Method for constructing engineered yeast for de novo synthesis of mogroside

Abstract

Provided is a method for constructing an engineered yeast for de novo synthesis of mogroside. The method comprises: inserting constructed cucurbitadienol synthase gene expression cassette Ptef1-cds-Tcyc1, epoxide hydrolase gene expression cassette Ptef1-eph3-Tcyc1, cytochrome P450 enzyme-encoding gene CYP and cytochrome P450 enzyme reductase gene expression cassette Ttdh3-cyp87d18-P gal1 / 10-atcpr2-Tcyc1, and glycosyltransferase-encoding gene expression cassette Tadh1-ugt74ac1-Pgal1 / 10-ugtms1-Tcyc1 into a yeast genome, so as to obtain an engineered yeast for mogroside production. The engineered yeast can be used for de novo synthesis of mogroside by using glucose as a substrate for fermentation.

Description

A method for constructing an engineered yeast for synthesizing mogroside from scratch Technical Field

[0001] The present invention relates to a method for constructing an engineered yeast, in particular to a method for constructing an engineered brewer's yeast for synthesizing mogroside from scratch. Background Art

[0002] Mogroside is a type of cucurbitane-type tetracyclic triterpenoid secondary metabolite derived from Momordica grosvenori fruit. It uses mogroside alcohol as aglycone and has different numbers of sugar groups connected by different glycosidic bonds at C-3 and C-24, including mogroside I A1, mogroside II E, and mogroside III. x , simenoside I, mogroside IV A, mogroside V and other structures. Among the many mogrosides, mogroside V is the component with the highest content and sweetness, and is also the main source of sweetness in Luo Han Guo. Its chemical formula is C 60 H 102 O 29 , with a relative molecular mass of 1287.43. Mogroside V was certified as safe and non-toxic by the FDA in 1995, allowing its use as a food additive. Due to its high sweetness, low calories, and lack of fermentability, it does not cause obesity or dental caries, making it an ideal natural sucrose substitute. Due to the widespread anti-inflammatory and glucose and lipid metabolism effects of these substances, other mogrosides, even those less sweet than mogroside V, are widely used in pharmaceutical and cosmetic production. This demonstrates the broad potential of mogrosides.

[0003] Currently, the industrial production of mogrosides primarily relies on extraction from monk fruit. However, the average content of mogrosides is less than 1% of the dry weight of the fruit. Therefore, direct extraction methods require a high volume of monk fruit, but the planting area is limited and susceptible to soil and climatic conditions, resulting in low extraction efficiency, which not only increases costs but also causes waste. At the beginning of this century, the rise of synthetic biology broke the limitation of specific species to produce corresponding products, making the microbial synthesis of mogrosides a reality. Microbial synthesis of mogrosides is not constrained by natural conditions, has a short production cycle, and the product can be secreted and easily isolated outside the cell, resulting in minimal pollution, making it suitable for large-scale production.

[0004] Yeast is not only used for brewing wine and making foods like bread and steamed buns, but with the development of gene editing technology, it can also be used as a base cell for the production of natural products. Yeast plays an important role in genetic engineering due to its low pathogenicity, high stress resistance, and low probability of phage contamination. However, yeast lacks the metabolic pathway for mogroside synthesis, and currently, there are no reports on the de novo synthesis of mogroside using glucose as a substrate through microbial fermentation.

[0005] Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a method for constructing an engineered yeast for de novo synthesis of mogroside. The method involves integrating exogenous genes into the yeast genome and regulating and transforming the yeast through metabolic engineering, so that the yeast can achieve efficient de novo synthesis of mogroside using glucose as a substrate through fermentation.

[0007] Specifically, a method for constructing an engineered yeast for synthesizing mogroside from scratch is to construct a cucurbitadienol synthase gene expression cassette P tef1 -cds-T cyc1 , epoxide hydrolase gene expression cassette P tef1 -eph3-T cyc1 , cytochrome P450 enzyme encoding gene CYP and cytochrome P450 enzyme reductase gene expression frame T tdh3 -cyp87d18-P gal1 / 10 -atcpr2-T cyc1 and glycosyltransferase encoding gene expression cassette T adh1 -ugt74ac1-P gal1 / 10 -ugtms1-T cyc1 , inserted into the yeast genome to obtain mogroside-producing engineered yeast (hereinafter referred to as: recombinant engineered yeast).

