Saccharomyces cerevisiae engineering bacteria for producing cholesterol as well as construction method and application thereof

Abstract

The invention belongs to the technical field of genetic engineering and bioengineering, and particularly relates to a saccharomyces cerevisiae engineering bacterium for producing cholesterol as well as a construction method and application of the saccharomyces cerevisiae engineering bacterium. According to the saccharomyces cerevisiae engineering bacteria, a promoter of an ERG6 gene of wild-type saccharomyces cerevisiae is replaced by a promoter ERG7p, genes GgDHCR24 and StDWF5 are inserted into an X-3 site, genes tHMG1 and IDI1 are inserted into an X-4 site, genes ERG20 and ERG9 are inserted into an XII-4 site, and a gene INO2 is inserted into an XII-5 site. The yeast endogenous gene is knocked out or weakened in the saccharomyces cerevisiae, key enzymes of sterol C-24 reductase and sterol C-7 reductase for synthesizing cholesterol are optimized, precursor supply is improved, an endoplasmic reticulum size regulation factor INO2 is overexpressed, the yield of cholesterol is increased, and the effect of obtaining 60.63 mg / L cholesterol through fermentation for 72 h in a shake flask is achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering and bioengineering, and specifically relates to an engineering yeast for producing cholesterol, a construction method and an application thereof. Background Art

[0002] Cholesterol, also known as cholesterol, is a cyclopentane polyhydrophenanthrene derivative and an important component of animal cell membranes. In the field of medicine, since cholesterol has a natural steroidal nucleus skeleton, its side chain degradation can be used to prepare a variety of steroidal drug intermediates, such as pregnenolone, androst-4-ene-3,17-dione, diosgenin, etc. Steroidal drugs have important clinical effects in anti-inflammatory, contraceptive, immunomodulation, endocrine disorders and senile diseases. In the production process of mRNA vaccines, cholesterol is one of the key excipients of lipid nanoparticles (LNP). In the field of materials, cholesterol and its derivatives can also be used to prepare flexible liquid crystal materials, which have the advantage of extremely low power consumption. They have been widely used in high-end electronic paper and radio frequency tags, and are also one of the main technologies used in e-book displays. In the field of feed, cholesterol is used as an additive to animal feed, especially for shrimp feed. At present, most cholesterol on the market is extracted from animal brain tissue, spinal cord tissue and lanolin, but this extraction method has the risk of spreading diseases (such as mad cow disease and Streptococcus suis), limited raw material sources, complex extraction process, serious process pollution and other disadvantages, which cannot meet the needs of green and sustainable development. The reported chemical synthesis method of cholesterol has problems such as complex process flow, high pollution, and expensive catalysts, which is not suitable for industrial production.

[0003] In recent years, synthetic biology technology has developed rapidly, providing new ideas for the green production of high-value-added chemicals by designing and constructing efficient cell factories. As an important microbial chassis cell, Saccharomyces cerevisiae is increasingly widely used in the fields of medicine, energy, food, agriculture, etc. Therefore, constructing a Saccharomyces cerevisiae cell factory to heterologously synthesize cholesterol instead of traditional chemical extraction and chemical synthesis can achieve the goals of less pollution, high efficiency, no restrictions on raw materials, and reduced production costs, providing a safe method for the industrial production of cholesterol. In 2019, Daewong Pharmaceutical Co., Ltd. of South Korea announced a method for synthesizing cholesterol in Saccharomyces cerevisiae (recombinant yeast strains with sterol production ability, preparation methods and uses thereof [P]. Gyeonggi-do: CN201980063312.8, 2024-04-12.), which produced about 30 mg / L of cholesterol after 5 days of fermentation in YPD medium. However, the current output of this method is still a certain distance from the level of industrialization, so how to further increase the output of cholesterol by means of metabolic regulation is an important problem that researchers in this field need to solve. Summary of the invention

[0004] In order to increase the production of cholesterol and meet the needs of green and sustainable development, the present invention increases the production of cholesterol by knocking out or weakening endogenous yeast genes, optimizing the key enzymes for synthesizing cholesterol, sterol C-24 reductase and sterol C-7 reductase, increasing precursor supply and expanding organelles anchored by key genes, laying a solid research foundation for the green biomanufacturing and industrial application of cholesterol.

[0005] The present invention specifically adopts the following technical solutions:

[0006] The present invention first provides an engineered yeast for producing cholesterol, wherein the promoter of the ERG6 gene of the wild-type yeast is replaced by the promoter ERG7p, the genes GgDHCR24 and StDWF5 are inserted into the X-3 site, the genes tHMG1 and IDI1 are inserted into the X-4 site, the genes ERG20 and ERG9 are inserted into the XII-4 site, and the gene INO2 is inserted into the XII-5 site. The sequence of the promoter of the ERG6 gene is shown in SEQ ID NO:1, the sequence of the promoter ERG7p is shown in SEQ ID NO:2, the sequence of the gene GgDHCR24 is shown in SEQ ID NO:3, the sequence of the gene StDWF5 is shown in SEQ ID NO:4, the sequence of the gene tHMG1 is shown in SEQ ID NO:5, the sequence of the gene IDI1 is shown in SEQ ID NO:6, the sequence of the gene ERG20 is shown in SEQ ID NO:7, the sequence of the gene ERG9 is shown in SEQ ID NO:8, and the sequence of the gene INO2 is shown in SEQ ID NO:9.

