Engineered yeast for producing squalene as well as construction method and application of engineered yeast

Abstract

The invention provides a yeast engineering bacterium for producing squalene as well as a construction method and application thereof, and belongs to the technical field of gene engineering. According to the invention, a homologous recombination technology is utilized to realize knockout and knockin of a target gene on a genome, and during specific operation, a phospholipid synthesis related gene positioned in an endoplasmic reticulum is knocked out; and then integrating and regulating endoplasmic reticulum genes, and simultaneously integrating and expressing acetyl coenzyme A to all genes in a squalene synthesis route, so that the yield of squalene is increased. The shake flask fermentation yield of squalene of the constructed engineering saccharomyces cerevisiae strain can reach 582.3 + / -7.8 mg / L. According to the invention, the brewer's yeast is transformed to synthesize the terpenoid, and a new idea is provided for promoting industrial application.

Description

Technical Field

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

[0002] Squalene is a linear triterpenoid compound with the chemical formula C 30 H 50 , has physiological activities such as antioxidant, anti-radiation and detoxification, and has broad application prospects in the fields of medicine, nutritional health products, cosmetics, etc. At present, the synthesis methods of squalene mainly include plant extraction, chemical synthesis and microbial synthesis. Among them, the unsustainable development of natural extraction process and the non-green environmental protection of chemical synthesis process limit the production and application of squalene, while the mild microbial method of synthesizing squalene has a broad application market and good development prospects.

[0003] Saccharomyces cerevisiae has become a host for the production of a variety of natural chemicals, including squalene, due to its many advantages, such as clear genetic information, complete gene editing tools, and excellent robustness. For the research on the synthesis of squalene in Saccharomyces cerevisiae, the existing research strategies are mainly based on the use of acetyl-CoA produced by metabolism in organelles such as peroxisomes and mitochondria. However, integrating complex metabolic pathways into organelles may cause cellular metabolic load and toxicity, thereby affecting the normal metabolic network. The endoplasmic reticulum, as the organelle with the largest membrane area in eukaryotic cells, plays an important role in protein folding and transport, and many enzymes in the squalene synthesis pathway, such as hydroxymethylglutaryl-CoA reductase and squalene synthase, are located on the endoplasmic reticulum. However, the potential impact of endoplasmic reticulum engineering and the regulation of lipid metabolism on squalene synthesis remains unclear. Summary of the invention

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and provide an engineered Saccharomyces cerevisiae that promotes squalene synthesis by regulating lipid synthesis, expanding the endoplasmic reticulum area and strengthening precursor carbon flux, and a construction method thereof.

[0005] The technical solution of the present invention is summarized as follows:

[0006] 1. A yeast engineered bacterium for producing squalene, wherein the yeast engineered bacterium is biologically modified based on the genome of a starting strain, wherein the biological modification includes replacing phospholipid synthesis-related genes with endoplasmic reticulum regulatory genes in the starting strain and overexpressing squalene synthesis precursor pathway genes.

[0007] Preferably, the starting strain comprises Saccharomyces cerevisiae.

[0008] Preferably, the phospholipid synthesis-related genes in the starting strain include the gene NEM1 located in the endoplasmic reticulum and regulating the dephosphorylation process of phosphatidic acid phosphatase, and the gene DPP1 encoding phosphatidic acid phosphatase;

[0009] When the starting strain is yeast (Saccharomyces cerevisiae) CEN.PK2-1D, the nucleotide sequence of the gene NEM1 is shown as SEQ ID NO.1; the nucleotide sequence of the gene DPP1 is shown as SEQ ID NO.2.

[0010] Preferably, the endoplasmic reticulum regulating gene includes the transcription factor gene INO2 for expanding the endoplasmic reticulum volume, and the enhancer gene INO4; the nucleotide sequence of the gene INO2 is shown in SEQ ID NO.3; the nucleotide sequence of the gene INO4 is shown in SEQ ID NO.4;

[0011] The genes of the squalene synthesis precursor pathway include: the first module gene ERG10, gene ERG13, gene tHMG and gene ERG12, the second module gene ERG8, gene ERG19 and gene IDI1, and the squalene synthesis pathway gene ERG20 and gene ERG9.

