Saccharomyces cerevisiae genetically engineered bacterium for efficiently synthesizing mogroside V based on multi-compartment multi-metabolic pathway and construction method of saccharomyces cerevisiae genetically engineered bacterium

Abstract

The invention discloses a saccharomyces cerevisiae genetically engineered bacterium for efficiently synthesizing mogroside V based on multi-compartment multi-metabolic pathways and a construction method of the saccharomyces cerevisiae genetically engineered bacterium. The preparation method comprises the following steps: anchoring an MVA pathway and a squalene synthesis pathway on the surface of a peroxisome membrane, and enabling mogrol synthetase and epoxy squalene cyclization enzyme ERG1 to form a linear polywheat complex through polypeptide interaction; then, through overexpression of MVA pathway key enzyme and introduction of an isopentenol utilization pathway, supply of precursor isopentenyl pyrophosphate and dimethyl allyl pyrophosphate in cytoplasm is increased, and squalene epoxidase ERG1 and mogrol synthetase are anchored in transmembrane ABC efflux protein PDR11 through polypeptide interaction; then anchoring the mogrol synthetase in the lipid droplet subcells; finally, the glucosyltransferase is displayed on the surface of the yeast cell wall through an alpha-lectin system, so that extracellular mogrol is converted into MG-V. The engineering bacterium improves the yield of mogroside V synthesized from the beginning in microorganisms, and has great industrial application value.

Description

Technical Field

[0001] The present invention relates to a genetically engineered yeast strain for efficiently synthesizing mogroside V based on multi-compartmental and multi-metabolic pathways and a construction method thereof, and belongs to the field of bioengineering. Background Art

[0002] Mogrosides, known for their high sweetness, low calorie content, safety, fat-reducing properties, and resistance to tooth decay, have become a new generation of sweeteners, satisfying consumer demand for sweetness and attracting increasing attention. Mogroside V (MG-V), the primary source of mogroside sweetness, boasts a sweetness 425 times greater than 5% sucrose at 1 / 10,000th of its original concentration, making it a highly sought-after next-generation functional sweetener both domestically and internationally. However, currently, mogroside V is extracted entirely from monk fruit (Shenggu), a resource that is limited by the availability of monk fruit (Shenggu) and climate, hindering large-scale production. Leveraging advanced synthetic biology techniques and microbial fermentation to produce high-purity MG-V, enabling low-cost, large-scale production, is poised to revolutionize traditional monk fruit extraction and production methods.

[0003] MG-V is a cytoplasmic triterpenoid saponin. Saccharomyces cerevisiae is a safe eukaryotic model organism with a well-defined genetic background and mature genetic engineering techniques. It also possesses a mevalonate (MVA) pathway for the biosynthesis of terpenoids. IPP and DMAPP are sequentially catalyzed by farnesyl pyrophosphate synthase ERG20 and squalene synthase ERG9 to synthesize the triterpenoid precursor squalene, which is then oxidized to diepoxysqualene (2,3;22,23-diepoxysqualene) by squalene epoxidase ERG1. Acetyl-CoA is a key intermediate in the yeast mogroside V biosynthesis pathway. In Saccharomyces cerevisiae, acetyl-CoA distribution is highly compartmentalized, primarily in the cytoplasm, peroxisomes, and nucleus. While some squalene in Saccharomyces cerevisiae enters the ergosterol pathway to synthesize ergosterol, the majority is temporarily stored as a lipid-soluble component in lipid droplet subcellular organelles. The present invention first anchors the yeast natural mevalonic acid (MVA) pathway and squalene synthesis pathway on the peroxisome membrane surface, and forms a linear polycomplex with mogroside synthase and epoxysqualene cyclase ERG1 through polypeptide interaction to promote the conversion of acetyl-CoA in peroxisomes into mogroside; then, by overexpressing key enzymes of the MVA pathway and introducing the isopentenol utilization pathway (IUP), the precursors isopentenyl diphosphate (IPP) and dimethylallyl pyrophosphate (DMP) in the cytoplasm are increased. diphosphate (DMAPP), and through polypeptide interactions, squalene epoxidase ERG1 and mogroside synthase are anchored in the transmembrane ABC efflux protein PDR11, thereby promoting the synthesis and efflux of mogroside in the cytoplasm; then, mogroside synthases are anchored in lipid droplet subcellular compartments, promoting the synthesis of mogroside from squalene in lipid droplets; finally, glucosyltransferases are displayed on the surface of yeast cell walls through the α-lectin system, thereby converting extracellular mogroside into MG-V, thus establishing a genetically engineered Saccharomyces cerevisiae strain that efficiently synthesizes mogroside V based on multi-compartmental and multi-metabolic pathways. The yield of mogroside V produced by shake flask fermentation of this engineered strain reached 234.1 mg / L, which is the highest level of de novo synthesis of mogroside V reported in microorganisms so far, and has significant industrial application value. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a genetically engineered yeast of Saccharomyces cerevisiae that can efficiently synthesize mogroside V based on multi-compartmental and multi-metabolic pathways and a construction method thereof.

