Saccharomyces cerevisiae genetic engineering strain with high squalene yield and application thereof

Abstract

The invention discloses a saccharomyces cerevisiae genetic engineering strain with high squalene yield and application thereof. According to the invention, an engineering strain SquM13 is taken as a starting strain, a plurality of modification strategies associated with organelles are tried, a plurality of saccharomyces cerevisiae genetic engineering strains SY04, SY12, SY19, SY39, SY49 and SY51 are screened from the engineering strain SquM13, the result of the strain SY51 is most prominent, and the yield of squalene fermented by a 5-liter bioreactor is up to 54.62 g / L. According to the saccharomyces cerevisiae genetic engineering strain with high squalene yield and the application of the saccharomyces cerevisiae genetic engineering strain, the yield of squalene is obviously improved, and large-scale squalene industrialization becomes possible.

Description

Technical Field

[0001] The invention relates to the field of biosynthesis applications, and more specifically to a genetically engineered strain of Saccharomyces cerevisiae with high squalene production and application thereof. Background Art

[0002] Squalene, also known as 30 carbon hexaene, has a chemical formula of C 30 H 50 It is a polyunsaturated hydrocarbon produced in the metabolic process of cholesterol synthesis in the human body. It contains 6 isoprene double bonds and belongs to terpenoid compounds. It is a colorless or slightly yellow clear oily liquid at room temperature. It is insoluble in water, difficult to dissolve in methanol, ethanol and glacial acetic acid, and easily soluble in organic solvents such as ether, petroleum ether, acetone, and carbon tetrachloride. The relative density is 0.8592, the refractive index is 1.4954, the freezing point is -75℃, and the boiling point is 429.3℃. Squalene has the characteristics of high biological activity, strong antioxidant ability, good stability, good permeability, wide sources, multiple physiological functions and high safety. Therefore, it is widely used in many fields such as cosmetics, food additives, and medicine.

[0003] The traditional method of producing squalene is mainly through extraction from deep-sea shark livers, which have a high content of squalene. The average content of squalene in some shark liver oils is about 40% or more of the total content, and can even reach up to 89%. However, due to the scarcity of deep-sea shark resources and animal protection reasons, the method of extracting squalene from shark livers is unsustainable. Although many vegetable oils contain a certain amount of squalene, the content is usually low and difficult to meet the needs of industrial applications. In contrast, the use of microorganisms to synthesize squalene has become a more economical and sustainable alternative, making large-scale squalene industrialization possible.

[0004] Among eukaryotic organisms, Saccharomyces cerevisiae, as a food-grade microorganism, has a long history of industrial application, and its fermentation process and technology are very mature and stable. At present, organelle compartmentalization strategies, including mitochondria and peroxisomes, have been widely used in the microbial cell production of squalene. As the power plant of cells and the main site of TCA cycle, mitochondria can provide 20-30 times more acetyl-CoA precursors than cytoplasm to synthesize terpenoids. However, with the increase of squalene products, the morphology and function of mitochondria are seriously adversely affected, resulting in the inhibition of the overall energy metabolism of cells. Peroxisome compartmentalization concentrates the accumulation of squalene inside peroxisomes, reduces the toxicity of squalene's physiological activity to cells, and expands the storage space of squalene. However, the storage space of peroxisomes is limited. Therefore, with the increase of products, it is necessary to explore new storage space. Summary of the invention

[0005] The purpose of the present invention is to provide a high-yield engineered strain for producing squalene and application thereof, so as to solve the problem of low yield of squalene produced by fermenting saccharomyces cerevisiae in the prior art.

[0006] In order to solve the above problems, the present invention adopts the following technical solutions:

[0007] According to a first aspect of the present invention, a genetically engineered yeast strain of Saccharomyces cerevisiae with high squalene production is provided, including: a genetically engineered yeast strain SY04 obtained by taking the engineering strain SquM13 as a starting strain and knocking out the PAH1 and OPI1 genes by regulating its lipid synthesis, wherein the NCBI accession number of the gene PAH1 is: NC_001145.3, and the NCBI accession number of the gene OPI1 is: NC_001140.6; a genetically engineered yeast strain SY12 obtained by taking the engineering strain SquM13 as a starting strain and knocking out the gene HRD1; wherein the NCBI accession number of the gene HRD1 is: NC_001147.6; and a genetically engineered yeast strain SY19 obtained by taking the engineering strain SquM13 as a starting strain and knocking out the UTH1 and ECM33 genes; wherein the NCBI accession number of the gene UTH1 is: NC_001143.9, and the NCBI accession number of the gene ECM33 is: NC_001134.8.

