Saccharomyces cerevisiae engineering strain with enhanced isopentenol tolerance and conversion ability and construction method and application thereof

Abstract

The application belongs to the field of microbial metabolic engineering, and discloses a Saccharomyces cerevisiae engineering strain with enhanced isopentenol tolerance and conversion capacity, and a construction method and application thereof. The Saccharomyces cerevisiae engineering strain is obtained by modifying an isopentenol utilization pathway-dependent Saccharomyces cerevisiae strain, and the energy metabolism of the Saccharomyces cerevisiae strain is enhanced to synthesize a large amount of ATP for the isopentenol utilization pathway; and the antioxidant stress resistance of the Saccharomyces cerevisiae strain is enhanced to relieve the oxidative damage caused by the isopentenol substrate. The Saccharomyces cerevisiae engineering strain is the strain with the highest efficiency in converting isopentenol into terpenes.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial metabolic engineering, and particularly relates to a Saccharomyces cerevisiae engineering strain with enhanced isoprenol tolerance and conversion capacity, and a construction method and application thereof. BACKGROUND

[0002] Terpenoids (isoprenoids) are one of the most structurally diverse and widely distributed natural products in nature, containing more than 90,000 known molecules from monoterpene to polyterpene. This class of substances undertakes key physiological functions such as signal transduction, antioxidant defense and cell structure assembly in organisms. Terpenoid biosynthesis mainly relies on the classical pathways of mevalonate pathway (MVA) and methylerythritol 4-phosphate (MEP). However, the terpenoid synthesis system based on the natural pathway has some inherent defects, such as low carbon flow transmission efficiency, more than 20 steps of reactions are required to generate C5 precursors from glucose via the MVA pathway, and the long path leads to carbon flux loss; path regulation is difficult, the rate-limiting enzymes of the two pathways are inhibited by multiple intermediate products, and multiple cofactors are required.

[0003] Isoprenol utilization (IU) pathway is a newly emerging non-natural synthesis route, which can efficiently convert isoprenol or prenol into C5 precursors IPP and DMAPP through two-step ATP-dependent phosphorylation reaction. The pathway generates IP or DMAP through initial phosphorylation by phosphokinase, and further phosphorylation by isoprenyl phosphate kinase (IPK), which is simple, only depends on ATP, and is more efficient and convenient than the natural pathway. As an important platform for synthesizing terpenoids, Saccharomyces cerevisiae has advantages such as similar cell structure to plants, strong genetic stability, and perfect tool system, which is suitable for introduction and expression of IU pathway. Previous studies have shown that the construction of IU pathway in Saccharomyces cerevisiae can realize the synthesis of various terpenoids, including monoterpene, sesquiterpene, diterpene, triterpene and tetraterpene, which shows good application prospect.

[0004] Isoprenol belongs to short-chain alcohol, which can inhibit the respiration of Saccharomyces cerevisiae and cause the low efficiency of artificial pathway. In order to solve this problem, a strategy of constructing an artificial pathway-dependent (IUPD) strain is proposed, that is, using an artificial pathway to replace the natural pathway. This strategy makes the growth of the strain coupled with the artificial pathway, and "forces" the cell to enhance the respiration to maintain the growth and reproduction, while the efficiency of the artificial pathway is significantly improved. Because the MVA pathway is blocked, acetyl-CoA can no longer be used to synthesize terpenoids, and a large amount of redundant acetyl-CoA can be redirected to the TCA cycle to promote the synthesis of ATP by the cell to provide coenzymes for the IU pathway. In addition, isoprenol as a short-chain alcohol can damage the structure of the mitochondrial membrane, causing electron leakage and ROS generation. When the accumulation of ROS exceeds the clearance capacity of the endogenous antioxidant system, oxidative stress is triggered, leading to protein denaturation, lipid peroxidation and DNA damage, and thus causing cell function decline and even cell death. Therefore, improving the ATP regeneration of the IUPD strain and enhancing the antioxidant stress of the strain to enhance the tolerance to isoprenol are crucial for the synthesis of terpenoids. SUMMARY

[0005] The primary purpose of the present application is to overcome the shortcomings and deficiencies of the prior art, and to provide a Saccharomyces cerevisiae engineering strain with enhanced isoprenol tolerance and conversion capacity.

[0006] Another purpose of the present application is to provide a construction method of the Saccharomyces cerevisiae engineering strain with enhanced isoprenol tolerance and conversion capacity.

[0007] Still another purpose of the present application is to provide an application of the Saccharomyces cerevisiae engineering strain with enhanced isoprenol tolerance and conversion capacity.