[0005] The method for constructing a genetically engineered yeast strain of Saccharomyces cerevisiae for efficiently synthesizing mogroside V based on a multi-compartmental and multi-metabolic pathway, which is used to solve the above technical problems, is constructed by the following method:

[0006] (1) The mogroside alcohol metabolism enzymes, including 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, cytochrome P450 enzyme reductase AtCPR1, and leucine selection marker Leu2, were integrated into the Saccharomyces cerevisiae transcriptional repressor GAL80 locus;

[0007] (2) Integrate truncated 3-hydroxy-3-methylglutaryl-CoA reductase tHMG1, prenyl pyrophosphate isomerase IDI1, epoxysqualene cyclase ERG1, ABC efflux protein PDR11, choline kinase ScCK, isopentenyl phosphate kinase AtIPK, geranyl pyrophosphate synthase ERG20, squalene synthase ERG9, hydroxymethylglutaryl-CoA reductase EfHMGR, hydroxymethylglutaryl-CoA reductase SpHMGR, cucurbitadienol synthase SgCDS, epoxyepoxide hydrolase SgEPH3, cytochrome P450 enzyme CYP87D18, cytochrome P450 enzyme reductase AtCPR1, and histidine selection marker His3 at the Saccharomyces cerevisiae glycoside hydrolase Exg1 site;

[0008] (3) Integrate squalene cyclase ERG1, cucurbitadienol synthase SgCDS, epoxide hydrolase SgEPH3, cytochrome P450 enzyme CYP87D18, cytochrome P450 enzyme reductase AtCPR1, glycosyltransferase UGTMG1, sucrose synthase Susy and glycosyltransferase mutant SgUGT94-289-3 into the Saccharomyces cerevisiae glycoside hydrolase EGH1 site V148M / G152A and the lysine selection marker Lys2.

[0009] In the step (1):

[0010] 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. The carboxyl termini of the endogenous squalene synthase ERG9 (NCBI Sequence ID: NP_012060.1) and the epoxysqualene cyclase ERG1 (NCBI Sequence ID: NP_011691.1) were fused to the natural anchor motif Pex15 (amino acid sequence shown in SEQ ID NO. 1) via a flexible linker (GGGGS) 3; the amino terminus of the epoxysqualene cyclase ERG1 was fused to the erythromycin polyketide synthase polypeptide interaction tag D5 via a flexible linker (GGGGS) 3 N DD (nucleotide sequence shown in SEQ ID NO.2) fusion.

[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 such 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 cytochrome P450 enzyme reductase AtCPR1 (NCBI Sequence ID: NP_194183.1) is fused to the carboxyl terminus of CYP87D18 (NCBI Sequence ID: K7NBR2.1) after truncating 46 amino acid residues at the amino terminus. The carboxyl terminus interacts with the chlortetracycline polyketide synthase polypeptide via a flexible linker (GGGGS) 3. C DD (nucleotide sequence such as SEQ ID NO.7) fusion.

[0014] In the step (2):

[0015] The amino end and carbon end of the epoxysqualene cyclase ERG1 are respectively interacted with the chlortetracycline polyketide synthase polypeptide tag A2 through a flexible linker (GGGGS) 3 N DD (nucleotide sequence as SEQ ID NO.8) and polypeptide interaction tag RIDD (nucleotide sequence as SEQ ID NO.9) are fused.

