Saccharomyces cerevisiae engineering bacteria for efficiently synthesizing mogroside V based on cytoplasm and peroxisome double-cell-region compartment and construction method of saccharomyces cerevisiae engineering bacteria

Abstract

The invention discloses a saccharomyces cerevisiae engineering bacterium for efficiently synthesizing mogroside V based on cytoplasm and peroxisome double-cell-region indoor and a construction method of the saccharomyces cerevisiae engineering bacterium. Wherein the construction method comprises the following steps: integrating acetoacetyl coenzyme A thiolase ERG10, 3-hydroxy-3-methylglutaryl coenzyme A synthetase ERG13, truncated 3-hydroxy-3-methylglutaryl coenzyme A reductase tHMG1, phosphomevalonate kinase ERG8, mevalonate pyrophosphate decarboxylase ERG19, pentenyl pyrophosphate isomerase IDI1 and geranyl pyrophosphate synthase ERG20 at a site of a saccharomyces cerevisiae genome GAL80, and carrying out enzymolysis, so as to obtain a saccharomyces cerevisiae strain; a squalene synthase ERG9, an epoxy squalene cyclization enzyme ERG1 and the like; aBC efflux protein PDR11, isopentene phosphokinase AtIPK, geranyl pyrophosphate synthase ERG20, endogenous squalene synthetase ERG9, epoxy squalene cyclization enzyme ERG1 and the like are integrated at a site Exg1 of a saccharomyces cerevisiae genome. The yield of MG-V synthesized by shake flask fermentation of the saccharomyces cerevisiae engineering bacteria constructed by the invention reaches 117.6 mg / L, and the saccharomyces cerevisiae engineering bacteria have the advantages of high yield, strong metabolic flux directionality, wide industrial application prospect and the like.

Description

Technical Field

[0001] The present invention relates to an engineered yeast of saccharomyces cerevisiae for efficiently synthesizing mogroside V based on cytoplasm and peroxisome dual-cell compartmentalization and a construction method thereof, and belongs to the field of bioengineering. Background Art

[0002] Mogroside, a cucurbitane-type triterpenoid saponin, is not only the main active ingredient in the traditional Chinese medicine Momordica grosvenori (Lotus Grosvenori) but also a non-sugar, natural sweetener. Pharmacological studies have shown that mogroside exhibits antitussive and expectorant, lung-moistening and liver-protecting, anti-inflammatory, anti-cancer, and blood sugar-lowering effects. It has become one of the few novel sweeteners with therapeutic properties discovered from traditional Chinese medicine. Mogroside V is the most abundant and sweetest component of mogrosides. However, current purification and production technologies for mogroside V are relatively low, resulting in increasingly prominent problems such as high production costs, long production times, and low production efficiency. This has made it difficult to meet market demand for mogroside V, necessitating urgent upgrades to the mogroside V industry to reduce production costs and improve efficiency.

[0003] Based on synthetic biology technology, the construction of a synthetic pathway for plant-derived terpenes in a microbial chassis host to achieve efficient synthesis of terpenes has become a new green and efficient production model and is the inevitable path for future development. As a generally recognized safe model microorganism (GRAS), Saccharomyces cerevisiae can endogenously synthesize terpenoid precursors and has a complete membrane structure. It has been widely used in the synthesis of various plant-derived terpenes. At present, the use of Saccharomyces cerevisiae to synthesize artemisinin precursor artemisinic acid and chemical raw material farnesene has been industrialized, which can be regarded as a milestone in the metabolic engineering of microorganisms to produce terpenoid natural products. Mogroside V is a triterpenoid saponin product in the cytoplasm, and acetyl-CoA is a key intermediate in the biosynthetic pathway of yeast mogroside V. Acetyl-CoA produced by β-oxidation of fatty acids in the peroxisome subcellular organelle can serve as a precursor for the biosynthesis of mogroside V. To increase the biosynthetic flux of mogroside V in yeast, this study constructs the isopentenol utilization pathway (IUP) and overexpresses key enzymes in the native mevalonate (MVA) pathway to enhance isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Natural yeast squalene primarily synthesizes ergosterol, where epoxysqualene cyclase ERG1 is a key enzyme in the synthesis of MG-V and ergosterol. By utilizing peptide interactions, one molecule of epoxysqualene cyclase ERG1 and two molecules of cucurbitadienol synthase SgCDS form a tri-enzyme complex, reducing competition within the ergosterol synthesis pathway and promoting the biosynthesis of mogroside V. Mogroside V is synthesized on the peroxisome membrane using oxisome surface display technology and a multi-enzyme complex directed assembly strategy. The α-lectin system is used to display glucosyltransferase on the surface of the yeast cell wall, and the ABC efflux pump PDR11 is used to transport mogroside alcohol to the extracellular space to synthesize MG-V, thereby constructing an engineered Saccharomyces cerevisiae strain that efficiently synthesizes mogroside V based on the dual compartmentalization of the cytoplasm and peroxisomes. The strain synthesizes MG-V at a yield of 117.6 mg / L through shake flask fermentation, which has the advantages of high yield, strong metabolic flow directionality, and broad prospects for industrial application, providing a new technical route for the microbial production of natural sweeteners. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide an engineered yeast of Saccharomyces cerevisiae for efficiently synthesizing mogroside V based on dual compartmentalization of cytoplasm and peroxisome and a construction method thereof.

[0005] The method for constructing an engineered yeast strain of Saccharomyces cerevisiae for efficiently synthesizing mogroside V based on the dual compartmentalization of cytoplasm and peroxisome to solve the above technical problems is as follows:

[0006] (1) Acetoacetyl-CoA thiolase ERG10, 3-hydroxy-3-methylglutaryl-CoA synthetase ERG13, truncated 3-hydroxy-3-methylglutaryl-CoA reductase tHMG1, mevalonate kinase ERG12, phosphomevalonate kinase ERG8, mevalonate pyrophosphate decarboxylase ERG19, pentenyl pyrophosphate isomerase IDI1, geranyl pyrophosphate synthase ERG20, squalene synthase ERG9, epoxysqualene cyclase ERG1, cucurbitadienol synthase SgCDS, epoxyepoxide hydrolase SgEPH3, cytochrome P450 enzyme CYP87D18, and cytochrome P450 enzyme reductase AtCPR1 were integrated into the GAL80 locus of the Saccharomyces cerevisiae genome;

[0007] (2) The ABC efflux protein PDR11, choline kinase ScCK, isopentenyl phosphate kinase AtIPK, geranyl pyrophosphate synthase ERG20, cucurbitadienol synthase SgCDS, endogenous squalene synthase ERG9, epoxysqualene cyclase ERG1, glycosyltransferase UGTMG1, sucrose synthase Susy and glycosyltransferase SgUGT94-289-3 were integrated into the Exg1 locus of the Saccharomyces cerevisiae genome.

