Saccharomyces cerevisiae engineering bacteria for efficiently synthesizing mogroside V based on peroxisome 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 peroxisome subcellular organelles and a construction method of the saccharomyces cerevisiae engineering bacteria. A construction method of the engineering bacterium comprises the following steps: (1) integrating mogrol metabolic enzyme and leucine selection marker Leu2 at a site of a transcription inhibition factor GAL80 of saccharomyces cerevisiae; and (2) integrating peroxisome membrane protein Pex11, carnitine acetyltransferase Cat2, phosphoglucose mutase PGM1, alpha-phosphoglucose mutase PGM2, uridine diphosphate glucose pyrophosphorylase UGP1, glycosyl transferase UGTMG1, glycosyl transferase SgUGT94-289-3 and a histidine selection marker His3 at the site of the saccharomyces cerevisiae glucoside hydrolase Exg1. The engineering strain has the advantages of high yield, strong metabolic flux directionality, wide industrial application prospect and the like, and a new technical route is provided for microbial manufacturing of natural sweeteners.

Description

Technical Field

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

[0002] Mogroside V (M5) is a highly abundant and sweet component of mogrosides. It exhibits numerous pharmacological activities, including clearing the lungs and relieving cough, relieving sore throat, promoting thirst, and promoting bowel movements. It has become a natural, non-sugar sweetener with therapeutic benefits and holds broad market potential. However, monk fruit cultivation is strictly restricted by climate and limited planting area, making large-scale expansion difficult in the short term. Furthermore, the content of M5 in fresh fruit is only 0.55% to 0.65% (w / w). Analogs such as mogroside IV and simanoside I are complex products that are difficult to purify, making large-scale production impossible through extraction from monk fruit.

[0003] Mogroside V(C 60 H 102 O 29 ) belongs to terpenoid compounds. Currently, the traditional methods of obtaining most terpenoid compounds are mainly plant extraction and chemical synthesis. However, the synthesis amount of terpenoid compounds in plants is low, the growth cycle is long, and they are affected by seasons and geographical locations, resulting in expensive products. The chemical synthesis process is complex, has many three-dimensional structures, is difficult to separate, and requires the use of a large amount of organic reagents, which is not environmentally friendly. Synthetic biology is a science that has emerged in recent years to redesign, engineer and apply life systems and processes. By constructing the synthesis pathway of plant-derived terpenes in microbial chassis hosts, the efficient synthesis of terpenes has become a new green and efficient production model. As a model microorganism recognized as safe (GRAS), Saccharomyces cerevisiae can endogenously synthesize terpenoid precursors and has a complete membrane structure. It has been widely used in the synthesis of a variety of plant-derived terpenes. At present, the use of Saccharomyces cerevisiae to synthesize artemisinin precursor artemisinic acid and chemical raw material farnesene has been industrialized, which can be regarded as a milestone in the metabolic engineering of microorganisms to produce terpene natural products. 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 peroxisomal subcellular organelles and a construction method thereof.

[0005] Specifically, the method for constructing an engineered yeast strain of Saccharomyces cerevisiae that efficiently synthesizes mogroside V based on the peroxisomal subcellular organelle comprises the following steps:

[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, endogenous 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) The peroxisomal membrane protein Pex11, carnitine acetyltransferase Cat2, phosphoglucomutase PGM1, α-phosphoglucomutase PGM2, uridine diphosphate glucose pyrophosphorylase UGP1, glycosyltransferase UGTMG1, glycosyltransferase SgUGT94-289-3, and histidine selection marker His3 were integrated into the Saccharomyces cerevisiae glycoside hydrolase Exg1 site.

[0008] In step (1):

[0009] The acetoacetyl-CoA thiolase ERG10 (NCBI Sequence ID: NP_015297.1), 3-hydroxy-3-methylglutaryl-CoA synthetase ERG13 (NCBI Sequence ID: NP_013580.1), HMG1 (NCBI Sequence ID: NP_013636.1) N-terminal truncation of 527 amino acids tHMG1, mevalonate kinase ERG12 (NCBI Sequence ID: NP_013935.1), phosphomevalonate kinase ERG8 (NCBI Sequence ID: NP_013947.1), mevalonate pyrophosphate decarboxylase ERG19 (NCBI Sequence ID: NP_014441.1), pentenyl pyrophosphate isomerase IDI1 (NCBI Sequence ID: NP_015208.1), geranyl pyrophosphate synthase ERG20 (NCBI Sequence ID: NP_014430.1), and pyrophosphate decarboxylase ERG19 (NCBI Sequence ID: NP_014431.1) were also studied. 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 as shown in SEQ ID NO.1) through a flexible linker (GGGGS) 3.