[0016] The carboxyl terminus of the ABC efflux protein PDR11 (NCBI Sequence ID: NP_012252.1) interacts with the chlortetracycline polyketide synthase polypeptide tag A1 through a flexible linker (GGGGS) 3 C DD (nucleotide sequence such as SEQ ID NO.10) fusion;

[0017] The amino and carboxyl ends of the cucurbitacin synthase SgCDS are respectively interacted with the rapamycin polyketide synthase polypeptide tag R11 through a flexible linker (GGGGS) 3. N DD (nucleotide sequence as SEQ ID NO.11) and polypeptide interaction tag RIDD (nucleotide sequence as SEQ ID NO.12) are fused.

[0018] The amino and carboxyl ends of the epoxide hydrolase SgEPH3 are respectively connected to the rapamycin polyketide synthase polypeptide interaction tag R5 through a flexible linker (GGGGS) 3. N DD (nucleotide sequence as SEQ ID NO.13) and R10 C DD (nucleotide sequence such as SEQ ID NO.14) fusion.

[0019] The cytochrome P450 enzyme reductase AtCPR1 is truncated with 46 amino acid residues at the amino terminal and then fused to the carboxyl terminal of CYP87D18, and the carboxyl terminal interacts with the rapamycin polyketide synthase polypeptide tag R4 through a flexible linker (GGGGS) 3. C DD (nucleotide sequence such as SEQ ID NO.15) fusion.

[0020] In the step (3):

[0021] The amino and carbon ends of the epoxysqualene cyclase ERG1 interact with the natural anchor motif PLN1 (NCBI Sequence ID: NP_012972.3) and the tacrolimus polyketide synthase peptide tag F4 through a flexible linker (GGGGS) 3, respectively. C DD (nucleotide sequence as SEQ ID NO.16) fusion.

[0022] The amino terminal of the cucurbitadienol synthase SgCDS is fused to the natural anchor motif PLN1 through a flexible linker (GGGGS) 3, and the carboxyl terminal of the SgCDS is fused to the cytochrome P450 enzyme reductase AtCPR1 with 46 amino acid residues truncated at the amino terminal.

[0023] The amino terminus of the cycloepoxide hydrolase SgEPH3 interacts with the tacrolimus polyketide synthase polypeptide through a flexible linker (GGGGS) 3 and a tag F7. N DD (nucleotide sequence as SEQ ID NO. 17).

[0024] 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. 18) subunits of α-agglutinin, respectively.

[0025] 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.

[0026] 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.

[0027] The epoxysqualene cyclase ERG1 is additionally expressed in three copies, of which one copy of ERG1 is anchored on the peroxidase membrane surface, one copy of ERG1 is anchored on the ABC efflux protein PDR11, and another copy of ERG1 is anchored in the lipid droplet subcellular compartment;

[0028] The synthesis of mogroside V is divided into two stages: extracellular and intracellular. The precursor mogroside alcohol is synthesized in the cytoplasm, lipid droplets and peroxidase surface, and mogroside V is synthesized on the cell wall surface.

[0029] The epoxysqualene cyclase ERG1 forms a linear multi-enzyme complex with the epoxy hydrolase SgEPH3, the cucurbitadienol synthase SgCDS, the cytochrome P450 enzyme CYP87D18 and the cytochrome P450 enzyme reductase AtCPR1 through polypeptide interaction on the peroxidase surface.

[0030] The epoxysqualene cyclase ERG1 and the cucurbitadienol synthase SgCDS in the cytoplasm form a three-enzyme complex with one ERG1 molecule and two SgCDS molecules through the polypeptide interaction tags RIAD and RIDD.

[0031] The epoxysqualene cyclase ERG1 forms a linear multiprotein complex in the cytoplasm through polypeptide interactions with ABC efflux protein PDR11, epoxy hydrolase SgEPH3, cucurbitadienol synthase SgCDS, cytochrome P450 enzyme CYP87D18 and cytochrome P450 enzyme reductase AtCPR1.

[0032] The epoxysqualene cyclase ERG1 forms a three-enzyme complex with the epoxy hydrolase SgEPH3 and the cucurbitadienol synthase SgCDS in the lipid droplet subcellular organelle through polypeptide interaction.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.19).

[0038] The present invention also provides a genetically engineered yeast strain of Saccharomyces cerevisiae that efficiently synthesizes mogroside V based on multi-compartmental and multi-metabolic pathways, which is constructed by the above-mentioned construction method.