[0007] In a specific embodiment, the starting strain of the engineered Saccharomyces cerevisiae bacteria is a wild-type Saccharomyces cerevisiae CEN.PK2-1C strain.

[0008] The present invention also provides a method for constructing the engineered yeast Saccharomyces cerevisiae, comprising the following steps:

[0009] (1) Using CRISPR-Cas9 technology, the promoter of the ERG6 gene of wild-type Saccharomyces cerevisiae was replaced with the promoter ERG7p to obtain recombinant bacteria 1.

[0010] (2) CRISPR-Cas9 technology was used to integrate the cholesterol synthesis genes GgDHCR24 and StDWF5 into the X-3 site of recombinant bacteria 1 to obtain recombinant bacteria 2.

[0011] (3) Using CRISPR-Cas9 technology, the genes tHMG1 and IDI1 were integrated into the X-4 site of recombinant strain 2 to obtain recombinant strain 3;

[0012] (4) Using CRISPR-Cas9 technology, genes ERG20 and ERG9 were integrated into the XII-4 site of recombinant strain 3 to obtain recombinant strain 4;

[0013] (5) The CRISPR-Cas9 technology was used to integrate the gene INO2 into the XII-5 site of the recombinant strain 4 to obtain the recombinant strain 5, which is an engineered Saccharomyces cerevisiae strain that produces cholesterol.

[0014] The present invention constructs a cholesterol synthesis pathway by expressing sterol C-24 reductase and sterol C-7 reductase with high catalytic activity; the present invention replaces the promoter of the ERG6 gene with the promoter of the ERG7 gene to reduce the synthesis of ergosterol in the competitive pathway; the present invention expands the area of ​​the endoplasmic reticulum by overexpressing the INO2 gene, improves the synthesis and folding ability of the endoplasmic reticulum protein, and thus increases the cholesterol production.

[0015] As a specific implementation, step (1) includes:

[0016] The promoter ERG7p was used as a DNA donor and a plasmid containing the Cas9 protein gene and gRNA (pCas-Promoter-ERG6, used to cut the promoter region of ERG6 in the wild-type Saccharomyces cerevisiae and allow the DNA donor to be inserted) was transferred into the wild-type Saccharomyces cerevisiae to obtain recombinant bacteria 1.

[0017] The promoter ERG7p can be obtained by the following method: using the wild-type Saccharomyces cerevisiae genome as a template, PCR amplification is performed to obtain the gene fragment of the promoter ERG7p (using Perg7-ERG6-donor-F and Perg7-ERG6-donor-R as primers). The preparation method of the plasmid pCas-Promoter-ERG6 is as follows: using the plasmid pST.URA as a template, PCR amplification is performed to obtain a fragment of the gRNA gene containing the ERG7p promoter (using Perg6-URA-1F and Perg6-URA-2R as primers). Then, the Golden-Gate reaction is used to insert the fragment of the gRNA gene containing the ERG7p promoter into the plasmid pCas9 to obtain the plasmid pCas-Promoter-ERG6.

[0018] As a specific implementation, step (2) includes:

[0019] The GgDHCR24-StDWF5 gene fragment was used as a DNA donor and a plasmid containing the Cas9 protein gene and gRNA (pCas-X-3, used to cut the X-3 site in the recombinant bacteria 1 and then allow the DNA donor to insert) was transferred into the recombinant bacteria 1 to obtain the recombinant bacteria 2.

[0020] The GgDHCR24-StDWF5 gene fragment can be obtained by the following method: using plasmid pSU-GgDHCR24-StDWF5 as a template, PCR amplification is performed to obtain the GgDHCR24-StDWF5 gene fragment (using X-3-donor-F and X-3-donor-R as primers). The preparation method of the plasmid pSU-GgDHCR24-StDWF5 is as follows: inserting the gene StDWF5 between PGK1p and ADH1t of the plasmid pSU, and inserting the gene GgDHCR24 between TEF1p and CYC1t of the plasmid pSU.

[0021] As a specific implementation, step (3) includes:

[0022] The tHMG1-IDI1 gene fragment was used as a DNA donor and a plasmid containing the Cas9 protein gene and gRNA (pCas-X-4, used to cut the X-4 site in the recombinant bacteria 2 and then allow the DNA donor to insert) was transferred into the recombinant bacteria 2 to obtain the recombinant bacteria 3.

[0023] The tHMG1-IDI1 gene fragment can be obtained by the following method: using plasmid pSU-tHMG1-IDI1 as a template, PCR amplification is performed to obtain the tHMG1-IDI1 gene fragment (using X-4-donor-F and X-4-donor-R as primers). The preparation method of the plasmid pSU-tHMG1-IDI1 is as follows: using the Saccharomyces cerevisiae genome as a template, PCR amplification is performed to obtain a fragment of the tHMG1 gene (using tHMG1-F and tHMG1-R as primers) and a fragment of the IDI1 gene (using IDI1-F and IDI1-R as primers). The tHMG1 gene fragment is inserted between the EcoRI and SacI restriction sites of the pSU vector, and the IDI1 gene fragment is inserted between the BamHI and NheI restriction sites of the pSU vector to obtain the plasmid pSU-tHMG1-IDI1.