[0008] In the step (1):

[0009] The acetoacetyl-CoA thiolase ERG10 (NCBI Sequence ID: NP_015297.1), 3-hydroxy-3-methylglutaryl-CoA synthetase ERG13 (NCBI Sequence ID: NP_013580.1), HMG1 (NCBI Sequence ID: NP_013636.1) N-terminal truncation of 527 amino acids tHMG1, mevalonate kinase ERG12 (NCBI Sequence ID: NP_013935.1), phosphomevalonate kinase ERG8 (NCBI Sequence ID: NP_013947.1), mevalonate pyrophosphate decarboxylase ERG19 (NCBI Sequence ID: NP_014441.1), pentenyl pyrophosphate isomerase IDI1 (NCBI Sequence ID: NP_015208.1), geranyl pyrophosphate synthase ERG20 (NCBI Sequence ID: NP_014430.1), and pyrophosphate decarboxylase ERG19 (NCBI Sequence ID: NP_014431.1) were also studied. ID: NP_012368.1), endogenous squalene synthase ERG9 (NCBI Sequence ID: NP_012060.1).

[0010] The amino and carboxyl ends of the epoxysqualene cyclase ERG1 (NCBI Sequence ID: NP_011691.1) are respectively connected to the erythromycin polyketide synthase polypeptide interaction tag D5 through a flexible linker (GGGGS) 3 N DD (nucleotide sequence shown in SEQ ID NO. 1) and the natural anchor motif Pex15 (amino acid sequence shown in SEQ ID NO. 2) are fused.

[0011] The amino and carboxyl ends of the cucurbitacinol synthase SgCDS (NCBI Sequence ID: K7NBZ9.1) interact with the chlortetracycline polyketide synthase polypeptide tag A3 through a flexible linker (GGGGS) 3. N DD (nucleotide sequence shown in SEQ ID NO.3) and erythromycin polyketide synthase polypeptide interaction tag D2 C DD (nucleotide sequence as SEQ ID NO.4) fusion.

[0012] The amino and carboxyl ends of the epoxide hydrolase SgEPH3 (NCBI Sequence ID: P0DO70.1) are respectively connected to the erythromycin polyketide synthase polypeptide interaction tag D3 through a flexible linker (GGGGS) 3. N DD (nucleotide sequence as SEQ ID NO.5) and D4 C DD (nucleotide sequence as SEQ ID NO.6) fusion.

[0013] The amino terminus of the cytochrome P450 enzyme CYP87D18 (NCBI Sequence ID: K7NBR2.1) interacts with the chlortetracycline polyketide synthase polypeptide tag A2 via a flexible linker (GGGGS) 3 N DD (nucleotide sequence as SEQ ID NO.7) fusion.

[0014] The cytochrome P450 enzyme reductase AtCPR1 (NCBI Sequence ID: NP_194183.1) is truncated at the amino terminus by 46 amino acid residues and fused to the carboxyl terminus of CYP87D18. The carboxyl terminus interacts with the chlortetracycline polyketide synthase polypeptide via a flexible linker (GGGGS) 3. Tag A2 C DD (nucleotide sequence as SEQ ID NO.8) fusion.

[0015] In the step (2):

[0016] The amino and carboxyl termini of the isopentenyl phosphate kinase AtIPK (NCBI Sequence ID: NP_173986.2) interact with the tacrolimus polyketide synthase polypeptide through a flexible linker (GGGGS) 3. N DD (nucleotide sequence as SEQ ID NO.9) and F6 C DD (nucleotide sequence such as SEQ ID NO.10) fusion.

[0017] The carboxyl terminus of the choline kinase ScCK (NCBI Sequence ID: NP_013234.1) is connected to the tacrolimus polyketide synthase peptide interaction tag F4 through a flexible linker (GGGGS) 3 C DD (nucleotide sequence such as SEQ ID NO.11) fusion.

[0018] The amino and carboxyl ends of the geranyl pyrophosphate synthase ERG20 are respectively connected to the tacrolimus polyketide synthase polypeptide interaction tag F7 through a flexible linker (GGGGS) 3 N DD (nucleotide sequence as SEQ ID NO.12) and rapamycin polyketide synthase peptide interaction tag R4 C DD (nucleotide sequence such as SEQ ID NO.13) fusion.

[0019] The amino and carboxyl ends of the cucurbitacin synthase SgCDS are respectively interacted with the chlortetracycline polyketide synthase polypeptide tag A3 through a flexible linker (GGGGS) 3 N DD (nucleotide sequence shown in SEQ ID NO.14) and polypeptide interaction tag RIDD (nucleotide sequence shown in SEQ ID NO.15) are fused.

[0020] The amino and carboxyl ends of the epoxysqualene cyclase ERG1 are respectively interacted with the erythromycin polyketide synthase polypeptide tag D5 through a flexible linker (GGGGS) 3. N DD (nucleotide sequence shown in SEQ ID NO. 16) and polypeptide interaction tag RIAD (nucleotide sequence shown in SEQ ID NO. 17) are fused.

[0021] The amino and carboxyl ends of the squalene synthase ERG9 are respectively interacted with the rapamycin polyketide synthase peptide tag R5 through a flexible linker (GGGGS) 3 N DD (nucleotide sequence such as SEQ ID NO.18) and R10 C DD (nucleotide sequence such as SEQ ID NO.19) fusion.

[0022] The carboxyl terminus and amino terminus of the sucrose synthase Susy (NCBI Sequence ID: WP_011381564.1) are fused to the Aga1p (NCBI Sequence ID: NP_014442.1) and Aga2p (nucleotide sequence shown in SEQ ID NO. 20) subunits of α-agglutinin, respectively.

[0023] The carboxyl terminus and amino terminus of the glycosyltransferase UGTMG1 (NCBI Sequence ID: 6L8W_A) are fused to the Aga1p and Aga2p subunits of α-agglutinin, respectively.