[0039] The present invention also provides a method for producing mogroside V.

[0040] Specifically, a method for producing mogroside V comprises fermenting the above-mentioned engineered yeast Saccharomyces cerevisiae to produce mogroside V.

[0041] The method for producing mogroside V uses 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 as a fermentation medium.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] (1) The yeast natural mevalonic acid (MVA) pathway and squalene synthesis pathway were anchored on the peroxisomal membrane surface, and through peptide interaction, mogroside synthase and epoxysqualene cyclase ERG1 formed a linear multienzyme complex to promote the conversion of acetyl-CoA in peroxisomes to mogroside alcohol;

[0044] (2) Overexpression of key enzymes of the MVA pathway and introduction of the isopentenol utilization pathway (IUP) to increase the supply of precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) in the cytoplasm;

[0045] (3) Using peptide interactions, squalene epoxidase ERG1 and mogroside synthase are anchored to the transmembrane ABC efflux protein PDR11, thereby promoting the synthesis and efflux of mogroside in the cytoplasm;

[0046] (4) Anchoring mogroside alcohol synthases in lipid droplet subcells to promote the synthesis of mogroside alcohol from squalene in lipid droplets;

[0047] (5) Through the α-lectin system, glucosyltransferase is displayed on the surface of yeast cell walls, thereby converting extracellular mogroside alcohol into MG-V.

[0048] Based on the above technical advantages, the brewer's genetically engineered bacteria constructed by the method described in the present invention, which can efficiently synthesize mogroside V based on multi-compartmental and multi-metabolic pathways, has a synthesized mogroside V content of 234.1 mg / L, which is the highest level of mogroside V production reported so far in microorganisms for de novo synthesis, and has significant industrial application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 Schematic diagram of integration into the GAL80 site of the Saccharomyces cerevisiae genome.

[0050] Figure 2 Schematic diagram of integration into the Exg1 locus of the Saccharomyces cerevisiae genome.

[0051] Figure 3 Schematic diagram of integration into the EGH1 locus of the Saccharomyces cerevisiae genome. DETAILED DESCRIPTION

[0052] 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.

[0053] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0054] Unless otherwise specified, the test materials used in the following examples were purchased from conventional biochemical reagent stores.

[0055] Saccharomyces cerevisiae BY4742 and Saccharomyces cerevisiae CEN.PK2-1D (NK2 yeast) are described in the following literature: Carrie Bakerbrachmann et al., 1998, YEAST, 14:115-132. They are publicly available from Guilin Medical University (i.e., the applicant). Hereinafter, Saccharomyces cerevisiae BY4742 is referred to as Saccharomyces cerevisiae.

[0056] 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.

[0057] Yeast selection medium SD-His-Leu-Lys-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.

[0058] 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:

[0059] Table 1 List of artificial genes

[0060]

[0061]

[0062] Example 2 Backbone plasmid construction

[0063] The information of the backbone plasmids involved in the construction of the following examples is shown in Table 2.

[0064] Table 2 Backbone plasmid list

[0065] 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

[0066] (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.

[0067] 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.

[0068] 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.

[0069] Table 3 Primer list

[0070]

[0071]

[0072] (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.

[0073] 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.

[0074] (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.

[0075] 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.

[0076] 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.

[0077] Similarly, pM351, pM352, pM353, pM354, pM355, and pM356 plasmids were constructed.

[0078] Example 2 Expression cassette plasmid construction

[0079] The plasmid information for constructing the expression cassettes involved in the following examples is shown in Table 4.

[0080] Table 4 Expression cassette plasmids

[0081]

[0082]

[0083] (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.

[0084] 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.

[0085] 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.

[0086] (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.

[0087] 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.

[0088] 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.

[0089] (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.

[0090] 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.

[0091] (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.

[0092] (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.

[0093] 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.

[0094] (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.

[0095] (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.

[0096] (8) Use T4 DNA ligase to ligate the AGA2-UGTMG1-AGA1-PacI:SbfI sticky end fragment and the pM355-PacI:SbfI linear vector.

[0097] (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.

[0098] Using bacterial cells as templates, the recombinant plasmid pM355-CPL3-3 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 bacterial culture, 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.