[0024] As a specific implementation, step (4) includes:

[0025] The ERG20-ERG9 gene fragment was used as a DNA donor and a plasmid containing the Cas9 protein gene and gRNA (pCas-XII-4, used to cut the XII-4 site of recombinant bacteria 3 and then allow the DNA donor to insert) was transferred into recombinant bacteria 3 to obtain recombinant bacteria 4.

[0026] The ERG20-ERG9 gene fragment can be obtained by the following method: using plasmid pSU-ERG20-ERG9 as a template, PCR amplification is performed to obtain the ERG20-ERG9 gene fragment (using XII-4-donor-F and XII-4-donor-R as primers). The preparation method of the plasmid pSU-ERG20-ERG9 is as follows: using the Saccharomyces cerevisiae genome as a template, PCR amplification is performed to obtain the ERG20 gene fragment (using ERG20-F and ERG20-R as primers) and the ERG9 gene fragment (using ERG9-F and ERG9-R as primers). The ERG20 gene fragment is inserted between the EcoRI and SacI restriction sites of the pSU vector, and the ERG9 gene fragment is inserted between the BamHI and NheI restriction sites of the pSU vector to obtain the plasmid pSU-ERG20-ERG9.

[0027] As a specific implementation, step (5) includes:

[0028] The INO2 gene fragment was used as a DNA donor and a plasmid containing the Cas9 protein gene and gRNA (pCas-XII-5, used to cut the XII-5 site of recombinant bacteria 4 and then allow the DNA donor to insert) was transferred into recombinant bacteria 4 to obtain recombinant bacteria 5.

[0029] The INO2 gene fragment can be obtained by the following method: using plasmid pSU-INO2 as a template, PCR amplification to obtain the INO2 gene fragment (using XII-5-donor-F and XII-5-donor-R as primers). The preparation method of the plasmid pSU-INO2 is as follows: using the genome of Saccharomyces cerevisiae as a template, PCR amplification to obtain a fragment of the INO2 gene (using INO2-F and INO2-R as primers) and inserting the INO2 gene fragment between the EcoRI and SacI restriction sites of the pSU vector to obtain the plasmid pSU-INO2.

[0030] The present invention also provides the use of the engineered yeast Saccharomyces cerevisiae in the production of cholesterol. As an embodiment, wild-type Saccharomyces cerevisiae CEN.PK2-1C was used as the starting strain to prepare the engineered yeast Saccharomyces cerevisiae, and a fermentation test of the engineered yeast Saccharomyces cerevisiae was conducted, and it was found that 60.63 mg / L cholesterol was obtained after fermentation for 72 hours in a shake flask.

[0031] The beneficial effects of the present invention are:

[0032] The present invention improves the cholesterol production by knocking out or weakening yeast endogenous genes in cerevisiae, optimizing the key enzymes sterol C-24 reductase and sterol C-7 reductase for cholesterol synthesis, increasing the supply of precursors and overexpressing the endoplasmic reticulum size regulator INO2, achieving the effect of obtaining 60.63 mg / L of cholesterol after 72 hours of fermentation in a shake flask, laying a solid research foundation for the green biomanufacturing and industrial application of cholesterol. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Schematic diagram of the plasmid pCas-Promoter-ERG5.

[0034] Figure 2 Schematic diagram of the plasmid pCas-Promoter-ERG6.

[0035] Figure 3 Schematic diagram of the plasmid pCas-ERG5.

[0036] Figure 4 Schematic diagram of the plasmid pCas-ERG6 map. Figure 1 , 2 The plasmids shown in , 3, and 4 have the same size structure framework, with only the 20 bp gRNA being different.

[0037] Figure 5 Schematic diagram of the plasmid pSU-tHMG1-IDI1.

[0038] Figure 6 Schematic diagram of the plasmid pSU-ERG20-ERG9.

[0039] Figure 7 Schematic diagram of the plasmid pSU-INO2.

[0040] Figure 8 Schematic diagram of the cholesterol synthesis pathway in Saccharomyces cerevisiae.

[0041] Fig. 9 Cholesterol production in Saccharomyces cerevisiae under different conditions.

[0042] Fig.10 To strengthen the premise supply and expand the endoplasmic reticulum area on cholesterol production. DETAILED DESCRIPTION

[0043] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution in the embodiment of the present invention will be clearly described below in conjunction with the drawings in the embodiment of the present invention. Obviously, the described embodiment is a part of the embodiment of the present invention, not all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0044] Example 1

[0045] 1. Experimental Materials and Methods

[0046] 1.1 Strains, plasmids and primers

[0047] The starting strain used in this example is Saccharomyces cerevisiae CEN.PK2-1C, which is used to construct an engineered strain of Saccharomyces cerevisiae that produces cholesterol. The strains involved in this example are shown in Table 1, the plasmids involved are shown in Table 2, and the primers used in the construction of the plasmids are shown in Table 3. The genes used were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and the primers used were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0048] Table 1. Strains involved in this example

[0049]

[0050]

[0051] Table 2. Plasmids involved in this example

[0052]

[0053] Table 3. Primers used in this example

[0054]

[0055]

[0056]

[0057] 1.2 Culture medium and culture conditions

[0058] YPD medium (1% yeast extract, 2% peptone, 2% anhydrous glucose) was used for activation and culture of Saccharomyces cerevisiae strains, and LBA medium (1% tryptone, 0.5% yeast extract, 1% sodium chloride, ampicillin 100 μg / mL) was used for culture of Escherichia coli. The medium was sterilized by high pressure steam at 121°C for 20 min and stored at room temperature.

