technology. ADH1 -P GAL10 -P GAL1 -D HCR24 -T CYC1 They were merged into a single complete fragment and named YPL062W-GAL1-DHCR24; (4) The fragment YPL062W-GAL1-DHCR24, together with the target gRNA plasmid, was transformed into the SquP1 engineered strain using the Saccharomyces cerevisiae transformation kit (ZYMO FROZEN-EZ YEASTTRANSFORMATION Ⅱ KIT). Positive transformants were screened using Leu-Ura-auxotrophic plates (yeast SD complete medium without leucine and uracil + 2% (w / v) agar powder). Transformants were confirmed by colony PCR. Validation primers were designed on both sides of the homologous arm and nucleic acid sequencing was used to confirm whether the gene modification was completed. (5) The genetically engineered bacteria with the introduced gene DHCR24 after successful sequencing were streaked on a Leu-deficient solid plate containing 5-FoA (the solid medium consists of yeast SD complete medium without leucine + 1 mg / mL 5-fluoroorotic acid + 2% (w / v) agar powder) to cause the gRNA plasmid to be lost. This strain was named DC1.

[0037] 2.2 YPD shake-flask fermentation Single colonies of the engineered strain DC1 obtained from section 2.1 on the plate were picked and inoculated into 5 mL YPD tubes for overnight culture. The culture was then transferred from the tubes to 250 mL Erlenmeyer flasks containing 15 mL YPD medium and cultured for 14–16 h, controlling the initial OD. 600 The initial OD was 0.2, and then transferred to a 250 mL Erlenmeyer flask containing 50 mL of YPD medium to control the initial OD. 600 The concentration was 0.2, and the culture conditions were 30℃ and 220 rpm. Growth OD was measured after 5 days of culture. 600 And cholesterol production analysis.

[0038] 2.3 Cholesterol Measurement Take 500 μL of fermentation broth and centrifuge at 12000 rpm for 5 min in a 2 ml disruption tube to remove the supernatant. Wash the bacteria twice with distilled water, then weigh 1.5 g of magnetic beads into the disruption tube, add 1 mL of 1.5 M KOH methanol solution, and grind 10 times at 55 Hz for 300 s using a cell disruptor. Place the mixture at 80 ℃ for 3 h, let it stand until it reaches room temperature, add 500 μL of n-heptane, and grind 5 times at 55 Hz for 300 s using a cell disruptor. Then centrifuge at 12000 rpm for 10 min and perform gas chromatography quantitative analysis.

[0039] Cell concentration was determined by spectrophotometry at 600 nm.

[0040] The test results showed that the engineered strain DC1 produced 17.5 mg / L of 7-dehydrocholesterol through YPD shake-flask fermentation.

[0041] Example 3: Construction of engineered bacteria DC4 Starting with the DC1 engineered strain, the promoter of the ERG6 gene was replaced with the promoter of the ERG7 gene to obtain the engineered strain DC4.

[0042] 3.1 Construction of engineered bacteria The specific procedures are the same as those for engineered strain DC1, the difference being the primers used, as detailed below: Using DC1 engineered bacteria as the chassis strain, P ERG6 The upstream homologous arm primers for gene amplification were pErg6-UF and pErg6-UR, and the downstream homologous arm primers were pErg6-DF and pErg6-DR. Both the upstream and downstream homologous arms were obtained from the CEN.PK2-1C genome via PCR amplification. ERG7 The gene amplification primers are pErg7-F and pErg7-R; 3.2 YPD shake-flask fermentation Same as 2.2 in Example 2.

[0043] 3.3 Cholesterol Measurement Same as 2.3 in Example 2.

[0044] The test results showed that the engineered strain DC4 produced 7-dehydrocholesterol at a yield of 71.5 mg / L through YPD shake-flask fermentation.

[0045] Example 4: Construction of the genetically engineered Saccharomyces cerevisiae CC1 Using the engineered strain DC4 obtained in Example 3 as the starting strain, P was integrated at its NEM1 site. GAL1 and T CYC1For promoters and terminators Solanum lycopersium The source of δ(7)-cholesterol reductase DWF5 gene P GAL1 -DWF5-T CYC1 A genetically engineered strain of Saccharomyces cerevisiae, CC1, capable of synthesizing cholesterol, was obtained.