[0024] The glycosyltransferase SgUGT94-289-3 (NCBI Sequence ID: 8HJO_A) mutant SgUGT94-289-3 V148M / G152A The carboxyl terminus and amino terminus were fused to the Aga1p and Aga2p subunits of α-agglutinin, respectively.

[0025] The acetoacetyl-CoA thiolase ERG10, 3-hydroxy-3-methylglutaryl-CoA synthetase ERG13, truncated 3-hydroxy-3-methylglutaryl-CoA reductase tHMG1, mevalonate kinase ERG12, phosphomevalonate kinase ERG8, mevalonate pyrophosphate decarboxylase ERG19, pentenyl pyrophosphate isomerase IDI1, geranyl pyrophosphate synthase ERG20, squalene synthase ERG9 and epoxysqualene cyclase ERG1 are anchored and displayed on the peroxisomal membrane surface through the natural anchoring motif Pex15.

[0026] The cycloepoxide hydrolase SgEPH3, cucurbitadienol synthase SgCDS, cytochrome P450 enzyme CYP87D18 and cytochrome P450 enzyme reductase AtCPR1 are directed to assemble into a multi-enzyme complex with epoxysqualene cyclase ERG1 through polypeptide interaction, and are displayed on the surface of the peroxisome membrane with epoxysqualene cyclase ERG1 as an anchoring base point.

[0027] The epoxysqualene cyclase ERG1 and the cucurbitacadienol synthase SgCDS construct a three-enzyme complex in the cytoplasm in the form of one ERG1 molecule and two SgCDS molecules through the polypeptide interaction tags RIAD and RIDD.

[0028] The choline kinase ScCK and the isopentenyl phosphate kinase AtIPK form a multi-enzyme complex with two molecules of geranyl pyrophosphate synthase ERG20, two molecules of squalene synthase ERG9, two molecules of epoxysqualene cyclase ERG1 and two molecules of cucurbitadienol synthase SgCDS through polypeptide interaction, and are displayed on the surface of the peroxisome membrane with epoxysqualene cyclase ERG1 as the anchor base point.

[0029] The synthesis of mogroside V is carried out in two different regions, wherein the precursor mogroside alcohol is synthesized in the peroxisome, and mogroside V is synthesized on the cell wall surface.

[0030] The precursor mogroside is transported from the cytoplasm to the extracellular space via the ABC efflux protein PDR11.

[0031] The glycosyltransferase UGTMG1, sucrose synthase Susy and glycosyltransferase SgUGT94-289-3 are displayed on the surface of yeast cell wall through the α-lectin system.

[0032] The mogroside V is synthesized by the glycosyltransferase UGTMG1, sucrose synthase Susy and glycosyltransferase SgUGT94-289-3 displayed on the surface of yeast cell walls to catalyze the synthesis of extracellular mogroside alcohol.

[0033] The uridine diphosphate glucose required for the synthesis of mogroside V is provided by sucrose in the culture medium catalyzed by sucrose synthase Susy displayed on the surface of yeast cell walls.

[0034] The coding genes of the MG-V anabolic enzymes are all expressed by the yeast galactokinase (GAL1) gene promoter (nucleotide sequence such as SEQ ID NO.21).

[0035] The present invention also provides an engineered yeast of Saccharomyces cerevisiae for efficiently synthesizing mogroside V based on the dual compartmentalization of cytoplasm and peroxisome, which is specifically constructed according to the above construction method.

[0036] The present invention also provides a method for producing mogroside V, which comprises fermenting and producing mogroside V using the engineered yeast Saccharomyces cerevisiae provided by the present invention.

[0037] In the present invention, when fermenting to produce mogroside V, YPD medium supplemented with 10 mM sucrose, 10 mM uridine diphosphate (UDP), 21 mM 3-methyl-3-butene-1-ol and 9 mM 3-methyl-2-butene-1-ol is used as the fermentation medium.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] Mogroside V (MG-V) is a triterpenoid. Acetyl-Coenzyme A (acetyl-CoA), isopentenyl diphosphate (IPP), dimethylallyl diphosphate (DMAPP), squalene, and mogroside alcohol are all key precursors for MG-V biosynthesis. β-Oxidation of long-chain fatty acids in yeast peroxisomes produces large amounts of acetyl-CoA, which provides a precursor for MG-V synthesis.

[0040] The isopentenol utilization pathway (IUP) was constructed and key enzymes of the native mevalonate (MVA) pathway were overexpressed to increase the production of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Therefore, the present invention increases the supply of the precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) in the cytoplasm by priming the heterologous isopentenol utilization pathway.

[0041] Natural yeast squalene is mainly synthesized along the ergosterol pathway, where epoxysqualene cyclase ERG1 is a key enzyme in the synthesis of MG-V and ergosterol. By utilizing polypeptide interactions, one molecule of epoxysqualene cyclase ERG1 and two molecules of cucurbitaciniol synthase SgCDS form a three-enzyme complex, reducing competition in the ergosterol synthesis pathway and promoting the biosynthesis of mogroside alcohol. The present invention utilizes a multi-enzyme complex directed assembly strategy to display squalene synthase and mogroside alcohol synthase on the peroxisome membrane surface, integrating squalene production and mogroside alcohol synthesis, thereby promoting the synthesis of mogroside alcohol.

[0042] The present invention synthesizes mogroside V on the peroxisome membrane surface through oxisome surface display technology and a multi-enzyme complex directed assembly strategy. The α-lectin system enables the display of glucosyltransferase on the yeast cell wall surface, and the ABC efflux pump PDR11 is used to transport mogroside V to the extracellular space for synthesis. This results in the construction of an engineered Saccharomyces cerevisiae strain that efficiently synthesizes mogroside V based on dual compartmentalization of the cytoplasm and peroxisomes. This strain achieves a yield of 117.6 mg / L of MG-V through shake flask fermentation. The strain boasts advantages such as high yield, strong metabolic flux orientation, and broad prospects for industrial application, providing a new technical route for the microbial production of natural sweeteners.

[0043] The present invention integrates the multi-enzyme complex directed assembly strategy, squalene, and mogroside alcohol synthesis, thereby facilitating the synthesis of mogroside alcohol. In addition, to reduce the effect of MG-V on yeast cell growth, the present invention utilizes the ABC efflux pump PDR11 to transport mogroside alcohol to the extracellular space for the synthesis of MG-V, thereby reducing the effect of MG-V on yeast cell growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 Schematic diagram of integration into the GAL80 locus of the Saccharomyces cerevisiae genome.