[0008] Furthermore, a truncated 3-hydroxy-3-methylglutaryl-CoA reductase gene expression cassette P was constructed. tef1 -tHMG1-T hmg1 , and inserted into the gal80 site in the recombinant yeast genome, removing the rate-limiting step of the MVA pathway. At the same time, it is not induced by galactose, and the synthesis pathway of mogroside is only regulated by glucose concentration.

[0009] Furthermore, the endogenous promoters of the hydroxymethylglutaryl-CoA synthase gene erg13 and the squalene synthase erg9 in the recombinant yeast were replaced with the strong promoter P tef1 , which strengthens the accumulation of mogroside precursors.

[0010] Furthermore, the copy number of the squalene epoxidase gene erg1 in the recombinant engineered yeast was increased to promote the flow of the intermediate squalene to the target product mogroside.

[0011] Furthermore, the key enzyme lanosterol synthase gene erg7 of the alternative lanosterol pathway was knocked out in the recombinant engineered yeast to avoid competition with the precursor substances of mogroside in the alternative pathway.

[0012] Furthermore, in order to achieve multi-copy expression of cyp87d18, the multi-copy plasmid pESC-G418-T was transformed into recombinant yeast. tdh3 -cyp87d18-P gal1 / 10 -atcpr2-T cyc1 , to obtain optimized engineered yeast.

[0013] In the present invention, the engineered yeast includes but is not limited to Saccharomyces cerevisiae, Pichia pastoris, Pichia fermentans, Hansenula anomala, Yarrowia lipolytica, Candida antarctica, Candida utilis, Candida tropicalis, Schizosaccharomyces pombe, and Rhodotorula glutinosus.

[0014] In one embodiment, the engineered yeast is Saccharomyces cerevisiae, and the cucurbitadienol synthase gene P tef1 -cds-T cyc1 The expression cassette of the epoxide hydrolase gene was inserted into the yeast genome at site 1622b; tef1 -eph3-T cyc1 Inserted into the yeast genome 911b site; cytochrome P450 enzyme encoding gene CYP and cytochrome P450 enzyme reductase gene expression cassette T tdh3 -cyp87d18-P gal1 / 10 -atcpr2-T cyc1 Inserted into the 106a site of the yeast genome; glycosyltransferase encoding gene expression cassette T adh1 -ugt74ac1-P gal1 / 10 -ugtms1-T cyc1 Inserted into the 308a site of the yeast genome; truncated 3-hydroxy-3-methylglutaryl coenzyme A reductase gene expression cassette P tef1 -tHMG1-T hmg1 Inserted into the gal80 site of the yeast genome.

[0015] The present invention also provides an engineering yeast for synthesizing mogroside from scratch.

[0016] Specifically, the mogroside-engineered yeast was synthesized from scratch, and the yeast genome contained the cucurbitadienol synthase gene expression cassette P tef1 -cds-T cyc1 , epoxide hydrolase gene expression cassette Ptef1 -eph3-T cyc1 , cytochrome P450 enzyme encoding gene CYP and cytochrome P450 enzyme reductase gene expression frame T tdh3 -cyp87d18-P gal1 / 10 -atcpr2-T cyc1 and glycosyltransferase encoding gene expression cassette T adh1 -ugt74ac1-P gal1 / 10 -ugtms1-T cyc1 .

[0017] The yeast genome contains a truncated 3-hydroxy-3-methylglutaryl coenzyme A reductase gene expression cassette P tef1 -tHMG1-T hmg1 .

[0018] The endogenous promoters of the hydroxymethylglutaryl-CoA synthase gene erg13 and the squalene synthase erg9 in the yeast are strong promoters. tef1 .

[0019] The yeast has three or more copies of the squalene epoxidase gene (erg1).

[0020] In the yeast, the lanosterol synthase gene erg7, a key enzyme in the lanosterol bypass pathway, was knocked out.

