A kind of brewer's yeast for producing squalene and its construction method and application

Abstract

The present invention discloses a saccharomyces cerevisiae for producing squalene and a construction method and application thereof. In the recombinant saccharomyces cerevisiae disclosed in the present invention, the yeast endogenous isopentenyl pyrophosphate (IPP) isomerase IDI1, the bifunctional farnesyl pyrophosphate (FPP) synthase ERG20 and the squalene synthase SQS are co-expressed by constructing a scaffold protein, and the expression of the yeast endogenous squalene epoxidase genes ERG1 and GAL80 is regulated by introducing a light-regulated expression system based on an EL222‑VP16‑NLS nuclear localization photosensitive element and a C120 DNA binding sequence. The squalene yield of the recombinant saccharomyces cerevisiae constructed by the present invention reaches 20.2 g / L.

Description

Technical Field

[0001] The invention belongs to the fields of synthetic biology and fermentation engineering, and relates to a squalene-producing yeast and a construction method and application thereof. Background Art

[0002] Squalene was first discovered in the livers of deep-sea sharks. In recent years, successful extraction of squalene from plants and oils has been successfully achieved. However, these traditional methods often suffer from complex extraction processes, low product yields, severe environmental pollution, and damage to wildlife and plant resources. To meet the high market demand for squalene, alternative green, efficient, and sustainable methods for its synthesis are urgently needed. Recently emerging methods using microorganisms to synthesize squalene offer the advantages of short production cycles, simple operation, high economic benefits, and freedom from raw material and environmental constraints.

[0003] Currently, most microbial biosynthesis of squalene uses Escherichia coli and yeast as the base cells. Escherichia coli can synthesize squalene through the 2-methylerythritol phosphate (MEP) pathway. However, the MEP pathway is inefficient in supplying isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) for product synthesis. Furthermore, the MEP pathway lacks a post-transcriptional modification mechanism, making it difficult to express cytochrome P450 monooxygenases (P450s), thus limiting the synthesis of terpenes such as squalene. In contrast, yeast not only possesses an endogenous mevalonate pathway and a sterol synthesis pathway, providing ample precursors for squalene synthesis, but also possesses a complete intracellular membrane system, making it highly susceptible to the expression of enzymes and P450 enzymes involved in squalene synthesis. Therefore, yeast has broad application prospects as a primary host for the biosynthesis of squalene.

[0004] The biosynthesis pathway of squalene in Saccharomyces cerevisiae involves the following steps: glucose undergoes glycolysis in the cytoplasm to produce pyruvate, which is then catalyzed by pyruvate dehydrogenase to produce acetyl-CoA. Using acetyl-CoA as a precursor, the pathway proceeds through seven enzyme steps: thiolase, HMG-CoA synthetase, HMG-CoA reductase, mevalonate kinase, 5-phosphomevalonate kinase, 5-phosphomevalonate decarboxylase, and IPP isomerases (ERG10, ERG13, HMG1, ERG12, ERG8, MVD1, and IDI1) to synthesize IPP and DMAPP, the five-carbon building blocks of terpenoids. Subsequently, two molecules of IPP and one molecule of DMAPP undergo a head-to-tail condensation to form FPP under the action of the bifunctional farnesyl pyrophosphate (FPP) synthase (ERG20). Finally, squalene is produced by squalene synthase (SQS). Due to the multi-step enzymatic reaction, the enzyme-catalyzed substrate dispersion leads to low catalytic efficiency and product yield. In addition, squalene in the cell is catalyzed in two steps by enzymes such as squalene epoxidase (ERG1) to produce ergosterol, which is one of the essential components of the cell membrane. If ERG1 is knocked out, the cells will not be able to grow normally.

[0005] With the advancement of transgenic technology, controllable gene expression systems have become indispensable tools in biomedical research and biotechnology. Over the past few decades, chemically regulated gene expression systems have been widely used to temporally regulate gene expression. However, precise spatial and temporal control of gene expression is difficult due to the free diffusion of these small molecule inducers, the difficulty of elimination, and potential off-target effects on cellular function. Light, however, is an ideal inducer of gene expression, offering strong controllability, no toxicity, and the ability to precisely control the expression of target genes in both space and time. The EL222 transcription factor, originally derived from a bacterial light-oxygen-barrier protein, dimerizes and binds to C120 DNA upon exposure to blue light, activating the expression of downstream genes. This system's light-dependent transcriptional activation requires only a few components: a light-sensitive domain (LOV) and a helix-turn-helix (HTH) DNA binding domain. In the dark, the LOV domain binds to the HTH domain, covering the HTH4α site required for dimerization and binding to C120 DNA. Irradiation with blue light triggers a photochemical reaction: the flavoprotein complex in the LOV domain interrupts the interaction between LOV and HTH, causing EL222 to dimerize and thus exert DNA-binding protein activity. Summary of the Invention

[0006] The purpose of the present invention is to genetically modify saccharomyces cerevisiae to enable it to efficiently produce squalene.

[0007] To achieve the above objectives, the present invention utilizes scaffold protein technology to co-express the three key enzymes IDI1, ERG20 and SQS for the synthesis of squalene. The synthesis pathway of squalene in Saccharomyces cerevisiae is as follows: Figure 1 As shown, a light-regulated expression system based on the EL222-VP16-NLS nuclear-localized light-sensitive element and the C120 DNA binding sequence was introduced to achieve segmented regulation of the squalene production process, thereby increasing the squalene production in recombinant Saccharomyces cerevisiae.

[0008] In the first aspect, the present invention provides a recombinant yeast (Saccharomyces cerevisiae), which coordinately expresses yeast endogenous isopentenyl pyrophosphate (IPP) isomerase IDI1, bifunctional farnesyl pyrophosphate (FPP) synthase ERG20 and squalene synthase SQS by constructing a scaffold protein, and regulates the expression of yeast endogenous squalene epoxidase genes ERG1 and GAL80 by introducing a light-regulated expression system based on the EL222-VP16-NLS nuclear-localized light-sensitive element and the C120 DNA binding sequence.

[0009] In the EL222-VP16-NLS nuclear-localized photosensor, EL222 is a light-sensitive transcription factor that, upon receiving light signals, binds to the promoter's C120 DNA binding sequence, initiating transcription of downstream genes. Gene transcription requires RNA polymerase, and VP16 is the transcription activation domain that recruits RNA polymerase. NLS is a nuclear localization signal peptide. The amino acid sequence of EL222-VP16-NLS is shown in SEQ ID NO:3, where positions 1-80 are EL222, positions 81-216 are VP16, and positions 217-223 are NLS.

[0010] In the presence of blue light, the EL222-VP16-NLS nuclear-localized photosensor activates the C120 promoter, leading to the expression of the ERG1 and GAL80 genes. ERG1 gene expression enables cells to convert squalene to ergosterol, promoting normal cell growth. GAL80 gene expression produces GAL80 protein, which binds to the intracellular GAL4 transcriptional activator, inactivating GAL4 and preventing it from activating the GAL1 and GAL10 promoters. Consequently, the IDI1, ERG20, and SQS genes are not expressed. When the blue light is removed, ERG1 gene expression is lost, cell growth arrests, and squalene begins to accumulate in the cells. GAL80 gene expression is lost, allowing the GAL4 transcriptional activator to activate the GAL1 and GAL10 promoters. This allows the normal expression of IDI1, ERG20, and SQS, promoting the accumulation of squalene. In short, the fundamental difference in segmented regulation is the presence or absence of blue light. When blue light is present, cells grow normally, consume squalene, increase cell OD, and do not accumulate squalene. When blue light is removed, the genes for synthesizing squalene are enhanced, cells do not consume squalene and stop growing, and squalene accumulates in the cells.

[0011] In some embodiments, in the above-mentioned recombinant Saccharomyces cerevisiae, the recombinant Saccharomyces cerevisiae is based on BY4742 as the starting strain.