[0046] 4.1 Construction of engineered bacteria The specific procedures are the same as those for engineered strain DC1, the difference being the primers used, as detailed below: Using DC4 engineered bacteria as the chassis strain, the DWF5 gene (NCBI Gene ID 101256596) was inserted at the NEM1 site. The nucleotide sequence of the codon-optimized DWF5 is shown in SEQ ID NO.4. Using CEN.PK2-1C as a template, upstream and downstream homologous arms were amplified using primers NEM1-UF / NEM1-UR and NEM1-DF / NEM1-DR, respectively; a P-type homologous gene was constructed. GAL1 For promoters and T CYC1 The DWF5 gene fragment P is the terminator. GAL1 -DWF5-T CYC1 The method is the same as 2.1 of Example 2; NEM1-UP, NEM1-DOWN and P GAL1 -DWF5-T CYC1 The engineered strain CC1 was obtained by fusing the fragments together and constructing it according to the method in Example 1.

[0047] 4.2 YPD shake-flask fermentation Same as 2.2 in Example 2.

[0048] 4.3 Cholesterol Measurement Same as 2.3 in Example 2.

[0049] The test results showed that the cholesterol production of engineered bacteria CC1 through YPD shake-flask fermentation reached 367.5 mg / L.

[0050] Example 5: Construction of the genetically engineered Saccharomyces cerevisiae CC11 Using the engineered strain CC1 obtained in Example 4 as the starting strain, the engineered strain CC11 was obtained by knocking out the TGL3 and TGL4 genes.

[0051] 5.1 Construction of engineered bacteria The specific procedures are the same as those for engineered strain DC1, the difference being the primers used, as detailed below: Using CC1 engineered strain as the chassis strain, the TGL3 gene was knocked out and the upstream homologous arm amplification primers were TGL3-UF and TGL3-UR, and the downstream homologous arm primers were TGL3-DF and TGL3-DR. The TGL4 gene was knocked out and the upstream homologous arm amplification primers were TGL4-UF and TGL4-UR, and the downstream homologous arm primers were TGL4-DF and TGL4-DR. Both upstream and downstream homologous arms were obtained from the genome of CEN.PK2-1C by PCR amplification. The two genes were knocked out sequentially to obtain the Saccharomyces cerevisiae engineered strain CC11. The NCBI ID of the TGL3 gene is 855361, and the NCBI Gene ID of the TGL4 gene is 853964.

[0052] 5.2 YPD shake-flask fermentation Same as 2.2 in Example 2.

[0053] 5.3 Cholesterol Measurement Same as 2.3 in Example 2.

[0054] The test results showed that the cholesterol production of engineered bacteria CC11 through YPD shake-flask fermentation reached 481.7 mg / L.

[0055] Example 6: Construction of the genetically engineered Saccharomyces cerevisiae CC16 Using the engineered strain CC11 obtained in Example 5 as the starting strain, the ROX1 gene was knocked out to obtain the engineered strain CC16.

[0056] 6.1 Construction of engineered bacteria The specific procedures are the same as those for engineered bacteria CC11, the difference being the primers used, as detailed below: Using CC11 engineered bacteria as the chassis strain, the upstream homologous arm primers for the knockout ROX1 gene amplification were ROX1-UF and ROX1-UR, and the downstream homologous arm primers were ROX1-DF and ROX1-DR. Both the upstream and downstream homologous arms were obtained from the genome of CEN.PK2-1C by PCR amplification; the Gene ID of the ROX1 gene was 856178.

[0057] 6.2 YPD shake-flask fermentation Same as 2.2 in Example 2.

[0058] 6.3 Cholesterol Measurement Same as 2.3 in Example 2.

[0059] The test results showed that the cholesterol production of engineered bacteria CC16 reached 598.2 mg / L through YPD shake-flask fermentation.

[0060] Example 7: Construction of the genetically engineered Saccharomyces cerevisiae CC32 Using the engineered bacteria CC16 obtained in Example 6 as the starting strain, P was integrated at its Int14 site. GAL1 and T CYC1 Following the FLX1 gene as both promoter and terminator, it integrates at its Int20 site with P GAL10 and T ADH1 The ZWF1 gene, which serves as both promoter and terminator, was used to generate the engineered strain CC32.