[0021] The saccharomyces cerevisiae also contains a multi-copy plasmid pESC-G418-T tdh3 -cyp87d18-P gal1 / 10 -atcpr2-T cyc1 .

[0022] Furthermore, the yeast is Saccharomyces cerevisiae, Pichia pastoris, Pichia fermentans, Hansenula anomala, Yarrowia lipolytica, Candida antarctica, Candida utilis, Candida tropicalis, Schizosaccharomyces pombe, or Rhodotorula glutinosus.

[0023] In one embodiment, the engineered yeast is Saccharomyces cerevisiae, in which the 1622b site of the genome is inserted into the cucurbitadienol synthase gene expression cassette P tef1 -cds-T cyc1 ; 911b site inserts the epoxide hydrolase gene expression cassette P tef1 -eph3-T cyc1 ; 106a site inserts the cytochrome P450 enzyme encoding gene CYP and cytochrome P450 enzyme reductase gene expression cassette T tdh3 -cyp87d18-P gal1 / 10 -atcpr2-T cyc1; 308a site inserts the glycosyltransferase encoding gene expression cassette T adh1 -ugt74ac1-P gal1 / 10 -ugtms1-T cyc1 gal80 site to insert the truncated 3-hydroxy-3-methylglutaryl coenzyme A reductase gene expression cassette P tef1 -tHMG1-T hmg1 .

[0024] The present invention also provides the application of engineered yeast for synthesizing mogroside from scratch, and particularly relates to the application of the engineered yeast in producing mogroside by biological fermentation.

[0025] In the present invention, the method for producing mogroside by fermentation with the engineered yeast comprises: taking the engineered yeast strain in a liquid YPD culture medium, culturing on a shaker at 30°C and 220 r / min for 18 hours to obtain a fermentation seed liquid; transferring the fermentation seed liquid into a fermentation culture medium at an inoculation rate of 3%, and culturing at 30°C and 220 r / min for 96 hours.

[0026] Beneficial effects of the present invention:

[0027] 1. The cerevisiae yeast provided by the present invention can produce mogroside by fermentation using glucose as a substrate, and the product can be secreted extracellularly, laying a good foundation for the downstream separation and purification steps. Saccharomyces cerevisiae has an endogenous MVA pathway, which can synthesize squalene, an important precursor of mogroside. Subsequently, squalene is synthesized into mogroside in sequence under the catalysis of exogenous squalene epoxidase, cucurbitadienol synthase, epoxide hydrolase, cytochrome P450 enzyme (and its companion protein cytochrome P450 reductase), and glycosyltransferase.

[0028] The final yeast strain Mogrol 09 provided by the present invention has a yield of mogroside V of 5.6 mg / L, which is 467 times that of the yeast strain Mogrol 04 that is initially capable of synthesizing mogroside V. The yields of other mogrosides, such as mogroside IIE, mogroside IIIx, mogroside IVA, and simanoside, of the final strain Mogrol 09 are also significantly higher than those of the strain Mogrol 04 that is initially capable of synthesizing mogroside V.

[0029] 2. The yeast construction method provided by the present invention is simple, easy to use, and has good prospects.

[0030] The present invention adopts the CRISPR Cas9 gene editing technology to integrate the key enzymes of the synthesis pathway, cucurbitadienol synthase CDS, epoxide hydrolase EPH, cytochrome P450 enzyme, cytochrome P450 enzyme reductase, glycosyltransferase UGT74AC1, and UGTMS1, that is, the synthetic metabolic pathway of mogroside, into yeast, so that the yeast can realize the de novo synthesis of mogroside by fermentation using glucose as a substrate.

[0031] 4. The yeast provided by the present invention is based on the promoter P tef1 、P gal1 / 10 Control the expression of genes in the mogroside synthesis pathway, integrate the truncated 3-hydroxy-3-methylglutaryl coenzyme A reductase tHMG1, increase the copy number of the squalene epoxidase gene erg1, and knock out the key enzyme lanosterol synthase gene erg7 in the bypass lanosterol pathway to avoid competition with the precursor substances of mogrosides in the bypass pathway and transform the multi-copy plasmid pESC-G418-T tdh3 -cyp87d18-P gal1 / 10 -atcpr2-T cyc1 , achieving multi-copy expression of cyp87d18, enabling yeast to efficiently synthesize mogroside from scratch using glucose as a substrate. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] FIG1 is a comparison of the yield of mogroside V between the Saccharomyces cerevisiae strain Mogrol 09 and the Saccharomyces cerevisiae strain Mogrol 04.