[0008] Preferably, the genetically engineered Saccharomyces cerevisiae bacteria with high squalene production provided by the present invention also includes: genetically engineered Saccharomyces cerevisiae bacteria SY39 obtained by knocking out the CIT2, HRD1, UTH1 and ECM33 genes using the genetically engineered Saccharomyces cerevisiae bacteria SY04 as the starting strain; wherein the NCBI accession number of the gene CIT2 is: NC_001135.5.

[0009] Preferably, the high-squalene-producing Saccharomyces cerevisiae genetically engineered bacteria provided by the present invention further comprises: using the Saccharomyces cerevisiae genetically engineered bacteria SY39 as the starting strain, knocking out the OCA5 and LYS1 genes at the same time, and integrating P GAL1 and T CYC1 ZWF1 gene P GAL1 -ZWF1-T CYC1 The obtained genetically engineered yeast SY49 has the NCBI accession number of gene OCA5: NC_001140.6; the NCBI accession number of gene LYS1: NC_001141.2; and the NCBI accession number of gene ZWF1: NC_001146.8.

[0010] Preferably, the squalene-producing Saccharomyces cerevisiae genetically engineered bacteria provided by the present invention further include: a Saccharomyces cerevisiae genetically engineered bacteria SY51 obtained by using the Saccharomyces cerevisiae gene SY49 as a starting strain and complementing the auxotrophic genes at the Int2 site, including Leu2, His3, Ura3 and Trp1, wherein the gene expression cassette is specifically: P His3 -His3-T ADH1 , P Leu2 -Leu2-T CYC1 , P Ura3 -Ura3-T PGK1 , P TRP1 -Trp1-T TPS1 .

[0011] Preferably, the first gene 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 gene expression cassette P Ura3 -Ura3-T PGK1 , P TRP1 -Trp1-T TPS1 The connection order is T PGK1 -Ura3-P Ura3 -P TRP1 -Trp1-T TPS1 .

[0012] According to a second aspect of the present invention, there is provided a use of the genetically engineered yeast Saccharomyces cerevisiae with high squalene production as described above in the production of squalene.

[0013] According to a preferred embodiment of the present invention, a single colony of genetically engineered yeast Saccharomyces cerevisiae on a plate was picked up, inoculated into a conical flask containing 5 mL of YPD medium, and cultured at 30°C, 220 rpm / min for 14 to 16 h, and then transferred to a conical flask containing 15 mL of YPD medium to control the initial OD 600 The fermentation seeds were inoculated into fresh YPD medium at an initial OD 600 The reaction mixture was cultured at 30°C and 220 rpm / min for 6 to 8 days.

[0014] The metabolic diagram of squalene biosynthesis in Saccharomyces cerevisiae is shown in Figure 2. Figure 1As shown. According to the present invention, we tried to alleviate the intracellular oxidative stress by regulating the size of the endoplasmic reticulum, and explored the possibility of storing squalene in lipid droplets to allow squalene to be overproduced. Since squalene accumulates on the cell membrane of microorganisms, as the output increases, the stress capacity on the cell wall gradually increases. When the cell wall is damaged, a large amount of cytoplasmic contents will leak, thereby limiting the production of squalene. Therefore, the present invention chooses to enhance the strength of the cell wall to increase the cell's tolerance to squalene. In addition, the present invention also reshapes the metabolic physiology of the engineered strain, including glycolysis and cofactor circulation.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] 1) The present invention uses the existing "cytoplasm-mitochondria" dual-regulated squalene engineering strain SquM13 in the laboratory, tries many transformation strategies related to organelles, and screens out multiple brewer's yeast genetic engineering strains, including: SY04, SY12, SY19, SY39, SY49, SY51, and the squalene production is significantly improved.

[0017] 2) Among the various genetically engineered strains of Saccharomyces cerevisiae with high squalene production obtained in the present invention, the result of strain SY51 was the most outstanding, with the highest squalene production reaching 54.62 g / L after fermentation in a 5-liter bioreactor.

