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

Abstract

The invention discloses saccharomyces cerevisiae engineering bacteria for efficiently synthesizing mogroside V on the basis of cytoplasm and lipid droplet double-cell area compartment and a construction method of the saccharomyces cerevisiae engineering bacteria. The construction method comprises the following steps: integrating truncated 3-hydroxy-3-methylglutaryl coenzyme A reductase tHMG1, pentenyl pyrophosphate isomerase IDI1, epoxy squalene cyclization enzyme ERG1, squalene synthetase ERG9, choline kinase ScCK, isopentenyl phosphokinase AtIPK, cucurbitadienol synthetase SgCDS, cyclic epoxy hydrolase SgEPH3, cytochrome P450 enzyme CYP87D18 and cytochrome P450 enzyme reductase AtCPR1 at the site of a saccharomyces cerevisiae genome GAL80; an ABC efflux protein PDR11, a glycosyl transferase UGTMG1, a sucrose synthase Susy and a glycosyl transferase SgUGT94-289-3 are integrated at a site Exg1 of a saccharomyces cerevisiae genome. The yield of mogroside V produced by shake flask fermentation of the saccharomyces cerevisiae engineering strain provided by the invention reaches 96.3 mg / L, so that mogroside V is efficiently synthesized from the beginning in the saccharomyces cerevisiae, and the saccharomyces cerevisiae engineering strain has important practical significance for promoting sustainable development of the mogroside industry.

Description

Technical Field

[0001] The present invention relates to an engineered yeast of Saccharomyces cerevisiae for efficiently synthesizing mogroside V based on cytoplasm and lipid droplet dual compartmentalization and a construction method thereof, belonging to the field of bioengineering. Background Art

[0002] Mogroside is a cucurbitane-type triterpenoid saponin primarily derived from Momordica grosvenori. Mogroside IV, mogroside V, and simanoside I are the three sweetest components of mogroside, 392, 425, and 563 times sweeter than sucrose, respectively. Currently, mogroside is primarily produced by extraction. However, due to limitations in upstream raw material cultivation area and climate, large-scale expansion in the short term is difficult. Therefore, there is an urgent need to upgrade the mogroside V industry to reduce production costs and improve efficiency.

[0003] Achieving efficient terpenoid synthesis by constructing plant-derived terpenoid synthesis pathways within microbial hosts based on synthetic biology techniques is a must for future development. Saccharomyces cerevisiae, a generally recognized as safe (GRAS) model microorganism, is capable of endogenously synthesizing terpenoid precursors and possesses an intact membrane structure, making it widely used in the synthesis of a variety of plant-derived terpenes. Currently, industrial production of the artemisinin precursor artemisinic acid and the chemical raw material farnesene using Saccharomyces cerevisiae has been achieved, marking a milestone in the metabolic engineering of microbial production of terpenoid natural products. Squalene and mogroside are key precursors for the synthesis of MG-V (mangostinol), directly impacting the yield of MG-V heterologously synthesized in Saccharomyces cerevisiae. In Saccharomyces cerevisiae, squalene is primarily sequestered in lipid droplets and primarily flows into the ergosterol synthesis pathway. The isopentenol utilization pathway (IUP) was constructed in the Saccharomyces cerevisiae cytoplasm to increase the levels of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), promoting squalene synthesis. Squalene epoxidase ERG1 is a key enzyme in the biosynthesis of ergosterol and MG-V. The present invention anchors squalene epoxidase ERG1 to lipid droplet subcellular organelles. Using a multi-enzyme complex-directed assembly strategy, cucurbitadienol synthase SgCDS, epoxide hydrolase SgEPH3, cytochrome P450 enzyme CYP87D18, and cytochrome P450 enzyme reductase AtCPR1 linearly assemble with ERG1 into a multi-enzyme complex in lipid droplets, reducing competition in the ergosterol pathway and promoting the synthesis of mogrosantheol. Through the ABC efflux pump PDR11 and yeast cell surface display technology, mogroside alcohol in the cytoplasm is excreted to the extracellular space and MG-V is synthesized extracellularly, reducing the cytotoxic effect of MG-V. Thus, an engineered bacterium that efficiently synthesizes mogroside V based on the dual compartmentalization of cytoplasm and lipid droplets was constructed. The engineered bacterium produced mogroside V at a yield of 96.3 mg / L through shake flask fermentation, thereby efficiently synthesizing mogroside V from scratch in brewer's yeast, which has important practical significance for promoting the sustainable development of the mogroside industry. 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 lipid droplets, and a construction method thereof.