[0012] In some embodiments, in any of the above-mentioned recombinant Saccharomyces cerevisiae, the scaffold protein is a GBD-SH3-PDZ scaffold protein, wherein the elements are GBD from rat N-WASP, SH3 from mouse Crk and PDZ from mouse α-syntrophin, the GBD domain, SH3 domain and PDZ domain are connected by a GS linker, and the GS linker has a total of 9 GS short peptide repeating units; the amino acid sequence of the GBD-SH3-PDZ scaffold protein is shown in SEQ ID NO: 2, wherein positions 1-501 are GBD, positions 520-823 are SH3, and positions 842-1346 are PDZ; the scaffold protein elements are referenced in “Dueber, JE, Wu, GC, Malmirchegini, GR, Moon, TS, Petzold, CJ, Ullal, AV, Prather, KL, Keasling, JD, 2009. Synthetic protein scaffolds provide modular control over metabolic flux. Nat. Biotechnol. 27, 753–759”.

[0013] When co-expressed, IDI1 binds to the GBD ligand in the scaffold protein GBD-SH3-PDZ, ERG20 binds to the SH3 ligand in the scaffold protein GBD-SH3-PDZ, and SQS binds to the PDZ ligand in the scaffold protein GBD-SH3-PDZ.

[0014] The isopentenyl pyrophosphate (IPP) isomerase IDI1 is shown in SEQ ID NO: 7;

[0015] The bifunctional farnesyl pyrophosphate (FPP) synthase ERG20 is shown in SEQ ID NO: 8;

[0016] The squalene synthase SQS is shown in SEQ ID NO:9.

[0017] In some embodiments, in any of the above-described recombinant Saccharomyces cerevisiae, the C120 DNA binding sequence is contained in the light-controlled promoter P C120 In the light-controlled promoter P C120 As shown in SEQ ID NO: 10, positions 501-1361, the light-controlled promoter P C120 The ERG1 promoter and the GAL80 promoter were replaced to achieve light-induced expression of the ERG1 gene and the GAL80 gene.

[0018] In some embodiments, in any of the above-described recombinant Saccharomyces cerevisiae, the EL222-VP16-NLS nuclear-localized light-sensitive element is driven by a strong constitutive promoter P PGK1 ERG1 is regulated by the light-activated promoter P C120 GAL80 is regulated by the light-controlled promoter P C120 The scaffold protein is regulated by the constitutive promoter P TEF1 The squalene pathway biosynthesis genes IDI1, ERG20, and SQS are regulated by P GAL1 / 10 and P GAL7 Promoter regulation.

[0019] In a second aspect, the present invention provides a method for constructing any of the above-mentioned recombinant Saccharomyces cerevisiae, comprising the following steps: replacing the promoters of the endogenous squalene epoxidase genes ERG1 and GAL80 of the starting strain with a light-controlled promoter containing a C120 DNA binding sequence, and then transferring the plasmid P1 expressing the scaffold protein and the EL222-VP16-NLS nuclear-localized light-sensitive element and the plasmid P2 expressing the endogenous isopentenyl pyrophosphate (IPP) isomerase IDI1, the bifunctional farnesyl pyrophosphate (FPP) synthase ERG20 and the squalene synthase SQS to obtain the recombinant Saccharomyces cerevisiae.

[0020] In some embodiments, in the above method, the starting strain is BY4742.

[0021] In some embodiments, in any of the above methods, the light-controlled promoter is P C120 The promoter is shown in positions 501-1361 of SEQ ID NO: 10. The promoters of the endogenous squalene epoxidase genes ERG1 and GAL80 of the starting strain are replaced with light-controlled promoters containing C120 DNA binding sequences by CRISPR technology, which can be specifically achieved by: replacing the target P ERG1 CRISPR plasmid C1 and fragment F1-P C120 -R1 was transformed into Saccharomyces cerevisiae BY4742 to obtain recombinant strain S1, which is a strain of P C120 The promoter was replaced with the promoter of the endogenous gene ERG1 ERG1 position to achieve light-induced expression of the ERG1 gene in recombinant bacteria; then target P GAL80 CRISPR plasmid C2 and fragment F2-P C120 -R2 was transformed into recombinant bacteria S1 to obtain recombinant bacteria S2, which is a strain that has P C120 The promoter was replaced with the promoter of the endogenous gene GAL80 GAL80 Position to achieve light-induced expression of the GAL80 gene in recombinant bacteria;

[0022] Wherein, the C1-gRNA sequence in the C1 plasmid is shown in SEQ ID NO: 11;

[0023] The fragment F1-P C120 - The sequence of R1 is shown in SEQ ID NO: 10;

[0024] The C2-gRNA sequence in the C2 plasmid is shown in SEQ ID NO: 13;

[0025] The fragment F2-P C120 The sequence of -R2 is shown in SEQ ID NO:12.

[0026] In some embodiments, in any of the above methods, the plasmid P1 is a plasmid comprising the plasmid P1 shown in SEQ ID NO: 1. TEF1 -GBD-(GS)9-SH3-(GS)9-PDZ-T ENO2 -P PGK1 -EL222-VP16-NLS-T ADH2 The fragment was inserted into the NotI site of the pRS415 vector, and the rest of the sequence remained unchanged.

[0027] In some embodiments, in any of the above methods, the plasmid P2 is a plasmid comprising the T sequence shown in SEQ ID NO: 6. HXT7 -IDI1-P GAL1 / 10 -ERG20-T CYC1 -P GAL7 -SQS-T PGI1 The fragment replaces the sequence between the XbaI and SacI restriction sites of the pRS416 vector, while the rest of the sequence remains unchanged.

[0028] In a third aspect, the present invention provides a method for preparing squalene by fermentation, comprising the step of fermenting and culturing any of the above-mentioned recombinant Saccharomyces cerevisiae.

[0029] In some embodiments, in the above fermentation method, blue light with a wavelength of 460nm to 500nm and a light intensity of 400 to 600lx is provided 0-30h after the start of fermentation culture, and no light is provided after 30h of fermentation until the end of 96 to 120h of fermentation.

[0030] In some embodiments, in any of the above fermentation methods, the fermentation culture conditions are as follows: initial OD600 = 0.2-0.5, temperature 28-32° C., and fermentation at 120-250 rpm for 96-120 h.

[0031] The present invention uses BY4742 as a starting strain, constructs a GBD-SH3-PDZ scaffold protein, and coordinately expresses three key enzymes, IDI1, ERG20, and ERG9, that participate in a continuous catalytic reaction in a squalene synthesis pathway. This shortens the spatial distance between the enzymes and improves the efficiency of squalene synthesis catalyzed by Saccharomyces cerevisiae. Furthermore, by introducing a light-regulated expression system based on an EL222-VP16-NLS nuclear-localized photosensor and a C120 DNA binding sequence, the expression and inhibition of squalene epoxidase ERG1 and GAL80 are induced in stages by controlling light conditions, thereby balancing the relationship between cell growth and squalene production and achieving a squalene yield of 20.2 g / L. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The biosynthesis pathway of squalene in Saccharomyces cerevisiae.

[0033] Figure 2 This is the HPLC spectrum of squalene standard.

[0034] Figure 3 This is the HPLC spectrum of the fermentation supernatant of Saccharomyces cerevisiae S3 in Example 6. DETAILED DESCRIPTION

[0035] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0036] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0037] The present invention will be further described below with reference to 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.

[0038] The pRS415 and pRS416 vectors are GENEWIZ products.

[0039] Saccharomyces cerevisiae BY4742 strain is a GENEWIZ product.

[0040] Determination of squalene production:

[0041] Agilent 1260 was used for high performance liquid chromatography detection, with a C18 ODS column (5 μm, 250×4.6 mm, Thermo Fisher Scientific); mobile phase: pure acetonitrile; flow rate: 0.6 mL / min; column temperature: 40°C; wavelength: 210 nm; injection volume: 10 μL.