[0018] In summary, the present invention obtains a variety of genetically engineered Saccharomyces cerevisiae bacteria that can efficiently produce squalene by combined regulation of multiple intracellular organelles, remodeling glycolysis and cofactor cycles, making large-scale squalene industrialization possible. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The schematic diagram of the metabolism of squalene biosynthesis in Saccharomyces cerevisiae;

[0020] Figure 2 The map information of the expression plasmid pUC20-GAL1-ZWF1;

[0021] Figure 3 The results of the squalene production test of the genetically engineered yeast SY51 are shown in Figure 2. DETAILED DESCRIPTION

[0022] The present invention will be further described below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional operations in the art, or according to the experimental methods recommended by the kit and instrument manufacturers. The reagents and materials used in the examples can be obtained from commercial sources unless otherwise specified.

[0023] The sources of the experimental materials used in the following examples are as follows:

[0024] Saccharomyces cerevisiae CEN.PK2-1C: a commercial strain, which can be obtained through common commercial channels.

[0025] Saccharomyces cerevisiae chassis strain SquM13: constructed according to the method disclosed in the document "Metabolic compartmentalization in yeast mitochondria: Burden and solution for squalene overproduction".

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

[0027] Table 1 Primer sequences for plasmid construction

[0028]

[0029]

[0030] Table 2 Primer sequences for engineering bacteria construction

[0031]

[0032]

[0033] The genetic engineering operation methods involved in the following examples, unless otherwise specified, are all conventional operations in the art, or are performed in accordance with the instructions of relevant products.

[0034] Example 1. Construction of expression cassette plasmid

[0035] 1.1. Construction of expression plasmid pUC20-GAL1-ZWF1

[0036] S1: First, primers for amplifying the target gene ZWF1 were designed: BamHI-ZWF1-F / XhoI-ZWF1-R, and the genome of CEN.PK2-1C bacteria was used as a template for amplification. The target fragment was recovered by gel electrophoresis.

[0037] S2: Using plasmid pESC-LEU (GenBank: AF063849.1) as a template, the empty plasmid backbone fragment was obtained by double restriction digestion with BamHI / XhoI, and the plasmid backbone fragment was recovered by gel electrophoresis.

[0038] S3: The plasmid backbone fragments obtained in S1 and S2 and the obtained target fragments were connected by seamless cloning, and the circular connection system was transformed into Escherichia coli DH5α competent cells. After Amp resistance plate screening and colony PCR and sequencing verification, the successfully constructed positive recombinant plasmid pUC20-GAL1-ZWF1 was obtained. The plasmid map is shown in Figure 2 shown.

[0039] 1.2. Construction of guide RNA plasmid

[0040] The construction method of plasmids PSCM-PAH1, PSCM-OPI1, PSCM-HRD1, PSCM-UTH1, PSCM-CIT2, PSCM-ECM33, PSCM-OCA5, PSCM-LYS1, PSCM-Int2, and PSCM-Int1 is as follows, taking PSCM-PAH1 as an example:

[0041] Primers gRNA-PAH1-F / R were used with the existing gRNA plasmid in the laboratory as a template to amplify the linearized gRNA plasmid fragment, which was recombined through seamless cloning. The ligation system was transformed into Escherichia coli DH5α competent cells, and two monoclonal colonies were randomly selected for sequencing verification through Amp resistance plate screening to obtain the successfully constructed positive recombinant plasmid PSCM-PAH1.

[0042] Example 2: Construction of genetically engineered yeast SY04

[0043] In previous studies, the inventor's laboratory had a "cytoplasm-mitochondria" dual-regulated squalene-producing engineered strain. On this basis, the endoplasmic reticulum lipid metabolism was regulated to alleviate metabolic limitations, and a brewer's yeast strain SY04 capable of synthesizing squalene was obtained, with a yield of 3.83g / L.

[0044] 2.1. Construction of engineered bacteria

[0045] Using SquM13 as the starting strain, SY04 was obtained by knocking out PAH1 and OPI1.

[0046] Among them, the NCBI accession number of the PAH1 gene is: NC_001145.3, and the NCBI accession number of the OPI1 gene is: NC_001140.6.