[0008] The purposes of the present application are achieved by the following technical solutions:

[0009] A Saccharomyces cerevisiae engineering strain with enhanced isoprenol tolerance and conversion capacity has the following characteristics (1) or the following characteristics (1) and (2):

[0010] (1) overexpressing at least one of acetyl-CoA synthetase ACS1, citrate synthase CIT2, citrate synthase CIT3 and alpha-ketoglutarate dehydrogenase KGD1; preferably overexpressing citrate synthase CIT2 and citrate synthase CIT3, or overexpressing acetyl-CoA synthetase ACS1 and alpha-ketoglutarate dehydrogenase KGD1;

[0011] (2) overexpressing at least one of superoxide dismutase SOD1, stress response transcriptional activator MSN2 and stress response transcriptional activator MSN4; preferably overexpressing stress response transcriptional activator MSN2 and stress response transcriptional activator MSN4, or overexpressing stress response transcriptional activator MSN4;

[0012] The starting strain of the Saccharomyces cerevisiae engineering strain with enhanced isoprenol tolerance and conversion ability is a Saccharomyces cerevisiae strain dependent on an isoprenol utilization pathway, which has the following characteristics: the mevalonate pathway is blocked, the 156th amino acid in the PRM10 gene is mutated from leucine to glutamine; and IDI1, SmDAGK S47A, L124A and AtIPK S270P, A272R are expressed.

[0013] The mevalonate pathway is blocked by deleting or generating a frameshift mutation in the ERG13 gene in the mevalonate pathway through gene editing technology.

[0014] The chassis cell of the Saccharomyces cerevisiae strain dependent on an isoprenol utilization pathway is preferably a Saccharomyces cerevisiae strain of the CEN.PK series; more preferably Saccharomyces cerevisiae CEN.PK2-1C.

[0015] The amino acid sequence of IDI1 is shown in accession number Genbank NP_015208, and there are 288 amino acids in total.

[0016] The SmDAGK S47A, L124A refers to the mutation of the 47th amino acid from serine S to alanine A and the 124th amino acid from leucine L to alanine A.

[0017] The amino acid sequence of SmDAGK is shown in accession number Genbank AAA26867.1, and there are 137 amino acids in total.

[0018] The AtIPK S270P, A272R refers to the mutation of the 270th amino acid from serine S to proline P and the 272nd amino acid from alanine A to arginine R.

[0019] The amino acid sequence of AtIPK is shown in accession number Genbank NP_173986.2, and there are 332 amino acids in total.

[0020] The copy number of the coding gene of IDI1 is preferably 3.

[0021] The copy number of the coding gene of SmDAGK S47A, L124A is preferably 3.

[0022] The coding gene of the AtIPK S270P, A272R The copy number of the coding gene of the AtIPK is preferably 3.

[0023] The starting strain of the Saccharomyces cerevisiae engineering strain with enhanced isopentenol tolerance and transformation ability is preferably strain IUP7.

[0024] The amino acid sequence of the acetyl-CoA synthetase ACS1 is shown in the accession number Genbank NP_009347.1, and the total number of amino acids is 713.

[0025] The sequence of the coding nucleic acid of the acetyl-CoA synthetase ACS1 is shown in SEQ ID NO. 1.

[0026] The amino acid sequence of the citrate synthase CIT2 is shown in the accession number Genbank NP_009931.1, and the total number of amino acids is 460.

[0027] The sequence of the coding nucleic acid of the citrate synthase CIT2 is shown in SEQ ID NO. 2.

[0028] The amino acid sequence of the citrate synthase CIT3 is shown in the accession number Genbank NP_015325.1, and the total number of amino acids is 486.

[0029] The sequence of the coding nucleic acid of the citrate synthase CIT3 is shown in SEQ ID NO. 3.

[0030] The amino acid sequence of the alpha-ketoglutarate dehydrogenase KGD1 is shown in the accession number Genbank NP_012141, and the total number of amino acids is 1014.

[0031] The sequence of the coding nucleic acid of the alpha-ketoglutarate dehydrogenase KGD1 is shown in SEQ ID NO. 4.

[0032] The amino acid sequence of the superoxide dismutase SOD1 is shown in the accession number Genbank NP_012638, and the total number of amino acids is 154.

[0033] The sequence of the coding nucleic acid of the superoxide dismutase SOD1 is shown in SEQ ID NO. 5.

[0034] The amino acid sequence of the stress response transcriptional activator MSN2 is shown in the accession number Genbank NP_013751.1, and the total number of amino acids is 704.

[0035] The sequence of the coding nucleic acid of the stress response transcriptional activator MSN2 is shown in SEQ ID NO. 6.

[0036] The amino acid sequence of the stress response transcriptional activator MSN4 is shown in Genbank NP_012861.1, which contains 630 amino acids.

[0037] The nucleic acid sequence encoding the stress response transcriptional activator MSN4 is shown in SEQ ID NO. 7.

[0038] The overexpression is to introduce the target gene into a host cell for expression.

[0039] The overexpression mode includes free expression and expression integrated in the genome.

[0040] The Saccharomyces cerevisiae engineering strain with enhanced isoprenol tolerance and conversion capacity preferably has the following characteristics: overexpression of acetyl-CoA synthase ACS1, alpha-ketoglutarate dehydrogenase KGD1, and stress response transcriptional activator MSN4; or overexpression of acetyl-CoA synthase ACS1, alpha-ketoglutarate dehydrogenase KGD1, overexpression of stress response transcriptional activator MSN2, and stress response transcriptional activator MSN4.