[0099] A similar method was adopted to obtain the plasmids listed in Table 4 through conventional molecular biology experimental techniques such as primer design, PCR amplification, OE-PCR fragment fusion, enzyme digestion and enzyme ligation.

[0100] Table 5 Primer list

[0101]

[0102] Example 3 Construction of p426-GAL80-Exg1-EGH1gRNA triple target plasmid

[0103] (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 EGH1 gRNA-2 fragments.

[0104] (2) Perform homologous recombination reactions on the GAL80 gRNA-1 and GAL80 gRNA-2 fragments, and the Exg1 gRNA-1 and EGH1 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.

[0105] (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.

[0106] 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-EGH1 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.

[0107] (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 25 and primer pair 26 shown in Table 6, and the corresponding PCR amplification products were recovered by gel cutting. The corresponding PCR amplification products were recovered by gel cutting to obtain GAL80 gRNA-3 and Exg1-EGH1 gRNA-3 fragments, and the GAL80 gRNA-3 and Exg1-EGH1 gRNA-3 fragments were homologously recombined using Seamless cloning Master Mix, transformed into TOP10 competent cells, and PCR verification was performed using primers gRNA-F and gRNA-R to obtain p426-GAL80-Exg1-EGH1 gRNA recombinant plasmid, and sequencing results of the recombinant plasmid showed that the recombinant plasmid contained GAL80gRNA (5'-gtcggtctcaacgcagccaa-3'), Exg1gRNA (5'-gaacaaattgaatggaagaa-3') and EGH1 gRNA (5'-gtggataggaatggctttgc-3').

[0108] Table 6 Primer list

[0109]

[0110] Example 4 Construction of recombinant yeast

[0111] (1) Using the plasmids shown in column 1 of Table 7 as templates, PCR amplification was performed using the primer pairs c shown in columns 3 and 4 of Table 7, and the corresponding PCR amplification products were recovered to obtain the screening marker Leu2, His3, Lys2 fragments and the modules CPL-M7-1, CPL-M7-2, and CPL-M4-3.

[0112] Table 7 Primer list

[0113]

[0114] (2) The plasmids shown in column 2 of Table 8 were double-digested with restriction endonucleases SmaI and StuI, respectively, and the double-digested products of the sizes shown in column 3 of Table 8 were recovered from the gel, thereby obtaining modules CPL-M1-1, CPL-M2-1, CPL-M3-1, CPL-M4-1, CPL-M5-1, CPL-M6-1, CPL-M7-1, CPL-M1-2, CPL-M2-2, CPL-M3-2, CPL-M4-2, CPL-M5-2, CPL-M6-2, CPL-M7-2, CPL-M1-3, CPL-M2-3, CPL-M3-3, and CPL-M4-3.

[0115] Table 8 Plasmid list

[0116]

[0117]

[0118] (3) Prepare Saccharomyces cerevisiae competent cells according to the instructions of the yeast competent cell preparation kit. To the competent cells of Saccharomyces cerevisiae, 0.1 μg of module CPL-M1-1, 0.1 μg of module CPL-M2-1, 0.1 μg of module CPL-M3-1, 0.1 μg of module CPL-M4-1, 0.1 μg of module CPL-M5-1, 0.1 μg of module CPL-M6-1, 0.1 μg of module CPL-M1-2, 0.1 μg of module CPL-M2-2, 0.1 μg of module CPL-M3-2, 0.1 μg of module CPL-M4-2, 0.1 μg of module CPL-M5-2, 0.1 μg of module CPL-M6-2, 0.1 μg of module CPL-M1-3, 0.1 μg of module CPL-M2-3 and 0.1 μg of module CPL-M3-3, and 0.2 μg of plasmid p426-GAL80-Exg1-EGH1 were added. gRNA, 0.2 μg of plasmid p414-Cas9, 0.1 μg of Leu2, 0.1 μg of His3, and 0.1 μg of Lys were electroporated at 2.7 kV. Then, 1 mL of mol / L sorbitol solution was added and the cells were allowed to recover at 30°C for 1 hour. The cells were then plated onto screening medium to obtain several single colonies and cultured at 30°C for at least 36 hours. The screening medium consisted of 0.8% SD-His-Leu-Lys-Ura, 2% glucose, and 2% agar powder.

