Saccharomyces cerevisiae engineering bacteria for efficiently synthesizing mogroside V based on lipid droplet subcellular organelles and construction method of saccharomyces cerevisiae engineering bacteria

Abstract

The invention discloses saccharomyces cerevisiae engineering bacteria for efficiently synthesizing mogroside V based on lipid droplet subcellular organelles and a construction method of the saccharomyces cerevisiae engineering bacteria. The construction method comprises the following steps: integrating squalene epoxidase ERG1, cucurbitadienol synthetase SgCDS, cyclic epoxy hydrolase SgEPH3, cytochrome P450 enzyme CYP87D18 and cytochrome P450 enzyme reductase AtCPR1 at a site GAL80 of a saccharomyces cerevisiae genome; the saccharomyces cerevisiae strain is characterized in that phosphoglucose mutase PGM1, alpha-phosphoglucose mutase PGM2, uridine diphosphate glucose pyrophosphorylase UGP1, glycosyl transferase UGTMG1 and glycosyl transferase SgUGT94-289-3 are integrated at a site Exg1 of a saccharomyces cerevisiae genome. The Saccharomyces cerevisiae engineering bacterium constructed by the invention realizes that the yield of MG-V synthesized by shake flask fermentation is 31.4 mg / L. The Saccharomyces cerevisiae engineering bacterium has the characteristics of strong metabolic flux directionality, high yield and wide industrial application prospect.

Description

Technical Field

[0001] The present invention relates to an engineered yeast of Saccharomyces cerevisiae capable of efficiently synthesizing mogroside V based on lipid droplet subcellular organelles and a construction method thereof, and belongs to the field of synthetic biology. Background Art

[0002] A high-sugar diet contributes to a range of modern health problems, including dental caries, obesity, diabetes, metabolic syndrome, and cardiovascular disease. Plant-derived mogrosides (MG) have emerged as a new generation of sweeteners, satisfying the need for sweetness, due to their high sweetness, low calories, safety, fat-reducing properties, and resistance to dental caries. Mogrosides are widely used as beverage and food additives. The national standard, "National Food Safety Standard for Food Additives" (GB 2760-2014), stipulates that mogrosides can be used in unlimited quantities. It is the only sweetener approved for use in infant and toddler foods, making it a new favorite in the global sweetener market. MG has currently received GRAS certification from the U.S. Food and Drug Administration (FDA) and has gained market access in over 20 countries, including the United States, Japan, South Korea, Singapore, and Australia. Against the backdrop of global sugar reduction efforts, MG holds enormous market potential.

[0003] Mogroside V is the main source of sweetness in mogrosides, but its content in the whole fruit is only 0.8% to 1.3%. Furthermore, the optimal cultivation area for monk fruit is narrow and biological resources are limited. Therefore, large-scale production of mogroside V cannot be achieved by extracting it from monk fruit. Using advanced synthetic biology techniques to construct microbial cell factories that synthesize mogroside V from scratch in microorganisms is a disruptive method for producing mogroside V in the future, enabling low-cost, large-scale production.

[0004] Mogroside V is a triterpenoid saponin produced in the cytoplasm. Its biosynthetic pathway can be roughly divided into four stages: the synthesis of the upstream precursors IPP and DMAPP, the formation of the midstream skeleton 24,25-epoxycucurbitadienol, the downstream production of the parent nucleus mogroside alcohol, and the formation of mogroside V. Saccharomyces cerevisiae is a safe eukaryotic model microorganism with a clear genetic background, mature genetic modification technology, and a mevalonate (MVA) pathway capable of biosynthesizing terpenes. IPP and DMAPP can be sequentially synthesized into the triterpene precursor squalene SQ under the catalysis of farnesyl pyrophosphate synthase ERG20 and squalene synthase ERG9.