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

[0006] (1) The truncated 3-hydroxy-3-methylglutaryl-CoA reductase tHMG1, prenyl pyrophosphate isomerase IDI1, epoxysqualene cyclase ERG1, squalene synthase ERG9, choline kinase ScCK, isopentenyl phosphate kinase AtIPK, cucurbitadienol synthase SgCDS, cycloepoxide 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, 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 HMG1 (NCBI Sequence ID: NP_013636.1) N-terminal truncation of 527 amino acids tHMG1, prenyl pyrophosphate isomerase IDI1 (NCBI Sequence ID: NP_015208.1), choline kinase ScCK (NCBI Sequence ID: NP_013234.1), isopentenyl phosphate kinase AtIPK (NCBI Sequence ID: NP_173986.2).

[0010] The number of copies of the epoxysqualene cyclase ERG1 (NCBI Sequence ID: NP_011691.1) is 2, and the amino terminus and carboxyl terminus of one copy of ERG1 are respectively connected to the erythromycin polyketide synthase polypeptide interaction tag D3 through a flexible linker (GGGGS) 3. N DD (nucleotide sequence as shown in SEQ ID NO.1) and the peptide interaction tag RIAD (nucleotide sequence as shown in SEQ ID NO.2) were fused; the amino and carboxyl termini of another copy of ERG1 were respectively linked to the natural anchor motif PLN1 (NCBI Sequence ID: NP_012972.3) and the rapamycin polyketide synthase peptide interaction tag R4 through a flexible linker (GGGGS) 3 C DD (nucleotide sequence as SEQ ID NO.3) fusion.

[0011] The copy number of the epoxide hydrolase SgEPH3 (NCBI Sequence ID: P0DO70.1) is 2, and the amino terminus and carboxyl terminus of one copy of SgEPH3 are respectively connected to the rapamycin polyketide synthase peptide interaction tag R5 through a flexible linker (GGGGS) 3. N DD (nucleotide sequence as SEQ ID NO.4) and R10C DD (nucleotide sequence as SEQ ID NO.5) fusion; another copy of SgEPH3 through a flexible linker (GGGGS) 3 and chlortetracycline polyketide synthase polypeptide interaction tag A2 N DD (nucleotide sequence shown in SEQ ID NO.6) and erythromycin polyketide synthase polypeptide interaction tag D4 C DD fusion (nucleotide sequence is shown in SEQ ID NO.7).

[0012] The copy number of the cucurbitadienol synthase SgCDS (NCBI Sequence ID: K7NBZ9.1) is 2, wherein the amino and carboxyl ends of one copy of SgCDS are respectively interacted with the erythromycin polyketide synthase polypeptide tag D5 through a flexible linker (GGGGS) 3. N DD (nucleotide sequence as shown in SEQ ID NO.8) and the peptide interaction tag RIDD (nucleotide sequence as shown in SEQ ID NO.9); the amino and carboxyl ends of another copy of SgCDS are respectively connected to the rapamycin polyketide synthase peptide interaction tag R11 through a flexible linker (GGGGS) 3 N DD (nucleotide sequence as SEQ ID NO.10) and tacrolimus polyketide synthase polypeptide interaction tag F6 C DD (nucleotide sequence such as SEQ ID NO.11) fusion.

[0013] The cytochrome P450 enzyme SgCYP87D18 (NCBI Sequence ID: K7NBR2.1) has two copies, one of which has an amino-terminal connection to a tacrolimus polyketide synthase polypeptide interaction tag F5 via a flexible linker (GGGGS) 3. N DD (nucleotide sequence as SEQ ID NO.12) fusion; another copy of SgCYP87D18 amino terminal through a flexible linker (GGGGS) 3 tacrolimus polyketide synthase polypeptide interaction tag F7 N DD (nucleotide sequence as SEQ ID NO.13) fusion.