[0047] The specific steps are as follows:

[0048] 1) First, guide RNA plasmid primers for knocking out the PAH1 and OPI1 genes were designed, namely: gRNA-PAH1-F / R and gRNA-OPI1-F / R to amplify the DNA fragment of the linearized gRNA and transform it into Escherichia coli DH5a competent cells;

[0049] 2) Regarding the knockout of the PAH1 gene, specifically: design primers PAH1-UF / UR and PAH1-DF / DR to amplify the upstream and downstream homology arms PAH1-UP / DOWN with the genome CEN.PK2-1C, and recover the DNA fragments by gel electrophoresis; Regarding the knockout of the OPI1 gene, specifically: design primers OPI1-UF / UR and OPI1-DF / DR to amplify the upstream and downstream homology arms OPI1-UP / DOWN with the genome CEN.PK2-1C, and recover the DNA fragments by gel electrophoresis;

[0050] 3) PAH1-UP and PAH1-DOWN were transformed into the SquM13 engineering strain together with the targeting gRNA plasmid using the Saccharomyces cerevisiae transformation kit (ZYMOFROZEN-EZ YEASTTRANSFORMATION II KIT), and the positive transformants were screened using Leu-Ura-auxotrophic plates. The transformants were confirmed by colony PCR, and verification primers were designed on both sides of the homology arms to confirm whether the knockout was successful by nucleic acid sequencing;

[0051] 4) The genetically engineered bacteria with the knockout gene PAH1 after successful sequencing are streaked on a solid plate of Leu-deficient culture medium containing 5-FoA to cause the gRNA plasmid to be lost.

[0052] 5) Knock out the OPI1 gene in the engineered strain (4) after successful sequencing and loss of the gRNA plasmid in the same manner as in step (3), and confirm whether the knockout is successful by the same method. The strain with the correct sequencing is subjected to step (4) to lose the gRNA plasmid. The engineered strain with the knockout genes PAH1 and OPI1 is named SY04.

[0053] 2.2 YPD shake flask fermentation

[0054] Pick a single colony of the engineered bacteria SY04 obtained in 2.1 on the plate, inoculate it into a 250 mL conical flask filled with 15 mL YPD medium and culture it for 14 to 16 h, then transfer it to a 250 mL conical flask filled with 50 mL YPD medium to control the initial OD 600 The culture conditions were 30°C and 220 rpm. During the culture process, 1 mL of sample was taken every 24 h for analysis of growth OD 600 and squalene production.

[0055] 2.3. Determination of Squalene

[0056] Weigh 1.5 g of magnetic beads into a 2 ml crushing tube, add 600 μL of fermentation broth and 600 μL of ethyl acetate, grind 8 times using a cell crusher at 55 Hz for 180 s, then centrifuge at 12000 rpm for 10 min, take the supernatant and mix with 1.25 g of anhydrous sodium sulfate to remove water, then centrifuge at 12000 rpm for 5 min, and then perform gas phase quantitative analysis.

[0057] The cell concentration was determined by spectrophotometry using the absorbance at 600 nm.

[0058] The test results showed that the squalene production of the engineered bacteria SY04 through YPD shake flask fermentation increased by 1.45 times compared with the strain SquM13, and the yield and yield reached 3.83g / L and 354.76mg / g DCW, respectively.

[0059] Example 3: Construction of engineered bacteria SY12

[0060] As squalene production increases further, the intracellular capacity is limited, which restricts the production of squalene and requires the expansion of more squalene storage space. We explored the possibility that lipid droplets are the storage site for squalene.

[0061] 3.1. Construction of engineered bacteria

[0062] The engineered bacteria SquM13 was used as the starting strain, and the HRD1 gene was knocked out to obtain the engineered bacteria SY12. The NCBI accession number of the gene HRD1 is: NC_001147.6.

[0063] The specific operation steps refer to the engineering bacteria SY04, the difference is that the primers used are different, as follows:

[0064] Using SquM13 as the chassis strain, the primers for amplifying the upstream homology arm were HRD1-UF and HRD1-UR, and the primers for the downstream homology arm were HRD1-DF and HRD1-DR. Both the upstream homology arm and the downstream homology arm were amplified by PCR from the genome of CEN.PK2-1C.

[0065] 3.2. YPD shake flask fermentation

[0066] Same as 2.2 of Example 2.