[0005] Thus, squalene is a key intermediate in the fermentation of mogroside V by Saccharomyces cerevisiae. Its content and utilization efficiency determine the yield of mogroside V produced by Saccharomyces cerevisiae. While some Saccharomyces cerevisiae squalene enters the ergosterol pathway, the majority is temporarily stored as a fat-soluble component in lipid droplet subcellular organelles. Squalene epoxidase ERG1 is a key enzyme in the biosynthesis of ergosterol and MG-V. The present invention anchors squalene epoxidase ERG1 in lipid droplets and, through a peptide interaction tag, assembles a multi-enzyme complex with the epoxide hydrolase SgEPH3 in a 1:2 ratio, pulling squalene toward MG-V synthesis. This results in a Saccharomyces cerevisiae engineering project for efficient MG-V synthesis within lipid droplet subcellular organelles. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide an engineered yeast of Saccharomyces cerevisiae that can efficiently synthesize mogroside V based on lipid droplet subcellular organelles and a construction method thereof.

[0007] The method for constructing an engineered yeast strain of Saccharomyces cerevisiae for efficiently synthesizing mogroside V based on lipid droplet subcellular organelles comprises the following steps:

[0008] (1) Squalene epoxidase ERG1, cucurbitadienol synthase SgCDS, epoxide hydrolase SgEPH3, cytochrome P450 enzyme CYP87D18, and cytochrome P450 enzyme reductase AtCPR1 were integrated into the GAL80 locus of the Saccharomyces cerevisiae genome;

[0009] (2) Integrate phosphoglucomutase PGM1, α-phosphoglucomutase PGM2, uridine diphosphate glucose pyrophosphorylase UGP1, glycosyltransferase UGTMG1, and glycosyltransferase SgUGT94-289-3 into the Exg1 locus of the Saccharomyces cerevisiae genome.

[0010] In step (1):

[0011] The amino terminus of the epoxysqualene cyclase ERG1 (NCBI Sequence ID: NP_011691.1) was fused to the natural anchor motif PLN1 (NCBI Sequence ID: NP_012972.3) via a flexible linker (GGGGS) 3.

[0012] The carboxyl end of the epoxysqualene cyclase ERG1 is fused to the polypeptide interaction tag RIAD (nucleotide sequence shown in SEQ ID NO.1) via a flexible linker (GGGGS) 3.

[0013] 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. 2) and erythromycin polyketide synthase polypeptide interaction tag D2 C DD (nucleotide sequence as SEQ ID NO.3) fusion.

[0014] 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 such as SEQ ID NO.4) and polypeptide interaction tag RIDD (nucleotide sequence such as SEQ ID NO.5) are fused.

[0015] 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.6) fusion.

[0016] 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.7) fusion.

[0017] In step (2):

[0018] The amino and carboxyl ends of the phosphoglucomutase PGM1 (NCBI Sequence ID: NP_012795.1) are respectively connected to the rapamycin polyketide synthase peptide interaction tag R5 through a flexible linker (GGGGS) 3 N DD (nucleotide sequence such as SEQ ID NO.8) and R10 C DD (nucleotide sequence as SEQ ID NO.9) fusion.

[0019] The carboxyl terminus of the α-phosphoglucomutase PGM2 (NCBI Sequence ID: NP_013823.1) interacts with the rapamycin polyketide synthase peptide tag R4 through a flexible linker (GGGGS) 3C DD (nucleotide sequence such as SEQ ID NO.10) fusion.

[0020] The amino and carboxyl ends of the uridine diphosphate glucose pyrophosphorylase UGP1 (NCBI Sequence ID: NP_012889.3) 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.11) and tacrolimus polyketide synthase peptide interaction tag F4 C DD (nucleotide sequence such as SEQ ID NO.12) fusion.

[0021] The amino and carboxyl ends of the glycosyltransferase UGTMG1 (NCBI Sequence ID: 6L8W_A) are respectively connected to the tacrolimus polyketide synthase polypeptide interaction tag F5 through a flexible linker (GGGGS) 3. N DD (nucleotide sequence as SEQ ID NO.13) and F6 C DD (nucleotide sequence such as SEQ ID NO.14) fusion.