[0014] The cytochrome P450 enzyme reductase AtCPR1 (NCBI Sequence ID: NP_194183.1) has two copies, one of which is truncated at the amino terminus by 46 amino acid residues and fused to the carboxyl terminus of SgCYP87D18, and the carboxyl terminus interacts with the chlortetracycline polyketide synthase polypeptide tag A2 via a flexible linker (GGGGS) 3. CDD (nucleotide sequence such as SEQ ID NO.14) is fused; the other copy of AtCPR1 is truncated with 46 amino acid residues at the amino terminus and then fused to the carboxyl terminus of SgCYP87D18.

[0015] In the step (2):

[0016] The carboxyl terminus of the ABC efflux protein PDR11 (NCBI Sequence ID: NP_012252.1) is connected to the tacrolimus polyketide synthase polypeptide interaction tag F4 via a flexible linker (GGGGS) 3 C DD (nucleotide sequence such as SEQ ID NO.15) fusion.

[0017] The carboxyl terminus and amino terminus of the glycosyltransferase UGTMG1 (NCBI Sequence ID: 6L8W_A) are fused to the Aga1p (NCBI Sequence ID: NP_014442.1) and Aga2p (amino acid sequence shown in SEQ ID NO. 16) subunits of α-agglutinin, respectively.

[0018] The carboxyl terminus and amino terminus of the sucrose synthase Susy (NCBI Sequence ID: WP_011381564.1) were fused to the Aga1p and Aga2p subunits of α-agglutinin, respectively.

[0019] 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.

[0020] The synthesis of mogroside V is carried out in two different regions, wherein the precursor mogroside alcohol is synthesized in the cytoplasm and lipid droplets, and mogroside V is synthesized on the surface of the cell wall.

[0021] The precursor mogroside is transported from the cytoplasm to the extracellular space via the ABC efflux protein PDR11.

[0022] The epoxysqualene cyclase ERG1 and the cucurbitadienol synthase SgCDS construct a three-enzyme complex in the cytoplasm in the form of one ERG1 molecule and two SgCDS molecules through the peptide interaction tags RIAD and RIDD.

[0023] In the cytoplasm, epoxysqualene cyclase ERG1, cucurbitadienol synthase SgCDS, epoxyepoxide hydrolase SgEPH3, cytochrome P450 enzyme CYP87D18 and cytochrome P450 enzyme reductase AtCPR1 form a linear directional multi-enzyme complex in the cell membrane matrix side through a polypeptide interaction tag and ABC efflux protein PDR11.

[0024] The epoxysqualene cyclase ERG1 is anchored in the lipid droplet subcell via a natural anchoring motif PLN1.

[0025] Cucurbitadienol synthase SgCDS, cycloepoxide hydrolase SgEPH3, cytochrome P450 enzyme CYP87D18 and cytochrome P450 enzyme reductase AtCPR1 in the lipid droplets form a linear directional multi-enzyme complex through polypeptide interaction with epoxysqualene cyclase ERG1 as an anchoring base point, thereby synthesizing mogroside alcohol in the lipid droplets.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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.17).

[0030] The present invention also provides an engineered yeast strain of Saccharomyces cerevisiae for efficiently synthesizing mogroside V based on the dual compartmentalization of cytoplasm and lipid droplets, which is specifically constructed by the above-mentioned construction method.

[0031] 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.

[0032] 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.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] Mogroside V (MG-V) is a triterpenoid compound, and squalene is the key precursor for its biosynthesis. Most squalene in yeast is mainly stored in lipid droplet subcellular compartments, and squalene mainly flows into the ergosterol synthesis pathway. Among them, squalene epoxidase ERG1 is the key enzyme in the biosynthesis of ergosterol and MG-V, which directly affects the yield of MG-V heterologously synthesized in Saccharomyces cerevisiae.