[0041] The overexpression mode is expression integrated in the genome; preferably expression integrated in the genome DPP1 site and XI-3 site, wherein acetyl-CoA synthase ACS1 and alpha-ketoglutarate dehydrogenase KGD1 are integrated in the genome DPP1 site, stress response transcriptional activator MSN4 is integrated in the genome XI-3 site, or stress response transcriptional activator MSN2 and stress response transcriptional activator MSN4 are integrated in the genome XI-3 site.

[0042] The method for constructing the Saccharomyces cerevisiae engineering strain with enhanced isoprenol tolerance and conversion capacity comprises the following steps:

[0043] 1) Constructing a fragment capable of expressing acetyl-CoA synthase ACS1 and alpha-ketoglutarate dehydrogenase KGD1, and introducing it into an isoprenol utilization pathway-dependent Saccharomyces cerevisiae strain, and through homologous recombination or gene editing technology, integrating the fragment capable of expressing acetyl-CoA synthase ACS1 and alpha-ketoglutarate dehydrogenase KGD1 into the DPP1 site of the genome to obtain strain A;

[0044] 2) Constructing a fragment capable of expressing stress response transcriptional activator MSN4, and introducing it into strain A, and through homologous recombination or gene editing technology, integrating the fragment capable of expressing stress response transcriptional activator MSN4 into the XI-3 site of the genome to obtain strain IUP16;

[0045] 3) Constructing a fragment capable of expressing stress response transcriptional activator MSN2 and stress response transcriptional activator MSN4, transforming into strain A, and integrating the fragment capable of expressing stress response transcriptional activator MSN2 and stress response transcriptional activator MSN4 into the XI-3 site of the genome by homologous recombination or gene editing technology to obtain strain IUP17.

[0046] The acetyl-CoA synthase ACS1 and the alpha-ketoglutarate dehydrogenase KGD1 in the fragment described in step 1) are independently expressed, and the structure is preferably as follows: T CYC1 - ACS1 - P GAL1 - P GAL10 - KGD1 - T ADH1 .

[0047] The isopentenol utilization pathway-dependent Saccharomyces cerevisiae strain described in step 1) is preferably strain IUP7.

[0048] The structure of the fragment described in step 2) is as follows: P GAL1 - MSN4 - T ADH1 .

[0049] The stress response transcriptional activator MSN2 and the stress response transcriptional activator MSN4 in the fragment described in step 3) are independently expressed, and the structure is preferably as follows: T ADH1 - MSN4 - P GAL1 - P GAL10 - MSN2 - T CYC1 .

[0050] The sequence of the nucleic acid encoding the acetyl-CoA synthase ACS1 is shown in SEQ ID NO. 1.

[0051] The sequence of the nucleic acid encoding the citrate synthase CIT2 is shown in SEQ ID NO. 2.

[0052] The sequence of the nucleic acid encoding the citrate synthase CIT3 is shown in SEQ ID NO. 3.

[0053] The sequence of the nucleic acid encoding the alpha-ketoglutarate dehydrogenase KGD1 is shown in SEQ ID NO. 4.

[0054] The sequence of the nucleic acid encoding the superoxide dismutase SOD1 is shown in SEQ ID NO. 5.

[0055] The sequence of the nucleic acid encoding the stress response transcriptional activator MSN2 is shown in SEQ ID NO. 6.

[0056] The sequence of the coding nucleic acid of the stress response transcriptional activator MSN4 is shown as SEQ ID NO. 7.

[0057] The Saccharomyces cerevisiae engineering strain with enhanced isoprenol tolerance and conversion capacity can tolerate an environment with a 3-methyl-3-buten-1-ol (isoprenol) concentration of 4 g / L or above; preferably an environment with a 3-methyl-3-buten-1-ol (isoprenol) concentration of 6-12 g / L or above; more preferably an environment with a 3-methyl-3-buten-1-ol (isoprenol) concentration of 8-12 g / L or above.

[0058] The Saccharomyces cerevisiae engineering strain with enhanced isoprenol tolerance and conversion capacity is applied in the preparation of flavonoids and terpenoids; preferably applied in the preparation of flavonoids and terpenoids with 3-methyl-3-buten-1-ol (isoprenol) as a substrate.

[0059] The 3-methyl-3-buten-1-ol has a concentration of 4-12 g / L; preferably 6-12 g / L; more preferably 8-12 g / L.

[0060] The terpenoid is preferably squalene.

[0061] The present application has the following advantages and effects relative to the prior art:

[0062] (1) The engineering strain provided by the present application has strong energy metabolism activity, can synthesize a large amount of ATP for the IU pathway to provide energy, and is the engineering strain with the highest efficiency in converting isoprenol into terpenoids at present.

[0063] (2) The engineering strain provided by the present application has high isoprenol tolerance, and can maintain high cell growth and squalene accumulation under a high concentration of isoprenol. BRIEF DESCRIPTION OF DRAWINGS

[0064] Figure 1 is a result graph of the influence of overexpression of key genes of central carbon metabolism in different strains on squalene accumulation; wherein a is the determination result of squalene accumulation of the recombinant strain obtained by taking IUP5 as the starting strain, and b is the determination result of squalene accumulation of the recombinant strain obtained by taking IUP7 as the starting strain.