[0042] Example 1: Construction of scaffold protein plasmid P1

[0043] (1) Artificially synthesized gene fragments

[0044] P TEF1 -GBD-(GS)9-SH3-(GS)9-PDZ-T ENO2 -P PGK1 -EL222-VP16-NLS-T ADH2 , and add 20 bp of homology arms of the pRS415 plasmid at both ends of the fragment, the sequence of which is shown in SEQ ID NO: 1, wherein positions 21-422 are constitutive promoter P TEF1 , positions 423-1925 are the coding genes for GBD from rat N-WASP, positions 1926-1979 are the coding genes for GS linkers, positions 1980-2891 are the coding genes for SH3 from mouse Crk, positions 2892-2945 are the coding genes for GS linkers, positions 2946-4463 are the coding genes for PDZ from mouse α-syntrophin, and positions 4464-4863 are the terminator T ENO2 , positions 4864-5641 are constitutive promoter P PGK1, positions 5642-5881 are the coding genes for the light-sensitive transcription factor EL222, positions 5882-6289 are the coding genes for the transcription activator VP16, positions 6290-6313 are the coding genes for the nuclear localization signal peptide NLS, and positions 6314-6641 are the terminator T ADH2 .

[0045] P TEF1 -GBD-(GS)9-SH3-(GS)9-PDZ-T ENO2 -P PGK1 -EL222-VP16-NLS-T ADH2 This fragment encodes the GBD-SH3-PDZ scaffold protein and the EL222-VP16-NLS nuclear-localized light-sensitive element. The amino acid sequence of the GBD-SH3-PDZ scaffold protein is shown in SEQ ID NO:2, where positions 1-501 are GBD, positions 520-823 are SH3, and positions 842-1346 are PDZ. The amino acid sequence of the EL222-VP16-NLS is shown in SEQ ID NO:3, where positions 1-80 are EL222, positions 81-216 are VP16, and positions 217-223 are NLS.

[0046] (2) The pRS415 vector was digested with NotI-HF and the linear vector was recovered by gel.

[0047] (3) The gene fragment of step (1) and the linear vector of step (2) were ligated at 37°C for 30 minutes under the action of SE ligase to obtain recombinant plasmid P1.

[0048] (4) The recombinant plasmid P1 was transformed into competent E. coli TOP10, spread on LB plates (containing 100 mg / L ampicillin), and cultured at 37°C for 16 h.

[0049] (5) Use primers M13F and M13R to perform PCR on the colonies grown on the plate. The strain with a target band of approximately 6600 bp is the correct strain.

[0050] M13F: 5'-gtaaaacgacggccagt-3' (SEQ ID NO: 4);

[0051] M13R: 5'-gtcatagctgtttcctg-3' (SEQ ID NO: 5).

[0052] (6) Cultivate the correct strain in LB medium for 16 to 20 hours and extract the recombinant plasmid P1.

[0053] Example 2: Construction of squalene synthesis plasmid P2

[0054] (1) Artificially synthesized gene fragment T HXT7 -IDI1-P GAL1 / 10 -ERG20-T CYC1 -P GAL7 -SQS-T PGI1 Sequence, and add 20bp of homology arms of pRS416 plasmid at both ends of the fragment, the sequence of which is shown in SEQ ID NO: 6, wherein positions 21-420 are the terminator T HXT7 , positions 421-1287 are from the IDI1 gene of Saccharomyces cerevisiae BY4742, and positions 1288-1955 are from the inducible promoter P GAL1 / 10 , positions 1956-3014 are the ERG20 gene from Saccharomyces cerevisiae BY4742, and positions 3015-3256 are the terminator T CYC1 , positions 3257-3657 are the inducible promoter P GAL7 , positions 3658-4992 are the SQS gene from Saccharomyces cerevisiae BY4742, and positions 4993-5442 are the terminator T PGI1 .

[0055] T HXT7 -IDI1-P GAL1 / 10 -ERG20-T CYC1 -P GAL7 -SQS-T PGI1 The fragment may encode the isopentenyl pyrophosphate (IPP) isomerase IDI1 shown in SEQ ID NO:7, the bifunctional farnesyl pyrophosphate (FPP) synthase ERG20 shown in SEQ ID NO:8, and the squalene synthase SQS shown in SEQ ID NO:9.

[0056] (2) The pRS416 vector was digested with XbaI and SacI, and the linear vector was recovered by gel chromatography.

[0057] (3) The gene fragment of step (1) and the linear vector of step (2) were ligated at 37°C for 30 minutes under the action of SE ligase to obtain recombinant plasmid P2.

[0058] (4) The recombinant plasmid P2 was transformed into competent E. coli TOP10, spread on LB plates (containing 100 mg / L ampicillin), and cultured at 37°C for 16 h.

[0059] (5) Use primers M13F and M13R to perform PCR on the colonies grown on the plate. The strain with a target band of approximately 5400 bp is the correct strain.

[0060] (6) Cultivate the correct strain in LB medium for 16 to 20 hours and extract the recombinant plasmid P2.

[0061] Example 3: Construction of Saccharomyces cerevisiae S1

[0062] (1) Artificially synthesized DNA fragment F1-P C120 -R1, the length of F1 in the fragment is 500bp, and the length of R1 is 500bp. F1-P C120 The sequence of -R1 is shown in SEQ ID NO: 10, wherein positions 1-500 are F1, positions 501-1361 are P C120 , positions 1362-1861 are R1.

[0063] (2) Artificially synthesized targeted P ERG1 CRISPR plasmid C1, which

[0064] The C1-gRNA sequence is: 5'-taatgcgatactgccgtagcggg-3' (SEQ ID NO: 11).

[0065] (3) Plasmid C1 and fragment F1-P C120 -R1 was transformed into Saccharomyces cerevisiae BY4742, plated on SD URA plates, and cultured at 30°C for 48 h.

[0066] (4) Pick a single colony from the SD URA plate and place it in YPD liquid medium. Culture it at 30°C and 220 rpm for 72 h. Take 10 μl of the diluted 100,000-fold and spread it on a YPD plate. Culture it at 30°C for 48 h.

[0067] (5) Select a single colony on the YPD plate and streak it on both YPD and SD URA plates. The strain that grows only on the YPD plate is the correct strain and is named S1. This strain is the strain that P C120 The promoter was replaced with the promoter of the endogenous gene ERG1 ERG1 Position to achieve light-induced expression of the ERG1 gene in recombinant bacteria.

[0068] Example 4: Construction of Saccharomyces cerevisiae S2

[0069] (1) Artificially synthesized DNA fragment F2-P C120 -R2, the length of F2 in the fragment is 500bp, and the length of R2 is 500bp. C120 The sequence of -R2 is shown in SEQ ID NO: 12, wherein positions 1-500 are F2, positions 501-1361 are P C120 , positions 1362-1861 are R2.

[0070] (2) Artificially synthesized targeted P GAL80 CRISPR plasmid C2, which

[0071] The C2-gRNA sequence is: 5'-aagaaaatcacacgagcgcccgg-3' (SEQ ID NO: 13).

[0072] (3) Plasmid C2 and fragment F2-P C120 -R2 was transformed into competent Saccharomyces cerevisiae S1, plated on SD URA plates, and cultured at 30°C for 48 h.

[0073] (4) Pick a single colony from the SD URA plate and place it in YPD liquid medium. Culture it at 30°C and 220 rpm for 72 h. Take 10 μl of the diluted 100,000-fold and spread it on a YPD plate. Culture it at 30°C for 48 h.

[0074] (5) Select a single colony on the YPD plate and streak it on both YPD and SD URA plates. The one that grows only on the YPD plate is the correct strain and is named S2. This strain is the P C120 The promoter was replaced with the promoter of the endogenous gene GAL80 GAL80 Position to achieve light-induced expression of the GAL80 gene in recombinant bacteria.

[0075] Example 5: Construction of Squalene-Synthesizing Saccharomyces cerevisiae S3

[0076] (1) Both recombinant plasmids P1 and P2 were transformed into competent cells of Saccharomyces cerevisiae S2, plated on SD URALEU plates, and cultured at 30°C for 48 h.

[0077] (2) Pick a single colony on the plate as the target strain and name it S3.