[0059] The transformation, strain screening and plate streaking in the yeast strain construction process used SC-URA nutrient-deficient medium with glucose as the carbon source, the culture temperature of Saccharomyces cerevisiae was 30°C, and the culture temperature of Escherichia coli was 37°C.

[0060] 1.3 Plasmid construction

[0061] (1) Construction of plasmids pCas-Promoter-ERG5 and pCas-Promoter-ERG6: Using plasmid pST.URA as a template, Perg5-URA-1F and Perg5-URA-2R, Perg6-URA-1F and Perg6-URA-2R as primers, PCR amplification was performed, and two gRNA gene fragments containing the ERG7p promoter were obtained after gel recovery. Then, the two gRNA gene fragments containing the ERG7p promoter were inserted into the BsaI restriction site of the plasmid pCas9 by Golden-Gate reaction, and then E. coli transformation and plasmid extraction were performed to obtain plasmid pCas-Promoter-ERG5 (the schematic diagram of the plasmid map is shown in FIG. Figure 1 ) and pCas-Promoter-ERG6 (plasmid map shown in Figure 2 The correct plasmid was used for the subsequent construction of integration strains.

[0062] (2) Construction of plasmids pCas-ERG5 and pCas-ERG6: Using plasmid pST.URA as a template, ERG5-URA-1F and ERG5-URA-2R, ERG6-URA-1F and ERG6-URA-2R as primers, PCR amplification was performed, and two fragments containing gRNA genes of ERG5 and ERG6 were obtained after gel recovery. Then, the two fragments containing gRNA genes of ERG5 and ERG6 were inserted into the BsaI restriction site of plasmid pCas9 by Golden-Gate reaction, and then E. coli transformation and plasmid extraction were performed to obtain plasmid pCas-ERG5 (the schematic diagram of the plasmid map is shown in FIG. Figure 3 ) and pCas-ERG6 (the plasmid map is shown in Figure 4 The correct plasmid was used for the subsequent construction of integration strains.

[0063] (3) Construction of plasmid pSU-tHMG1-IDI1: Using the genome of Saccharomyces cerevisiae CEN.PK2-1C as a template, PCR amplification was performed with tHMG1-F and tHMG1-R as primers, and the fragment of the tHMG1 gene was obtained after gel recovery. Then the plasmid pSU was double-digested with EcoRI and SacI, gel recovered, and the pSU vector digested fragment was obtained. Finally, the tHMG1 gene fragment and the pSU vector digested fragment were homologously ligated, E. coli transformed, plasmid extracted, and sequenced to obtain the plasmid pSU-tHMG1. Next, using the genome of Saccharomyces cerevisiae CEN.PK2-1C as a template, PCR amplification was performed with IDI1-F and IDI1-R as primers, and the fragment of the IDI1 gene was obtained after gel recovery. Then the plasmid pSU-tHMG1 was double-digested with BamHI and NheI, gel recovered, and the pSU-tHMG1 vector digested fragment was obtained. Finally, the IDI1 gene fragment and the pSU-tHMG1 vector restriction fragment were homologously connected, E. coli was transformed, and the plasmid was extracted to obtain the plasmid pSU-tHMG1-IDI1 (the plasmid map is shown in the figure). Figure 5 The correct plasmid was used for the subsequent construction of integration strains.

[0064] (4) Construction of plasmid pSU-ERG20-ERG9: Using the genome of Saccharomyces cerevisiae CEN.PK2-1C as a template, PCR amplification was performed with ERG20-F and ERG20-R as primers, and the ERG20 gene fragment was obtained after gel recovery. Then the plasmid pSU was double-digested with EcoRI and SacI, and gel recovery was performed to obtain the pSU vector digestion fragment. Finally, the ERG20 gene fragment and the pSU vector digestion fragment were homologously ligated, E. coli was transformed, the plasmid was extracted, and sequencing was performed to obtain the plasmid pSU-ERG20. Next, using the genome of Saccharomyces cerevisiae CEN.PK2-1C as a template, PCR amplification was performed with ERG9-F and ERG9-R as primers, and the ERG9 gene fragment was obtained after gel recovery. Then the plasmid pSU-ERG20 was double-digested with BamHI and NheI, and gel recovery was performed to obtain the pSU-ERG20 vector digestion fragment. Finally, the ERG9 gene fragment and the pSU-ERG20 vector restriction fragment were homologously connected, transformed into E. coli, and the plasmid was extracted to obtain the plasmid pSU-ERG20-ERG9 (the plasmid map is shown in the figure). Figure 6 The correct plasmid was used for the subsequent construction of integration strains.

[0065] (5) Construction of plasmid pSU-INO2: Using the genome of Saccharomyces cerevisiae CEN.PK2-1C as a template and primers INO2-F and INO2-R for PCR amplification, the INO2 gene fragment was obtained after gel recovery. Then the plasmid pSU was double-digested with EcoRI and SacI, gel recovered, and the pSU vector enzyme fragment was obtained. Finally, the INO2 gene fragment and the pSU vector enzyme fragment were homologously ligated, E. coli was transformed, and the plasmid was extracted to obtain the plasmid pSU-INO2 (the schematic diagram of the plasmid map is shown in FIG. Figure 7 The correct plasmid was used for the subsequent construction of integration strains.