[0067] 3.3. Determination of Squalene

[0068] Same as 2.3 of Example 2.

[0069] The test results showed that the squalene production and yield of the engineered bacteria SY12 through YPD shake flask fermentation reached 3.17 g / L and 337.18 mg / g DCW, respectively.

[0070] Example 4: Construction of engineered bacteria SY19

[0071] The accumulation of squalene on the yeast cell membrane puts tremendous pressure on the cells, causing autolysis of the yeast in the late fermentation period, which is not conducive to product production. Therefore, strengthening the cell wall has become a potential strategy to improve cell robustness.

[0072] 4.1. Construction of engineered bacteria

[0073] The engineered bacteria SquM13 was used as the starting strain, and the UTH1 and ECM33 genes were knocked out together on this basis to obtain the engineered bacteria SY19. The primers for amplifying the upstream homologous arm are UTH1-UF and UTH1-UR, and the primers for the downstream homologous arm are UTH1-DF and UTH1-DR. The upstream homologous arm and the downstream homologous arm are obtained by PCR amplification from the genome of CEN.PK2-1C; the primers for amplifying the upstream homologous arm are ECM33-UF and ECM33-UR, and the primers for the downstream homologous arm are ECM33-DF and ECM33-DR. The upstream homologous arm and the downstream homologous arm are obtained by PCR amplification from the genome of CEN.PK2-1C; SY19 was obtained after knocking out the two genes in sequence, and specific reference was made to steps (3) to (5) of 2.1 of Example 2. Among them, the NCBI accession number of gene UTH1 is: NC_001143.9, and the NCBI accession number of gene ECM33 is: NC_001134.8.

[0074] 4.2. YPD shake flask fermentation

[0075] Same as 2.2 of Example 2.

[0076] 4.3. Determination of Squalene

[0077] Same as 2.3 of Example 2.

[0078] The test results showed that the squalene production and yield of the engineered bacteria SY19 through YPD shake flask fermentation reached 4.21g / L and 416.78mg / g DCW, respectively.

[0079] Example 5. Construction of engineered bacteria SY39

[0080] The engineered bacteria SY04 obtained in Example 2 was used as the starting strain, and the CIT2, HRD1, UTH1 and ECM33 genes were knocked out to obtain the CIT2 gene, wherein the NCBI accession number of the CIT2 gene is: NC_001135.5.

[0081] 5.1. Construction of engineered bacteria

[0082] The specific operation steps refer to the engineering bacteria SY04, the difference is that the primers used are different, as follows:

[0083] Using SY04 as the chassis strain, the genes CIT2, UTH1 and ECM33 were knocked out in sequence. The primers for amplifying the upstream homology arm of the CIT2 gene were CIT2-UF and CIT2-UR, and the primers for the downstream homology arm were CIT2-DF and CIT2-DR. Both the upstream homology arm and the downstream homology arm were amplified by PCR from the genome of CEN.PK2-1C.

[0084] 5.2. YPD shake flask fermentation

[0085] Same as 2.2 of Example 2.

[0086] 5.3. Determination of Squalene

[0087] Same as 2.3 of Example 2.

[0088] The cell concentration was determined by spectrophotometry using the absorbance at 600 nm.

[0089] The test results showed that the squalene production and yield of the engineered bacteria SY39 through YPD shake flask fermentation reached 5.93g / L and 629.24mg / g DCW, respectively.

[0090] Example 6: Construction of engineered bacteria SY49

[0091] The engineered bacteria SY39 obtained in Example 5 was used as the starting strain, and after knocking out the OCA5 and LYS1 genes, the P GAL1 as promoter and T CYC1 The engineered bacteria SY49 was obtained by using the ZWF1 gene as the terminator. The NCBI accession number of the gene ZWF1 is: NC_001146.8.