[0033] FIG2 shows the yields of other mogrosides by Saccharomyces cerevisiae strains Mogrol 09 and Mogrol 04. DETAILED DESCRIPTION

[0034] The following describes specific embodiments of the present invention in more detail. These embodiments are provided to enable a more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art.

[0035] The sample processing method of the embodiment of the present invention:

[0036] The fermentation broth was collected and centrifuged at 8000 rpm for 5 minutes. 1 mL of the supernatant was added to chromatographic-grade formic acid to a final concentration of 3% (v / v). The mixture was allowed to stand at 4°C for 4-8 hours to remove proteins. The mixture was centrifuged at 8000 rpm for 5 minutes. 0.5 mL of the supernatant was collected and filtered through a 0.22 μm aqueous filter. The supernatant was then added to a liquid chromatography assay bottle for yield analysis.

[0037] The detection method of the embodiment of the present invention:

[0038] High performance liquid chromatography-mass spectrometry (LC-MS) detection method: Waters (MICROMASS QUATTRO MICRO), BEH-C18 column, mobile phase acetonitrile and 0.1% formic acid aqueous solution, column temperature 45°C, injection volume 1 μL.

[0039] Example 1 Construction of a mogroside-producing strain

[0040] According to the amino acid sequences of key enzymes for mogroside synthesis published on NCBI or derived from references, the amino acid sequences include cucurbitadienol synthase CDS (NCBI ID: AUC64917.1), the amino acid sequence of which is shown in SEQ ID NO.1; epoxide hydrolase EPH, the amino acid sequence of which is shown in SEQ ID NO.2; cytochrome P450 enzyme CYP87D18 (NCBI ID: K7NBR2.1), the amino acid sequence of which is shown in SEQ ID NO.3; cytochrome P450 enzyme reductase AtCPR2 (NCBI ID: UTD45115.1), the amino acid sequence of which is shown in AA SEQ ID NO.4; glycosyltransferase UGT74AC1, the amino acid sequence of which is shown in SEQ ID NO.5; and glycosyltransferase UGTMS1, the amino acid sequence of which is shown in SEQ ID NO.6.

[0041] The truncated 3-hydroxy-3-methylglutaryl-CoA reductase tHMG1 is obtained by removing the transmembrane domain consisting of amino acids 2-530 at the N-terminus of the endogenous Saccharomyces cerevisiae enzyme HMG1. The amino acid sequence of the truncated 3-hydroxy-3-methylglutaryl-CoA reductase tHMG1 is shown in SEQ ID NO.7.

[0042] Codon optimization and whole gene synthesis were performed according to the codon preference of Saccharomyces cerevisiae.

[0043] The 20bp PAM sequence of CRISPR Cas9 was selected based on the gene sequences of the integration sites 1622b, 911b, 106a, and 308a of the Saccharomyces cerevisiae genome. Primers for amplifying the upstream and downstream homologous arms of the gene expression frame were designed on both sides of the PAM sequence. The upstream and downstream homologous arms were amplified by PCR using Saccharomyces cerevisiae genomic DNA as a template.

[0044] The primer conditions are shown in Table 1. The PCR conditions are shown in Table 2.

[0045] Table 1 Primers required for homology arm amplification

[0046] Table 2 PCR amplification conditions

[0047] According to the overlapping derivatization PCR primer design method, primers were designed to amplify the key enzymes in the mogroside biosynthesis pathway and the promoters and terminators involved. The corresponding gene fragments were amplified using the fully synthesized cds, eph3, cyp87d18, atcpr2, ugtac1, and ugtms1 plasmids as templates. The promoter P was amplified using the genomic DNA of Saccharomyces cerevisiae as a template. tef1 、P gal1 / 10 , terminator T cyc1 、T adh1 、T tdh3 The gene expression cassette was constructed by overlap extension PCR.