[0078] Example 6: Preparation of squalene by shake flask fermentation

[0079] Given the significant growth burden caused by the initial accumulation of squalene, a two-stage fermentation strategy consisting of a cell growth phase and a squalene accumulation phase was implemented by regulating light intensity to achieve precise control of cell and product production. The first stage, the cell growth phase, involved providing blue light to the cell growth environment to enable normal yeast cell growth. The second stage, the product production phase, involved fermentation in the dark, which enabled normal expression of the GAL promoter in the yeast and thus the accumulation of squalene.

[0080] Fermentation method:

[0081] A single colony of the S3 strain was inoculated into 10 mL of SD URALEU liquid culture medium and cultured at 30°C and 220 rpm for 24 hours to obtain a seed solution; the seed solution was inoculated into a 250 mL conical flask containing 50 mL of SD URA LEU liquid culture medium. After inoculation, the initial OD600 was maintained at 0.5, and blue light with a wavelength of 460 nm to 500 nm was provided 0-30 hours after the start of fermentation, with a light intensity of 500 lx. No light was provided from 30 hours to the end of fermentation for 96 hours.

[0082] In the presence of blue light, the EL222-VP16-NLS nuclear-localized photosensor activates the C120 promoter, leading to the expression of the ERG1 and GAL80 genes. ERG1 gene expression enables cells to convert squalene to ergosterol, promoting normal cell growth. GAL80 gene expression produces GAL80 protein, which binds to the intracellular GAL4 transcriptional activator, inactivating GAL4 and preventing it from activating the GAL1 and GAL10 promoters. Consequently, the IDI1, ERG20, and SQS genes are not expressed. When the blue light is removed, ERG1 gene expression is lost, cell growth arrests, and squalene begins to accumulate in the cells. GAL80 gene expression is lost, allowing the GAL4 transcriptional activator to activate the GAL1 and GAL10 promoters. This allows the normal expression of IDI1, ERG20, and SQS, promoting the accumulation of squalene. In short, the fundamental difference in segmented regulation is the presence or absence of blue light. When blue light is present, cells grow normally, consume squalene, increase cell OD, and do not accumulate squalene. When blue light is removed, the genes for synthesizing squalene are enhanced, cells do not consume squalene and stop growing, and squalene accumulates in the cells.

[0083] Extraction method:

[0084] After the fermentation, 1 mL of fermentation broth was taken, 1 mL of ethyl acetate and 0.5 g of glass beads were added for grinding and crushing, and the mixture was centrifuged at 12000 rpm for 10 min. The supernatant was taken for HPLC detection to determine the squalene content.

[0085] The HPLC spectra of squalene standard and fermentation broth supernatant are as follows: Figure 2 and Figure 3 shown.

[0086] The retention time of squalene standard was 5.3 min, and the retention time of squalene in the fermentation broth supernatant was 5.3 min.

[0087] The results showed that the squalene production of the finally constructed Saccharomyces cerevisiae S3 reached 2 g / L through shake flask fermentation.

[0088] Example 7: Preparation of Squalene in a 5L Fermentation Tank

[0089] Fermentation medium: YPD, supplemented with 20 g / L potassium dihydrogen phosphate, 2 g / L ferrous sulfate, and 1 mg / L vitamin A.

[0090] The tank culture process is as follows:

[0091] First, Saccharomyces cerevisiae S3 was inoculated into a tube containing 5 mL of YPD and cultured at 30° C. and 220 rpm for 18 hours to prepare a seed culture, ie, a first-stage culture.

[0092] Next, 0.15 mL of the first-stage culture was transferred to a 250 mL Erlenmeyer flask containing 15 mL of YPD for subculture and cultured at 30°C and 220 rpm for 14 h to obtain a second-stage culture.

[0093] Again, 3 mL of the second-stage culture was transferred to a 500 mL flask containing 100 mL of YPD for subculture and cultured at 30°C and 230 rpm for 24 h to obtain a third-stage seed culture.

[0094] Finally, the third-stage seed culture was inoculated into a 5-L bioreactor at an inoculum size of 10% (v / v), containing 2.7 L of fermentation medium, with an initial OD600 of 0.5, and fermented at 30°C and 150 rpm. Blue light with a wavelength of 460 nm to 500 nm and an intensity of 500 lx was provided 0-30 h after the start of fermentation. No light was provided from 30 h to the end of fermentation at 96 h.

[0095] After the fermentation, 1 mL of the fermentation liquid was taken, 1 mL of ethyl acetate and 0.5 g of glass beads were added for grinding and crushing, and the mixture was centrifuged at 12000 rpm for 10 min. The supernatant was taken for HPLC detection to determine the squalene content.

[0096] The results showed that after 5L fermentation, the squalene production of the finally constructed Saccharomyces cerevisiae S3 was 20.2g / L.

[0097] sequence

[0098] SEQ ID NO: 1

[0099] actcactatagggcgaattgaatgtttctactcctttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaag

[0100] cacagcatactaaatttcccctctttcttctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctt

[0101] tttcttcgtcgaaaaaggcaataaaaatttttatcacgtttcttttcttgaaaatttttttttttgattttttctctttcgatgacctccattgatatttaagt

[0102] taataaacggtcttcaatttctcaagtttcagtttcattttcttgttctattacaactttttttacttcttgctcattagaaagaaagcatagcaatctaat

[0103] ctaagttttaattacaaaatgagctcgggccagcagcccccggggagggtcaccaacgtgggctccctgctgctcaccccgcaagagaac

[0104] gagtctctcttctcccttctcggcaagaaatgtgtgactatgtcttcagcagtggtgcagttgtatgcagcagatcggaactgtatgtgggcaaa

[0105] gaagtgcagtggtgtcgcttgtctggttaaggaacaatcctcagagatcttttttttaagaatatttgacattaaggatgggaaattactgtgggaa

[0106] caagagctatacaataactttgtatataatagtcctagaggatattttcatacctttgctggagatacttgtcaagtagctcttaatttgccaatgaa

[0107] gaagagcaaaaaagttccgaaaagcagttacagacctgttgggccgacgacaaaggaaatctgaaaaaagacgagatgctccaaatggt

[0108] cccaatctacccatggctacagttgacataaaaaatccagaaatcacaacaaatagatattttatggttcacaagtcaacaacatctcccacaca

[0109] aagaaaaagaaaggaaaaagctaaaagaagagattaaccaaggcagatattggaacaccaagtaatttccagcacattgggcatgttgg

[0110] atgggatccaaatacaggttttgatctaaataatttggatccagaattaaagaatctttttgatatgtgtgggatctctgaggcccagcttaaagac

[0111] agaaacatcaaaagttatttatgactttatgaaaaacaggaggtgtagaagctgttaaaacgaactccgaagacaagcaccaccacc

[0112] acctccaccctcgagaggacctccccctcctcctccccctcctcatagctcaggtcctcctccccctcctgcccgaggaagggggggct

[0113] cctctccccccaccttcaagagctcctactgctgcacctccaccctcaccctctttaggcctggtgttgttgttcctccgcccccctccaaacag

[0114] gatgtaccctcctccaccaccagcgctgcctcttcagcaccttcaggccctccaccaccctcaccctccatctatggcagggtccacagcac

[0115] caccacctctcctcaccctccgcccccaccagggccaccacctcccctggcctgccttctgatggtgaccatcaagttccagctccttcag

[0116] gaaacaaagcagctcttttggatcaaattagagagggtgctcagctgaaaaaagtggaacagaatagccggcccgtgtcctgctcaggaag

[0117] ggatgcgcttttagaccagatacgacagggcattcaattgaaatctgtgtctgatggccaagagtccacaccaccaacacccgcacccacttc

[0118] aggaattgtgggtgcgctgatggaagtgatgcagaaaaggagcaaagccattcattcctcagatgaagatgaagacgatgatgatgaagaa

[0119] gattttgaggatgatgatgagtgggaagacggaagcggaagcggaagcggaagcggaagcggaagcggaagcggaagcggaagcat

[0120] ggcgggcaacttcgactcggaggagcggagtagctggtactggggccgcctgagccggcaggaggcggtggcgctattgcagggcca