[0119] (4) The single clones were first verified by PCR amplification using the primer pairs in the first column of Table 9, and then verified by DNA sequencing. The sequencing results showed that the modules CPL-M1-1, CPL-M2-1, CPL-M3-1, CPL-M4-1, CPL-M5-1, CPL-M6-1, CPL-M7-1 and the screening marker Leu2 were correctly inserted into the GAL80 site of the yeast genome; CPL-M1-2, CPL-M2-2, CPL-M3-2, CPL-M4-2, CPL-M5-2, CPL-M6-2, CPL-M7-2 and the screening marker His3 were correctly inserted into the Exg1 site of the yeast genome; CPL-M1-3, CPL-M2-3, CPL-M3-3, CPL-M4-3 and the screening marker Lys2 were inserted into the EGH1 site of the yeast.

[0120] Table 9 Primer list

[0121]

[0122]

[0123] Example 6: Fermentation of engineered yeast Saccharomyces cerevisiae and detection of mogroside V

[0124] (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.

[0125] (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.

[0126] (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.

[0127] (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 the engineered yeast Saccharomyces cerevisiae via shake flask fermentation was 234.1 mg / L.

[0128] 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 a genetically engineered yeast strain of Saccharomyces cerevisiae for efficiently synthesizing mogroside V based on multi-compartmental and multi-metabolic pathways, characterized by: The following steps are involved: (1) The mogroside alcohol metabolism enzymes, including 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, cytochrome P450 enzyme reductase AtCPR1, and leucine selection marker Leu2, were integrated into the Saccharomyces cerevisiae transcriptional repressor GAL80 locus; (2) Integrate truncated 3-hydroxy-3-methylglutaryl-CoA reductase tHMG1, prenyl pyrophosphate isomerase IDI1, epoxysqualene cyclase ERG1, ABC efflux protein PDR11, choline kinase ScCK, isopentenyl phosphate kinase AtIPK, geranyl pyrophosphate synthase ERG20, squalene synthase ERG9, hydroxymethylglutaryl-CoA reductase EfHMGR, hydroxymethylglutaryl-CoA reductase SpHMGR, cucurbitadienol synthase SgCDS, epoxyepoxide hydrolase SgEPH3, cytochrome P450 enzyme CYP87D18, cytochrome P450 enzyme reductase AtCPR1, and histidine selection marker His3 at the Saccharomyces cerevisiae glycoside hydrolase Exg1 site; (3) Integrate squalene cyclase ERG1, cucurbitadienol synthase SgCDS, epoxide hydrolase SgEPH3, cytochrome P450 enzyme CYP87D18, cytochrome P450 enzyme reductase AtCPR1, glycosyltransferase UGTMG1, sucrose synthase Susy and glycosyltransferase mutant SgUGT94-289-3 into the Saccharomyces cerevisiae glycoside hydrolase EGH1 site V148M / G152 A and the lysine selection marker Lys2.

2. The method for constructing an engineered yeast of Saccharomyces cerevisiae for efficiently synthesizing mogroside V based on peroxisomal subcellular organelles according to claim 1, characterized in that: In the step (1): The carboxyl termini of 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, endogenous squalene synthase ERG9, and epoxysqualene cyclase ERG1 are fused to the natural anchor motif Pex15 via a flexible linker (GGGGS) 3; the amino terminus of the epoxysqualene cyclase ERG1 is fused to the erythromycin polyketide synthase polypeptide interaction tag D5 via a flexible linker (GGGGS) 3. N DD fusion; 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 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 of Saccharomyces cerevisiae for efficiently synthesizing mogroside V based on peroxisomal subcellular organelles according to claim 1, characterized in that: In the step (2): The amino end and carbon end of the epoxysqualene cyclase ERG1 are respectively interacted with the chlortetracycline polyketide synthase polypeptide tag A2 through a flexible linker (GGGGS) 3 N DD and peptide interaction tag RIDD fusion; The carboxyl end of the ABC efflux protein PDR11 interacts with the chlortetracycline polyketide synthase polypeptide through a flexible linker (GGGGS) 3. C DD fusion; The amino and carboxyl ends of the cucurbitacin synthase SgCDS are respectively interacted with the rapamycin polyketide synthase polypeptide tag R11 through a flexible linker (GGGGS) 3. N DD and peptide interaction tag RIDD fusion; The amino and carboxyl ends of the epoxide hydrolase SgEPH3 are respectively connected to the rapamycin polyketide synthase polypeptide interaction tag R5 through a flexible linker (GGGGS) 3. N DD and R10 C DD fusion; The cytochrome P450 enzyme reductase AtCPR1 is truncated with 46 amino acid residues at the amino terminal and then fused to the carboxyl terminal of CYP87D18, and the carboxyl terminal interacts with the rapamycin polyketide synthase polypeptide tag R4 through a flexible linker (GGGGS) 3. C DD fusion.