[0022] The glycosyltransferase SgUGT94-289-3 V148M / G152A The amino and carboxyl termini of the mutant SgUGT94-289-3 (NCBI Sequence ID: 8HJO_A) were linked to the tacrolimus polyketide synthase peptide interaction tag F7 through flexible linkers (GGGGS) 3, respectively. N DD (nucleotide sequence as SEQ ID NO.15) and chlortetracycline polyketide synthase polypeptide interaction tag A1 C DD (nucleotide sequence such as SEQ ID NO.16) fusion.

[0023] The epoxysqualene cyclase ERG1 is anchored in the lipid droplet subcellular compartment via the natural anchoring motif PLN1.

[0024] The one molecule of epoxysqualene cyclase ERG1 and the two molecules of epoxyhydrolase SgEPH3 are assembled into a multi-enzyme complex in lipid droplets through the polypeptide interaction tags RIAD and RIDD.

[0025] The cycloepoxide hydrolase SgEPH3, cucurbitadienol synthase SgCDS, cytochrome P450 enzyme CYP87D18, cytochrome P450 enzyme reductase AtCPR1, glycosyltransferase SgUGT94-289-3, glycosyltransferase UGTMG1, uridine diphosphate glucose pyrophosphorylase UGP1, phosphoglucomutase PGM1 and α-phosphoglucomutase PGM2 are linearly assembled into a multi-enzyme complex in lipid droplets through an erythromycin polyketide synthase module, a chlortetracycline polyketide synthase module, a tacrolimus polyketide synthase module and a rapamycin polyketide synthase module.

[0026] The coding genes of the MG-V anabolic enzymes are all driven to express by the yeast galactokinase (GAL1) gene promoter (nucleotide sequence such as SEQ ID NO.17).

[0027] The Saccharomyces cerevisiae engineered bacteria that efficiently synthesizes mogroside V based on lipid droplet subcellular organelles is constructed by the above-mentioned construction method.

[0028] The present invention also provides a method for producing mogroside V.

[0029] Specifically, a method for producing mogroside V comprises fermenting the above-mentioned engineered yeast Saccharomyces cerevisiae to produce mogroside V.

[0030] The method for producing mogroside V uses YPD culture medium as fermentation medium.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] Mogroside V (MG-V) is a triterpenoid compound, and squalene is a key precursor in its biosynthesis. Squalene epoxidase ERG1 is a key enzyme in the biosynthesis of ergosterol and MG-V. To increase the biosynthetic flux of mogroside V, the present invention anchors the squalene cyclase ERG1 in lipid droplets. Through a peptide interaction tag, a multi-enzyme complex is assembled with the epoxide hydrolase SgEPH3 at a 1:2 ratio, pulling squalene toward MG-V synthesis. Moreover, the epoxide hydrolase SgEPH3 and downstream metabolic enzymes, such as cucurbitadienol synthase SgCDS, cytochrome P450 enzyme CYP87D18, cytochrome P450 enzyme reductase AtCPR1, phosphoglucomutase PGM1, α-phosphoglucomutase PGM2, uridine diphosphate glucose pyrophosphorylase UGP1, glycosyltransferase UGTMG1, and glycosyltransferase SgUGT94-289-3, are linearly assembled into a multi-enzyme complex in lipid droplets, thereby allowing more squalene in lipid droplets to flow to the MG-V anabolic flow. In addition, glycosyltransferase UGTMG1 and glycosyltransferase SgUGT94-289-3 assemble into an enzyme complex, which brings different mogroside glycosyltransferases closer in space, strengthens the approach effect and directional effect of glycosyltransferases, improves the adaptability of exogenous glycosyltransferases in the lipid droplet subcellular organelle microenvironment, increases the activity of glycosyltransferases, and also increases the specificity of glycosyltransferases in catalyzing the formation of mogroside V, thereby avoiding the production of other glycosides. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Schematic diagram of integration into the GAL80 locus of the Saccharomyces cerevisiae genome.