[0065] Figure 2 is a result graph of the influence of combined expression of key genes of central carbon metabolism in the chromosome on squalene accumulation.

[0066] Figure 3Figure 1 is a graph showing the results of central carbon metabolism strengthening to increase ATP synthesis to enhance isoprenol tolerance; wherein a is the determination result of squalene accumulation amount, and b is the determination result of growth amount.

[0067] Figure 4 Figure 2 is a graph showing the influence of overexpression of antioxidant stress factors on squalene accumulation.

[0068] Figure 5 Figure 3 is a graph showing the influence of combined expression of antioxidant stress factors in the chromosome on squalene accumulation and growth of the strain; wherein a is the determination result of squalene accumulation amount, and b is the determination result of growth amount. DETAILED DESCRIPTION

[0069] The application will be further described in conjunction with the embodiments and the accompanying drawings, but the embodiments of the application are not limited thereto.

[0070] In the following embodiments, if the specific test conditions are not specified, the general test conditions or the test conditions recommended by the reagent company are generally used. If not specified, the materials, reagents, etc. used are reagents and materials obtained from commercial channels.

[0071] Routine PCR amplification: high-fidelity PrimeSTAR Max DNA polymerase (Takara, Japan) was used.

[0072] PCR product recovery: after product recovery using SanPrep column PCR product purification kit (Shanghai Sangon Biotech), the DNA concentration was determined using ultramicro spectrophotometer (K5600C, Beijing Kai Ao Technology Development Co., Ltd.).

[0073] Fusion PCR: 10 ng of each fragment needed for fusion was taken, and the upstream primer of the first segment and the downstream primer of the end were added, and the amplification system and PCR conditions were consistent with routine PCR amplification.

[0074] Gibson assembly: the specific operation was performed according to the 2X MultiF Seamless Assembly Mix instruction manual of Abudantam Biotech.

[0075] DH5α competent cells: purchased from Shanghai Sangon Biotech, and the transformation method was described in the product instruction manual.

[0076] Fermentation of engineering strains: a single colony was selected and inoculated in 12 mL culture tube containing 2 mL YPD (containing 2 g / L isoprenol) medium, and seed liquid was obtained by overnight culture at 30°C and 220 rpm. An appropriate amount of seed liquid was inoculated in each well of 48-well plate containing 1 mL YPD (containing different concentrations of isoprenol) medium, and fermented for 4 days.

[0077] Squalene assay: Take 0.2 mL of fermentation broth, add 0.7 g of 0.5 mm glass beads and 1 mL of ethyl acetate into a 2 mL homogenizer tube, break the cells by a biological sample homogenizer (Bioprep-24R, Hangzhou Aosheng Instrument Co., Ltd.), and centrifuge at 10000 g for 1 min. Take the upper ethyl acetate phase, filter through a 0.22 μm nylon organic filter membrane, and perform HPLC analysis. The instrument uses Shimadzu LC-16 (Shimadzu Corporation, Japan) equipped with an SPD-16 detector; the chromatographic column is Agilent Poroshell 120EC-C18 (2.1 × 100 mm); the mobile phase is pure acetonitrile for isocratic elution, the flow rate is 0.5 mL / min, the injection volume is 2 μL, and the elution time is 7.5 min.

[0078] The primers used in the application are shown in Tables 1 and 2:

[0079] Table 1 Primers

[0080]

[0081] Table 2 Primers

[0082]

[0083] Example 1: Free overexpression of central carbon metabolism key genes

[0084] After the MVA pathway of the IUPD strain is blocked, AcCoA is no longer used for terpenoid synthesis, and there is abundant carbon resource available for utilization in central carbon metabolism. According to the metabolic pathway, the genes GUT1, GUT2 involved in glycerol conversion and ADH2, ALD4, ALD6, ACS1, ACS2 involved in ethanol conversion are overexpressed, which can improve the utilization of fermentation products by Saccharomyces cerevisiae; the redundant AcCoA can flow to the TCA cycle through the proteins encoded by CIT1, CIT2 and CIT3; overexpression of IDP2 and KGD1 can promote the TCA cycle to produce more reducing power for energy metabolism; overexpression of NADH dehydrogenase NDE1 and NDE2 on the inner mitochondrial membrane is often used to strengthen the electron transport chain to promote ATP synthesis. The endogenous gene sequences of Saccharomyces cerevisiae can be downloaded from the yeast genome database (https: / / www.yeastgenome.org).