4. The method for constructing an engineered yeast of Saccharomyces cerevisiae for efficiently synthesizing mogroside V based on peroxisomal subcellular organelles according to claim 1, wherein: In the step (3): The amino and carbon ends of the epoxysqualene cyclase ERG1 interact with the natural anchor motif PLN1 and the tacrolimus polyketide synthase peptide tag F4 respectively through a flexible linker (GGGGS) 3. C DD fusion; The amino terminus of the cucurbitadienol synthase SgCDS is fused to the natural anchor motif PLN1 via a flexible linker (GGGGS) 3, and the carboxyl terminus of the SgCDS is fused to the cytochrome P450 enzyme reductase AtCPR1 with 46 amino acid residues truncated at the amino terminus; The amino terminus of the cycloepoxide hydrolase SgEPH3 interacts with the tacrolimus polyketide synthase polypeptide through a flexible linker (GGGGS) 3 and a tag F7. N DD; 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.

5. The method for constructing a genetically engineered yeast strain of Saccharomyces cerevisiae for efficiently synthesizing mogroside V based on multi-compartmental and multi-metabolic pathways according to claim 1, characterized in that: The epoxysqualene cyclase ERG1 was additionally co-expressed in three copies, of which one copy of ERG1 was anchored on the peroxidase membrane surface and formed a linear multi-enzyme complex with the epoxy hydrolase SgEPH3, the cucurbitadienol synthase SgCDS, the cytochrome P450 enzyme CYP87D18 and the cytochrome P450 enzyme reductase AtCPR1 through peptide interactions; one copy of ERG1 was anchored on the ABC efflux protein PDR11 and formed a linear multi-protein complex with the epoxy hydrolase SgEPH3, the cucurbitadienol synthase SgCDS, the cytochrome P450 enzyme CYP87D18 and the cytochrome P450 enzyme reductase AtCPR1 through peptide interactions; and one copy of ERG1 was anchored in the lipid droplet subcellular compartment and formed a three-enzyme complex with the epoxy hydrolase SgEPH3 and the cucurbitadienol synthase SgCDS through peptide interactions.

6. The method for constructing a genetically engineered yeast strain of Saccharomyces cerevisiae for efficiently synthesizing mogroside V based on multi-compartmental and multi-metabolic pathways according to claim 1, characterized in that: The synthesis of mogroside V is divided into two stages: extracellular and intracellular. The precursor mogroside alcohol is synthesized in the cytoplasm, lipid droplets and peroxidase surface, and mogroside V is synthesized on the cell wall surface.

7. The method for constructing a genetically engineered yeast strain of Saccharomyces cerevisiae for efficiently synthesizing mogroside V based on multi-compartmental and multi-metabolic pathways 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 wall through an α-lectin system, and jointly catalyze the conversion of mogroside alcohol into mogroside V.

8. The method for constructing a genetically engineered yeast strain of Saccharomyces cerevisiae for efficiently synthesizing mogroside V based on multi-compartmental and multi-metabolic pathways according to claim 1, characterized in that: The coding genes of the MG-V anabolic enzymes are all expressed by the yeast galactokinase GAL1 gene promoter.

9. A genetically engineered strain of Saccharomyces cerevisiae that efficiently synthesizes mogroside V based on a multi-compartmental and multi-metabolic pathway, 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 above-mentioned engineered yeast Saccharomyces cerevisiae was used to ferment and produce mogroside V, using YPD medium supplemented with 10 mM sucrose, 10 mM uridine diphosphate, 21 mM 3-methyl-3-butene-1-ol and 9 mM 3-methyl-2-butene-1-ol as the fermentation medium.

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