[0045] Figure 2 Schematic diagram of integration into the Exg1 locus of the Saccharomyces cerevisiae genome. DETAILED DESCRIPTION

[0046] The present invention will be further described in detail below with reference to the accompanying drawings and examples, but the protection scope of the present invention is not limited to these examples.

[0047] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0048] Unless otherwise specified, the test materials used in the following examples were purchased from conventional biochemical reagent stores.

[0049] Saccharomyces cerevisiae BY4742 is described in the following document: Carriebaker brachmann et al., 1998, YEAST, 14: 115-132., and is publicly available from Guilin Medical University (the applicant). Hereinafter, Saccharomyces cerevisiae BY4742 is referred to as Saccharomyces cerevisiae.

[0050] The p414-TEF1p-Cas9-CYC1t (abbreviated as p414-Cas9, prokaryotic anti-Amp; screening marker is Trp) plasmid, p426-SNR52p-gRNA.CAN1.Y-SUP4t (abbreviated as p426, prokaryotic anti-Amp; screening marker is URA3) plasmid, yeast high-copy plasmid pRS425 (prokaryotic anti-Amp; screening marker is Leu2) and low-copy plasmid pRS313 (prokaryotic anti-Amp; screening marker is His3) were purchased from Beina Chuanglian Biotechnology Co., Ltd.

[0051] Yeast selection medium SD-His-Leu-Trp-Ura is a product of Beijing Coolaibo Technology Co., Ltd. The pLB-Simple Vector vector is a product of Tiangen Biochemical Technology (Beijing) Co., Ltd. Seamless cloning MasterMix, SpeedyCut SmaI, SpeedyCut StuI, SpeedyCut AscI, SpeedyCut SacII, SpeedyCutPacI, SpeedyCut SbfI, 2*SanTaq PCR Mix, and yeast competent cell preparation kit are products of Shanghai Sangon Biotechnology Co., Ltd. KOD-Plus-Neo is a product of Toyobo (Shanghai) Biotechnology Co., Ltd. T4 DNALigase is a product of NEB (New England Biolabs). Agarose gel DNA recovery kit, high-purity plasmid mini-extraction kit, large-scale DNA product purification kit, and yeast genomic DNA extraction kit are products of Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0052] In the examples, except for the endogenous genes of Saccharomyces cerevisiae, all other exogenous genes were synthesized by Shanghai Sangon Biotechnology Co., Ltd. after codon optimization, and the genes were cloned into the vector pUC57-Ampicillin to construct plasmids, as shown in Table 1:

[0053] Table 1 List of artificial genes

[0054]

[0055]

[0056] Example 2 Backbone plasmid construction

[0057] The information of the backbone plasmids involved in the construction of the following examples is shown in Table 2.

[0058] Table 2 Backbone plasmid list

[0059] Plasmid name Basic Information pM350 Contains the SmaI-TDH1t-SacII-AscI-pGAL1,10-PacI-SbfI-GPM1t-StuI backbone pM351 Contains the SmaI-ADH1t-SacII-AscI-pGAL1,10-PacI-SbfI-ADH2t-StuI backbone pM352 Contains the SmaI-TDH2t-SacII-AscI-pGAL1,10-PacI-SbfI-TDH3t-StuI backbone pM353 Contains the SmaI-CYC1t-SacII-AscI-pGAL1,10-PacI-SbfI-TEF1t-StuI backbone pM354 Contains the SmaI-TPI1t-SacII-AscI-pGAL1,10-PacI-SbfI-FBA1t-StuI backbone pM355 Contains the SmaI-ACT1t-SacII-AscI-pGAL1,10-PacI-SbfI-PGI1t-StuI backbone pM356 Contains the PmeI-pGAL1-PacI-SbfI-ENO1t-StuI backbone pM358 Contains the PmeI-pGAL1-PacI-SbfI-ADH2t-StuI backbone

[0060] (1) Using yeast genomic DNA as a template, PCR amplification was performed using the primers listed in columns 3 and 4 of Table 3, respectively, and the corresponding PCR amplification products were recovered, namely, the amplification products of the promoter pGAL1, terminators TDH1t, GPM1t, ADH1t, ADH2t, TDH2t, TDH3t, CYC1t, TEF1t, TPI1t, FBA1t, ACT1t, PGI1t, and ENO1t were obtained.

[0061] The reaction system is 50 μL, including 32 μL of distilled water, 5 μL of 10× buffer for KOD-Plus, 5 μL of dNTP mix, 2 μL of MgSO4, 1.5 μL of each primer, 2 μL of template, and 1 μL of KOD-Plus.

[0062] The reaction conditions were as follows: pre-denaturation at 94°C for 2 min; denaturation at 94°C for 15 s, annealing at 58°C for 30 s, extension at 68°C for 1 min, 25 cycles; and extension at 72°C for 10 min.

[0063] Table 3 Primer list

[0064]

[0065]

[0066] (2) The amplified product of promoter pGAL1 was double-digested with restriction endonucleases SacII and SbfI; the amplified products of TDH1t and GPM1t were single-digested with restriction endonucleases SacII and SbfI, respectively. The digested products were purified using a PCR product purification kit.

[0067] The enzyme digestion reaction system (50 μL) consists of 5 μL of 10× SpeedyOne Buffer, 2 μL of endonuclease, 2000 ng of PCR product, and distilled water to 50 μL. Digestion was performed at 37°C for 2 hours. The PCR product was purified using a purification kit.

[0068] (3) Use T4 DNA ligase to ligate the digested products of TDH1t, pGAL1, and GPM1t. 10 μL of the T4 DNA ligase reaction system consists of: 1 μL of 10× T4 DNA Ligase Buffer, 30 ng of each fragment, 0.5 μL of T4 DNA Ligase, and distilled water to make up to 10 μL. Incubate at room temperature (25°C) for 2 hours.

[0069] Add 10 μL of the T4 DNA ligase reaction product to 100 μL of TOP10 competent cells and incubate on ice for 30 minutes. Incubate at 42°C for 90 seconds, then immediately place on ice for 3 minutes. Add 500 μL of preheated LB medium at 37°C and shake at 150 rpm for 45 minutes. Pipette 100 μL onto LB solid agar medium containing ampicillin and gently spread the cells evenly with a sterile curved rod. Once the surface of the plate is dry, invert the plate and incubate at 37°C for 12–16 hours.