[0012] Preferably, the squalene synthesis precursor pathway gene is an endogenous gene of the starting strain, and the first module gene is knocked into the gene site H1 in the genome, the second module gene is knocked into the gene site H2 in the genome; the squalene synthesis pathway gene is knocked into the gene site H3 in the genome;

[0013] When the starting strain is Saccharomyces cerevisiae CEN.PK2-1D, the nucleotide sequence of the gene ERG10 is shown in SEQ ID NO.5, the nucleotide sequence of the gene ERG13 is shown in SEQ ID NO.6; the nucleotide sequence of the gene tHMG is shown in SEQ ID NO.7; the nucleotide sequence of the gene ERG12 is shown in SEQ ID NO.8; the nucleotide sequence of the gene ERG8 is shown in SEQ ID NO.9; the nucleotide sequence of the gene ERG19 is shown in SEQ ID NO.10; the nucleotide sequence of the gene IDI1 is shown in SEQ ID NO.11; the nucleotide sequence of the gene ERG20 is shown in SEQ ID NO.12; and the nucleotide sequence of the gene ERG9 is shown in SEQ ID NO.13.

[0014] 2. A method for constructing an engineered yeast strain, comprising the following steps: connecting the endoplasmic reticulum regulating gene to a strong expression constitutive promoter, and using the upstream and downstream nucleotide sequences of the phospholipid synthesis-related gene as homology arms, integrating the recombinant fragment into the genome of the starting strain, and obtaining an engineered Saccharomyces cerevisiae intermediate strain;

[0015] The first module genes in the squalene synthesis precursor pathway genes are connected to strong expression constitutive promoters and integrated into the gene site H1 in the genome of the engineered Saccharomyces cerevisiae intermediate bacteria; the second module genes are connected to strong expression constitutive promoters and integrated into the gene site H2 in the genome; the squalene synthesis pathway genes are connected to strong expression constitutive promoters and integrated into the gene site H3 in the genome to obtain yeast engineering bacteria.

[0016] Preferably, the strong expression constitutive promoter comprises a nucleotide sequence as shown in SEQ ID NO.14. TEF1 , the nucleotide sequence is as shown in SEQ ID NO.15 TDH3 , the nucleotide sequence is as shown in SEQ ID NO.16 PGK1 , the nucleotide sequence is as shown in SEQ ID NO.17 FBA1 .

[0017] Preferably, the method comprises: respectively linking the gene INO2 and the gene INO4 to a strong expression constitutive promoter P TEF1 connect;

[0018] The first module gene ERG10, ERG13, tHMG and ERG12 of the MVA pathway were linked to the strong constitutive promoter P TEF1 , P TDH3 , P PGK1 and P FBA1 connect;

[0019] The second module gene ERG8, ERG19 and IDI1 of the MVA pathway were linked to the strong constitutive promoter P TEF1 , P TDH3 and P PGK1 connect;

[0020] The squalene biosynthesis pathway genes ERG20 and ERG9 were linked to the strong constitutive promoter P TEF1 and P PGK1 connect.

[0021] The present invention uses the genome of the starting strain CEN.PK2-1D as a template, utilizes fragment PCR to amplify the gene and promoter sequence respectively, and utilizes overlap extension PCR to obtain the recombinant gene fragment;

[0022] The recombinant gene fragment was further integrated into the yeast genome using the lithium acetate yeast transformation method, and the correctness of the engineered strain was verified using the Ura auxotrophic tag. The nucleotide sequence of the Ura auxotrophic tag is shown in SEQ ID NO.18.