[0010] The amino terminus of the epoxysqualene cyclase ERG1 interacts with the erythromycin polyketide synthase polypeptide through 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 CDD (nucleotide sequence as SEQ ID NO.6) fusion.

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

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

[0015] In step (2):

[0016] The carnitine acetyltransferase Cat2 (NCBI Sequence ID: NP_013670.1) carboxyl terminus was fused to Pex15 via a flexible linker (GGGGS) 3.

[0017] 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.9) and R10 C DD (nucleotide sequence such as SEQ ID NO.10) fusion.

[0018] 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) 3 C DD (nucleotide sequence such as SEQ ID NO.11) fusion.

[0019] 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.12) and tacrolimus polyketide synthase peptide interaction tag F4 CDD (nucleotide sequence such as SEQ ID NO.13) fusion.

[0020] 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.14) and F6 C DD (nucleotide sequence such as SEQ ID NO.15) fusion.

[0021] The glycosyltransferase SgUGT94-289-3 (NCBI Sequence ID: 8HJO_A) mutant SgUGT94-289-3 V148M / G152A The amino and carboxyl ends are respectively linked to the tacrolimus polyketide synthase peptide interaction tag F7 through a flexible linker (GGGGS) 3 N DD (nucleotide sequence as SEQ ID NO.16) and chlortetracycline polyketide synthase polypeptide interaction tag A1 C DD (nucleotide sequence such as SEQ ID NO.17) fusion.

[0022] The acetoacetyl-CoA thiolase ERG10, 3-hydroxy-3-methylglutaryl-CoA synthetase ERG13, truncated 3-hydroxy-3-methylglutaryl-CoA reductase tHMG1, mevalonate kinase ERG12, phosphomevalonate kinase ERG8, mevalonate pyrophosphate decarboxylase ERG19, pentenyl pyrophosphate isomerase IDI1, geranyl pyrophosphate synthase ERG20, endogenous squalene synthase ERG9, epoxysqualene cyclase ERG1 and carnitine acetyltransferase Cat2 are anchored to the cytoplasmic side of the peroxisome membrane surface through the natural anchoring motif Pex15.

[0023] 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 through erythromycin (mPKSeal), chlortetracycline (mAURSeal), tacrolimus (mFKBSeal) and rapamycin (mRAPSeal) polyketide synthase modules and anchored to the epoxysqualene cyclase ERG1 site.

[0024] 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.18).

[0025] The present invention also provides an engineered yeast of Saccharomyces cerevisiae for efficiently synthesizing mogroside V based on peroxisomal subcellular organelles.

[0026] The Saccharomyces cerevisiae engineered bacteria that efficiently synthesizes mogroside V based on the peroxisome subcellular organelle is constructed by the above-mentioned construction method.

[0027] The present invention also provides a method for producing mogroside V.

[0028] Specifically, a method for producing mogroside V comprises fermenting the above-mentioned engineered yeast Saccharomyces cerevisiae to produce mogroside V.

[0029] The method for producing mogroside V uses YPD culture medium as fermentation medium.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] Mogroside V is a triterpenoid saponin product found in the cytoplasm, and acetyl-CoA is a key intermediate in the yeast mogroside V biosynthesis pathway. Based on the unique metabolic properties of yeast peroxisomes—degrading long-chain and very-long-chain fatty acids through β-oxidation to produce acetyl-CoA, and transporting some of this acetyl-CoA to the cytoplasm as acetylcarnitine—the present invention has developed a peroxisome surface display technology platform. This platform achieves efficient synthesis of MG-V by combining peroxisome engineering with polyketide synthase assembly strategies: (1) Directed assembly of multi-enzyme complexes: squalene synthase is precisely anchored to the peroxisome membrane surface, and erythromycin (mPKSeal), chlortetracycline (mAURSeal), tacrolimus (mFKBSeal) and rapamycin (mRAPSeal) polyketide synthase assembly strategies are used to linearly assemble mogrosanol synthase and MG-V synthase into a multi-enzyme complex and anchor it to the epoxysqualene cyclase ERG1 site, significantly improving the metabolic flux of precursor squalene to MG-V synthesis; (2) Peroxisome engineering: overexpression of the key regulatory protein Pex11 for peroxisome biosynthesis significantly expands the peroxisome membrane area; overexpression of the peroxisome surface carnitine acetyltransferase Cat2 promotes the efficient transport of acetyl-CoA from the peroxisome matrix to the cytoplasm. This engineered strain has the advantages of high yield, strong metabolic flow directionality, and broad prospects for industrial application, providing a new technical route for the microbial production of natural sweeteners. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1Schematic diagram of integration of mogroside alcohol metabolizing enzymes into the Saccharomyces cerevisiae transcriptional repressor GAL80 locus.