[0070] Using bacterial cells as templates, the recombinant plasmid pM350 was obtained using primers pLB-F (5'-cgactcactatagggagagcgtc-3') and pLB-R (5'-aagaacatcgcttttcgatggcag-3'). The PCR verification system (25 μL) consisted of 12.5 μL of 2× SanTaq PCR Mix, 1 μL of liquid buffer, 1 μL each of pLB-F and pLB-R, and 11.5 μL of distilled water. Sequencing of the recombinant plasmid pM350 revealed a SmaI-TDH1t-SacII-AscI-pGAL1,10-PacI-SbfI-GPM1t-StuI backbone.

[0071] Similarly, pM351, pM352, pM353, pM354, pM355, and pM356 plasmids were constructed.

[0072] Example 2 Expression cassette plasmid construction

[0073] The plasmid information for constructing the expression cassettes involved in the following examples is shown in Table 4.

[0074] Table 4 Expression cassette plasmids

[0075]

[0076]

[0077] (1) Using the plasmid or yeast genomic DNA shown in column 3 of Table 5 as a template, PCR amplification was performed using the primers shown in columns 4 and 5 of Table 5, and the corresponding PCR amplification products were recovered by gel excision to obtain the DNA fragments shown in column 2 of Table 5.

[0078] The reaction system is 50 μL, including 32 μL of distilled water, 5 μL of 10× buffer for KOD-Plus, 5 μL of dNTP mix, 2 μL of MgSO4, 1.5 μL of each primer, 2 μL of template, and 1 μL of KOD-Plus.

[0079] The reaction conditions were as follows: pre-denaturation at 94°C for 2 min; denaturation at 94°C for 15 s, annealing at 58°C for 30 s, extension at 68°C for 20 s to 2 min, 25 cycles; and extension at 72°C for 10 min.

[0080] (2) Overlap extension PCR (OE-PCR) was performed using the AGA1-1 fragment, Susy fragment, and AGA2-1 fragment in the second column of Table 5 as templates. The OE-PCR amplification product was recovered by gel excision to obtain the AGA2-Susy-AGA1 fragment.

[0081] The reaction system is 50 μL, including 32 μL of distilled water, 5 μL of 10× buffer for KOD-Plus, 5 μL of dNTP mix, 2 μL of MgSO4, 1.5 μL of each primer, 1 μL of each DNA fragment template, and 1 μL of KOD-Plus.

[0082] The reaction conditions were as follows: pre-denaturation at 94°C for 2 min; denaturation at 94°C for 15 s, annealing at 58°C for 30 s, extension at 68°C for 2 min, 25 cycles; and extension at 72°C for 10 min.

[0083] (3) The AGA2-Susy-AGA1 fragment and the pM355 plasmid were double-digested with restriction endonucleases AscI and SacII, respectively. The digested products were purified using a PCR product purification kit to obtain the AGA2-Susy-AGA1-AscI:SacII sticky end fragment and the pM355-AscI:SacII linear vector.

[0084] The enzyme digestion reaction system is 50 μL: 5 μL of 10× SpeedyOne Buffer, 2 μL of endonuclease, 2000 ng of DNA fragment or plasmid, and distilled water to 50 μL. Incubate at 37°C for 2 hours. Purify the PCR product using a purification kit.

[0085] (4) Use T4 DNA ligase to ligate the AGA2-Susy-AGA1-AscI:SacII sticky-end fragment and the pM355-AscI:SacII linear vector. 10 μL of the T4 DNA ligase reaction system consists of: 1 μL of 10×T4 DNA Ligase Buffer, 30 ng of the AGA2-Susy-AGA1-AscI:SacII sticky-end fragment and the pM355-AscI:SacII linear vector, 0.5 μL of T4 DNA Ligase, and distilled water to 10 μL. Incubate at room temperature (25°C) for 2 hours.

[0086] (5) Add 10 μL of T4 DNA ligase reaction to 100 μL of TOP10 competent cells and place on ice for 30 minutes. After incubating at 42°C for 90 seconds, immediately place on ice for 3 minutes. Add 500 μL of LB medium preheated at 37°C and shake at 150 rpm at 37°C for 45 minutes. Pipette 100 μL and add it to LB solid agar medium containing ampicillin. Use a sterile curved glass rod to gently spread the cells evenly. After the surface of the plate is dry, invert the plate and incubate at 37°C for 12 to 16 hours.

[0087] Using bacterial cells as templates, the recombinant plasmid pM355-AGA2-Susy-AGA1 was obtained using primers ACT1t-f1 (5'-ctgaaaagcgatgaagagatgat-3') and pGAL1-r1 (5'-gacgaggacgcacggaggagagt-3'). The PCR verification system (25 μL) consisted of 12.5 μL of 2× SanTaq PCR Mix, 1 μL of liquid buffer, 1 μL each of ACT1t-f1 and pGAL1-r1, and 11.5 μL of distilled water. Sequencing of the recombinant plasmid pM355-AGA2-Susy-AGA1 revealed the presence of the SmaI-ACT1t-SacII-AGA1-Susy-AGA2-AscI-pGAL1,10-PacI-SbfI-PGI1t-StuI expression cassette.

[0088] (6) OE-PCR was performed using the AGA2-2, UGTMG1, and AGA1-2 fragments in the second column of Table 5 as templates. The OE-PCR amplification products were recovered by gel excision to obtain the AGA2-UGTMG1-AGA1 fragment.

[0089] (7) The AGA2-UGTMG1-AGA1 fragment and the pM355-AGA2-Susy-AGA1 plasmid were double-digested with restriction endonucleases PacI and SbfI, respectively. The digested products were purified using a PCR product purification kit to obtain the AGA2-UGTMG1-AGA1-PacI:SbfI sticky end fragment and the pM355-PacI:SbfI linear vector.

[0090] (8) Use T4 DNA ligase to ligate the AGA2-UGTMG1-AGA1-PacI:SbfI sticky end fragment and the pM355-PacI:SbfI linear vector.