[0066] 1.4 Construction of Saccharomyces cerevisiae strains

[0067] (1) Construction of strain CEN.PK2-1C-Werg5: First, the CEN.PK2-1C genome was used as a template and Perg7-ERG5-donor-F and Perg7-ERG5-donor-R were used as primers for PCR amplification. After gel recovery, the gene fragment of the promoter ERG7p was obtained as a DNA donor. Then, the plasmid pCas-Promoter-ERG5 containing the Cas9 protein gene and gRNA and the DNA donor were chemically transformed into Saccharomyces cerevisiae CEN.PK2-1C. The primers Perg7-ERG5-CK-F and Perg7-ERG5-CK-R were used to verify whether the transformant was correct. After the plasmid was lost, the strain CEN.PK2-1C-Werg5 was obtained.

[0068] (2) Construction of strain CEN.PK2-1C-Werg6: First, the CEN.PK2-1C genome was used as a template and Perg7-ERG6-donor-F and Perg7-ERG6-donor-R were used as primers for PCR amplification. After gel recovery, the gene fragment of the promoter ERG7p was obtained as a DNA donor. Then, the plasmid pCas-Promoter-ERG6 containing the Cas9 protein gene and gRNA and the DNA donor were transferred into the cerevisiae CEN.PK2-1C by chemical transformation. The primers Perg7-ERG6-CK-F and Perg7-ERG6-CK-R were used to verify whether the transformant was correct. After the plasmid was lost, the strain CEN.PK2-1C-Werg6 was obtained.

[0069] (3) Construction of strain CH-1: First, plasmid pSU-GgDHCR24-StDWF5 was used as a template (GeneBank of gene GgDHCR24: NP_001026459.1; GeneBank of StDWF5: XP_006362628.1), and EGR5-donor-F and EGR5-donor-R were used as primers for PCR amplification. After gel recovery, the GgDHCR24-StDWF5 gene fragment (containing PGK1p-StDWF5-ADH1t-TEF1p-GgDHCR24-CYC1t) was obtained as a DNA donor. Then, the plasmid pCas-ERG5 containing the Cas9 protein gene and gRNA and the DNA donor were chemically transformed into Saccharomyces cerevisiae CEN.PK2-1C, and the primers ERG5-CK-F and ERG5-CK-R were used to verify whether the transformant was correct. After the plasmid was lost, strain CH-1 was obtained.

[0070] (4) Construction of strain CH-2: First, PCR amplification was performed using plasmid pSU-GgDHCR24-StDWF5 as a template and EGR6-donor-F and EGR6-donor-R as primers. The GgDHCR24-StDWF5 gene fragment was obtained after gel recovery and used as a DNA donor. Then, the plasmid pCas-ERG6 containing the Cas9 protein gene and gRNA and the DNA donor were chemically transformed into Saccharomyces cerevisiae CEN.PK2-1C. Primers ERG6-CK-F and ERG6-CK-R were used to verify whether the transformant was correct. After the plasmid was lost, strain CH-2 was obtained.

[0071] (5) Construction of strain CH-3: First, PCR amplification was performed using plasmid pSU-GgDHCR24-StDWF5 as a template and primers X-3-donor-F and X-3-donor-R. The GgDHCR24-StDWF5 gene fragment was obtained after gel recovery and used as a DNA donor. Then, the plasmid pCas-X-3 containing the Cas9 protein gene and gRNA and the DNA donor were chemically transformed into Saccharomyces cerevisiae CEN.PK2-1C. Primers X-3-CK-F and X-3-CK-R were used to verify whether the transformant was correct. After the plasmid was lost, strain CH-3 was obtained.

[0072] (6) Construction of strain CH-4: First, PCR amplification was performed using plasmid pSU-GgDHCR24-StDWF5 as a template and primers X-3-donor-F and X-3-donor-R. After gel recovery, the GgDHCR24-StDWF5 gene fragment was obtained as a DNA donor. Then, the plasmid pCas-X-3 containing the Cas9 protein gene and gRNA and the DNA donor were chemically transformed into Saccharomyces cerevisiae CEN.PK2-1C-Werg5. Primers X-3-CK-F and X-3-CK-R were used to verify whether the transformant was correct. After losing the plasmid, strain CH-4 was obtained.

[0073] (7) Construction of strain CH-5: First, PCR amplification was performed using plasmid pSU-GgDHCR24-StDWF5 as a template and primers X-3-donor-F and X-3-donor-R. After gel recovery, the GgDHCR24-StDWF5 gene fragment was obtained as a DNA donor. Then, the plasmid pCas-X-3 containing the Cas9 protein gene and gRNA and the DNA donor were chemically transformed into Saccharomyces cerevisiae CEN.PK2-1C-Werg6. Primers X-3-CK-F and X-3-CK-R were used to verify whether the transformant was correct. After the plasmid was lost, strain CH-5 was obtained.