[0035] In Saccharomyces cerevisiae, squalene is primarily sequestered in lipid droplets and primarily flows into the ergosterol biosynthesis pathway. By constructing an isopentenol utilization pathway (IUP) in the Saccharomyces cerevisiae cytoplasm, the levels of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) are increased, promoting squalene synthesis. Squalene epoxidase ERG1 is a key enzyme in the biosynthesis of ergosterol and MG-V. This study anchors squalene epoxidase ERG1 to lipid droplet subcellular organelles. Using a multi-enzyme complex-directed assembly strategy, cucurbitadienol synthase SgCDS, cycloepoxide hydrolase SgEPH3, cytochrome P450 enzyme CYP87D18, and cytochrome P450 reductase AtCPR1 linearly assemble with ERG1 into a multi-enzyme complex within lipid droplets, reducing competition with the ergosterol pathway and promoting mogrosanol synthesis. Specifically, the present invention overexpresses two copies of epoxysqualene cyclase ERG1, wherein one copy of epoxysqualene cyclase ERG1 is anchored in lipid droplets and, through polypeptide interactions, interacts with epoxy hydrolase SgEPH3 and downstream metabolic enzymes such as cucurbitadienol synthase SgCDS, cytochrome P450 enzyme CYP87D18 and cytochrome P450 enzyme reductase AtCPR1 to form a linear multi-enzyme complex, thereby facilitating the conversion of squalene in lipid droplets into mogroside alcohol; the other copy of epoxysqualene cyclase ERG1 is anchored in lipid droplets and interacts with epoxy hydrolase SgEPH3 and downstream metabolic enzymes such as cucurbitadienol synthase SgCDS, cytochrome P450 enzyme CYP87D18 and cytochrome P450 enzyme reductase AtCPR1 to form a linear multi-enzyme complex. The squalene cyclase ERG1 is expressed in the cytoplasm and assembles a multi-enzyme complex with the epoxide hydrolase SgEPH3 in a 1:2 ratio through a peptide interaction tag. It further assembles into a linear multi-enzyme complex with the epoxide hydrolase SgEPH3 and downstream metabolic enzymes such as cucurbitadienol synthase SgCDS, cytochrome P450 enzyme CYP87D18 and cytochrome P450 enzyme reductase AtCPR1 in the cytoplasm, thereby promoting the conversion of cytoplasmic squalene to mogroside alcohol.

[0036] In addition, the present invention uses the ABC efflux pump PDR11 and yeast cell surface display technology to excrete mogroside alcohol in the cytoplasm to the extracellular space and synthesize MG-V extracellularly, thereby reducing the cytotoxic effect of MG-V. That is, the ABC efflux pump protein PDR11 and the mogroside alcohol synthase are assembled into a multi-enzyme complex to form an integrated synthesis and efflux of mogroside alcohol, thereby promoting the transport of mogroside alcohol to the extracellular space to synthesize MG-V, and reducing the effect of MG-V on yeast cell growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Schematic diagram of integration into the GAL80 locus of the Saccharomyces cerevisiae genome.

[0038] Figure 2 Schematic diagram of integration into the Exg1 locus of the Saccharomyces cerevisiae genome. DETAILED DESCRIPTION

[0039] 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.

[0040] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0041] Unless otherwise specified, the test materials used in the following examples were purchased from conventional biochemical reagent stores.

[0042] 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.

[0043] 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.

[0044] 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, SpeedyCutSmaI, SpeedyCutStuI, SpeedyCutAscI, SpeedyCutSacII, SpeedyCutPacI, SpeedyCutSbfI, 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 DNA Ligase 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.

[0045] 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:

[0046] Table 1 List of artificial genes

[0047]

[0048] Example 2 Backbone plasmid construction

[0049] The information of the backbone plasmids involved in the construction of the following examples is shown in Table 2.

[0050] Table 2 Backbone plasmid list

[0051]

[0052]

[0053] (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.

[0054] 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.

[0055] 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.

[0056] Table 3 Primer list

[0057]

[0058]

[0059] (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.

[0060] 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.

[0061] (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.

[0062] 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.

[0063] 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.

[0064] Similarly, plasmids pM351, pM352, pM353, pM354, pM355, pM356, and pM357 were constructed.

[0065] Example 2 Expression cassette plasmid construction

[0066] The plasmid information for constructing the expression cassettes involved in the following examples is shown in Table 4.

[0067] Table 4 Expression cassette plasmids

[0068]

[0069] (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.

[0070] 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.

[0071] 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.

[0072] (2) Take R4 in the second column of Table 5 C Overlap extension PCR (OE-PCR) was performed on the DD, ERG1 and PLN1 fragments, and the OE-PCR amplification products were recovered by gel cutting to obtain PLN1-ERG1-R4 C DD fragment.