[0034] Figure 2 Schematic diagram of integration into the Exg1 locus of the Saccharomyces cerevisiae genome. DETAILED DESCRIPTION

[0035] 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.

[0036] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0037] Unless otherwise specified, the test materials used in the following examples were purchased from conventional biochemical reagent stores.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] In the embodiment, 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. The specific artificial plasmids were: pUC57-RIAD-RIDD plasmid (including RIAD and RIDD polypeptide interaction tags), pUC57-mPAFR plasmid (including erythromycin (mPKSeal), chlortetracycline (mAURSeal), tacrolimus (mFKBSeal) and rapamycin (mRAPSeal) polyketide synthase polypeptide interaction tags), pU C57-SgCDS plasmid (containing the gene encoding the cucurbitadienol synthase SgCDS from Momordica grosvenori), pUC57-SgEPH3 plasmid (containing the gene encoding the cycloepoxide hydrolase SgEPH3 from Momordica grosvenori), pUC57-SgCYP87D18 plasmid (containing the gene encoding the cytochrome P450 enzyme CYP87D18 from Momordica grosvenori), pUC57-AtCPR1 plasmid (containing the gene encoding the cytochrome P450 enzyme reductase AtCPR1 from Arabidopsis thaliana), pUC57-UGTMG1 plasmid (containing the gene encoding the glycosyltransferase mutant UGT74AC1 from Momordica grosvenori) T79Y / L48M / R28H / L109I / S15A / M76L / H47R encoding gene) and pUC57-SgUGT94-289-3 V148M / G152A (including the glycosyltransferase mutant SgUGT94-289-3 from Momordica grosvenori V148M / G152A coding genes).

[0042] The information of the backbone plasmids involved in the construction of the following examples is shown in Table 1.

[0043] Table 1 Backbone plasmid list

[0044] Plasmid name Basic Information 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 pM357 Contains the PmeI-pGAL1-PacI-SbfI-FBA1tt-StuI backbone

[0045] (1) Using yeast genomic DNA as a template, PCR amplification was performed using the primer pairs listed in column 1 of Table 2, and the corresponding PCR amplification products were recovered, namely, the amplification products of the promoter pGAL1, terminators TPI1t, FBA1t, ACT1t, PGI1t, and ENO1t were obtained.

[0046] 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.

[0047] 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.

[0048] Table 2 Primer list

[0049]

[0050]

[0051] (2) The amplified product of promoter pGAL1 was double-digested with restriction endonucleases SacII and SbfI; the amplified products of TPI1t and FBA1t were single-digested with restriction endonucleases SacII and SbfI, respectively. The digested products were purified using a PCR product purification kit.

[0052] 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.

[0053] (3) Use T4 DNA ligase to ligate the digested products of TPI1t, pGAL1, and FBA1t. 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.

[0054] 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.

[0055] Using bacterial cells as templates, the recombinant plasmid pM354 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 pM354 revealed a SmaI-TPI1t-SacII-AscI-pGAL1,10-PacI-SbfI-FBA1t-StuI backbone.

[0056] Similarly, construct pM355, pM356, and pM357 backbone plasmids.

[0057] Example 2 Expression cassette plasmid construction

[0058] The expression cassette plasmid information involved in the construction example is shown in Table 3.

[0059] Table 3 Expression cassette plasmid list

[0060]

[0061] (1) Using the plasmid or yeast genomic DNA shown in column 3 of Table 4 as templates, PCR amplification was performed using the primer pairs shown in Table 4. The corresponding PCR amplification products were recovered by gel excision to obtain the DNA fragments shown in column 2 of Table 5.

[0062] 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.

[0063] 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.

[0064] (2) Take R10 in the second column of Table 4 C DD fragment, PGM1 fragment and R5 N Overlap extension PCR (OE-PCR) was performed on the DD fragment, and the OE-PCR amplification product was recovered by gel cutting to obtain R5 N DD-PGM1-R10 C DD fragment.