[0092] 6.1. Construction of engineered bacteria

[0093] The specific operation steps for knocking out the OCA5 and LYS1 genes refer to the engineered bacteria SY49, the difference lies in the different primers used, as follows:

[0094] Using SY39 as the chassis strain, the primers for knocking out the OCA5 gene and amplifying the upstream homology arm are OCA5-UF and OCA5-UR, and the primers for the downstream homology arm are OCA5-DF and OCA5-DR; the primers for knocking out the LYS1 gene and amplifying the upstream homology arm are LYS1-UF and LYS1-UR, and the primers for the downstream homology arm are LYS1-DF and LYS1-DR; after two rounds of gene knockout operations and eliminating the gRNA plasmid, the ZWF1 gene was inserted at the Int1 site. GAL1 as promoter and T CYC1 The ZWF1 gene expression plasmid with a terminator as the terminator has been constructed in Example 1. P was amplified from Example 1 using primers Int1-TADH1-F and Int1-TCYC1-R. GAL1 -ZWF1-T CYC1 Expression cassette; using CEN.PK2-1C as a template, primers Int1-UF / Int1-TADH1-UR and Int1-TCYC1-DF / Int1-DR were used to amplify the upstream and downstream homology arms. The expression cassette and the upstream and downstream homology arms were recovered by gel electrophoresis. The engineering strain was constructed according to the method in Example 1.

[0095] 6.2. YPD shake flask fermentation

[0096] Same as 2.2 of Example 2.

[0097] 6.3. Determination of Squalene

[0098] Same as 2.3 of Example 2.

[0099] The cell concentration was determined by spectrophotometry using the absorbance at 600 nm.

[0100] The test results showed that the squalene production and yield of the engineered bacteria SY49 through YPD shake flask fermentation reached 6.24g / L and 649.26mg / g DCW, respectively.

[0101] Example 7: Construction and fermentation of engineered bacteria SY51

[0102] 7.1. Construction of engineered bacteria

[0103] The engineered bacteria SY 49 obtained in Example 6 was used as the starting strain, and the missing four amino acid genes HIS3, LEU2, URA3 and TRP1 were complemented at the Int2 site. The upstream homology arm of the Int2 site was amplified by PCR using CNE.PK2-1C as a template and primers Int2-UF / Int2-TADH1-UR; the downstream homology arm was amplified by PCR using CNE.PK2-1C as a template and primers Int2-TTPS1-DF / Int2-DR; the first expression cassette PHis3 -His3-T ADH1 , P Leu2 -Leu2-T CYC1 The connection order is T ADH1 -His3-P His3 -P Leu2 -Leu2-T CYC1 , which was amplified from Int2-TADH1-F / TCYC1-TPGK1-R using the existing plasmid PZT20 in the laboratory (see Chinese invention patent application 202111145425.8); the second expression cassette P Ura3 -Ura3-T PGK1 , P TRP1 -Trp1-T TPS1 The connection order is T PGK1 -Ura3-P Ura3 -P TRP1 -Trp1-T TPS1 , amplified by TPGK1-TCYC1-F / TTPS1-Int2-R using the existing plasmid PZT110 in the laboratory (see Chinese invention patent application 202111145425.8).

[0104] 7.2 Shake flask fermentation

[0105] YPD medium: 10g / L yeast extract, 20g / L peptone, 20g / L glucose (YPD solid medium, additional 20g / L agar powder is required)

[0106] First, pick a single colony of engineered bacteria growing on a YPD plate, inoculate the yeast colony into a 5 mL YPD tube, and culture it at 30°C and 220 rpm / min for 12 h. Then transfer the culture solution from the tube to 15 mL YPD medium, with an initial bacterial density of 600 nm (OD 600 ) was 0.1, and the culture was carried out at 30°C and 220 rpm / min for about 14 h. Finally, the fermented seeds were inoculated into 50 ml of fresh YPD medium, and the initial OD 600 The reaction mixture was cultured at 30°C and 220 rpm / min for 7 days.

[0107] 7.3. Squalene detection method

[0108] For the extraction of squalene, 600 μL of cultured cells and 600 μL of ethyl acetate were mixed with 1.5 g of magnetic beads with a diameter of 0.5 mm, placed in a 2 mL microcentrifuge tube, and shaken at 55 Hz and 4 °C for 30 min using a cryo-grinder (Jingxin, Shanghai, China). The shaken mixture was centrifuged at 12,000 rpm for 10 min, dehydrated with anhydrous sodium sulfate, and the upper organic phase was used for gas chromatography detection. The gas chromatography system (Agilent 7890a, USA) was equipped with an HP-5 column (30 m × 0.25 mm, 0.25 μm film thickness) and a flame ionization detector (FID). The temperature was kept at 290 °C for 8 min, and squalene was quantified by the external standard method. All results are reported as the mean of three representatives. Statistical analysis was performed using the Student's t-test (one-tailed; two-sample unequal variance; *p<0.05, **p<0.01, ***p<0.001). All data represent the mean of at least three independent samples, and the error bars represent the standard deviation.