[0074] (8) Construction of strain CH-6: First, PCR amplification was performed using plasmid pSU-tHMG1-IDI1 as a template and primers X-4-donor-F and X-4-donor-R. After gel recovery, the tHMG1-IDI1 gene fragment was obtained as a DNA donor. Then, the plasmid pCas-X-4 containing the Cas9 protein gene and gRNA and the DNA donor were chemically transformed into Saccharomyces cerevisiae CH-5. Primers X-4-CK-F and X-4-CK-R were used to verify whether the transformant was correct. After the plasmid was lost, strain CH-6 was obtained.

[0075] (9) Construction of strain CH-7: First, plasmid pSU-ERG20-ERG9 was used as a template and primers XII-4-donor-F and XII-4-donor-R were used for PCR amplification. After gel recovery, the ERG20-ERG9 gene fragment was obtained as a DNA donor. Then, the plasmid pCas-XII-4 containing the Cas9 protein gene and gRNA and the DNA donor were transferred into Saccharomyces cerevisiae CH-6 by chemical transformation. Primers XII-4-CK-F and XII-4-CK-R were used to verify whether the transformant was correct. After the plasmid was lost, strain CH-7 was obtained.

[0076] (10) Construction of strain CH-8: First, plasmid pSU-INO2 was used as a template and primers XII-5-donor-F and XII-5-donor-R were used for PCR amplification. After gel recovery, the INO2 gene fragment was obtained as a DNA donor. Then, the plasmid pCas-XII-5 containing the Cas9 protein gene and gRNA and the DNA donor were transferred into Saccharomyces cerevisiae CH-7 by chemical transformation. Primers XII-5-CK-F and XII-5-CK-R were used to verify whether the transformant was correct. After the plasmid was lost, strain CH-8 was obtained.

[0077] 1.5 Chemical transformation of Saccharomyces cerevisiae

[0078] In this embodiment, the method for transferring the plasmid into Saccharomyces cerevisiae adopts the lithium acetate / salmon sperm / polyethylene glycol method (LiOAc / ssDNA / PEG) reported in the literature (Dong G, Zhao Y, Ding W, et al. Metabolic engineering of Saccharomyces cerevisiae for de novo production of odd-numbered medium-chain fatty acids [J]. Metabolic Engineering. 2024, 82: 100-109.), and the transformation system is shown in Table 4.

[0079] Table 4. Saccharomyces cerevisiae transformation system

[0080] Reagents Dosage 1M Lithium Acetate (LiOAc) 36μL 2mg / mL single-stranded DNA (ssDNA) 50μL 50% Polyethylene glycol (PEG3350) 240μL Plasmids 300-500ng <![CDATA[Supplementary ddH 2 O to]]> 360μL

[0081] 1.6 Saccharomyces cerevisiae fermentation culture

[0082] Pick the single colony with the correct transformation on the plate, streak it on the corresponding solid plate, and place it in a 30℃ constant temperature incubator for enrichment culture; then inoculate it into 3mL of YPD medium and culture it overnight in a shaker at 220rpm and 30℃; the next day, according to the initial OD 600 The ratio of 0.2 was transferred to a 100 mL shake flask containing YPD medium and the culture was carried out in a shaker at 220 rpm and 30°C for 72 hours. Three replicates were set for each sample, and the fermentation broth was collected after 72 hours to determine the cholesterol content in the fermentation broth.

[0083] 1.7 Cholesterol detection method

[0084] Take 2 mL of fermentation broth, centrifuge at 12000g for 3 min, discard the supernatant, and wash twice with deionized water; then add an appropriate amount of acidic glass beads and oscillate on a vortex oscillator for 30 min; then add 3M NaOH-methanol solution and saponify in a water bath at 80°C for 1 h; after saponification, add 2 mL of n-hexane, oscillate on a vortex oscillator for 10 min, and then centrifuge at 12000g for 1 min; transfer the upper n-hexane phase to a new EP tube and evaporate to dryness in a vacuum concentrator; add 200 μL of silanization derivatization reagent, mix well, and place in a 60°C oven for reaction for 1 h; after the reaction is completed, add n-hexane to dilute the derivatized sample 10 times, take an appropriate amount into a gas phase injection vial, and use GC-MS (GC-MS QP2020, Shimadzu, Japan) for analysis and detection.

[0085] The detection conditions were as follows: DB-5MS column (30m×0.25mm×0.25μm, Agilent), EI source bombardment energy of 70eV, helium carrier gas, flow rate of 3.0mL / min, inlet temperature of 280℃, ion source temperature of 250℃, sample injection volume of 1μL, split ratio of 10:1, and temperature program: column temperature was maintained at 70℃ for 2min, increased to 250℃ at a rate of 20℃ / min, maintained at 250℃ for 2min, increased to 280℃ at a rate of 10℃ / min, and maintained at 280℃ for 15min.