[0061] 7.1 Construction of engineered bacteria The specific procedures are the same as those for engineered strain DC1, the difference being the primers used, as detailed below: Using CC16 engineered bacteria as the chassis strain, the FLX1 gene was inserted at the Int14 site. Using CEN.PK2-1C as a template, upstream and downstream homologous arms (Int14-UP, Int14-DOWN) were amplified using primers Int14-UF / Int14-UR and Int14-DF / Int14-DR, respectively; a P-type gene was constructed. GAL1 For promoters and T CYC1 The FLX1 gene fragment P is the terminator. GAL1 -FLX1-T CYC1 ; Int14-UP, Int14-DOWN and P GAL1 -FLX1-T CYC1 They were fused into a single, complete fragment. The FLX1 gene has an NCBI Gene ID of 854672.

[0062] After a round of gene knock-in and gRNA plasmid removal, the ZWF1 gene was inserted at the Int20 site. Using CEN.PK2-1C as a template, upstream and downstream homologous arms (Int20-UP, Int20-DOWN) were amplified using primers Int20-UF / Int20-UR and Int20-DF / Int20-DR, respectively; a gene structure was constructed using P... GAL10 For promoters and T ADH1 The ZWF1 gene fragment P is the terminator. GAL10 -ZWF1-T ADH1 The method is the same as 2.1 in Example 2; Int20-UP, Int20-DOWN and P GAL10 -ZWF1-T ADH1 The fragments were fused together to construct an engineered strain, following the method described in Example 1. The NCBI Gene ID of the ZWF1 gene is 855480.

[0063] 7.2 YPD shake-flask fermentation Same as 2.2 in Example 2.

[0064] 7.3 Cholesterol Measurement Same as 2.3 in Example 2.

[0065] The test results showed that the cholesterol production of engineered bacteria CC32 through YPD shake-flask fermentation reached 682.8 mg / L.

[0066] Example 8: Construction of engineered bacteria CC37 Using CC32 engineered bacteria as the starting strain, the promoter of the HXK1 gene was replaced with the promoter of the ADH2 gene, and then integrated at its Int10 site using P. GAL1 and T CYC1 The ERG11 gene, which serves as both a promoter and a terminator, was used to generate the engineered strain CC37. The NCBI Gene ID of the ERG11 gene is 856398.

[0067] 8.1 Construction of engineered bacteria The specific procedure is the same as that for engineered bacteria DC4, the difference being the primers used, as detailed below: Using CC32 engineered bacteria as the chassis strain and CEN.PK2-1C as the template, upstream and downstream homologous arms were amplified using primers pHXK1-UF / pHXK1-UR and pHXK1-DF / pHXK1-DR, respectively. ADH2 The gene amplification primers were pADH2-F and pADH2-R. After one round of gene knock-in and elimination of the gRNA plasmid, the ERG11 gene was inserted at the Int10 site. Using CEN.PK2-1C as a template, upstream and downstream homologous arms were amplified using primers Int10-UF / Int10-UR and Int10-DF / Int10-DR, respectively; a gene structure was constructed using P... GAL1 For promoters and T CYC1 The ERG11 gene fragment P is the terminator. GAL1 -ERG11-T CYC1 The method is the same as 2.1 in Example 2; Int10-UP, Int10-DOWN and P GAL1 -ERG11-T CYC1 The strain was fused into a complete fragment and constructed using the method described in Example 1.

[0068] 8.2 YPD shake-flask fermentation Same as 2.2 in Example 2.

[0069] 8.3 Cholesterol Measurement Same as 2.3 in Example 2.

[0070] The test results showed that the cholesterol production of engineered bacteria CC37 through YPD shake-flask fermentation reached 805.3 mg / L.

[0071] Example 9: Construction of engineered bacteria CC37N 9.1 Construction of engineered bacteria Using the engineered strain CC37 obtained in Example 8 as the starting strain, the missing four amino acid genes HIS3, LEU2, URA3 and TRP1 were restored at the Int9 site.