[0121] gcggcacggggtgttcctggtgcgggactcgagcaccagccccggggactatgtgcttagcgtctccgaaaactcgcgcgtctcccactac

[0122] atcatcaacagcagcggcccgcgccctccagtgcctccgtcgcccgctcagcctccgccgggagtgagtccctccaggctccgaatagga

[0123] gatcaagaatttgattcattgcctgctttactggaattctacaaaatacactatttggacactacaacattgatagaaccagtggccagatcaagg

[0124] cagggtagtggagtgattctcaggcaggaggaggcagagtatgtgcgggccctctttgactttaatgggaatgatgaagaagatcttcccttt

[0125] aagaaaggagacatcctgagaatccgggataagcctgaagagcagtggtggaatgcagaggacagcgaaggaaagagggggatgattc

[0126] ctgtcccttacgtggagaagtatagacctgcctccgcctcagtatcggctctgattggaggtaaccaggagggttcccacccacagccactg

[0127] ggtgggccggagcctgggccctatgcccaacccagcgtcaacactccgctccctaacctccagaatgggcccatttatgccagggttatcc

[0128] agaagcgagtccctaatgcctacgacaagacagccttggctttggaggtcggtgagctggtaaaggttacgaagattaatgtgagtggtcag

[0129] tgggaaggggagtgtaatggcaaacgaggtcacttcccattcacacatgtccgtctgctggatcaacagaatcccgatgaggacttcagcgg

[0130] aagcggaagcggaagcggaagcggaagcggaagcggaagcggaagcggaagcatggcgtccggcaggcgcgccccgcgcaccgg

[0131] gctgctggaactgcgtgccggggcgggctcgggggccggcggcgagcggtggcagcgggtgctgctctcgctggcggaggacgtgct

[0132] gaccgtgagccccgccgacggcgagcccggcccggagcccggcgccccgcgggagcaggagcccgcgcagctcaacggcgccgc

[0133] cgagcccggctcggcgaccccccagctgcccgaggcgctgctgctccagcggcgccgcgtgacggtgcgcaaggccgacgccggtgg

[0134] gctgggtatcagcatcaaaggtggccgggagaacaaaatgcctattctcatttctaagatcttcaagggattggcggctgaccagacagagg

[0135] ccctctttgtgggggatgccatcctgtccgtgaatggggaagacttgtcctctgccacccacgatgaggctgtgcaggccctcaagaagaca

[0136] ggcaaggaggtggtgctggaggtcaagtacatgaaggaggtctcaccgtatttcaagaactctgctagcgggacctcggtcggctgggact

[0137] cgcctcccgcctcaccgcttcagcgacagccttcctcccctggccccccaccccggaaccttggtgaggccaaacacgtatccttgaagat

[0138] ggcatatgtctcgaggaggtgcacccctactgacccggagcccaggtacctggagatctgttcggcagatggccaagacaccctcttcctg

[0139] agggccaaggatgaagcaagcgcaaggtcgtgggcaggtgccattcaagcccaggtcaacgccctggtgccctgggtcaaggacgagc

[0140] tacaggcactgctggcagcttccggctctgctgggagccaggatatcaagcagattggctggctgactgagcagctgcccagtgggggca

[0141] cagcccccaccctggccctgctgaccgaaaaggagctgctcctctactgctgtctcccccagacccgagaggccttgagccggccggccc

[0142] gcaccgccccgctcatcgccaccagactggtgcactcgggcccctccaagggctcggtgccctacgacacagagctctcctttgccctacg

[0143] cacgggcacacgccatggtgtggacactcacctcttcagcgtggagtcaccgcaggagctggctgcctggacccgccagctagtggatgg

[0144] ctgtcaccgggccgccgagggtgtgcaggaggtgtcgacagcctgcacgtggaacggccgaccctgcagcctctctgtgcacatcgaca

[0145] agggcttcacactgtgggcggctgagcccggggcggcccgagctgtgctgctccgacagcccttcgagaagctgcagatgtcctcagatg

[0146] atggtgccagtctccttttcctggactttgggggtgctgaaggcgagatccagctggacctgcactcctgtcccaaaaccatggtcttcatcatc

[0147] cactccttcctctcggccaaagtcactcgcctggggctcttggcctagagtgcttttaactaagaattattagtcttttctgcttattttttcatcatag

[0148] tttagaacactttatattaacgaatagtttatgaatctatttaggtttaaaaattgatacagttttataagttactttttcaaagactcgtgctgtctattgc

[0149] ataatgcactggaaggggaaaaaaaaggtgcacacgcgtggctttttcttgaatttgcagtttgaaaaataactacatggatgataagaaaaca

[0150] tggagtacagtcactttgagaaccttcaatcagctggtaacgtcttcgttaattggatactcaaaaaagatggatagcatgaatcacaagatgga

[0151] aggaaatgcgggccacgaccacagtgatatgcatatgggagatggagatgataccttattttagattcctgacttcaactcaagacgcacaga

[0152] tattataacatctgcataataggcatttgcaagaattactcgtgagtaaggaaagagtgaggaactatcgcatacctgcatttaaagatgccgat

[0153] ttgggcgcgaatcctttattttggcttcaccctcatactattatcagggccagaaaaaggaagtgtttccctccttcttgaattgatgttaccctcat

[0154] aaagcacgtggcctcttatcgagaaagaaattaccgtcgctcgtgatttgtttgcaaaaagaacaaaactgaaaaaacccagacacgctcga

[0155] cttcctgtcttcctattgattgcagcttccaatttcgtcacacaacaaggtcctagcgacggctcacaggttttgtaacaagcaatcgaaggttct

[0156] ggaatggcgggaaagggtttagtaccacatgctatgatgcccactgtgatctccagagcaaagttcgttcgatcgtactgttactctctctctttc

[0157] aaacagaattgtccgaatcgtgtgacaacaacagcctgttctcacacactcttttcttctaaccaagggggtggtttagtttagtagaacctcgtg

[0158] aaacttacatttacatatatataaacttgcataaattggtcaatgcaagaaatacatatttggtcttttctaattcgtagtttttcaagttcttagatgctt

[0159] tctttttctcttttttacagatcatcaaggaagtaattatctactttttacaacaaatataaaacaatgaccgcagacctcagcaccatgccggcgg

[0160] cccaccgcagcacagaagccgtgtcgagcctttccccacgcgagactcaggtcctcgcttacatcgccgccggctacacccaccaccaaa

[0161] cgtccaagaggctgaacataagccagagctcggtcgagacctacctgcagcgcatccgcttcaagctcgatgtaccgacacgggcgcacc

[0162] tcatccgtgcagccgtcgaactggggctgacgacatacaaaacttctatttgggagaattacaagcaagggagaaatcatatgctactatgttt

[0163] tatggatactgtcgtgctctggcagagtatattagacagtcggcgattaaagatcttcgggacgctcgagtcgaagataaaaatattggtgcgt

[0164] gtagtaaagcacgacaatacatcgccgaaaggtattatcgagaagcggcaaggtttgcgaagctcttatatgtgcacttatacctttctaccac

[0165] gcgggatgtatcacagcgactcgaagcatctcaaatgggacgacaaaatatatttgtatatttgcaatgcgaatggttacaagaaagacacttt

[0166] cattgtttatttcaacctgtactttttaaccatggcgtacttattgtcgaagggcgtgtttccaaagaagaagcggaaagaatagagctttggactt

[0167] cttcgccagaggtttggtcaagtctccaatcaaggttgtcggcttgtctaccttgccagaaatttacgaaaagatggaaaagggtcaaatcgttg

[0168] gtagatacgttgttgacacttctaaataagcgaatttcttatgatttatgattttattattaaataagttataaaaaaaataagtgtatacaaattttaaa

[0169] gtgactcttaggttttaaaacgaaaattcttattcttgagtaactctttcctgtaggtcaggttgctttctcaggtatagcatgaggtcgctcttattga