[0091] (9) Add 10 μL of T4 DNA ligase reaction to 100 μL of TOP10 competent cells and place on ice for 30 minutes. After incubating at 42°C for 90 seconds, immediately place on ice for 3 minutes. Add 500 μL of LB medium preheated at 37°C and shake at 150 rpm at 37°C for 45 minutes. Pipette 100 μL and add it to LB solid agar medium containing ampicillin. Use a sterile curved glass rod to gently spread the cells evenly. After the surface of the plate is dry, invert the plate and incubate at 37°C for 12 to 16 hours.

[0092] Using bacterial cells as templates, the recombinant plasmid pM355-CP5 was obtained using primers pGAL1-f1 (5'-caaccataggatgataatgcgat-3') and PGI1t-r1 (5'-cacaataaagtcttcacgacgctaa-3'). The PCR verification system (25 μL) consisted of 12.5 μL of 2× SanTaq PCR Mix, 1 μL of liquid buffer, 1 μL each of pGAL1-f1 and PGI1t-r1, and 11.5 μL of distilled water. Sequencing of the recombinant plasmid revealed the SmaI-ACT1t-SacII-AGA1-UGTMG1-AGA2-AscI-pGAL1,10-PacI-AGA2-Susy-AGA1-SbfI-PGI1t-StuI expression cassette.

[0093] Similarly, the plasmids listed in Table 4 were obtained.

[0094] Table 5 Primer list

[0095]

[0096]

[0097] Example 3 Construction of p426-GAL80-Exg1 gRNA dual-target plasmid

[0098] (1) Using KOD-Plus high-fidelity enzyme and p426 plasmid as templates, primer pair 56, primer pair 57, primer pair 58, and primer pair 59 shown in Table 6 were used for PCR amplification, and the corresponding PCR amplification products were recovered by gel cutting to obtain GAL80 gRNA-1, GAL80 gRNA-2, Exg1 gRNA-1, and Exg1 gRNA-2 fragments.

[0099] (2) Perform homologous recombination reactions on the GAL80 gRNA-1 and GAL80 gRNA-2 fragments, and the Exg1 gRNA-1 and Exg1 gRNA-2 fragments using Seamless Cloning Master Mix. The homologous recombination reaction system (10 μL) consists of: 5 μL of Seamless Cloning Master Mix, 50 ng of each fragment, and distilled water to 10 μL. Incubate at 50°C for 20 minutes, then immediately cool on ice for 2 minutes.

[0100] (3) Add the homologous recombination reaction product to 100 μL of TOP10 competent cells and place them on ice for 30 minutes. After incubating at 42°C for 90 seconds, immediately place them on ice for 3 minutes. Add 500 μL of LB medium preheated at 37°C and shake at 150 rpm at 37°C for 45 minutes. Pipette 100 μL and add it to LB solid agar medium containing ampicillin. Use a sterile curved glass rod to gently spread the cells evenly. After the surface of the plate is dry, invert the plate and incubate at 37°C for 12 to 16 hours.

[0101] Using bacteria as templates, PCR verification was performed with primers gRNA-F (5'-cattaggcaccccaggcttt-3') and gRNA-R (5'-tttcggttagagcggatgtg-3'), resulting in the generation of recombinant plasmids p426-GAL80 gRNA and p426-Exg1 gRNA, respectively. Sequencing of the recombinant plasmids revealed that the recombinant plasmids contained GAL80 gRNA (5'-gtcggtctcaacgcagccaa-3') and Exg1 gRNA (5'-gaacaaattgaatggaagaa-3'), respectively.

[0102] (4) Using KOD-Plus high-fidelity enzyme, p426-GAL80 gRNA plasmid and p426-Exg1 gRNA plasmid as templates, PCR amplification was performed using primer pair 60 and primer pair 61 shown in Table 6, and the corresponding PCR amplification products were recovered by gel excision. The corresponding PCR amplification products were recovered by gel excision to obtain GAL80 gRNA-3 and Exg1 gRNA-3 fragments, and the GAL80 gRNA-3 and Exg1 gRNA-3 fragments were homologously recombined using Seamless cloning Master Mix. OP10 competent cells were transformed and PCR verification was performed using primers gRNA-F and gRNA-R to obtain p426-GAL80-Exg1 gRNA recombinant plasmid. The sequencing results of the recombinant plasmid showed that the recombinant plasmid contained GAL80 gRNA (5'-gtcggtctcaacgcagccaa-3') and Exg1 gRNA (5'-gaacaaattgaatggaagaa-3').

[0103] Table 6 Primer list

[0104]

[0105] Example 4 Construction of recombinant yeast

[0106] (1) Using the plasmids shown in column 1 of Table 7 as templates, PCR amplification was performed using the primers shown in columns 3 and 4 of Table 7, and the corresponding PCR amplification products were recovered to obtain the screening markers Leu2 and His3 fragments and the modules CP-M7 and CP-M6'.

[0107] Table 7 Primer list

[0108]

[0109]

[0110] (2) The plasmids shown in column 2 of Table 8 were double-digested with the restriction endonucleases shown in column 3 of Table 8, and the double-digested products of the sizes shown in column 4 of Table 8 were recovered from the gel, thereby obtaining modules CP-M1, CP-M2, CP-M3, CP-M4, CP-M5, CP-M6 and CP-M1', CP-M2', CP-M3', CP-M4' and CP-M5'.

[0111] Table 8 Plasmid list

[0112] Module Plasmid name Endonuclease name Size (bp) CP-M1 pM350-CP1 SmaI and StuI 5050 CP-M2 pM351-CP2 SmaI and StuI 4945 CP-M3 pM352-CP3 SmaI and StuI 4706 CP-M4 pM353-CP4 SmaI and StuI 4197 CP-M5 pM354-CP5 SmaI and StuI 5414 CP-M6 pM355-CP6 SmaI and StuI 5674 CP-M1 pM358-CP1 PmeI and StuI 5333 CP-M2 pM352-CP2 SmaI and StuI 5259 CP-M3 pM353-CP3 SmaI and StuI 4074 CP-M4 pM354-CP4 SmaI and StuI 4140 CP-M5 pM355-CP5 SmaI and StuI 8891

[0113] (3) Prepare Saccharomyces cerevisiae competent cells according to the instructions of the yeast competent cell preparation kit. Add 0.1 μg module CP-M1, 0.1 μg module CP-M2, 0.1 μg module CP-M3, 0.1 μg module CP-M4, 0.1 μg module CP-M5, 0.1 μg module CP-M6, 0.1 μg module CP-M7, 0.1 μg module CP-M1`, 0.1 μg module CP-M2`, 0.1 μg module CP-M3`, 0.1 μg module CP-M4`, 0.1 μg module CP-M5` and 0.1 μg module CP-M6`, 0.2 μg plasmid p426-GAL80-Exg1 gRNA, 0.2 μg plasmid p414-Cas9, 0.1 μg fragment Leu2 and 0.1 μg fragment His3 to the Saccharomyces cerevisiae competent cells, and add 1 mL after 2.7 kV electric shock. mol / L sorbitol solution, resuscitated at 30°C for 1 hour, plated onto screening medium to obtain several single colonies, and cultured at 30°C for at least 36 hours. The screening medium consists of 0.8% SD-His-Leu-Trp-Ura, 2% glucose, and 2% agar powder.