[0072] The upstream homologous arm of the Int9 site was obtained by PCR amplification using CNE.PK2-1C as a template and primers Int9-UF / Int9-UR; the downstream homologous arm was obtained by PCR amplification using CNE.PK2-1C as a template and primers Int9-DF / Int9-DR; the first expression cassette P His3 -His3-T ADH1 P Leu2 -Leu2-T CYC1 The connection order is T ADH1 -His3-P His3 -P Leu2 -Leu2-T CYC1 The second expression cassette, P, was amplified from cas-TADH1-F / tPGK1-tCYC1-R using the existing laboratory plasmid PZT20 (see Chinese invention patent application 202111145425.8). Ura3 -Ura3-T PGK1 P Trp1 -Trp1-T TPS1 The connection order is T PGK1 -Ura3-P Ura3 -P Trp1 -Trp1-T TPS1 It was obtained by amplification of tCYC1-tPGK1-F / Int9-DR using the laboratory-existing plasmid PZT110 (see Chinese invention patent application 202111145425.8).

[0073] 9.2 Shake-flask fermentation Optimized YPD medium: 10 g / L yeast extract, 20 g / L peptone, 40 g / L glucose (YPD solid medium, with an additional 20 g / L agar powder added). First, a single yeast colony growing on a YPD plate is picked and inoculated into a 5 mL YPD tube and cultured overnight (14-16 h) at 30°C and 220 rpm / min. Then, the culture is transferred from the tube to a 250 mL Erlenmeyer flask containing 15 mL of YPD medium and cultured for 14-16 h, controlling the initial OD. 600 The initial OD was 0.2, then transferred to a 250 mL Erlenmeyer flask containing 50 mL of YPD medium to control the initial OD. 600 The concentration was 0.2, and the mixture was incubated at 30℃ and 220rpm for 5 days.

[0074] 9.3 Cholesterol Detection Methods For cholesterol extraction, 500 μL of fermentation broth was centrifuged at 12000 rpm for 5 min in a 2 ml disruption tube to remove the supernatant. After washing the bacteria twice with distilled water, 1.5 g of magnetic beads were weighed into the disruption tube, and 1 mL of 1.5 M KOH methanol solution was added. The mixture was then ground 10 times at 55 Hz for 300 s using a cryogenic grinder (Shanghai Jingxin, China), and placed at 80 ℃ for 3 h. After standing to room temperature, 500 μL of n-heptane was added, and the mixture was ground 5 times at 55 Hz for 300 s using a cryogenic grinder (Shanghai Jingxin, China). The mixture was then centrifuged at 12000 rpm for 10 min, and then subjected to gas chromatography quantitative analysis.

[0075] A gas chromatography system (Agilent 8860, USA) equipped with an HP-5 column (30 m × 0.32 mm, 0.25 μm film thickness) and a flame ionization detector (FID). The injection temperature was 300 °C, the detector temperature was 310 °C, the carrier gas was nitrogen, the flow rate was 2 ml / min, the initial temperature was 260 °C held for 5 min, then increased to 300 °C at a rate of 10 °C / min and held for 5 min for time-dependent qualitative analysis. Cholesterol was quantified using the external standard method. All results are reported as the mean of three representatives. Statistical analysis was performed using Student's t-test (one-tailed; two-sample unequal variances; *p < 0.05, **p < 0.01, ***p < 0.001). All data represent the mean of at least three independent samples, and error bars indicate standard deviation.

[0076] 9.4 Cholesterol Test Results like Figure 3 As shown in the figure, among the engineered bacteria that produced high cholesterol, strain CC37N showed the most outstanding results, achieving a cholesterol yield of up to 6.23 g / L after fermentation in a 5-liter bioreactor.

[0077] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the invention. Various variations can be made to the above embodiments of the present invention. All simple and equivalent changes and modifications made in accordance with the claims and description of this application fall within the protection scope of the claims of this patent. All aspects not described in detail in this invention are conventional technical content.