[0065] The reaction system is 50 μL, including 10× buffer for KOD-Plus 5 μL, dNTP mix 5 μL, MgSO4 2 μL, 1.5 μL each primer, 1 μL each DNA fragment template, KOD-Plus 1 μL, and distilled water to 50 μL.

[0066] 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.

[0067] (3) Double digestion of R5 with restriction endonucleases AscI and SacII N DD-PGM1-R10 C The DD fragment and pM354 plasmid were purified by PCR product purification kit to obtain R5 N DD-PGM1-R10 C DD-AscI:SacII sticky-end fragment and pM354-AscI:SacII linear vector.

[0068] 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.

[0069] (4) Use T4 DNA ligase to ligate R5 N DD-PGM1-R10 C The DD-AscI:SacII sticky end fragment and the pM354-AscI:SacII linear vector were connected. The T4 DNA ligase reaction system 10μL is: 10×T4 DNA Ligase Buffer 1μL, R5 N DD-PGM1-R10 C 30 ng of the DD-AscI:SacII sticky-end fragment and pM354-AscI:SacII linear vector, 0.5 μL of T4 DNA Ligase, and distilled water were added to make up to 10 μL. The mixture was allowed to stand at room temperature (25°C) for 2 hours.

[0070] (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.

[0071] Using bacteria as templates, primers TPI11t-f1 (5'-cgccttgctttttgttgtatcac-3') and pGAL1-r1 (5'-gacgaggacgcacggaggagagt-3') were used for verification to obtain the recombinant plasmid pM354-R5. N DD-PGM1-R10 C DD. PCR verification system 25μL: 2×SanTaq PCR Mix 12.5μL, drop solution 1μL, TPI11t-f1 and pGAL1-r1 1μL each, distilled water 11.5μL. N DD-PGM1-R10 C DD was sequenced, and the sequencing results showed that the recombinant plasmid contained SmaI-TPI11t-SacII-R10 C DD-PGM1-R5 N D-AscI-pGAL1,10-PacI-SbfI-FBA1t-StuI expression cassette.

[0072] (6) Using PacI-PGM2-F and SbfI-R4cDD-R shown in columns 4 and 5 of Table 4 as primers, and PGM2 and R4 in column 2 of Table 4 as primers, 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 PGM2-R4. C DD fragment.

[0073] (7) PGM2-R4 was double-digested with restriction endonucleases PacI and SbfI. C DD fragment and pM354-R10 C DD-PGM1-R5 N DD plasmids, and the enzyme digestion products were purified using a PCR product purification kit to obtain PGM2-R4 C DD-PacI:SbfI sticky-end fragment and pM354-R5 N DD-PGM1-R10 CDD-PacI:SbfI linearized vector.

[0074] (8) Use T4 DNA ligase to ligate PGM2-R4 C DD-PacI:SbfI sticky-end fragment and pM354-R5 N DD-PGM1-R10 C Ligate the DD-PacI:SbfI linear vector.

[0075] (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.

[0076] Using bacterial cells as templates and primers pGAL1-f1 (5'-caaccataggatgataatgcgat-3') and FBA1t-r1 (5'-tcagaagaaaagagccgaccaat-3'), the recombinant plasmid pM354-M1 was obtained. The PCR verification system (25 μL) consisted of 12.5 μL of 2× SanTaq PCR Mix, 1 μL of liquid buffer, 1 μL of pGAL1-f1 and FBA1t-r1, and 11.5 μL of distilled water. Sequencing of the recombinant plasmid revealed the presence of the SmaI-TPI1t-SacII-R10CDD-PGM1-R5NDD-AscI-pGAL1,10-PacI-PGM2-R4CDD-SbfI-FBA1t-St uI expression cassette.

[0077] In a similar manner, the plasmids listed in Table 3 were obtained through conventional experimental methods such as primer design, PCR amplification, OE-PCR, enzyme digestion and enzyme ligation.