[0170] ccacacctctaccggcatgcagcttttgttccctttagt

[0171] SEQ ID NO:2

[0172] MSSGQQPPRRVTNVGSLLLTPQENESLFSFLGKKCVTMSSAVVQLYAADR

[0173] NCMWAKKCSGVACLVKDNPQRSYFLRIFDIKDGKLLWEQELYNNFVYNSP

[0174] RGYFHTFAGDTCQVALNFANEEEAKKFRKAVTDLLGRRQRKSEKRRDAPN

[0175] GPNLPMATVDIKNPEITTNRFYGSQVNNISHTKEKKKGKAKKKRLTKADI

[0176] GTPSNFQHIGHVGWDPNTGFDLNNLDPELKNLFDMCGISEAQLKDRETSK

[0177] VIYDFIEKTGGVEAVKNELRRQAPPPPPPSRGGPPPPPPPPHSSGPPPPP

[0178] ARGRGAPPPPPSRAPTAAPPPPPPSRPGVVVPPPPPNRMYPPPPPALPSS

[0179] APSGPPPPPPPSMAGSTAPPPPPPPPPPPGPPPPPGLPSDGDHQVPAPSG

[0180] NKAALLDQIREGAQLKKVEQNSRPVSCSGRDALLDQIRQGIQLKSVSDGQ

[0181] ESTPPTPAPTSGIVGALMEVMQKRSKAIHSSDEDEDDDDEEDFEDDDEWE

[0182] DGSGSGSGSGSGSGSGSGSMAGNFDSEERSSWYWGRLSRQEAVALLQGQR

[0183] HGVFLVRDSSTSPGDYVLSVSENSRVSHYIINSSGPRPPVPPSPAQPPPG

[0184] VSPSRLRIGDQEFDSLPALLEFYKIHYLDTTTLIEPVARSRQGSGVILRQ

[0185] EEAEYVRALFDFNGNDEEDLPFKKGDILRIRDKPEEQWWNAEDSEGKRGM

[0186] IPVPYVEKYRPASASVSALIGGNQEGSHPQPLGGPEPGPYAQPSVNTPLP

[0187] NLQNGPIYARVIQKRVPNAYDKTALALEVGELVKVTKINVSGQWEGECNG

[0188] KRGHFPFTHVRLLDQQNPDEDFSGSGSGSGSGSGSGSGSGSMASGRRAPR

[0189] TGLLELRAGAGSGAGGERWQRVLLSLAEDVLTVSPADGEPGPEPGAPREQ

[0190] EPAQLNGAAEPGSATPQLPEALLLQRRRVTVRKADAGGLGISIKGGRENK

[0191] MPILISKIFKGLAADQTEALFVGDAILSVNGEDLSSATHDEAVQALKKTG

[0192] KEVVLEVKYMKEVSPYFKNSASGTSVGWDSPPASPLQRQPSSPGPPPRNL

[0193] GEAKHVSLKMAYVSRRCTPTDPEPRYLEICSADGQDTLFLRAKDEASARS

[0194] WAGAIQAQVNALVPWVKDELQALLAASGSAGSQDIKQIGWLTEQLPSGGT

[0195] APTLALLTEKELLLYCCLPQTREALSRPARTAPLIATRLVHSGPSKGSVP

[0196] YDTELSFALRTGTRHGVDTHLFSVESPQELAAWTRQLVDGCHRAAEGVQE

[0197] VSTACTWNGRPCSLSVHIDKGFTLWAAEPGAARAVLLRQPFEKLQMSSDD

[0198] GASLLFLDFGGAEGEIQLDLHSCPKTMVFIIHSFLSAKVTRLGLLA

[0199] SEQ ID NO:3

[0200] MTADLSTMPAAHRSTEAVSSLSPRETQVLAYIAAGYTHHQTSKRLNISQS

[0201] SVETYLQRIRFKLDVPTRAHLIRAAVELGLTTYKTSIWENYKQGRNHMLL

[0202] CFMDTVVLWQSILDSRRLKIFGTLESKIKILVRVVKHDNTSPKGIIEKRQ

[0203] GLRSSYMCTYTFLPRGMYHSDSKHLKWDDKIYLYICNANGYKKDTFIVYF

[0204] NLYFLTMAYLLSKGVFPKKKRKE

[0205] SEQ ID NO:6

[0206] gggggatccactagttctagtttgcgaacacttttattaattcatgatcacgctctaatttgtgcatttgaaatgtactctaattctaattttatattttta

[0207] atgatatcttgaaaagtaaatacgtttttaatatatacaaaataatacagtttaattttcaagtttttgatcatttgttctcagaaagttgagtgggacg

[0208] gagacaaagaaactttaaagagaaatgcaaagtgggaagaagtcagttgtttaccgaccgcactgttattcacaaatattccaattttgcctgc

[0209] agacccacgtctacaaattttggttagtttggtaaatggtaaggatatatagtagagcctttttgaaatgggaaatatcttctttttctgtatcccgcttc

[0210] aaaagtgtctaatgagtcagttatttatagcattctatgaatttgcctgtcattttccacttcagaaaggtcatctaattgctcccaccagttgaata

[0211] agtaattctcgcaaataatcttaaaccaaggcgtaaacttgtaacttgggtcagcaaacatagttttcaaatcatttggtgaaacccatttgaagtc

[0212] tctaacttcattgacgtttgggttgacagtcaagttttctttagcgttgatcttataaaataggatgtaatcaatttcatgttcaccccatggttcattgc

[0213] ttggtgccatgtaatggattctgtttaaaaagtgaaacttaccccttgtcttagtttcatcttctggaatacctaattcatgatctagttttctcaccgc

[0214] cgcagtaatagcgcccttaatcttatcgtctagcttacccttcaaacctaattcgtcatcaatacatagtggatgagagcagcatgtgttagtcca

[0215] aagatcagggaaagttattttttcagtggctctttgttgtaaaagtaattcaccttgttcattgaaaataaagacggagaatgcacgatgtagtaaa

[0216] ccctttcaatattttccattaaatgacaaactttcttggtaccggcaccaatagcattatcgtcccaatccaaaacaatacaattttcattcattaact

[0217] taatttgctcctcatcatgaccagaaaaacatgtttctccgctttcgtcatttgacgtctcactagatcgggtattaggtctttgttgtaatggaataa

[0218] tttcaggaaactcttccaaaatgtcttcaggtgtttggttttgcactaatttggcgtaactagatactgcaccatggggcatactattgttgtcggca

[0219] gtcattatagttttttctccttgacgttaaagtatagaggtatattaacaattttttgttgatacttttatgacatttgaataagaagtaatacaaaccga

[0220] aaatgttgaaagtattagttaaagtggttatgcagcttttgcatttatatatctgttaatagatcaaaaatcatcgcttcgctgattaattaccccaga

[0221] aataaggctaaaaaactaatcgcattattatcctatggttgttaatttgattcgttgatttgaaggtttgtggggccaggttactgccaatttttcctct

[0222] tcataaccataaaagctagtattgtagaatctttattgttcggagcagtgcggcgcgaggcacatctgcgtttcaggaacgcgaccggtgaag

[0223] accaggacgcacggaggagagtcttccgtcggagggctgtcgcccgctcggcggcttctaatccgtacttcaatatagcaatgagcagttaa

[0224] gcgtattactgaaagttccaaagagaaggtttttttaggctaagataatggggctctttacatttccacaacataataagtaagattagatatggata

[0225] tgtatatggtggtattgccatgtaatatgattattaaacttctttgcgtccatccaaaaaaaaagtaagaatttttgaaaattcaataataaatggcttc

[0226] agaaaaagaaattaggagagagagattcttgaacgttttccctaaattagtagaggaattgaacgcatcgcttttggcttacggtatgcctaagg

[0227] aagcatgtgactggtatgcccactcattgaactacaacactccaggcggtaagctaaatagaggtttgtccgttgtggacacgtatgctattctc

[0228] tccaacaagaccgttgaacaattggggcaagaagaatacgaaaaggttgccattctaggttggtgcattgagttgttgcaggcttacttcttggt

[0229] cgccgatgatatgatggacaagtccattaccagaagaggccaaccatgttggtacaaggttcctgaagttggggaaattgccatcaatgacg