[0085] Taking the construction of ACS1 overexpression plasmid vector as an example, p426-T CYC1 -P GPD -P TEF1 -T ADH1The plasmid (disclosed in supplementary file 5 in the literature “Li GJ, et al. Yeast metabolism adaptation for efficient terpenoids synthesis via isopentenol utilization. Nature Communications, 2024, Vol 15, Issue 1”) was used as a template to amplify the plasmid backbone using the primer pair p426-F / p426-R. The Saccharomyces cerevisiae CEN.PK2-1C genome was used as a template to amplify the ACS1 gene using the primer pair ACS1-F / ACS1-R. The nucleotide sequence of the amplified product is shown as SEQ ID NO. 1. After the products were recovered, the Gibson assembly method was used to obtain p426-ACS1-T TEF1 -ACS1-T ADH1 plasmid. The overexpression plasmid vectors of GUT1, GUT2, ADH2, ALD4, ALD6, ACS2, CIT1, CIT2, CIT3, IDP2, KGD1, NDE1 and NDE2 genes were obtained in the same way, which were p426-P TEF1 -GUT1-T ADH1 , p426-P TEF1 -GUT2-T ADH1 , p426-P TEF1 -ADH2-T ADH1 , p426-P TEF1 -ALD4-T ADH1 , p426-P TEF1 - ALD6-T ADH1 , p426-P TEF1 -ACS2-T ADH1 , p426-P TEF1 -CIT1-T ADH1 , p426-P TEF1 -CIT2-T ADH1 , p426-P TEF1 -CIT3-T ADH1 , p426-P TEF1 -IDP2-T ADH1 , p426-P TEF1 -KGD1-T ADH1 , p426-P TEF1 -NDE1-T ADH1 , p426-P TEF1 -NDE2-T ADH1 , wherein the primers used are shown in Table 1, and the template is the Saccharomyces cerevisiae CEN.PK2-1C genome. The constructed plasmids and p426-TCYC1 -P GPD -P TEF1 -T ADH1 The control plasmids were transformed into IUP5 strains (which have been disclosed in the literature "Li GJ, et al. Yeast metabolism adaptation for efficient terpenoids synthesis via isopentenol utilization. Nature Communications, 2024, Vol 15, Issue 1") respectively, fermented using YPD medium containing 2 g / L isoprenol, and the squalene content was determined. The results are shown in Figure Figure 1 As shown in a of Figure 1, strengthening cell energy metabolism cannot promote the conversion of isoprenol, and also causes metabolic disorders to affect cell growth, ultimately reducing the flux of the IU pathway. It shows that the ATP supply of IUP5 strain is sufficient to maintain the flux of IU pathway, at this time, energy metabolism is not the rate-limiting factor, and the problem of low IU pathway flux needs to be solved first.

[0086] In order to improve the flux of IU pathway, the strategy of increasing the gene copy number of isoprenol utilization pathway was adopted. IUP7 strain was used as host, which was derived from IUP5 (which has been disclosed in the literature "Li GJ, et al. Yeast metabolism adaptation for efficient terpenoids synthesis via isopentenol utilization. Nature Communications, 2024, Vol 15, Issue 1") and the copy number of SmDAGK S47A, L124A , AtIPK S270P, A272R and IDI1 genes on the chromosome was increased to three, with higher isoprenol conversion efficiency. The above plasmids and p426-T CYC1 -P GPD -P TEF1 -T ADH1 The control plasmids were transformed into IUP7 respectively, fermented using YPD medium containing 2 g / L isoprenol, and the squalene content was determined. The results are shown in b of Figure Figure 1 Compared with the control group, overexpression of ACS1, CIT2, CIT3 and KGD1 genes can increase the squalene accumulation of the strain by 17.3%, 26.7%, 34.6% and 35.2% respectively.

[0087] Example 2: Chromosomal integration of central carbon metabolism key genes for overexpression

[0088] To get the best combination of central carbon metabolism key genes expression, different combinations of genes were integrated into the chromosome of IUP7 strain, CIT2+CIT3, KGD1+ACS1, CIT2+CIT3+ACS1, CIT2+CIT3+ACS1+KGD1 were integrated respectively.

[0089] pRS415 (obtained from addgene global plasmid sharing platform) as template, primer pair pRS415-F / pRS415-R, Leu2-F / Leu2-R were used to amplify pRS415 backbone and Leu2 tag respectively; Saccharomyces cerevisiae CEN.PK2-1C genome as template, primer pair LPP1up-F / LPP1up-R, LPP1down-F / LPP1down-R, CIT2-F / CIT2-R, P GAL1, 10 -F / P GAL1, 10 -R were used to amplify LPP1up, LPP1down, CIT2 gene and P GAL1, 10 bidirectional promoter respectively; p426-T CYC1 -P GPD -P TEF1 -T ADH1 plasmid as template, primer pair T CYC1 -F / T CYC1 -R were used to amplify T CYC1 terminator; p426-P TEF1 -CIT3-T ADH1 as template, primer pair CIT3-T ADH1 -F / CIT3-T ADH1 -R were used to amplify CIT3-T ADH1 . Using fusion PCR method, T CYC1 terminator, Leu2 tag and homologous arm LPP1down were fused to obtain T CYC1 -Leu2-LPP1down fragment, CIT3-T ADH1 and homologous arm LPP1up were fused to obtain CIT3-T ADH1 -LPP1up fragment. Using Gibson assembly method, T CYC1 -Leu2-LPP1down fragment, CIT3-T ADH1 -LPP1up fragment, pRS415 backbone, P GAL1, 10 bidirectional promoter and CIT2 gene were assembled into pRS415-T CYC1 -CIT2-P GAL1 -P GAL10 -CIT3-T ADH1Plasmid for chromosomal homologous recombination of CIT2 and CIT3 genes.