[0086] 2. Results and Discussion

[0087] 2.1 Construction of the de novo cholesterol synthesis pathway

[0088] In order to achieve de novo cholesterol synthesis in Saccharomyces cerevisiae, it is necessary to express the key enzymes sterol C-24 reductase and sterol C-7 reductase. The synthesis pathway is as follows Figure 8As shown. Therefore, this embodiment selected the chicken-derived C-24 reductase GgDHCR24 and the potato-derived C-7 reductase StDWF5 to be expressed in the Saccharomyces cerevisiae CEN.PK2-1C strain. Since the yeast endogenous ergosterol synthesis pathway is a competitive pathway for synthesizing cholesterol, in order to reduce the impact of the competitive pathway, it is necessary to knock out or weaken the key genes ERG5 or ERG6 for synthesizing ergosterol. By the CRISPR-Cas9 method (reference Zhang Y, Wang J, Wang Z, et al. A gRNA-tRNA array for CRISPR-Cas9based rapid multiplexed genome editing in Saccharomyces cerevisiae [J]. Nature Communications, 2019, 10 (1): 1053.), the cholesterol synthesis genes GgDHCR24 and StDWF5 were integrated into the ERG5 or ERG6 sites of the CEN.PK2-1C strain to obtain strains CH-1 and CH-2. At the same time, in order to investigate the effects of knocking out or weakening the ergosterol synthesis pathway on yeast strain growth and cholesterol production, the promoters of the ERG5 and ERG6 genes were replaced with the weak promoter ERG7p to obtain strains CEN.PK2-1C-Werg5 and CEN.PK2-1C-Werg6, respectively. Then, the cholesterol synthesis genes GgDHCR24 and StDWF5 were integrated into the X-3 site of strains CEN.PK2-1C, CEN.PK2-1C-Werg5 or CEN.PK2-1C-Werg6 to obtain strains CH-3, CH-4 and CH-5.

[0089] The five strains (CH-1, CH-2, CH-3, CH-4, and CH-5) were fermented in YPD liquid medium, and the 72-hour fermentation broth was pretreated with derivatization. Finally, the cholesterol content in the samples was analyzed and detected by GC-MS. The test results are as follows: Fig. 9 As shown. Fig. 9 It can be seen that cholesterol was detected in all strains. Compared with strain CH-3, the cholesterol content in strains CH-1 and CH-2 was increased to a certain extent, indicating that knocking out the key genes ERG5 or ERG6 for synthesizing ergosterol is beneficial to the synthesis of cholesterol. The cholesterol content in strains CH-4 and CH-5 was further improved, and the cholesterol content in strain CH-5 reached 12.83 mg / L, indicating that weakening the expression of the key genes ERG5 or ERG6 for synthesizing ergosterol is more conducive to the synthesis of cholesterol and does not affect the growth of the strain. Therefore, strain CH-5 was selected for further optimization and improvement.

[0090] 2.2 Effect of enhanced precursor supply on cholesterol production

[0091] In the mevalonate pathway, tHmg1p and Idi1p are two key rate-limiting enzymes. Therefore, in order to increase the production of cholesterol, it is necessary to adjust the expression of key enzymes and genes in the mevalonate pathway to promote the maximum metabolic flow to the target product. We integrated the genes tHMG1 and IDI1 into the X-4 site of strain CH-5 to obtain strain CH-6. In addition, ERG20 and ERG9 are key genes for the synthesis of cholesterol precursor squalene. We integrated the above two genes into the XII-4 site of strain CH-6 to obtain strain CH-7. Strains CH-6 and CH-7 were then fermented in YPD liquid medium, and the 72h fermentation broth was taken for derivatization pretreatment. Finally, the cholesterol content in the sample was analyzed and detected by GC-MS. The test results are shown as follows. Fig.10 As shown. Fig.10 It can be seen that the cholesterol content in strains CH-6 and CH-7 has been significantly increased, reaching 38.41 mg / L and 51.86 mg / L, respectively, which is 1.99 times and 3.04 times higher than that of strain CH-5, respectively. The above results show that the production of cholesterol in Saccharomyces cerevisiae can be effectively increased by overexpressing key genes in the mevalonate pathway.

[0092] 2.3 Effect of expanding the endoplasmic reticulum area on cholesterol production

[0093] In the metabolic pathway for synthesizing cholesterol, the enzymes after the mevalonate pathway are all located in the endoplasmic reticulum. Therefore, the size of the endoplasmic reticulum space area has an important influence on the correct folding and expression of related proteases. INO2 is a regulatory gene for yeast phospholipid biosynthesis, which can effectively expand the surface area of ​​the endoplasmic reticulum, thereby enhancing the synthesis and folding ability of endoplasmic reticulum proteins. In order to further increase the production of cholesterol, we integrated the INO2 gene into the XII-5 site of strain CH-7 to obtain strain CH-8. Strain CH-8 was then fermented in YPD liquid medium, and the 72h fermentation broth was taken for derivatization pretreatment. Finally, the cholesterol content in the sample was analyzed and detected by GC-MS. The test results are as follows Fig.10 As shown. Fig.10 It can be seen that the cholesterol content in strain CH-8 was further increased to 60.63 mg / L, which was 3.72 times higher than that in strain CH-5. Therefore, increasing the area of ​​the endoplasmic reticulum by overexpressing the INO2 gene is an effective strategy to increase the cholesterol production in Saccharomyces cerevisiae.

[0094] The present invention uses multiple optimization strategies to increase the cholesterol production in brewer's yeast, and the obtained strain CH-8 can be used for subsequent further optimization and improvement, while also providing a solid research foundation for the biosynthesis and industrial production of cholesterol and its derivatives.

[0095] References:

[0096] [1]Xu S, Qiao W, Wang Z, et al. Exploiting a heterologous construction of the3-hydroxypropionic acid carbon fixation pathway with mesaconate as anindicator in Saccharomyces cerevisiae[J]. Bioresources and Bioprocessing, 2023,10:33.