[0230] cattcatgttagaggctgctatctacaagcttttgaaatctcacttcagaaacgaaaaatactacatagatatcaccgaattgttccatgaggtca

[0231] ccttccaaaccgaattgggccaattgatggacttaatcactgcacctgaagacaaagtcgacttgagtaagttctccctaaagaagcactcctt

[0232] catagttactttcaagactgcttactattctttctacttgcctgtcgcattggccatgtacgttgccggtatcacggatgaaaaggatttgaaacaa

[0233] gccagagatgtcttgattccattgggtgaatacttccaaattcaagatgactacttagactgcttcggtaccccagaacagatcggtaagatcg

[0234] gtacagatatccaagataacaaatgttcttgggtaatcaacaaggcattggaacttgcttccgcagaacaaagaaagactttagacgaaaatta

[0235] cggtaagaaggactcagtcgcagaagccaaatgcaaaaagattttcaatgacttgaaaattgaacagctataccacgaatatgaagagtctat

[0236] tgccaaggatttgaaggccaaaatttctcaggtcgatgagtctcgtggcttcaaagctgatgtcttaactgcgttcttgaacaaagtttacaaga

[0237] gaagcaaatagcatgtaattagttatgtcacgcttacattcacgccctcctcccacatccgctctaaccgaaaaggaaggagttagacaacctg

[0238] aagtctaggtccctatttattttttttaatagttatgttagtattaagaacgttatttatatttcaaatttttctttttttttctgtacaaacgcgtgtacgcatgt

[0239] aacattatactgaaaaccttgcttgagaaggttttgggacgctcgaaggcttttggctattgaagcctgcgagtttttgcacgcctttgcaacgag

[0240] cccaaatattcctgaacatatcttacaaccatatgttaaggatatcgtgccaatattattatcgaaaatggtctataacgaagaatccatcgttctcc

[0241] tggaagcttctaatgatgatgatgcattcttggaggataaagatgaggacatcaagcccattgcaccccgtattgtgaaaaagaaagaggcag

[0242] gaaatggagaggatgcagatgacaacgaagatgatgatgatgatgatgatgatgaagatggcgatgttgatacgcaatggaatttgagaaaa

[0243] tgttccgcggcaacgctagatgtaatgacgaatattttacctcatcaagtgatggatatagcgttcccatttttaatgggaaagctattacaattgg

[0244] cattgcatccggtcgagatgaaggcagctttgaagctgaagttttgcagaacaccgctattctccatctatgatcagtccacgtctccatatctct

[0245] tgcactgtttcgaactgttgaacttgacctccagatcgtttgctgctgtgatcagagagctgcatccagaattgagaaactgtgttactctcttttat

[0246] ttgattttaagggctttggataccatcgaagacgatatgtccatcgaacacgatttgaaaattgacttgttgcgtcacttccacgagaaattgttgtt

[0247] aactaaatggagtttcgacggaaatgcccccgatgtgaaggacagagccgttttgacagatttcgaatcgattcttattgaattccacaaattga

[0248] aaccagaatatcaagaagtcatcaaggagatcaccgagaaaatgggtaatggtatggccgactacatctttagatgaaaattacaacttgaatg

[0249] ggttgcaaaccgtccacgactacgacgtgtactgtcactacgtagctggtttggtcggtgatggtttgaccgtttgattgtcattgccaagtttg

[0250] ccaacgaatcttgtgtattctaatgagcaattgtatgaaagcatgggtcttttcctacaaaaaaccaacatcatcagagattacaatgaagatttggt

[0251] cgatggtagatccttctggcccaaggaaatctggtcacaatacgctcctcagttgaaggacttcatgaaacctgaaaacgaacaactggggtt

[0252] ggactgtataaaccacctcgtcttaaacgcattgagtcatgttatcgatgtgttgacttatttggccggtatccacgagcaatccactttccaatttt

[0253] gtgccattccccaagttatggccattgcaaccttggctttggtattcaacaaccgtgaagtgctacatggcaatgtaaagattcgtaagggtact

[0254] acctgctatttaattttgaaatcaaggactttgcgtggctgtgtcgagatttttgactattacttacgtgatatcaaatctaaattggctgtgcaagat

[0255] ccaaatttcttaaaattgaacattcaaatctccaagatcgaacagtttatggaagaaatgtaccaggataaattacctcctaacgtgaagccaaat

[0256] gaaactccaattttcttgaaagttaaagaaagatccagatacgatgatgaattggttccaacccaacaagaagaagagtacaagttcaatatgg

[0257] ttttatctatcatcttgtccgttcttcttgggttttattatatatacactttacacagagcgtgaacaaatcgctcttaaatatatacctaaagaacatta

[0258] aagctatattataagcaaagatacgtaaattttgcttatattattatacacatatcatatttctatatttttaagatttggttatataatgtacgtaatgcaa

[0259] aggaaataaattttatacattattgaacagcgtccaagtaactacattatgtgcactaatagtttagcgtcgtgaagactttattgtgtcgcgaaaa

[0260] gtaaaaattttaaaatagagcaccttgaacttgcgaaaaggttctcatcactgtttaaaggaggatatcaggtcctatttctgacaaacaa

[0261] tatacaaatttagttcaaagatgaatcagtgcgcgaaggacataactcatgaagcctccagtatacccatcgatttgcaagaaagatactcgc

[0262] actggaagaaaacactaaactacagctccagctttgttccct

[0263] SEQ ID NO:7

[0264] MTADNNSMPHGAVSSYAKLVQNQTPEDILEEFPEIIPLQRPNTRSSETSNDESGETCFSGH

[0265] DEEQIKLMNENCIVLDWDDNAIGAGTKKVCHLMENIEKGLLHRAFSVFIFNEQGELLLQ

[0266] QRATEKITFPDLWTNTCCSHPLCIDDELGLKGKLDDKIKGAITAAVRKLDHELGIPEDETK

[0267] TRGKFHFLNRIHYMAPSNEPWGEHEIDYILFYKINAKENLTVNPNVNEVRDFKWVSPND

[0268] LKTMFADPSYKFTPWFKIICENYLFNWWEQLDDLLSEVENDRQIHRML

[0269] SEQ ID NO:8

[0270] MASEKEIRRERFLNVFPKLVEELNASLLAYGMPKEACDWYAHSLNYNTPGGKLNRGLSV

[0271] VDTYAILSNKTVEQLGQEEYEKVAILGWCIELLQAYFLVADDMMDKSITRRGQPCWYKV

[0272] PEVGEIAINDAFMLEAAIYKLLKSHFRNEKYYIDITELFHEVTFQTELGQLMDLITAPEDK

[0273] VDLSKFSLKKHSFIVTFKTAYYSFYLPVALAMYVAGITDEKDLKQARDVLIPLGEYFQIQD

[0274] DYLDCFGTPEQIGKIGTDIQDNKCSWVINKALELASAEQRKTLDENYGKKDSVAEAKCK

[0275] KIFNDLKIEQLYHEYEESIAKDLKAKISQVDESRGFKADVLTAFLNKVYKRSK

[0276] SEQ ID NO:9

[0277] MGKLLQLALHPVEMKAALKLKFCRTPLFSIYDQSTSPYLLHCFELLNLTSRSFAAVIRELH

[0278] PELRNCVTLFYLILRALDTIEDDMSIEHDLKIDLLRHFHEKLLLTKWSFDGNAPDVKDRA

[0279] VLTDFESILIEFHKLKPEYQEVIKEITEKMGNGMADYILDENYNLNGLQTVHDYDVYCHY

[0280] VAGLVGDGLTRLIVIAKFANESLYSNEQLYESMGLFLQKTNIIRDYNEDLVDGRSFWPKEI

[0281] WSQYAPQLKDFMKPENEQLGLDCINHLVLNALSHVIDVLTYLAGIHEQSTFQFCAIPQVM

[0282] AIATLALVFNNREVLHGNVKIRKGTTCYLILKSRTLRGCVEIFDYYLRDIKSKLAVQDPNF

[0283] LKLNIQISKIEQFMEEMYQDKLPPNVKPNETPIFLKVKERSRYDDELVPTQQEEEYKFNM

[0284] VLSIILSVLLGFYYIYTLHRA

[0285] SEQ ID NO:10

[0286] ggataagctgttcacaaaatctcttctcgagtatgggttcgatgtggctattccaagcaacgcaagaaggtttttaccaaacgatgaagagttaa