[0078] Table 4 Primer list

[0079]

[0080] Example 3 Construction of p426-GAL80-Exg1 gRNA dual-target plasmid

[0081] (1) Using KOD-Plus high-fidelity enzyme and p426 plasmid as template, PCR amplification was performed with primer pair 12, primer pair 13, primer pair 14, and primer pair 15 shown in Table 5, respectively. 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.

[0082] (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.

[0083] (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.

[0084] 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.

[0085] (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 16 and primer pair 17 shown in Table 5, 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. TOP10 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').

[0086] Table 5 Primer list

[0087]

[0088] Example 4 Construction of recombinant yeast

[0089] (1) Using the plasmids shown in the first column of Table 6 as templates, PCR amplification was performed using primer pair 18, primer pair 19, primer pair 20, and primer pair 21 in Table 6, and the corresponding PCR amplification products were recovered to obtain the screening marker Leu2 and His3 fragments and modules M3 and M3'.

[0090] Table 6 Primer list

[0091]

[0092]

[0093] (2) The plasmids in column 2 of Table 7 were double-digested using the restriction endonucleases listed in column 3 of Table 7, and the digestion products of the sizes shown in column 4 of Table 7 were recovered to obtain modules M1, M2, M1′, and M2′.

[0094] Table 7 Double enzyme digestion plasmid list

[0095] Module Plasmid name Double enzyme digestion name Size (bp) M1 pM357-M1 plasmid PmeI and StuI 3722 M2 pM355-M2 plasmid SmaI and StuI 5587 M1` pM354-M1 plasmid SmaI and StuI 6053 M2` pM355-M2 plasmid SmaI and StuI 5299

[0096] (3) Prepare Saccharomyces cerevisiae competent cells according to the instructions of the yeast competent cell preparation kit. Add 0.1 μg module M1, 0.1 μg module M2, 0.1 μg module M3, 0.1 μg module M1', 0.1 μg module M2' and 0.1 μg module M3', 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. After 2.7 kV electric shock, add 1 mL mol / L sorbitol solution, resuscitate at 30°C for 1 hour, spread on the screening medium, obtain several single clones, and culture at 30°C for more than 36 hours. The screening medium is: 0.8% SD-His-Leu-Trp-Ura, 2% glucose and 2% agar powder.

[0097] (4) Single clones were first verified by PCR amplification using the primers shown in columns 3 and 4 of Table 8, and then verified by DNA sequencing. The sequencing results showed that modules M1, M2, M3 and the screening marker Leu2 were correctly inserted into the GAL80 site of the yeast genome, and M1', M2', M3' and the screening marker were correctly inserted into the Exg1 site of the yeast genome.

[0098] Table 8 Primer list

[0099]

[0100]

[0101] Example 5: Fermentation of engineered yeast Saccharomyces cerevisiae and detection of mogroside V

[0102] (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.

[0103] (2) Fermentation: Inoculate 100 mL of YPD liquid medium with a 1% to 5% inoculum of the seed culture medium, incubate at 30°C, 220 rpm for 96 h, and collect the fermentation broth.

[0104] (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.

[0105] (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 31.4 mg / L.