[0023] 3. A method for producing squalene using yeast engineering bacteria or yeast engineering bacteria obtained by the above construction method, comprising the following steps: activating and multiplying the yeast engineering bacteria to obtain seed liquid, inoculating the seed liquid into a fermentation medium for fermentation to obtain squalene.

[0024] Preferably, the medium used for fermentation and expansion is YPD medium, and the fermentation medium is YPD medium;

[0025] The activation, propagation and fermentation temperatures are all 28-30°C, accompanied by shaking at 220-240 rpm / min.

[0026] 4. The present invention uses Saccharomyces cerevisiae CEN.PK2-1D as the starting strain, and the engineered bacteria after the above biological transformation is named engineered Saccharomyces cerevisiae Sc-Sq03;

[0027] 5. The present invention provides a method for producing squalene, comprising the following steps:

[0028] The engineered brewer's yeast Sc-Sq03 was inoculated into the primary seed culture medium, and cultured for 16-18 hours at 28-30° C. and 220-240 rpm / min to prepare the primary seed solution;

[0029] The prepared first-level seed solution is inoculated into the second-level seed culture medium at an inoculation ratio of 1-2%, and cultured at 28-30° C. and 220-240 rpm / min for 14-16 hours to prepare the second-level seed solution;

[0030] The prepared secondary seed solution was inoculated into the shake flask fermentation medium to control the initial OD 600 The pH value is 0.15-0.20, and the culture is carried out under the culture conditions of 28-30°C and 220-240rpm / min for 4-5 days to produce squalene.

[0031] The primary seed culture medium, the secondary seed culture medium and the shaking flask fermentation culture medium are all YPD liquid culture medium.

[0032] Beneficial effects:

[0033] The present invention regulates the lipid metabolism process by knocking out phospholipid synthesis-related genes NEM1 and DPP1, and expands the endoplasmic reticulum membrane area, enhances the ability of the endoplasmic reticulum to synthesize proteins, improves protein abundance, and alleviates the potential metabolic limitation of intracellular squalene synthesis by knocking out the endoplasmic reticulum genes INO2 and INO4 in situ in the phospholipid genes; further, the present invention systematically strengthens the gene expression level of the complete pathway from acetyl-CoA to squalene synthesis by overexpressing all genes of the MVA pathway and its downstream squalene synthesis pathway, redirects the carbon metabolic flux to the squalene synthesis pathway, and the constructed engineered brewer's yeast Sc-Sq03 has a yield of 582.3±7.8 mg / L in shake flask fermentation, showing greater advantages in squalene production and having good application prospects.

[0034] The present invention conducts synthetic biology transformation on Saccharomyces cerevisiae, systematically improves the production of squalene from the perspectives of organelles and metabolic engineering, and provides a new idea and direction for Saccharomyces cerevisiae to synthesize terpenoid compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 is the OD after the shake flask fermentation of the starting strain and the engineered Saccharomyces cerevisiae 600 value;

[0036] Figure 2 Squalene production under shake flask fermentation conditions of starting strain and engineered Saccharomyces cerevisiae. DETAILED DESCRIPTION

[0037] In order to further illustrate the present invention, the technical solutions provided by the present invention are described in detail below in conjunction with embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0038] In the embodiment of the present invention, Saccharomyces cerevisiae CEN.PK2-1D, referred to as Sc-CEN.PK2-1D, is used as a starting strain and is transformed by synthetic biology to produce squalene.

[0039] The culture medium involved in the embodiments of the present invention is as follows:

[0040] (1) YPD medium described in the examples: 20 g / L glucose, 20 g / L tryptone, 10 g / L yeast extract;

[0041] (2) SC-ura medium described in the examples: glucose 20 g / L, amino-free yeast nitrogen source 6.7 g / L, dropout 2 g / L, tryptophan 0.02 g / L, leucine 0.1 g / L, histidine 0.02 g / L;

[0042] (3) SC+5-FoA medium described in the examples: glucose 20 g / L, amino-free yeast nitrogen source 6.7 g / L, Dropout 2 g / L, tryptophan 0.02 g / L, leucine 0.1 g / L, histidine 0.02 g / L, uracil 0.02 g / L, 5-FoA 0.1 g / L.