[0033] Figure 2 Schematic diagram of the integration of mogroside V metabolizing enzymes into the Saccharomyces cerevisiae glycoside hydrolase Exg1 site. DETAILED DESCRIPTION

[0034] 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.

[0035] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0036] Unless otherwise specified, the test materials used in the following examples were purchased from conventional biochemical reagent stores.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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:

[0041] Table 1 List of artificial plasmids

[0042]

[0043] Example 1 Backbone plasmid construction

[0044] The information of the backbone plasmids involved in the construction of the following examples is shown in Table 2.

[0045] Table 2 Backbone plasmid list

[0046]

[0047]

[0048] (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.

[0049] 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.

[0050] 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.

[0051] Table 3 Primer list

[0052]

[0053]

[0054] (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.

[0055] 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.

[0056] (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.

[0057] 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 solid LB 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.

[0058] 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.

[0059] Similarly, pM351, pM352, pM353, pM354, pM355, and pM356 plasmids were constructed.

[0060] Example 2 Expression cassette plasmid construction

[0061] The plasmid information for constructing the expression cassettes involved in the following examples is shown in Table 4.

[0062] Table 4 Expression cassette plasmid list

[0063]

[0064] (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.

[0065] 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.

[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 20 s to 2 min, 25 cycles; and extension at 72°C for 10 min.

[0067] (2) Overlap extension PCR (OE-PCR) was performed using SacII-Pex15-f and AscI-ERG10-r shown in columns 4 and 5 of Table 5 as primers and the Pex15-1 fragment and ERG10 fragment shown in column 2 of Table 5 as templates. The OE-PCR amplification product was recovered by gel excision to obtain the ERG10-Pex15 fragment.

[0068] 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.

[0069] 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.

[0070] (3) The ERG10-Pex15 fragment and the pM350 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 ERG10-Pex15-AscI:SacII sticky end fragment and the pM350-AscI:SacII linear vector.

[0071] 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.

[0072] (4) Use T4 DNA ligase to ligate the ERG10-Pex15-AscI:SacII sticky-end fragment and the pM350-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 ERG10-Pex15-AscI:SacII sticky-end fragment and the pM350-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.

[0073] (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.

[0074] Using bacterial cells as templates, the recombinant plasmid pM350-ERG10-Pex15 was obtained using primers TDH1t-f1 (5'-tgcgttctttgccaatagtcaca-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 bacterial suspension, 1 μL each of TDH1t-f1 and pGAL1-r1, and 11.5 μL of distilled water. Sequencing of the recombinant plasmid pM350-ERG10-Pex15 revealed the presence of the SmaI-TDH1t-SacII-Pex15-ERG10-AscI-pGAL1,10-PacI-SbfI-GPM1t-StuI expression cassette.

[0075] (6) OE-PCR was performed using PacI-ERG13-f and SbfI-Pex15-r shown in columns 4 and 5 of Table 5 as primers and the ERG13 and Pex15-2 fragments in column 2 of Table 5 as templates. The OE-PCR amplification product was recovered by gel excision to obtain the ERG13-Pex15 fragment.

[0076] (7) The ERG13-Pex15 fragment and the pM350-ERG10-Pex15 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 ERG13-Pex15-PacI:SbfI sticky end fragment and the pM350-PacI:SbfI linear vector.

[0077] (8) Use T4 DNA ligase to ligate the ERG13-Pex15-PacI:SbfI sticky end fragment and the pM350-PacI:SbfI linear vector.

[0078] (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.

[0079] Using bacterial cells as templates, the recombinant plasmid pM350-P1 was obtained using primers pGAL1-f1 (5'-caaccataggatgataatgcgat-3') and GPM1t-r1 (5'-ttccaacatactgtccctga-3'). The PCR verification system (25 μL) consisted of 12.5 μL of 2× SanTaq PCR Mix, 1 μL of bacterial suspension, 1 μL each of pGAL1-f1 and GPM1t-r1, and 11.5 μL of distilled water. Sequencing of the recombinant plasmid revealed the SmaI-TDH1t-SacII-Pex15-ERG10-AscI-pGAL1,10-PacI-ERG13-Pex15-SbfI-GPM1t-StuI expression cassette.

[0080] A similar method was adopted to obtain the plasmids listed in Table 4 through conventional molecular biology experimental techniques such as primer design, PCR cloning, OE-PCR fragment splicing, enzyme digestion and enzyme ligation.

[0081] Table 5 Primer list

[0082]

[0083] Example 3 Construction of p426-GAL80-Exg1 gRNA dual-target plasmid

[0084] (1) Using KOD-Plus high-fidelity enzyme and p426 plasmid as templates, primer pair 19, primer pair 20, primer pair 21, and primer pair 22 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.

[0085] (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.