[0048] The primer conditions are shown in Table 3. The PCR amplification conditions are shown in Table 2.

[0049] Table 3 Primers required for fragment amplification

[0050] Gene expression cassettes include: cucurbitadienol synthase gene expression cassette P tef1 -cds-T cyc1 , epoxide hydrolase gene expression cassette P tef1 -eph3-T cyc1 , cytochrome P450 enzyme encoding gene CYP and cytochrome P450 enzyme reductase gene expression frame T tdh3 -cyp87d18-P gal1 / 10 -atcpr2-T cyc1 and glycosyltransferase encoding gene expression cassette T adh1 -ugt74ac1-P gal1 / 10 -ugtms1-T cyc1 .

[0051] Promoter P tef1 It is a constitutive promoter endogenous to Saccharomyces cerevisiae, and its nucleotide sequence is shown as NA SEQ ID NO.8. gal1 / 10 It is a bidirectional promoter composed of the promoters gal1 and gal10. gal1 / 10 The nucleotide sequence of is shown in NA SEQ ID NO.9. The terminator T cyc1 、T adh1 、T tdh3 They are endogenous terminators of Saccharomyces cerevisiae, and their nucleotide sequences are shown as NA SEQ ID NO.10, NA SEQ ID NO.11, and NA SEQ ID NO.12, respectively.

[0052] The sgRNA plasmid of CRISPR Cas9 was constructed. The plasmid pML104 was used as a template and circular PCR was performed with a primer containing a 20 bp PAM sequence (as shown in NA SEQ ID NO. 13). The plasmid pML104-PAM with the corresponding PAM sequence was obtained by sequencing.

[0053] The constructed pML104-PAM and gene expression cassette were transformed into competent cells of the host strain Saccharomyces cerevisiae CEN PK2-1C MATa; ura3-52; trp1-289; leu2-3,112; his3Δ1; MAL2-8C; SUC2, and the correct transformants by colony PCR were picked for sequencing verification, finally obtaining the recombinant Saccharomyces cerevisiae strain Mogrol 04 with the mogroside synthesis pathway.

[0054] Example 2 Strengthening the accumulation of key intermediates

[0055] Mogroside synthesis belongs to secondary metabolism, and the accumulation of precursors is relatively low. In order to improve the synthesis of precursors and relieve the restriction of hydroxymethylglutaryl-CoA reductase, the rate-limiting enzyme in the mevalonate pathway, the N-terminal endoplasmic reticulum localization signal peptide of the gene hmg1 was removed to obtain thmg1. tef1 -thmg1-T hmg1 , transformed into strain Mogrol 04, and inserted into the gal80 locus in the Saccharomyces cerevisiae genome, i.e., adding one copy of thmg1 to the gal80 locus, resulting in a strain named Mogrol 05. Strain Mogrol 05 eliminates the rate-limiting step of the MVA pathway. Furthermore, the strain is not induced by galactose, and the activation of the mogroside synthesis pathway is regulated only by glucose concentration.

[0056] On this basis, the key enzymes in the mevalonate pathway were overexpressed, and the endogenous promoters of the endogenous hydroxymethylglutaryl-CoA synthase gene erg13 and squalene synthase erg9 in Saccharomyces cerevisiae were replaced with the strong promoter P tef1 , strain Mogrol 06 was obtained.

[0057] Example 3 Strengthening of the Mogroside Synthesis Pathway

[0058] To promote the flow of the intermediate squalene to mogroside, strain Mogrol 06 was used as the starting strain, and the copy number of the squalene epoxidase gene erg1 was increased, specifically by 3 copies, to obtain strain Mogrol 07. To prevent squalene from flowing into the bypass pathway and thus competing with the precursor substances of the mogroside synthesis pathway, strain Mogrol 07 was used as the starting strain, and the lanosterol synthase gene erg7, a key enzyme in the bypass lanosterol pathway, was knocked out to obtain strain Mogrol 08. Due to the low expression level of cytochrome P450, to increase the expression level of cyp87d18, strain Mogrol 08 was used as the starting strain and transformed with the multi-copy plasmid pESC-G418-T tdh3 -cyp87d18-P gal1 / 10 -atcpr2-T cyc1 , strain Mogrol 09 was obtained.