[0043] The PCR amplification reaction system for obtaining endogenous genes and promoters in the embodiment of the present invention is as follows: Phanta Flash Master Mix (2×) 25 μL, template 1 μL, upstream primer and downstream primer 2 μL each and ddH2O2 2 μL.

[0044] The squalene extraction method involved in the embodiment of the present invention is as follows:

[0045] (1) Pipette 1 mL of the fermented bacterial suspension and centrifuge at 4°C, 8000 rpm for 2 min to collect the bacteria;

[0046] (2) Add 1 mL of sterile water to resuspend the cells, and centrifuge at 8000 rpm at 4°C for 2 min to wash the cells;

[0047] (3) Add 3 M NaOH to the bacteria, boil at 100°C for 3 min, and then centrifuge at 4°C, 8000 rpm for 2 min to collect the bacteria;

[0048] (4) Add 1 mL of sterile water to resuspend the cells, and centrifuge at 8000 rpm at 4°C for 2 min to fully wash the cells;

[0049] (5) Add 1 mL of acetone to the cells, vortex for 30-40 min, and centrifuge at 4°C, 12,000 rpm for 2 min;

[0050] (6) Take the upper acetone extract, dilute it to an appropriate multiple and put it into a liquid phase bottle for testing.

[0051] The squalene content detection method involved in the embodiment of the present invention is as follows:

[0052] A Waters 2695 liquid chromatograph, a 2998 UV detector and a Waters (250×4.6 mm, 5 μm) C18 column were used for detection; the liquid chromatography conditions were as follows: isocratic elution, a mobile phase of 60% methanol and 40% acetonitrile, a flow rate of 1.0 mL / min, a detection wavelength of 205 nm, a column temperature of 40°C, and a detection time of 20 min.

[0053] The primer sequences used in this example are shown in Table 1.

[0054] Table 1 Primer sequences used in the examples

[0055]

[0056]

[0057]

[0058] Example 1: A method for constructing an engineered Saccharomyces cerevisiae for producing squalene

[0059] (1) Constructing an engineered yeast Sc-Sq01 that regulates lipid synthesis and expands the endoplasmic reticulum membrane area, comprising the following steps:

[0060] The Saccharomyces cerevisiae genome was used as a template and NEM1 R -U is upward, NEM1 R -D was used as the down primer to amplify the upstream homology arm of gene NEM1; F -U is upward, NEM1 F -D was used as the down primer to amplify the downstream homology arm of the gene NEM1; INO2-U was used as the up primer and INO2-D was used as the down primer to amplify the gene fragment INO2; DPP1 R -U is upward, DPP1 R -D was used as the down primer to amplify the upstream homologous arm of the gene DPP1; F -U is upward, DPP1 F -D was used as the down primer to amplify the downstream homology arm of the gene DPP1; INO4-U was used as the up primer and INO4-D was used as the down primer to amplify the gene fragment INO4; P TEF1 -U is upward, P TEF1 -D is the down primer, amplifying the promoter fragment P TEF1 The nutritional deficiency gene Ura was obtained by artificial synthesis. Subsequently, the homologous arms, promoter and gene fragments were assembled by overlap extension PCR to obtain the recombinant gene fragments NEM1 and NEM2. F -P TEF1 -INO2-NEM1 R -Ura-NEM1 R and DPP1 F -P TEF1 -INO4-DPP1 R -Ura-DPP1 R .

[0061] Furthermore, the two recombinant gene fragments were knocked into the genome of the starting bacteria using the lithium acetate yeast transformation method. The obtained positive transformant single colony was inoculated into the liquid culture medium of SC-ura, cultured at 30°C for 16-18 hours, and then spread on the 5-FoA solid culture medium until the correct single colony grew, and the engineered Saccharomyces cerevisiae Sc-Sq01 was obtained.