[0086] (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.

[0087] 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.

[0088] (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 23 and primer pair 24 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').

[0089] Table 6 Primer list

[0090]

[0091] Example 4 Construction of recombinant yeast

[0092] (1) Using the plasmids shown in column 1 of Table 7 as templates, PCR amplification was performed using the primer pairs 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 modules M7 and M4'.

[0093] Table 7 Primer list

[0094]

[0095]

[0096] (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 M1, M2, M3, M4, M5, M6, M1′, M2′, and M3′.

[0097] Table 8 Plasmid list

[0098] Module Plasmid name Size (bp) M1 pM350-P1 5050 M2 pM351-P2 4945 M3 pM352-P3 4706 M4 pM353-P4 4197 M5 pM354-P5 5414 M6 pM355-P6 5674 M1` pM353-P1 4746 M2` pM354-P2 6053 M3` pM355-P3 5299

[0099] (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 M4, 0.1 μg module M5, 0.1 μg module M6, 0.1 μg module M7, 0.1 μg module M1`, 0.1 μg module M2`, 0.1 μg module M3`, 0.1 μg module M4`, 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 consisted of: 0.8% SD-His-Leu-Trp-Ura, 2% glucose and 2% agar powder.

[0100] (4) Single clones were first verified by PCR amplification using the primers shown in columns 3 and 4 of Table 9, and then verified by DNA sequencing. The sequencing results showed that modules M1, M2, M3, M4, M5, M6 and the screening marker Leu2 were correctly inserted into the GAL80 site of the yeast genome, and M1', M2', M3', M4' and the screening marker were correctly inserted into the Exg1 site of the yeast genome.

[0101] Table 9 Primer list

[0102]

[0103]

[0104] Example 5: Fermentation of engineered yeast Saccharomyces cerevisiae and detection of mogroside V

[0105] (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.

[0106] (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.

[0107] (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.

[0108] (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 55.7 mg / L.

[0109] 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 that can efficiently synthesize mogroside V based on peroxisomal subcellular organelles, characterized in that: 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, endogenous 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) The peroxisomal membrane protein Pex11, carnitine acetyltransferase Cat2, phosphoglucomutase PGM1, α-phosphoglucomutase PGM2, uridine diphosphate glucose pyrophosphorylase UGP1, glycosyltransferase UGTMG1, glycosyltransferase SgUGT94-289-3, and histidine selection marker His3 were integrated into the Saccharomyces cerevisiae glycoside hydrolase Exg1 site.

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 interacts with the erythromycin polyketide synthase polypeptide through 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 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 peroxisomal subcellular organelles according to claim 1, characterized in that: In the step (2): The carnitine acetyltransferase Cat2 carboxyl terminus is fused to Pex15 via a flexible linker (GGGGS) 3; 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 mutant SgUGT94-289-3 of the glycosyltransferase SgUGT94-289-3 V148M / G152A The amino and carboxyl ends are respectively linked to the tacrolimus polyketide synthase peptide interaction tag F7 through a flexible linker (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 peroxisomal subcellular organelles according to claim 1, characterized in that: The acetoacetyl-CoA thiolase ERG10, 3-hydroxy-3-methylglutaryl-CoA synthetase ERG13, truncated 3-hydroxy-3-methylglutaryl-CoA reductase tHMG1, mevalonate kinase ERG12, phosphomevalonate kinase ERG8, mevalonate pyrophosphate decarboxylase ERG19, pentenyl pyrophosphate isomerase IDI1, geranyl pyrophosphate synthase ERG20, endogenous squalene synthase ERG9, epoxysqualene cyclase ERG1 and carnitine acetyltransferase Cat2 are anchored to the cytoplasmic side of the peroxisome membrane surface through the natural anchoring motif Pex15.

5. The method for constructing an engineered yeast strain of Saccharomyces cerevisiae for efficiently synthesizing mogroside V based on peroxisomal subcellular organelles according to claim 1, characterized in that: 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 through erythromycin, chlortetracycline, tacrolimus and rapamycin polyketide synthase modules and anchored to the epoxysqualene cyclase ERG1 site.

6. The method for constructing an engineered yeast strain of Saccharomyces cerevisiae for efficiently synthesizing mogroside V based on peroxisomal subcellular organelles 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.

7. An engineered yeast strain of Saccharomyces cerevisiae that efficiently synthesizes mogroside V based on peroxisomal subcellular organelles, characterized by: Constructed by the construction method according to any one of claims 1 to 6.

8. A method for producing mogroside V, characterized in that: The engineered yeast Saccharomyces cerevisiae according to claim 7 is used to ferment and produce mogroside V.

9. The method for producing mogroside V according to claim 8, wherein: YPD medium was used as the fermentation medium.

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