[0106] Although the present invention discloses the preferred embodiments as 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 lipid droplet subcellular organelles, characterized in that: The following steps are involved: (1) Squalene epoxidase ERG1, cucurbitadienol synthase SgCDS, epoxide hydrolase SgEPH3, cytochrome P450 enzyme CYP87D18, and cytochrome P450 enzyme reductase AtCPR1 were integrated into the GAL80 locus of the Saccharomyces cerevisiae genome; (2) Integrate phosphoglucomutase PGM1, α-phosphoglucomutase PGM2, uridine diphosphate glucose pyrophosphorylase UGP1, glycosyltransferase UGTMG1, and glycosyltransferase SgUGT94-289-3 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 in lipid droplet subcellular organelles according to claim 1, characterized in that: In the step (1): The amino terminus of the epoxysqualene cyclase ERG1 is fused to the natural anchor motif PLN1 via a flexible linker (GGGGS) 3; The carboxyl end of the epoxysqualene cyclase ERG1 is fused to the polypeptide interaction tag RIAD via a flexible linker (GGGGS) 3; 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 peptide interaction tag RIDD 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 of Saccharomyces cerevisiae for efficiently synthesizing mogroside V based on lipid droplet subcellular organelles according to claim 1, characterized in that: In the step (2): The amino and carboxyl ends of the phosphoglucomutase PGM1 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; The carboxyl terminus of the α-phosphoglucomutase PGM2 interacts with the rapamycin polyketide synthase peptide tag R4 through a flexible linker (GGGGS) 3 C DD fusion; The amino and carboxyl ends of the uridine diphosphate glucose pyrophosphorylase UGP1 are respectively connected to the rapamycin polyketide synthase peptide interaction tag R11 through a flexible linker (GGGGS) 3 N DD and Tacrolimus Polyketide Synthase Peptide Interaction Tag F4 C DD fusion; The amino and carboxyl ends of the glycosyltransferase UGTMG1 are respectively connected to the tacrolimus polyketide synthase polypeptide interaction tag F5 through a flexible linker (GGGGS) 3 N DD and F6 C DD fusion; The glycosyltransferase SgUGT94-289-3 V148M / G152A The amino and carboxyl termini of the SgUGT94-289-3 mutant were respectively tagged with the tacrolimus polyketide synthase polypeptide through flexible linkers (GGGGS) 3. N DD and chlortetracycline polyketide synthase peptide interaction tag A1 C DD fusion.

4. The method for constructing an engineered yeast strain of Saccharomyces cerevisiae for efficiently synthesizing mogroside V based on lipid droplet subcellular organelles according to claim 1, characterized in that: The epoxysqualene cyclase ERG1 is anchored to lipid droplets via the native anchoring motif PLN1.

5. The method for constructing an engineered yeast strain of Saccharomyces cerevisiae for efficiently synthesizing mogroside V based on lipid droplet subcellular organelles according to claim 1, characterized in that: Through the peptide interaction tags RIAD and RIDD, one molecule of squalene cyclase ERG1 and two molecules of epoxide hydrolase SgEPH3 assembled into a multi-enzyme complex in lipid droplets.

6. The method for constructing an engineered yeast strain of Saccharomyces cerevisiae for efficiently synthesizing mogroside V based on lipid droplet subcellular organelles according to claim 1, characterized in that: Through the erythromycin polyketide synthase module, chlortetracycline polyketide synthase module, tacrolimus polyketide synthase module and rapamycin polyketide synthase module, cycloepoxide hydrolase SgEPH3, cucurbitadienol synthase SgCDS, cytochrome P450 enzyme CYP87D18, cytochrome P450 enzyme reductase AtCPR1, glycosyltransferase SgUGT94-289-3, glycosyltransferase UGTMG1, uridine diphosphate glucose pyrophosphorylase UGP1, phosphoglucomutase PGM1 and α-phosphoglucomutase PGM2 are linearly assembled into a multi-enzyme complex in lipid droplets.

7. The method for constructing an engineered yeast strain of Saccharomyces cerevisiae for efficiently synthesizing mogroside V based on lipid droplet subcellular organelles according to claim 1, characterized in that: The coding genes of the MG-V anabolic enzymes are all driven to express by the yeast galactokinase GAL1 gene promoter.

8. An engineered yeast strain of Saccharomyces cerevisiae that efficiently synthesizes mogroside V based on lipid droplet subcellular organelles, characterized by: Constructed by the construction method according to any one of claims 1 to 7.

9. A method for producing mogroside V, characterized in that: The engineered yeast Saccharomyces cerevisiae according to claim 8 is used to ferment and produce mogroside V.

10. The method for producing mogroside V according to claim 9, wherein: YPD medium was used as the fermentation medium.

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