[0114] (4) Single clones were first verified by PCR amplification using the primers shown in columns 3 and 4 of Table 9, and then verified by DNA sequencing. The sequencing results showed that modules CP-M1, CP-M2, CP-M3, CP-M4, CP-M5, CP-M6, CP-M7 and the screening marker Leu2 were correctly inserted into the GAL80 site of the yeast genome, and CP-M1`, CP-M2`, CP-M3`, CP-M4`, CP-M5`, CP-M6` and the screening marker His3 were correctly inserted into the Exg1 site of the yeast genome.

[0115] Table 9 Primer list

[0116]

[0117]

[0118] Example 6: Fermentation of engineered yeast Saccharomyces cerevisiae and detection of mogroside V

[0119] (1) Preparation of seed culture: Pick a single colony of engineered Saccharomyces cerevisiae and transfer it to 5 mL of YPD liquid medium. Cultivate at 30°C and 220 rpm for 12-15 h until the cells reach the logarithmic growth phase.

[0120] (2) Fermentation culture: The seed culture was inoculated into YPD medium supplemented with 10 mM sucrose, 10 mM uridine diphosphate (UDP), 21 mM 3-methyl-3-butene-1-ol, and 9 mM 3-methyl-2-butene-1-ol at an inoculum size of 1% to 5%. The culture was carried out at 30°C and 220 rpm for 96 h, and the fermentation broth was collected.

[0121] (3) Fermentation Sample Processing: After centrifugation at 8000 rpm for 5 min, the supernatant was collected and chromatographic-grade formic acid was added to the supernatant to a final concentration of 3% (v / v). The supernatant was allowed to stand at 4°C for 5 h and then centrifuged at 8000 rpm for 5 min. The supernatant was collected and filtered through a 0.22 μm aqueous filter before being added to a liquid chromatography detection bottle for testing.

[0122] (4) Detection of Mogroside V: Detection was performed using a high performance liquid chromatography (HPLC) instrument under the following conditions: chromatographic column: C18 column, column length 250 nm, inner diameter 4.6 mm, filled with C18 filler, particle size 5 μm; mobile phase: water + acetonitrile (79 + 21); flow rate: 1 mL / min; detection wavelength: 203 nm; injection volume: 10 μL; column temperature: 30°C. The test results showed that the yield of mogroside V produced by shake flask fermentation of engineered Saccharomyces cerevisiae was 117.6 mg / L.

[0123] Although the present invention discloses the preferred embodiments as described above, it is not intended to limit the present invention. Any technician familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for constructing an engineered yeast strain of Saccharomyces cerevisiae for efficiently synthesizing mogroside V based on dual compartmentalization of cytoplasm and peroxisome, characterized by: The following steps are involved: (1) Acetoacetyl-CoA thiolase ERG10, 3-hydroxy-3-methylglutaryl-CoA synthetase ERG13, truncated 3-hydroxy-3-methylglutaryl-CoA reductase tHMG1, mevalonate kinase ERG12, phosphomevalonate kinase ERG8, mevalonate pyrophosphate decarboxylase ERG19, pentenyl pyrophosphate isomerase IDI1, geranyl pyrophosphate synthase ERG20, squalene synthase ERG9, epoxysqualene cyclase ERG1, cucurbitadienol synthase SgCDS, epoxyepoxide hydrolase SgEPH3, cytochrome P450 enzyme CYP87D18, and cytochrome P450 enzyme reductase AtCPR1 were integrated into the GAL80 locus of the Saccharomyces cerevisiae genome; (2) The ABC efflux protein PDR11, choline kinase ScCK, isopentenyl phosphate kinase AtIPK, geranyl pyrophosphate synthase ERG20, cucurbitadienol synthase SgCDS, endogenous squalene synthase ERG9, epoxysqualene cyclase ERG1, glycosyltransferase UGTMG1, sucrose synthase Susy and glycosyltransferase SgUGT94-289-3 were integrated into the Exg1 locus of the Saccharomyces cerevisiae genome.

2. The method for constructing an engineered yeast strain of Saccharomyces cerevisiae for efficiently synthesizing mogroside V based on dual compartmentalization of cytoplasm and peroxisome according to claim 1, characterized in that: In the step (1): The acetoacetyl-CoA thiolase ERG10, 3-hydroxy-3-methylglutaryl-CoA synthetase ERG13, tHMG1 with 527 amino acids truncated at the N-terminus of HMG1, mevalonate kinase ERG12, phosphomevalonate kinase ERG8, mevalonate pyrophosphate decarboxylase ERG19, pentenyl pyrophosphate isomerase IDI1, geranyl pyrophosphate synthase ERG20, and endogenous squalene synthase ERG9; The amino and carboxyl ends of the epoxysqualene cyclase ERG1 are respectively interacted with the erythromycin polyketide synthase polypeptide tag D5 through a flexible linker (GGGGS) 3 N DD is fused with the natural anchor motif Pex15; The amino and carboxyl ends of the cucurbitacinol synthase SgCDS are respectively interacted with the chlortetracycline polyketide synthase polypeptide tag A3 through a flexible linker (GGGGS) 3 N DD and erythromycin polyketide synthase peptide interaction tag D2 C DD fusion; The amino and carboxyl ends of the cycloepoxide hydrolase SgEPH3 are respectively interacted with the erythromycin polyketide synthase polypeptide tag D3 through a flexible linker (GGGGS) 3 N DD and D4 C DD fusion; The amino terminus of the cytochrome P450 enzyme CYP87D18 interacts with the chlortetracycline polyketide synthase polypeptide via a flexible linker (GGGGS) 3. N DD fusion; The cytochrome P450 enzyme reductase AtCPR1 is truncated with 46 amino acid residues at the amino terminal and fused to the carboxyl terminal of CYP87D18, and the carboxyl terminal interacts with the chlortetracycline polyketide synthase polypeptide tag A2 through a flexible linker (GGGGS) 3. C DD fusion.