[0059] Example 4 Production of Mogroside by Fermentation with Recombinant Saccharomyces cerevisiae

[0060] The recombinant strain of Saccharomyces cerevisiae having the mogroside biosynthesis pathway was streaked onto SD plates (supplemented with 50 mg / L uracil) and cultured at 30° C. until a large number of colonies grew.

[0061] Inoculate a loop of a single colony into the seed culture medium and culture at 30°C and 220 rpm for 18-20 h until the cells reach the early logarithmic stage of growth.

[0062] The seed culture was inoculated into the fermentation medium at an initial inoculum volume of 3%, and cultured at 30°C and 220 r / min for 96 h. The bacterial culture was collected every 12 h and the OD was measured. 600 After 96 hours, stop the culture and transfer the bacterial suspension to a 50mL centrifuge tube. Centrifuge at 8000 rpm for 5 minutes at room temperature. Take 1mL of the supernatant and add chromatography-grade formic acid to a final concentration of 3% (v / v). Let it stand at 4°C for 4-8 hours to remove proteins. Centrifuge at 8000 rpm for 5 minutes. Take 0.5mL of the supernatant, filter it through an aqueous filter, and add it to a liquid chromatography detection bottle for yield detection.

[0063] The seed culture medium is liquid YPD medium. The medium composition is: glucose 20g / L, peptone 20g / L, yeast powder 10g / L.

[0064] The fermentation medium is liquid YPD medium with adjusted glucose concentration. The medium composition is: glucose 30g / L, peptone 20g / L, yeast powder 10g / L.

[0065] The yields of mogroside V and other mogrosides are shown in Figures 1 and 2, respectively. The recombinant strain Mogrol09 produced 5.6 mg / L of mogroside V, which is 467 times the yield of the originally constructed strain Mogrol 04 capable of synthesizing mogroside V. The yields of other mogrosides in S. cerevisiae Mogrol 09 were also significantly increased compared to Mogrol 04, with the yields of mogroside IIE, mogroside IIIx, mogroside IVA, and simanoside I reaching 150.2 μg / L, 212.3 μg / L, 183.5 μg / L, and 110.4 μg / L, respectively.

Claims

1. A method for constructing an engineered yeast for de novo synthesis of mogrosides, characterized in that The constructed expression cassette P of cucurbitadienol synthase gene tef1 -cds-T cyc1 , the expression cassette P of epoxide hydrolase gene tef1 -eph3-T cyc1 , the expression cassette T of cytochrome P450 enzyme-encoding gene CYP and cytochrome P450 enzyme reductase gene tdh3 -cyp87d18-P gal1 / 10 -atcpr2-T cyc1 and the expression cassette T of glycosyltransferase-encoding gene adh1 -ugt74ac1-P gal1 / 10 -ugtms1-T cyc1 were inserted into the yeast genome to obtain the engineering yeast for mogroside production.

2. The construction method of the engineered yeast for de novo synthesis of mogroside according to claim 1, characterized in that, Construct a truncated 3-hydroxy-3-methylglutaryl coenzyme A reductase gene expression cassette P tef1 -tHMG1-T hmg1 , and insert it into the gal80 locus in the genome of the engineered yeast for mogroside production.

3. The construction method of the engineered yeast for de novo synthesis of mogroside according to claim 2, characterized in that the replacement The endogenous promoters of hydroxymethylglutaryl-CoA synthase gene erg13 and squalene synthase erg9 in the engineered yeast for producing mogroside are strong promoter P tef1 .

4. The construction method of the engineered yeast for de novo synthesis of mogroside according to claim 3, characterized in that, Increase the copy number of the squalene epoxidase gene erg1 in the engineered yeast for producing mogroside, and the copy number is more than 3.