Claims

1. A genetically engineered strain of *Saccharomyces cerevisiae*, characterized in that, The strain described is based on the Saccharomyces cerevisiae strain SquP1, which integrates at the YPL062W site with P... GAL1 For promoter and with T CYC1 The δ(24)-sterol reductase DHCR24 gene P, a terminator, is used for this purpose. GAL1 -DHCR24-T CYC1 The obtained Saccharomyces cerevisiae genetically engineered strain DC1; The NCBI Gene ID of the DHCR24 gene is 424661.

2. A genetically engineered strain of *Saccharomyces cerevisiae*, characterized in that, The strain is based on strain DC1 as described in claim 1, with P... ERG6 Replace the promoter with P ERG7 The promoter-derived Saccharomyces cerevisiae strain DC4.

3. A genetically engineered strain of *Saccharomyces cerevisiae*, characterized in that, The strain is based on strain DC4 as described in claim 2, and integrates at the NEM1 site using P... GAL1 For promoter and with T CYC1 For the termination child Solanum lycopersium The source of δ(7)-cholesterol reductase DWF5 gene P GAL1 -DWF5-T CYC1 The obtained Saccharomyces cerevisiae genetically engineered strain CC1; The NCBIGene ID of the DWF5 gene is 101256596.

4. A genetically engineered strain of *Saccharomyces cerevisiae*, characterized in that, The strain is a genetically engineered Saccharomyces cerevisiae strain CC11 obtained by simultaneously knocking out the TGL3 and TGL4 genes, using the strain CC1 as the starting strain according to claim 3. The NCBI ID of the TGL3 gene is 855361. The NCBIGene ID of the TGL4 gene is 853964.

5. A genetically engineered strain of *Saccharomyces cerevisiae*, characterized in that, The strain is CC16, a genetically engineered Saccharomyces cerevisiae strain obtained by knocking out ROX1, starting with strain CC11 as the strain described in claim 4. The NCBI Gene ID of the ROX1 gene is 856178.

6. A genetically engineered strain of *Saccharomyces cerevisiae*, characterized in that, The strain is based on strain CC16 as described in claim 5, and integrates P at the Int14 site. GAL1 For promoter and with T CYC1 The FLX1 gene P is a terminator. GAL1 -FLX1-T CYC1 And integrated at the Int20 site with P GAL1 For promoter and with T CYC1 The ZWF1 gene P is the terminator. GAL10 -ZWF1-T ADH1 The obtained Saccharomyces cerevisiae genetically engineered strain CC32; The NCBI Gene ID of the FLX1 gene is 854672. The NCBIGene ID of the ZWF1 gene is 855480.

7. A genetically engineered strain of *Saccharomyces cerevisiae*, characterized in that, The strain is based on the strain CC32 described in claim 6 as the starting strain, with P HXK1 Replace the promoter with P ADH2 The promoter is integrated at the Int10 site with P GAL1 For promoter and with T CYC1 The ERG11 gene P is the terminator. GAL1 -ERG11-T CYC1 The obtained Saccharomyces cerevisiae genetically engineered strain CC37; The NCBIGene ID of the ERG11 gene is 856398.

8. A genetically engineered strain of *Saccharomyces cerevisiae*, characterized in that, The strain is the Saccharomyces cerevisiae genetically engineered strain CC37N obtained by using the strain CC37 described in claim 7 as the starting strain and adding the auxotrophic gene at the Int9 site. The auxotrophic gene expression cassette includes: His3 gene expression cassette, specifically P His3 -His3-T ADH1 ; Leu2 gene expression cassette, specifically P Leu2 -Leu2-T CYC1 ; Ura3 gene expression cassette, specifically P Ura3 -Ura3-T PGK1 ; Trp1 gene expression cassette, specifically P Ura3 -Ura3-T PGK1 .

9. The Saccharomyces cerevisiae genetically engineered strain according to claim 8, characterized in that, The connection sequence of the His3 gene expression cassette and the Leu2 gene expression cassette is T. ADH1 -His3-P His3 -P Leu2 -Leu2-T CYC1 ; The connection sequence of the Ura3 gene expression cassette and the Trp1 gene expression cassette is T. PGK1 -Ura3-P Ura3 -P Trp1 -Trp1-T TPS1 .

10. The use of a genetically engineered strain of *Saccharomyces cerevisiae* as described in any one of claims 1 to 9 in the synthesis of cholesterol.

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