[0062] (2) Constructing an engineered Saccharomyces cerevisiae Sc-Sq02 overexpressing the MVA pathway, comprising the following steps:

[0063] The Saccharomyces cerevisiae genome was used as a template and H1 R -U is upward, H1 R -D is used as the down primer to amplify the upstream homology arm of gene locus H1; F -U is upward, H1 F -D was used as the lower primer to amplify the downstream homology arm of gene locus H1; ERG10-U was used as the upper primer and ERG10-D was used as the lower primer to amplify the gene fragment ERG10; ERG13-U was used as the upper primer and ERG13-D was used as the lower primer to amplify the gene fragment ERG13; tHMG-U was used as the upper primer and tHMG-D was used as the lower primer to amplify the gene fragment tHMG; ERG12-U was used as the upper primer and ERG12-D was used as the lower primer to amplify the gene fragment ERG12; P TDH3 -U is upward, P TDH3 -D is the down primer, amplifying the promoter fragment P TDH3 ; PGK1 -U is upward, P PGK1 -D is the down primer, amplifying the promoter fragment P PGK1 ; FBA1 -U is upward, P FBA1 -D is the down primer, amplifying the promoter fragment P FBA1 ; The nutritional deficiency gene Ura was obtained by artificial synthesis. Subsequently, the homologous arms, promoter and gene fragments were assembled by overlap extension PCR to obtain the recombinant gene fragment H1 F -P TEF1 -ERG10-P TDH3 -ERG13-P PGK1 -tHMG-P FBA1 -ERG12-H1 R -Ura-H1 R .

[0064] Furthermore, the two recombinant gene fragments were knocked into the genome of engineered Saccharomyces cerevisiae Sc-Sq01 using the lithium acetate yeast transformation method. The obtained positive transformant single colony was inoculated into the liquid culture medium of SC-ura, cultured at 30°C for 16-18 hours, and then spread on 5-FoA solid culture medium until the correct single colony grew, and the engineered Saccharomyces cerevisiae intermediate bacteria were screened.

[0065] Subsequently, the second module gene ERG8, gene ERG19 and gene ERG19 of the MVA pathway were introduced into the above-mentioned engineered Saccharomyces cerevisiae. R -U is upward, H2 R -D is used as the down primer to amplify the upstream homology arm of gene locus H2; F -U is upward, H2 F -D was used as a down primer to amplify the downstream homology arm of gene locus H2; ERG8-U was used as an up primer and ERG8-D was used as a down primer to amplify the gene fragment ERG8; ERG19-U was used as an up primer and ERG19-D was used as a down primer to amplify the gene fragment ERG19; IDI1-U was used as an up primer and IDI1-D was used as a down primer to amplify the gene fragment IDI1; the auxotrophic gene Ura was obtained by artificial synthesis. Subsequently, the homology arms, promoters and gene fragments were assembled by overlap extension PCR to obtain the recombinant gene fragment H2 F -P TEF1 -ERG8-P TDH3 -ERG19-P PGK1 -IDI1-H2 R -Ura-H2 R .

[0066] Furthermore, the two recombinant gene fragments were knocked into the genome of the intermediate bacteria using the lithium acetate yeast transformation method. The obtained positive transformant single colony was inoculated into the liquid culture medium of SC-ura, cultured at 30°C for 16-18 hours, and then spread on the 5-FoA solid culture medium until the correct single colony grew, and the engineered brewer's yeast Sc-Sq02 was screened.