[0090] pRS414 backbone and TRP1 tag were amplified from pRS414 (obtained from addgene global plasmid sharing platform) using primer pairs pRS414-F / pRS414-R and TRP1-F / TRP1-R, respectively; DPP1 up, DPP1 down and ACS1 genes for DPP1 site integration were amplified from S. cerevisiae CEN.PK2-1C genome using primer pairs DPP1up-F / DPP1up-R, DPP1down-F / DPP1down-R and ACS1a-F / ACS1a-R, respectively; p426-P TEF1 -KGD1-T ADH1 As template, KGD1-T ADH1 -F / KGD1-T ADH1 -R were used to amplify KGD1-T ADH1 fragment. Using fusion PCR method, T CYC1 terminator, TRP1 tag and homology arm DPP1up were fused to obtain T CYC1 -TRP1-DPP1up fragment; KGD1-T ADH1 fragment and homology arm DPP1up were fused to obtain KGD1-T ADH1 -DPP1up fragment. Using Gibson assembly method, T CYC1 -TRP1-DPP1up fragment, KGD1-T ADH1 -DPP1up fragment, pRS414 backbone, P GAL1, 10 bidirectional promoter and ACS1 were assembled into pRS414-T CYC1 -ACS1-P GAL1 -P GAL10 -KGD1-T ADH1 Plasmid for chromosomal homologous recombination of ACS1 and KGD1 genes. pRS414-T CYC1 -ACS1-P GAL1 -P GAL10 -KGD1-T ADH1 Plasmid as template, P GAL1, 10 -F / T ADH1 (2)-R were used to amplify, and the PCR product was recovered to obtain pRS414-P GAL1 -ACS1-T CYC1 Plasmid.

[0091] pRS415-TCYC1 -CIT2-P GAL1 -P GAL10 -CIT3-T ADH1 The plasmid was used as a template, and a primer pair of LPP1up-F / LPP1down-R was used for amplification to obtain a homologous recombination fragment of CIT2 and CIT3 integrated into the LPP1 site of the engineered Saccharomyces cerevisiae strain; pRS414-P GAL1 -ACS1-T CYC1 The plasmid was used as a template, and a primer pair of DPP1down-F / DPP1up-R was used for amplification to obtain a homologous recombination fragment of ACS1 integrated into the DPP1 site of the engineered Saccharomyces cerevisiae strain; pRS414-T CYC1 -ACS1-P GAL1 -P GAL10 -KGD1-T ADH1 The plasmid was used as a template, and a primer pair of DPP1down-F / DPP1up-R was used for amplification to obtain a homologous recombination fragment of ACS1 and KGD1 integrated into the DPP1 site of the engineered Saccharomyces cerevisiae strain. The above fragments were transformed into the IUP7 strain using a Frozen-EZ Yeast Transformation II™ kit, and the transformation agar plates for the integration of the LPP1 and DPP1 sites were leucine-deficient and tryptophan-deficient YNB agar plates (2 g / L isoprenol was added). The engineered strains integrated with CIT2+CIT3, KGD1+ACS1, CIT2+CIT3+ACS1, and CIT2+CIT3+ACS1+KGD1 genes were named as IUP12, IUP13, IUP14, and IUP15 strains, respectively. Fermentation was performed in YPD medium containing 4 g / L isoprenol, and the results are shown in Figure 2 As shown in a of FIG. 6, the squalene accumulation of the IUP12 and IUP13 strains was 13.8% and 15.1% higher than that of the starting strain IUP7, which was 576.9 mg / L and 583.3 mg / L, respectively, and the squalene accumulation of the IUP14 and IUP15 strains was equivalent to or even slightly lower than that of IUP7; when fermentation was performed with the addition of 6 g / L isoprenol, the results are shown in Figure 3 As shown in b of FIG. 6, the squalene accumulation of the IUP7 strain was severely decreased, while the squalene accumulation of the IUP12, IUP13, IUP14, and IUP15 strains changed little, indicating that the IUP12, IUP13, IUP14, and IUP15 strains not only had a higher squalene accumulation, but also had a higher substrate tolerance. Figure 3

[0092] Example 3: Chromosomal integration overexpression of antioxidant stress-related genes

[0093] ​The mitochondrial electron transport chain is a significant source of endogenous reactive oxygen species (ROS). During periods of high energy metabolism, excessive activity in the electron transport chain can lead to electron leakage. Furthermore, isopentenol, an alcohol compound, readily disrupts the mitochondrial membrane structure, further exacerbating electron leakage and resulting in excessive ROS production. When the rate of ROS production exceeds the scavenging capacity of the endogenous antioxidant system, oxidative stress is induced, leading to protein denaturation, lipid peroxidation, and DNA damage, ultimately causing a gradual decline in cell function and inducing cell death. Cells possess endogenous antioxidant defense mechanisms, including antioxidant enzyme systems (such as superoxide dismutase, catalase, and glutathione peroxidase) and non-enzymatic antioxidants (such as glutathione). These components effectively scavenge excess ROS, preventing oxidative stress from damaging cells and thus maintaining normal cellular physiological functions. To mitigate the adverse effects of excessive ROS accumulation caused by the addition of isopentenol and active energy metabolism, eight genes, namely CCP1, HSP104, SOD1, SOD2, GSH1, YAP1, MSN2, and MSN4, were selected for overexpression to improve the intracellular redox environment. The relevant information of these genes is shown in Table 3.