[0097] [2] Zhang Y, Wang J, Wang Z, et al. AgRNA-tRNAarray for CRISPR-Cas9basedrapid multiplexed genome editing in Saccharomyces cerevisiae[J]. Nature Communications, 2019, 10(1):1053.

[0098] [3]Meng J, Qiu Y, Zhang Y, et al. CMI: CRISPR / Cas9based efficient multiplexed integration in Saccharomyces cerevisiae[J]. ACS synthetic biology, 2023, 12(5): 1408-1414.

[0099] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An engineered yeast strain for producing cholesterol, characterized in that: The engineered yeast strain of Saccharomyces cerevisiae is ERG6 The promoter of the gene was replaced with the promoter ERG7p, GgDHCR24 and StDWF5 Insertion into X-3 site, gene tHMG1 and IDI1 Insertion into X-4 site, gene ERG20 and ERG9 Insertion into XII-4 site, gene INO2 Insertion into XII-5 site; Said ERG6 The sequence of the promoter of the gene is shown in SEQ ID NO: 1, the sequence of the promoter ERG7p is shown in SEQ ID NO: 2, and the gene GgDHCR24 The sequence of gene is shown in SEQ ID NO:

3. StDWF5 The sequence of gene is shown in SEQ ID NO:

4. tHMG1 The sequence of gene is shown in SEQ ID NO:

5. IDI1 The sequence of gene is shown in SEQ ID NO:

6. ERG20 The sequence of gene is shown in SEQ ID NO:

7. ERG9 The sequence of gene is shown in SEQ ID NO:

8. INO2 The sequence is shown in SEQ ID NO:

9.

2. The cholesterol-producing Saccharomyces cerevisiae engineered bacteria according to claim 1, characterized in that: The starting strain of the engineered yeast Saccharomyces cerevisiae is the wild-type Saccharomyces cerevisiae CEN.PK2-1C strain.

3. The method for constructing the engineered yeast of Saccharomyces cerevisiae according to claim 1, characterized in that: The following steps are involved: (1) Using CRISPR-Cas9 technology, wild-type Saccharomyces cerevisiae ERG6 The promoter of the gene was replaced with promoter ERG7p to obtain recombinant bacteria 1; (2) Using CRISPR-Cas9 technology to clone the cholesterol synthesis gene GgDHCR24 and StDWF5 Integrated into the X-3 site of recombinant bacteria 1 to obtain recombinant bacteria 2; (3) Using CRISPR-Cas9 technology to modify genes tHMG1 and IDI1 It was integrated into the X-4 site of recombinant strain 2 to obtain recombinant strain 3; (4) Using CRISPR-Cas9 technology to modify genes ERG20 and ERG9 It was integrated into the XII-4 site of recombinant strain 3 to obtain recombinant strain 4; (5) Using CRISPR-Cas9 technology to modify genes INO2 The recombinant strain 4 was integrated at the XII-5 site to obtain the recombinant strain 5, which is an engineered Saccharomyces cerevisiae strain producing cholesterol.

4. The construction method according to claim 3, characterized in that: Step (1) includes: Using the wild-type Saccharomyces cerevisiae genome as a template, the promoter ERG7p fragment was obtained by PCR amplification, and it was used as a DNA donor and a plasmid containing the Cas9 protein gene and gRNA to be transferred into the wild-type Saccharomyces cerevisiae to obtain recombinant bacteria 1; The plasmid containing the Cas9 protein gene and gRNA is used to cut the promoter region of ERG6 in wild-type Saccharomyces cerevisiae.

5. The construction method according to claim 3, characterized in that: Step (2) includes: The GgDHCR24-StDWF5 gene fragment was used as a DNA donor and the plasmid containing the Cas9 protein gene and gRNA was transferred into the recombinant bacteria 1 to obtain the recombinant bacteria 2; The plasmid 2 containing the Cas9 protein gene and gRNA is used to cut the X-3 site in the recombinant bacteria 1.

6. The construction method according to claim 3, characterized in that: Step (3) includes: The tHMG1-IDI1 gene fragment was used as a DNA donor and the plasmid containing the Cas9 protein gene and gRNA was transferred into the recombinant bacteria 2 to obtain the recombinant bacteria 3; The plasmid three containing the Cas9 protein gene and gRNA is used to cut the X-4 site in the recombinant bacteria 2.

7. The construction method according to claim 3, characterized in that: Step (4) includes: The ERG20-ERG9 gene fragment was used as a DNA donor and plasmid 4 containing the Cas9 protein gene and gRNA was transferred into the recombinant bacteria 3 to obtain the recombinant bacteria 4; The plasmid four containing the Cas9 protein gene and gRNA is used to cut the XII-4 site in the recombinant bacteria 3.

8. The construction method according to claim 3, characterized in that: Step (5) includes: Will INO2 The gene fragment is used as a DNA donor and the plasmid 5 containing the Cas9 protein gene and gRNA is transferred into the recombinant bacteria 4 to obtain the recombinant bacteria 5; The plasmid 5 containing the Cas9 protein gene and gRNA is used to cut the XII-5 site in the recombinant bacteria 4.

9. Use of the engineered yeast Saccharomyces cerevisiae as claimed in claim 1 in producing cholesterol.

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