[0067] (3) Constructing an engineered Saccharomyces cerevisiae Sc-Sq03 that overexpresses the squalene synthesis pathway, comprising the following steps:

[0068] The Saccharomyces cerevisiae genome was used as a template and H3 R -U is upward, H3 R -D is used as the down primer to amplify the upstream homology arm of gene locus H3; F -U is upward, H3 F-D was used as a down primer to amplify the downstream homology arm of gene locus H3; ERG20-U was used as an up primer and ERG20-D was used as a down primer to amplify the gene fragment ERG20; ERG9-U was used as an up primer and ERG9-D was used as a down primer to amplify the gene fragment ERG9; the auxotrophic gene Ura was obtained by artificial synthesis. Subsequently, the homology arms, promoter and gene fragments were assembled by overlap extension PCR to obtain the recombinant gene fragment H3 F -P TEF1 -ERG20-P TDH3 -ERG9-H3 R -Ura-H3 R .

[0069] Furthermore, the two recombinant gene fragments were knocked into the genome of the engineered Saccharomyces cerevisiae Sc-Sq02 using the lithium acetate yeast transformation method. The obtained positive transformant single colony was inoculated into the liquid culture medium of SC-ura, cultured at 30°C for 16-18 hours, and then spread on the 5-FoA solid culture medium until the correct single colony grew, and the engineered Saccharomyces cerevisiae Sc-Sq03 was screened.

[0070] Example 2: Production of squalene by engineered Saccharomyces cerevisiae Sc-Sq03 under shake flask fermentation conditions

[0071] The engineered Saccharomyces cerevisiae Sc-Sq03 was inoculated into 5 mL of YPD liquid medium and cultured at 30°C and 250 rpm / min for 19 hours to prepare a primary seed solution;

[0072] Further, the prepared first-level seed solution was inoculated into 50 mL YPD liquid culture medium at a 1% inoculation ratio, and cultured at 30° C. and 250 rpm / min for 18 hours to prepare a second-level seed solution;

[0073] Further, the prepared secondary seed solution was inoculated into a liquid culture medium containing 50 mL YPD, and the initial OD value of the fermentation bacterial suspension was controlled. 600 =0.1, and then cultured at 30°C and 250 rpm / min for 120 hours to obtain a fermentation broth.

[0074] Further, the squalene production capacity of the engineered Saccharomyces cerevisiae Sc-Sq03 was evaluated according to the squalene extraction method and detection method, and the OD of the fermentation broth was detected using an ultraviolet spectrophotometer. 600 , to evaluate the growth level of the strain.

[0075] like Figure 1 and Figure 2As shown, after 120 hours of shake flask fermentation, the squalene production of the engineered Saccharomyces cerevisiae strain Sc-Sq03 reached 582.3±7.8 mg / L, and the final OD 600 It was 10.2, indicating that regulating the endoplasmic reticulum membrane area and phospholipid synthesis of Saccharomyces cerevisiae can effectively increase squalene production.

[0076] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A yeast engineering bacterium for producing squalene, characterized in that: The yeast engineered bacteria are biologically modified based on the genome of the starting strain, and the biological modification includes replacing phospholipid synthesis-related genes with endoplasmic reticulum regulating genes in the starting strain and overexpressing squalene synthesis precursor pathway genes.

2. The yeast engineering bacteria according to claim 1, characterized in that The starting strain includes Saccharomyces cerevisiae.

3. The yeast engineering bacteria according to claim 1 or 2, characterized in that: The phospholipid synthesis-related genes in the starting strain include the gene NEM1 located in the endoplasmic reticulum and regulating the dephosphorylation process of phosphatidic acid phosphatase, and the gene DPP1 encoding phosphatidic acid phosphatase; When the starting strain is yeast (Saccharomyces cerevisiae) CEN.PK2-1D, the nucleotide sequence of the gene NEM1 is shown as SEQ ID NO.1; the nucleotide sequence of the gene DPP1 is shown as SEQ ID NO.

2.

4. The yeast engineering bacteria according to claim 3, characterized in that: The endoplasmic reticulum regulating genes include the transcription factor gene INO2 that expands the endoplasmic reticulum volume, and the enhancer gene INO4; the nucleotide sequence of the gene INO2 is shown in SEQ ID NO.3; The nucleotide sequence of the gene INO4 is shown in SEQ ID NO.4; The genes of the squalene synthesis precursor pathway include: the first module gene ERG10, gene ERG13, gene tHMG and gene ERG12, the second module gene ERG8, gene ERG19 and gene IDI1, and the squalene synthesis pathway gene ERG20 and gene ERG9.