[0109] 7.4. Squalene test results

[0110] like Figure 3 Among the obtained high-yield squalene engineering bacteria, the strain SY51 had the most outstanding results, with the highest squalene production reaching 54.62 g / L after fermentation in a 5-liter bioreactor.

[0111] The above is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. The above embodiments of the present invention can also be modified in various ways. All simple, equivalent changes and modifications made according to the claims and the description of the present invention fall within the scope of protection of the claims of the present invention. The contents not described in detail in the present invention are all conventional technical contents.

Claims

1. A genetically engineered strain of Saccharomyces cerevisiae that produces high levels of squalene, characterized in that: include: The engineered strain SquM13 was used as the starting strain, and the PAH1 and OPI1 genes were knocked out by regulating its lipid synthesis to obtain the Saccharomyces cerevisiae genetically engineered strain SY04, wherein the NCBI accession number of the PAH1 gene is: NC_001145.3, and the NCBI accession number of the OPI1 gene is: NC_001140.6; The genetically engineered yeast strain SY12 of Saccharomyces cerevisiae was obtained by using the engineered strain SquM13 as the starting strain and knocking out the gene HRD1; wherein the NCBI accession number of the gene HRD1 is: NC_001147.6; The engineered strain SquM13 was used as the starting strain, and the UTH1 and ECM33 genes were knocked out to obtain the Saccharomyces cerevisiae genetically engineered strain SY19; wherein the NCBI accession number of the UTH1 gene is: NC_001143.9, and the NCBI accession number of the ECM33 gene is: NC_001134.

8.

2. The genetically engineered yeast of claim 1, characterized in that: Also includes: The Saccharomyces cerevisiae genetically engineered bacteria SY39 is obtained by knocking out the CIT2, HRD1, UTH1 and ECM33 genes using the Saccharomyces cerevisiae genetically engineered bacteria SY04 as the starting strain; wherein the NCBI accession number of the gene CIT2 is: NC_001135.

5.

3. The genetically engineered yeast of claim 2, characterized in that: Also includes: The SY39 genetically engineered yeast was used as the starting strain, and the OCA5 and LYS1 genes were knocked out at the same time, and the P GAL1 and T CYC1 ZWF1 gene P GAL1 -ZWF1-T CYC1 The obtained genetically engineered yeast SY49 has the NCBI accession number of gene OCA5: NC_001140.6; the NCBI accession number of gene LYS1: NC_001141.2; and the NCBI accession number of gene ZWF1: NC_001146.

8.

4. The genetically engineered yeast of claim 3, characterized in that: Also includes: The brewer's yeast gene SY49 was used as the starting strain, and the auxotrophic genes, including Leu2, His3, Ura3 and Trp1, were complemented at the Int2 site to obtain the brewer's yeast genetic engineering strain SY51, the gene expression cassette of which is specifically: His3 -His3-T ADH1 , P Leu2 -Leu2-T CYC1 , P Ura3 -Ura3-T PGK1 , P TRP1 -Trp1-T TPS1 .

5. The genetically engineered yeast of claim 4, characterized in that: The first gene 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 ; Second paragraph Gene expression cassette P Ura3 -Ura3-T PGK1 , P TRP1 -Trp1-T TPS1 The connection order is T PGK1 -Ura3-P Ura3 -P TRP1 -Trp1-T TPS1 .

6. Use of the genetically engineered yeast of Saccharomyces cerevisiae with high squalene production according to any one of claims 1 to 5 in the production of squalene.

7. The use according to claim 6, characterized in that: Pick a single colony of genetically engineered Saccharomyces cerevisiae on the plate, inoculate it into a conical flask containing 5 mL of YPD medium and culture it at 30°C, 220 rpm / min for 14-16 h, then transfer it to a conical flask containing 15 mL of YPD medium to control the initial OD 600 The fermentation seeds were inoculated into fresh YPD medium at an initial OD 600 The reaction mixture was cultured at 30°C and 220 rpm / min for 6 to 8 days.

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