[0287] gagattcttataaatacggctccaacgttggaggttcgcattacgcctatctaatctcctcattcgacattcccgaaggtgacaatgacaagtttt

[0288] ggagtcttgtcgaatactactatgaccgctttttagaatcgtacgacaacggtgaccacttgattggtctgggggtcctacaacttgattttatcgt

[0289] tgaaaacaagaatatagacagccttcttgccaactcttatttgcaccagcaaagaggcggtgcaatcatcagtaatacaggacttgtctcgcaa

[0290] gatacgaccaagccgtactacgttcgggatttaatcttctcgcagtctgcaggcgccttgagatttgcgttcggcctaaacgtttgctccacaaa

[0291] cgtgaatggtatgaacatggacaatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccag

[0292] aaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgag

[0293] agttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagca

[0294] actcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagaga

[0295] attatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttg

[0296] cacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatg

[0297] cctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcg

[0298] gataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggt

[0299] atcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatag

[0300] acagatcgctgagataggtgcctcactgattaagcattggtaaatgtctgctgttaacgttgcacctgaattgattaatgccgacaacacaatta

[0301] cctacgatgcgattgtcatcggtgctggtgttatcggtccatgtgttgctactggtctagcaagaaagggtaagaaagttcttatcgtagaacgt

[0302] gactgggctatgcctgatagaattgttggtgaattgatgcaaccaggtggtgttagagcattgagaagtctgggtatgattcaatctatcaacaa

[0303] catcgaagcatatcctgttaccggttataccgtctttttcaacggcgaacaagttgatattccatacccttacaaggccgatatccctaaagttga

[0304] aaaattgaaggacttggtcaaagatggtaatgacaaggtcttggaagacagcactattcacatcaaggattacgaagatgatgaaagagaaa

[0305] ggggtgttgcttttgttcatggtagattcttgaacaacttgagaaacattactgctcaagagccaaatgt

[0306] SEQ ID NO:12

[0307] gcctgtctacaggataaagacgggtcggatacctgcacaagcaatttggcacctgcataccccatttccccagtagataacttcaacacacac

[0308] atcaatgtccctcaccagtttatttccaaaagagacgctttttactacctgactagattttcattttgtttcttttggattgcgcttgcctttgtaggtgtg

[0309] tcgtttatcctttacgttttgacttggtgctcgaagatgctttcagagatggtgcttatcctcatgtcttttgggtttgtcttcaatacggcagccgttgt

[0310] cttgcaaacggccgcctctgccatggcaaagaatgctttccatgacgatcatcgtagtgcccaattgggtgcctctatgatgggtatggcttgg

[0311] gcaagtgtctttttatgtatcgtggaatttatcctgctggtcttctggtctgttagggcaaggttggcctctacttactccatcgacaattcaagatac

[0312] agaacctcctcatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtga

[0313] aagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccg

[0314] aagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccg

[0315] catacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgct

[0316] gccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatggg

[0317] ggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatg

[0318] gcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgca

[0319] ggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagca

[0320] ctggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctg

[0321] agataggtgcctcactgattaagcattggtaaatggactacaacaagagatcttcggtctcaaccgtgcctaatgcagctcccataagagtcg

[0322] gattcgtcggtctcaacgcagccaaaggatgggcaatcaagacacattaccccgccatactgcaactatcgtcacaatttcaaatcactgcctt

[0323] atacagtccaaaaattgagacttctattgccaccattcagcgtctaaaattgagtaatgccactgcttttcccactttagagtcatttgcatcatcttc

[0324] cactatagatatgatagtgatagctatccaagtggccagccattatgaagttgttatgcctctcttggaattctccaaaaataatccgaacctcaa

[0325] gtatcttttcgtagaatgggcccttgcatgttcactagatcaagccgaatccatttataaggctgctgctgaacgtggggttcaaaccatcatctc

[0326] tttacaaaggtcgtaaatcaccatatattttgagagcaaaagaattaaatatctca

Claims

1. A recombinant yeast (Saccharomyces cerevisiae), which co-expresses yeast endogenous isopentenyl pyrophosphate (IPP) isomerase IDI1, bifunctional farnesyl pyrophosphate (FPP) synthase ERG20 and squalene synthase SQS by constructing a scaffold protein, and regulates the expression of yeast endogenous squalene epoxidase genes ERG1 and GAL80 by introducing a light-regulated expression system based on EL222-VP16-NLS nuclear localization light-sensitive element and C120 DNA binding sequence; The scaffold protein is a GBD-SH3-PDZ scaffold protein, wherein the elements are GBD from rat N-WASP, SH3 from mouse Crk and PDZ from mouse α-syntrophin; The C120 DNA binding sequence is contained in the light-controlled promoter P C120 In the C120 The ERG1 promoter and the GAL80 promoter were replaced.

2. The recombinant Saccharomyces cerevisiae according to claim 1, characterized in that: The recombinant Saccharomyces cerevisiae uses BY4742 as the starting strain.

3. The recombinant Saccharomyces cerevisiae according to claim 1 or 2, characterized in that: When coordinately expressed, IDI1 binds to the GBD ligand in the scaffold protein GBD-SH3-PDZ, ERG20 binds to the SH3 ligand in the scaffold protein GBD-SH3-PDZ, and SQS binds to the PDZ ligand in the scaffold protein GBD-SH3-PDZ.

4. A method for constructing the recombinant Saccharomyces cerevisiae according to any one of claims 1 to 3, comprising the following steps: The promoters of the endogenous squalene epoxidase genes ERG1 and GAL80 of the starting strain are replaced with a light-controlled promoter containing a C120 DNA binding sequence, and then the plasmid P1 expressing the scaffold protein and the EL222-VP16-NLS nuclear localization light-sensitive element and the plasmid P2 expressing the endogenous isopentenyl pyrophosphate (IPP) isomerase IDI1, the bifunctional farnesyl pyrophosphate (FPP) synthase ERG20 and the squalene synthase SQS are transferred into the recombinant Saccharomyces cerevisiae; The scaffold protein is a GBD-SH3-PDZ scaffold protein, wherein the elements are GBD from rat N-WASP, SH3 from mouse Crk and PDZ from mouse α-syntrophin; The light-controlled promoter is P C120 Promoter.

5. The method according to claim 4, characterized in that: The starting strain is BY4742.

6. The method according to claim 4 or 5, characterized in that: The plasmid P1 is a plasmid of P shown in SEQ ID NO:

1. TEF1 -GBD-(GS)9-SH3-(GS)9-PDZ-T ENO2 -P PGK1 -EL222-VP16-NLS-T ADH2 The fragment is inserted into the NotI site of the pRS415 vector, and the rest of the sequence remains unchanged; and / or The plasmid P2 is a plasmid containing T shown in SEQ ID NO:

6. HXT7 -IDI1-P GAL1 / 10 -ERG20-T CYC1 -P GAL7 -SQS-T PGI1 The fragment replaced the sequence between the XbaI and SacI restriction sites of the pRS416 vector, and the rest of the sequence remained unchanged.

7. A method for preparing squalene by fermentation, comprising the step of fermenting and culturing the recombinant Saccharomyces cerevisiae according to any one of claims 1 to 3.

8. The method according to claim 7, characterized in that: Blue light with a wavelength of 460nm to 500nm and a light intensity of 400 to 600lx is provided 0-30h after the start of fermentation culture. No more light is provided from 30h to the end of fermentation.

9. The method according to claim 7 or 8, characterized in that: The fermentation culture conditions are as follows: initial OD600=0.2-0.5, temperature 28-32°C, and fermentation at 120-250 rpm for 96-120 hours.

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