[0073] 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.

[0074] 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.

[0075] (3) Double digestion of PLN1-ERG1-R4 with restriction endonucleases AscI and SacII C The DD fragment and pM354 plasmid were purified by PCR product purification kit to obtain PLN1-ERG1-R4 C DD-AscI:SacII sticky-end fragment and pM354-AscI:SacII linear vector.

[0076] 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.

[0077] (4) Use T4 DNA ligase to ligate PLN1-ERG1-R4 C Ligate the DD-AscI:SacII sticky-end fragment to the pM354-AscI:SacII linearized vector. A 10μL T4 DNA ligase reaction system consists of: 1μL 10× T4 DNA Ligase Buffer, 30ng of the ERG10-Pex15-AscI:SacII sticky-end fragment and the pM354-AscI:SacII linearized vector, 0.5μL T4 DNA Ligase, and distilled water to a total of 10μL. Incubate at room temperature (25°C) for 2 hours.

[0078] (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.

[0079] Using bacteria as templates, primers CYC1t-f1 (5'-tttcggttagagcggatgtg-3') and pGAL1-r1 (5'-gacgaggacgcacggaggagagt-3') were used for verification to obtain the recombinant plasmid pM354-PLN1-ERG1-R4. C DD. PCR verification system 25μL: 2×SanTaq PCR Mix 12.5μL, drop solution 1μL, CYC1t-f1 and pGAL1-r1 1μL each, distilled water 11.5μL. C DD was sequenced, and the sequencing results showed that the recombinant plasmid contained SmaI-CYC1t-SacII-PLN1-ERG1-R4 C DD-AscI-pGAL1,10-PacI-SbfI-TEF1t-StuI expression cassette.

[0080] (6) Take R5 in the second column of Table 5 N DD, SgEPH3, and R10 C The DD fragment was used as a template for OE-PCR, and the OE-PCR amplification product was recovered by gel cutting to obtain R5. N DD-SgEPH3-R10 C DD fragment.

[0081] (7) Double digestion of R5 with restriction endonucleases PacI and SbfI N DD-SgEPH3-R10 C DD fragment and pM354-PLN1-ERG1-R4 C DD plasmids, and the enzyme digestion products were purified using a PCR product purification kit to obtain R5 N DD-SgEPH3-R10 C DD-PacI:SbfI sticky-end fragment and pM354-PLN1-ERG1-R4 C DD-PacI:SbfI linearized vector.

[0082] (8) Use T4 DNA ligase to ligate R5 N DD-SgEPH3-R10 C DD-PacI:SbfI sticky-end fragment and pM354-PLN1-ERG1-R4 C Ligate the DD-PacI:SbfI linear vector.

[0083] (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.

[0084] Using bacterial cells as templates and primers pGAL1-f1 (5'-caaccataggatgataatgcgat-3') and TEF1t-r1 (5'-catttggactgtcgcctgtt-3') as validation, the recombinant plasmid pM353-CP4 was obtained. The PCR verification system (25 μL) consisted of: 12.5 μL of 2× SanTaq PCR Mix, 1 μL of liquid, 1 μL each of pGAL1-f1 and TEF1t-r1, and 11.5 μL of distilled water. The recombinant plasmid was sequenced, and the sequencing results showed that the recombinant plasmid contained SmaI-CYC1t-SacII-R4. C DD-ERG1-PLN1-AscI-pGAL1,10-PacI-R5 N DD-SgEPH3-R10 C DD-SbfI-TEF1t-StuI expression cassette.

[0085] Similarly, the plasmids listed in Table 4 were obtained.

[0086] Table 5 Primer list

[0087]

[0088] Example 3 Construction of p426-GAL80-Exg1 gRNA dual-target plasmid

[0089] (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.

[0090] (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.

[0091] (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.

[0092] 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.

[0093] (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').

[0094] Table 6 Primer list

[0095]

[0096] Example 4 Construction of recombinant yeast

[0097] (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-M3'.

[0098] Table 7 Primer list

[0099]

[0100] (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, CP-M1′, and CP-M2′.