3. The method for constructing an engineered yeast strain of Saccharomyces cerevisiae for efficiently synthesizing mogroside V based on dual compartmentalization of cytoplasm and peroxisome according to claim 1, characterized in that: In the step (2): The amino and carboxyl ends of the isopentenyl phosphate kinase AtIPK interact with the tacrolimus polyketide synthase polypeptide through a flexible linker (GGGGS) 3. N DD and F6 C DD fusion; The carboxyl terminus of the choline kinase ScCK interacts with the tacrolimus polyketide synthase peptide tag F4 through a flexible linker (GGGGS) 3 C DD fusion; The amino and carboxyl ends of the geranyl pyrophosphate synthase ERG20 are respectively connected to the tacrolimus polyketide synthase polypeptide interaction tag F7 through a flexible linker (GGGGS) 3 N DD and rapamycin polyketide synthase peptide interaction tag R4 C DD fusion; The amino and carboxyl ends of the cucurbitacin synthase SgCDS are respectively interacted with the chlortetracycline polyketide synthase polypeptide tag A3 through a flexible linker (GGGGS) 3 N DD and peptide interaction tag RIDD fusion; The amino and carboxyl ends of the epoxysqualene cyclase ERG1 are respectively interacted with the erythromycin polyketide synthase polypeptide tag D5 through a flexible linker (GGGGS) 3. N DD and peptide interaction tag RIAD fusion; The amino and carboxyl ends of the squalene synthase ERG9 are respectively interacted with the rapamycin polyketide synthase peptide tag R5 through a flexible linker (GGGGS) 3 N DD and R10 C DD fusion; The carboxyl terminus and amino terminus of the sucrose synthase Susy are fused to the Aga1p and Aga2p subunits of α-agglutinin respectively; The carboxyl terminus and amino terminus of the glycosyltransferase UGTMG1 are fused to the Aga1p and Aga2p subunits of α-agglutinin, respectively; The glycosyltransferase SgUGT94-289-3 mutant SgUGT94-289-3 V148M / G152A The carboxyl terminus and amino terminus were fused to the Aga1p and Aga2p subunits of α-agglutinin, respectively.

4. The method for constructing an engineered yeast strain of Saccharomyces cerevisiae for efficient synthesis of mogroside V based on dual compartmentalization of cytoplasm and peroxisome according to claim 1, characterized in that: The acetoacetyl-CoA thiolase ERG10, 3-hydroxy-3-methylglutaryl-CoA synthetase ERG13, truncated 3-hydroxy-3-methylglutaryl-CoA reductase tHMG1, mevalonate kinase ERG12, phosphomevalonate kinase ERG8, mevalonate pyrophosphate decarboxylase ERG19, pentenyl pyrophosphate isomerase IDI1, geranyl pyrophosphate synthase ERG20, squalene synthase ERG9 and epoxysqualene cyclase ERG1 are anchored and displayed on the peroxisomal membrane surface through the natural anchoring motif Pex15; The cycloepoxide hydrolase SgEPH3, cucurbitadienol synthase SgCDS, cytochrome P450 enzyme CYP87D18 and cytochrome P450 enzyme reductase AtCPR1 are directed to assemble into a multi-enzyme complex with epoxysqualene cyclase ERG1 through polypeptide interaction, and are displayed on the surface of the peroxisome membrane with epoxysqualene cyclase ERG1 as an anchoring base point.

5. The method for constructing an engineered yeast strain of Saccharomyces cerevisiae for efficient synthesis of mogroside V based on dual compartmentalization of cytoplasm and peroxisome according to claim 1, characterized in that: The epoxysqualene cyclase ERG1 and the cucurbitacadienol synthase SgCDS construct a three-enzyme complex in the cytoplasm in the form of one ERG1 molecule and two SgCDS molecules through the polypeptide interaction tags RIAD and RIDD.

6. The method for constructing an engineered yeast strain of Saccharomyces cerevisiae for efficient synthesis of mogroside V based on dual compartmentalization of cytoplasm and peroxisome according to claim 1, characterized in that: The choline kinase ScCK and the isopentenyl phosphate kinase AtIPK form a multi-enzyme complex with two molecules of geranyl pyrophosphate synthase ERG20, two molecules of squalene synthase ERG9, two molecules of epoxysqualene cyclase ERG1 and two molecules of cucurbitadienol synthase SgCDS through polypeptide interaction, and are displayed on the surface of the peroxisome membrane with epoxysqualene cyclase ERG1 as the anchor base point.

7. The method for constructing an engineered yeast strain of Saccharomyces cerevisiae for efficient synthesis of mogroside V based on dual compartmentalization of cytoplasm and peroxisome according to claim 1, characterized in that: The synthesis of mogroside V is carried out in two different regions, wherein the precursor mogroside alcohol is synthesized in the peroxisome and transported from the cytoplasm to the extracellular space through the ABC efflux protein PDR11, and mogroside V is synthesized on the cell wall surface.

8. The method for constructing an engineered yeast strain of Saccharomyces cerevisiae for efficient synthesis of mogroside V based on dual compartmentalization of cytoplasm and peroxisome according to claim 1, characterized in that: The glycosyltransferase UGTMG1, sucrose synthase Susy and glycosyltransferase SgUGT94-289-3 are displayed on the surface of yeast cell walls through an α-lectin system, and jointly catalyze the conversion of mogroside alcohol into mogroside V; the encoding genes of the MG-V anabolic enzymes are all expressed by the yeast galactokinase GAL1 gene promoter.

9. An engineered yeast strain of Saccharomyces cerevisiae that efficiently synthesizes mogroside V based on dual compartmentalization of cytoplasm and peroxisome, characterized by: Constructed by the construction method according to any one of claims 1 to 8.

10. A method for producing mogroside V, characterized in that: The cerevisiae engineered bacteria according to claim 9 are used to ferment and produce mogroside V, using YPD medium supplemented with 10mM sucrose, 10mM uridine diphosphate, 21mM 3-methyl-3-butene-1-ol and 9mM 3-methyl-2-butene-1-ol as the fermentation medium.

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