[0094] Table 3. Genes related to oxidative stress response in Saccharomyces cerevisiae

[0095]

[0096] Following Example 1, overexpression plasmid vectors for the genes CCP1, HSP104, SOD1, SOD2, GSH1, YAP1, MSN2, and MSN4 were constructed, resulting in plasmid p426-P. TEF1 -CCP1-T ADH1 p426-P TEF1 -HSP104-T ADH1 p426-P TEF1 -SOD1-T ADH1 p426-P TEF1 -SOD2-T ADH1 p426-P TEF1 -GSH1-T ADH1 p426-P TEF1 -YAP1-T ADH1 p426-P TEF1 -MSN2-T ADH1 p426-P TEF1 -MSN4-T ADH1 The primers are shown in Table 1, and the template is the *Saccharomyces cerevisiae* CEN.PK2-1C genome. The constructed plasmid and p426-T... CYC1 -P GPD -P TEF1-T ADH1 As control plasmids, they were transformed into IUP12 strain respectively, fermented in YPD medium containing 8 g / L isoprenol, and the squalene content was determined, and the results are shown in Table 1. Figure 4 Compared with the control group IUP12 strain, overexpression of SOD1, MSN2 and MSN4 genes respectively can increase the squalene accumulation of the strain by 9.7%, 30.2% and 20.3% respectively.

[0097] Example 4: Chromosomal integration overexpression of antioxidant stress related genes

[0098] p426-T CYC1 -P GPD -P TEF1 -T ADH1 The plasmid was used as a template, and primer pair T CYC1 -F / T ADH1 -R was used for amplification to obtain the p426 skeleton; the genome of Saccharomyces cerevisiae CEN.PK2-1C was used as a template, and primer pair MSN4(2)-F / MSN4(2)-R and primer pair MSN2(2)-F / MSN2(2)-R were used for amplification to obtain the MSN4 gene and the MSN2 gene. Using the Gibson assembly method, the p426 skeleton, the MSN4 gene, the MSN2 gene and the P GAL1, 10 bidirectional promoter in Example 3 were assembled into p426-T ADH1 -MSN4-P GAL1 -P GAL10 -MSN2-T CYC1 plasmid. The p426-T ADH1 -MSN4-P GAL1 -P GAL10 -MSN2-T CYC1 plasmid was used as a template, and primer pair T CYC1 -F / P GAL1, 10 (2)-R was used for amplification, and the PCR product was recovered to obtain the p426-P GAL1 -MSN4-T ADH1 plasmid.

[0099] Gene marker was performed using CRISPR-Cas9 tool, pYZ463 (obtained from addgene global plasmid sharing platform, Plasmid # 187971, TEF1p-Cas9-CYC1t and SNR52p-Not1-SUP4t) was used as template, primer pair XI-3sgRNA-F / XI-3sgRNA-R was used for amplification, E. coli DH5a was used for transformation, plasmid extraction and sequencing verification, finally, Crispr-Cas9 plasmid with 5'-GTAGAAATCAGACGCACGCT-3' as sgRNA was obtained, which was pYZ463-XI-3, targeting XI-3 site of S. cerevisiae. p426-PMSN4-T, p426-PMSN4-P, p426-PMSN2-T and p426-PMSN2-P were used as templates, primer pair DonorXI3-F / DonorXI3-R was used for amplification, and Donor DNA for integrating MSN4 gene and integrating MSN2 and MSN4 genes was obtained. The above fragments were transformed into IUP13 strain using Frozen-EZ Yeast Transformation II™ kit, and the plates used for transformation were YNB agar plates (adding 2 g / L isoprenol) with uracil deficiency, and IUP16 and IUP17 strains were obtained after colony PCR. GAL1 p426-PMSN4-T ADH1 p426-PMSN4-P ADH1 p426-PMSN2-T GAL1 p426-PMSN2-P GAL10 p426-PMSN4-T CYC1 p426-PMSN4-P Using the above fragments as templates, primer pair DonorXI3-F / DonorXI3-R was used for amplification, and Donor DNA for integrating MSN4 gene and integrating MSN2 and MSN4 genes was obtained. The above fragments were transformed into IUP13 strain using Frozen-EZ Yeast Transformation II™ kit, and the plates used for transformation were YNB agar plates (adding 2 g / L isoprenol) with uracil deficiency, and IUP16 and IUP17 strains were obtained after colony PCR.

[0100] The results are shown in Table 1. Figure 5 The starting strain IUP13 could not grow under the addition of high concentration of isoprenol; IUP16 strain obtained by overexpressing MSN4 in IUP13 had increased isoprenol tolerance and a small amount of squalene accumulation; IUP17 strain obtained by simultaneously overexpressing MSN4 and MSN2 in IUP13 had further increased isoprenol tolerance, and the growth of the strain and squalene accumulation were greatly enhanced.