[0101] Table 8 Plasmid list

[0102] Module Plasmid name Endonuclease name Size (bp) CP-M1 pM350-CP1 SmaI and StuI 4358 CP-M2 pM351-CP2 SmaI and StuI 3389 CP-M3 pM352-CP3 SmaI and StuI 4305 CP-M4 pM353-CP4 SmaI and StuI 5963 CP-M5 pM354-CP5 SmaI and StuI 4770 CP-M6 pM355-CP6 SmaI and StuI 4631 CP-M1 pM357-CP1 PmeI and StuI 5518 CP-M2 pM355-CP2 SmaI and StuI 8891

[0103] (3) Prepare Saccharomyces cerevisiae competent cells according to the instructions of the yeast competent cell preparation kit. To Saccharomyces cerevisiae competent cells, 0.1 μg of modules CP-M1, 0.1 μg of modules CP-M2, 0.1 μg of modules CP-M3, 0.1 μg of modules CP-M4, 0.1 μg of modules CP-M5, 0.1 μg of modules CP-M6, 0.1 μg of modules CP-M7, 0.1 μg of modules CP-M1`, 0.1 μg of modules CP-M2`, and 0.1 μg of modules CP-M3`, 0.2 μg of plasmid p426-GAL80-Exg1 gRNA, 0.2 μg of plasmid p414-Cas9, 0.1 μg of fragment Leu2, and 0.1 μg of fragment His3 were added. After 2.7 kV electroporation, 1 mL of mol / L sorbitol solution was added, and the cells were revived at 30°C for 1 hour. The cells were then plated onto screening medium to obtain several single clones, which were cultured at 30°C for at least 36 hours. The screening medium consisted of: 0.8% SD-His-Leu-Trp-Ura, 2% glucose and 2% agar powder.

[0104] (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 the modules CP-M1, M2, M3, M4, M5, M6, M7 and the screening marker Leu2 were correctly inserted into the GAL80 site of the yeast genome, and the M1`, M2`, M3` and His3 screening markers were correctly inserted into the Exg1 site of the yeast genome.

[0105] Table 9 Primer list

[0106]

[0107]

[0108] Example 6: Fermentation of engineered yeast Saccharomyces cerevisiae and detection of mogroside V

[0109] (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.

[0110] (2) Fermentation culture: The seed culture was inoculated at a 1% to 5% inoculum into YPD liquid 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. The culture was carried out at 30°C and 220 rpm for 96 h, and the fermentation broth was collected.

[0111] (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.

[0112] (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 96.3 mg / L.

[0113] 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 efficient synthesis of mogroside V based on dual compartmentalization of cytoplasm and lipid droplets, characterized by: The following steps are involved: (1) Integrate truncated 3-hydroxy-3-methylglutaryl-CoA reductase tHMG1, prenyl pyrophosphate isomerase IDI1, epoxysqualene cyclase ERG1, squalene synthase ERG9, choline kinase ScCK, isopentenyl phosphate kinase AtIPK, cucurbitadienol synthase SgCDS, cycloepoxide hydrolase SgEPH3, cytochrome P450 enzyme CYP87D18, and cytochrome P450 enzyme reductase AtCPR1 into the GAL80 locus of the Saccharomyces cerevisiae genome; (2) The ABC efflux protein PDR11, 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 lipid droplets according to claim 1, characterized in that: In the step (1): The HMG1 N-terminal truncation of 527 amino acids tHMG1, prenyl pyrophosphate isomerase IDI1, choline kinase ScCK, isopentenyl phosphate kinase AtIPK; The number of copies of the epoxysqualene cyclase ERG1 is 2, and the amino end and carboxyl end of one copy of ERG1 are respectively connected to the erythromycin polyketide synthase polypeptide interaction tag D3 through a flexible linker (GGGGS) 3. N DD is fused to the peptide interaction tag RIAD; the amino and carboxyl ends of another copy of ERG1 are respectively connected to the natural anchor motif PLN1 and the rapamycin polyketide synthase peptide interaction tag R4 through a flexible linker (GGGGS) 3 C DD fusion; The number of copies of the epoxide hydrolase SgEPH3 is 2, and the amino terminus and carboxyl terminus of one copy of SgEPH3 are respectively connected to the rapamycin polyketide synthase peptide interaction tag R5 through a flexible linker (GGGGS) 3. N DD and R10 C DD fusion; another copy of SgEPH3 interacts with the chlortetracycline polyketide synthase polypeptide tag A2 through a flexible linker (GGGGS) 3 N DD (and erythromycin polyketide synthase peptide interaction tag D4 C DD fusion; The copy number of the cucurbitacadienol synthase SgCDS is 2, wherein the amino terminus and carboxyl terminus of one copy of SgCDS are respectively connected to the erythromycin polyketide synthase polypeptide interaction tag D5 through a flexible linker (GGGGS) 3. N DD and peptide interaction tag RIDD are fused; the other copy of SgCDS amino and carboxyl ends are respectively connected to rapamycin polyketide synthase peptide interaction tag R11 through flexible linker (GGGGS) 3 N DD and Tacrolimus Polyketide Synthase Peptide Interaction Tag F6 C DD fusion; The number of copies of the cytochrome P450 enzyme SgCYP87D18 is 2, and the amino terminus of one copy of SgCYP87D18 interacts with the tacrolimus polyketide synthase polypeptide through a flexible linker (GGGGS) 3 and a tag F5. N DD fusion; another copy of SgCYP87D18 amino terminus interacts with tacrolimus polyketide synthase polypeptide through a flexible linker (GGGGS) 3 tag F7 N DD fusion; The cytochrome P450 enzyme reductase AtCPR1 has two copies, one of which is truncated at the amino terminal by 46 amino acid residues and fused to the carboxyl terminal of SgCYP87D18, and the carboxyl terminal interacts with the chlortetracycline polyketide synthase polypeptide through a flexible linker (GGGGS) 3. Tag A2 C DD fusion; the other copy of AtCPR1 was truncated at the amino terminus by 46 amino acid residues and then fused to the carboxyl terminus of SgCYP87D18.