5. The yeast engineering bacteria according to claim 4, characterized in that: The squalene synthesis precursor pathway genes are endogenous genes of the starting strain, and the first module gene is knocked into the gene site H1 in the genome, and the second module gene is knocked into the gene site H2 in the genome; the squalene synthesis pathway gene is knocked into the gene site H3 in the genome; When the starting strain is Saccharomyces cerevisiae CEN.PK2-1D, the nucleotide sequence of the gene ERG10 is shown in SEQ ID NO.5, the nucleotide sequence of the gene ERG13 is shown in SEQ ID NO.6; the nucleotide sequence of the gene tHMG is shown in SEQ ID NO.7; the nucleotide sequence of the gene ERG12 is shown in SEQ ID NO.8; the nucleotide sequence of the gene ERG8 is shown in SEQ ID NO.9; the nucleotide sequence of the gene ERG19 is shown in SEQ ID NO.10; the nucleotide sequence of the gene IDI1 is shown in SEQ ID NO.11; the nucleotide sequence of the gene ERG20 is shown in SEQ ID NO.12; and the nucleotide sequence of the gene ERG9 is shown in SEQ ID NO.

13.

6. The method for constructing the yeast engineering bacteria according to any one of claims 1 to 5, characterized in that: The following steps are involved: The endoplasmic reticulum regulatory genes were connected to strong expression constitutive promoters, and the upstream and downstream nucleotide sequences of phospholipid synthesis-related genes were used as homology arms, and the recombinant fragments were integrated into the genome of the starting strain to obtain the engineered Saccharomyces cerevisiae intermediate strain; The first module genes in the squalene synthesis precursor pathway genes are connected to strong expression constitutive promoters and integrated into the gene site H1 in the genome of the engineered Saccharomyces cerevisiae intermediate bacteria; the second module genes are connected to strong expression constitutive promoters and integrated into the gene site H2 in the genome; the squalene synthesis pathway genes are connected to strong expression constitutive promoters and integrated into the gene site H3 in the genome to obtain yeast engineering bacteria.

7. The construction method according to claim 6, characterized in that: The strong expression constitutive promoter includes a nucleotide sequence as shown in SEQ ID NO.

14. TEF1 , the nucleotide sequence is as shown in SEQ ID NO.15 TDH3 , the nucleotide sequence is shown in SEQ ID NO.16 PGK1 , the nucleotide sequence is as shown in SEQ ID NO.17 FBA1 .

8. The construction method according to claim 7, characterized in that: include: The genes INO2 and INO4 were linked to the strong constitutive promoter P TEF1 connect; The first module gene ERG10, ERG13, tHMG and ERG12 of the MVA pathway were linked to the strong constitutive promoter P TEF1 , P TDH3 , P PGK1 and P FBA1 connect; The second module gene ERG8, ERG19 and IDI1 of the MVA pathway were linked to the strong constitutive promoter P TEF1 , P TDH3 and P PGK1 connect; The squalene biosynthesis pathway genes ERG20 and ERG9 were linked to the strong constitutive promoter P TEF1 and P PGK1 connect.

9. A method for producing squalene using the yeast engineered bacteria according to any one of claims 1 to 5 or the yeast engineered bacteria obtained by the construction method according to any one of claims 6 to 8, characterized in that: The method comprises the following steps: activating and multiplying the yeast engineering bacteria to obtain seed liquid, inoculating the seed liquid into a fermentation medium for fermentation to obtain squalene.

10. The method according to claim 9, characterized in that: The culture medium used for fermentation and expansion is YPD culture medium, and the fermentation culture medium is YPD culture medium; The activation, propagation and fermentation temperatures are all 28-30°C, accompanied by shaking at 220-240 rpm / min.

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