[0101] The above embodiments are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above embodiments, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and are all included in the protection scope of the present application.

Claims

1. A strain of *Saccharomyces cerevisiae* with enhanced isopentenol tolerance and conversion ability, characterized in that... It has the following characteristics (1) or has the following characteristics (1) and (2): (1) Overexpression of citrate synthase CIT2 and citrate synthase CIT3, or overexpression of acetyl-CoA synthase ACS1 and α-ketoglutarate dehydrogenase KGD1; (2) Overexpression of stress response transcription activator MSN2 and stress response transcription activator MSN4, or overexpression of stress response transcription activator MSN4; The starting strain of the engineered Saccharomyces cerevisiae with enhanced isopentenol tolerance and transformation ability is strain IUP7.

2. The engineered Saccharomyces cerevisiae strain with enhanced isopentenol tolerance and conversion ability according to claim 1, characterized in that... It has the following characteristics: overexpression of acetyl-CoA synthase ACS1, α-ketoglutarate dehydrogenase KGD1, and stress response transcription activator MSN4; or overexpression of acetyl-CoA synthase ACS1, α-ketoglutarate dehydrogenase KGD1, and overexpression of stress response transcription activator MSN2 and stress response transcription activator MSN4.

3. The engineered Saccharomyces cerevisiae strain with enhanced isopentenol tolerance and conversion ability according to claim 2, characterized in that: The acetyl-CoA synthase ACS1 and the α-ketoglutarate dehydrogenase KGD1 are integrated into the DPP1 site of the genome; The stress response transcription activator MSN2 is integrated into the XI-3 site of the genome, or the stress response transcription activator MSN2 and stress response transcription activator MSN4 are integrated into the XI-3 site of the genome.

4. The engineered Saccharomyces cerevisiae strain with enhanced isopentenol tolerance and conversion ability according to any one of claims 1 to 3, characterized in that: The amino acid sequence of the acetyl-CoA synthase ACS1 is shown in Genbank accession number NP_009347.1; The amino acid sequence of the citrate synthase CIT2 is shown in Genbank accession number NP_009931.1; The amino acid sequence of the citrate synthase CIT3 is shown in Genbank accession number NP_015325.1; The amino acid sequence of the α-ketoglutarate dehydrogenase KGD1 is shown in Genbank accession number NP_012141.1; The amino acid sequence of the stress response transcription activator MSN2 is shown in Genbank accession number NP_013751.1; The amino acid sequence of the stress response transcription activator MSN4 is shown in Genbank accession number NP_012861.

1.

5. The method for constructing the engineered Saccharomyces cerevisiae strain with enhanced isopentenol tolerance and conversion ability as described in claim 3, characterized in that... Includes the following steps: 1) Construct a fragment that can express acetyl-CoA synthase ACS1 and α-ketoglutarate dehydrogenase KGD1, transform it into an isopentenol utilization pathway-dependent Saccharomyces cerevisiae strain, and integrate the fragment that can express acetyl-CoA synthase ACS1 and α-ketoglutarate dehydrogenase KGD1 into the DPP1 site of the genome through homologous recombination or gene editing technology to obtain strain A; 2) Construct a fragment that can express the stress response transcription activator MSN4, transform it into strain A, and integrate the fragment that can express the stress response transcription activator MSN4 into the XI-3 site of the genome through homologous recombination or gene editing technology to obtain strain IUP16; 3) Construct a fragment that can express stress response transcription activator MSN2 and stress response transcription activator MSN4, transform it into strain A, and integrate the fragment that can express stress response transcription activator MSN2 and stress response transcription activator MSN4 into the XI-3 site of the genome through homologous recombination or gene editing technology to obtain strain IUP17; The isopentenol utilization pathway-dependent Saccharomyces cerevisiae strain mentioned in step 1) is strain IUP7.

6. The method for constructing the engineered Saccharomyces cerevisiae strain with enhanced isopentenol tolerance and conversion ability according to claim 5, characterized in that: In the fragment described in step 1), acetyl-CoA synthase ACS1 and α-ketoglutarate dehydrogenase KGD1 are independently expressed; In the fragment described in step 3), stress response transcription activator MSN2 and stress response transcription activator MSN4 are expressed independently.

7. The method for constructing the engineered Saccharomyces cerevisiae strain with enhanced isopentenol tolerance and conversion ability according to claim 6, characterized in that: The structure of the fragment described in step 1) is as follows: T CYC1 -ACS1-P GAL1 -P GAL10 -KGD1-T ADH1 ; The structure of the fragment described in step 2) is as follows: P GAL1 -MSN4-T ADH1 ; The structure of the fragment described in step 3) is as follows: T ADH1 -MSN4-P GAL1 -P GAL10 -MSN2-T CYC1 .

8. The application of the engineered Saccharomyces cerevisiae strain with enhanced isopentenol tolerance and conversion ability as described in claim 2 or 3 in the preparation of squalene.

9. The application according to claim 8, characterized in that: The application described uses 3-methyl-3-buten-1-ol as a substrate; The concentration of the 3-methyl-3-buten-1-ol is 4–12 g / L.

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