3. The method for constructing an engineered yeast strain of Saccharomyces cerevisiae for efficiently synthesizing mogroside V based on dual compartmentalization of cytoplasm and lipid droplets according to claim 1, characterized in that: In the step (2): The carboxyl end of the ABC efflux protein PDR11 interacts with the tacrolimus polyketide synthase polypeptide through a flexible linker (GGGGS) 3 and a tag F4. C DD fusion; The carboxyl terminus and amino terminus of the glycosyltransferase UGTMG1 are fused to the Aga1p and Aga2p subunits of α-agglutinin, respectively; The carboxyl terminus and amino terminus of the sucrose synthase Susy 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 lipid droplets 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 cytoplasm and lipid droplets and transported from the cytoplasm to the extracellular space through the ABC efflux protein PDR11; mogroside V is synthesized on the cell wall surface.

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 lipid droplets according to claim 1, characterized in that: The epoxysqualene cyclase ERG1 and the cucurbitadienol synthase SgCDS construct a three-enzyme complex in the cytoplasm in the form of one ERG1 molecule and two SgCDS molecules through the peptide interaction tags RIAD and RIDD; In the cytoplasm, epoxysqualene cyclase ERG1, cucurbitadienol synthase SgCDS, epoxyepoxide hydrolase SgEPH3, cytochrome P450 enzyme CYP87D18 and cytochrome P450 enzyme reductase AtCPR1 form a linear directional multi-enzyme complex in the matrix side of the cell membrane through a polypeptide interaction tag with the ABC efflux protein PDR11; The epoxysqualene cyclase ERG1 is anchored in the lipid droplet subcell via a natural anchoring motif PLN1.

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 lipid droplets according to claim 1, characterized in that: Cucurbitadienol synthase SgCDS, cycloepoxide hydrolase SgEPH3, cytochrome P450 enzyme CYP87D18 and cytochrome P450 enzyme reductase AtCPR1 in the lipid droplets form a linear directional multi-enzyme complex through polypeptide interaction with epoxysqualene cyclase ERG1 as an anchoring base point, thereby synthesizing mogroside alcohol in the lipid droplets.

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 lipid droplets 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 an engineered yeast strain of Saccharomyces cerevisiae for efficient synthesis of mogroside V based on dual compartmentalization of cytoplasm and lipid droplets 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. An engineered yeast strain of Saccharomyces cerevisiae that efficiently synthesizes mogroside V based on dual compartmentalization of cytoplasm and lipid droplets, 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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