5. The construction method of the engineered yeast for de novo synthesis of mogroside according to claim 4, characterized in that, Knock out the lanosterol synthase gene erg7, which is the key enzyme in the bypass lanosterol pathway of the engineered yeast for producing mogroside.

6. The construction method of the engineered yeast for de novo synthesis of mogroside according to claim 5, characterized in that, Transforming the multi-copy plasmid pESC-G418-T into the Saccharomyces cerevisiae for mogroside production tdh3 -cyp87d18-P gal1 / 10 -atcpr2-T cyc1 。 7. The construction method of the engineered yeast for de novo synthesis of mogroside according to any one of claims 1-6, characterized in that, Its characteristics are as follows: The engineered yeast includes but is not limited to Saccharomyces cerevisiae, Pichia pastoris, Pichia fermentans, Hansenula anomala, Yarrowia lipolytica, Candida antarctica, Candida utilis, Candida tropicalis, Schizosaccharomyces pombe, Rhodotorula glutinis.

8. The method for constructing an engineered yeast for de novo synthesis of mogroside according to any one of claims 2-6, characterized in that, The engineered yeast is Saccharomyces cerevisiae, and the expression cassette of the cucurbitadienol synthase gene P tef1 -cds-T cyc1 is inserted into the 1622b site of the yeast genome; the expression cassette of the epoxide hydrolase gene P tef1 -eph3-T cyc1 is inserted into the 911b site of the yeast genome; the expression cassette of the cytochrome P450 enzyme-encoding gene CYP and the cytochrome P450 enzyme reductase gene T tdh3 -cyp87d18-P gal1 / 10 -atcpr2-T cyc1 is inserted into the 106a site of the yeast genome; the expression cassette of the glycosyltransferase-encoding gene T adh1 -ugt74ac1-P gal1 / 10 -ugtms1-T cyc1 is inserted into the 308a site of the yeast genome; the expression cassette of the truncated 3-hydroxy-3-methylglutaryl coenzyme A reductase gene P tef1 -tHMG1-T hmg1 is inserted into the gal80 site of the yeast genome.

9. An engineered yeast for de novo synthesis of mogroside, characterized in that the yeast The genome contains the cucurbitadienol synthase gene expression cassette P tef1 -cds-T cyc1 and the epoxide hydrolase gene expression cassette P tef1 -eph3-T cyc1 and the cytochrome P450 enzyme-encoding gene CYP and the cytochrome P450 enzyme reductase gene expression cassette T tdh3 -cyp87d18-P gal1 / 10 -atcpr2-T cyc1 and the glycosyltransferase-encoding gene expression cassette T adh1 -ugt74ac1-P gal1 / 10 -ugtms1-T cyc1 .

10. The engineered yeast for de novo synthesis of mogroside according to claim 9, characterized in that, The yeast genome contains a truncated 3-hydroxy-3-methylglutaryl coenzyme A reductase gene expression cassette P tef1 -tHMG1-T hmg1 .

11. The engineered yeast for de novo synthesis of mogroside according to claim 10, characterized in that, The endogenous promoters of hydroxymethylglutaryl-CoA synthase gene erg13 and squalene synthase erg9 in the yeast are strong promoter P tef1 .

12. The engineered yeast for de novo synthesis of mogroside according to claim 11, wherein, The copy number of the squalene epoxidase gene (erg1) in the yeast is more than 3.

13. The engineered yeast for de novo synthesis of mogroside according to claim 12, characterized in that, In the yeast, the lanosterol synthase gene erg7, which is the key enzyme in the bypass lanosterol pathway, is knocked out.

14. The engineered yeast for de novo synthesis of mogroside according to claim 13, wherein, The Saccharomyces cerevisiae also contains a multi-copy plasmid pESC-G418-T tdh3 -cyp87d18-P gal1 / 10 -atcpr2-T cyc1 .

15. The engineered yeast for de novo synthesis of mogroside according to any one of claims 9-14, characterized in that, The engineered yeast includes but is not limited to Saccharomyces cerevisiae, Pichia pastoris, Pichia fermentans, Hansenula anomala, Yarrowia lipolytica, Candida antarctica, Candida utilis, Candida tropicalis, Schizosaccharomyces pombe, Rhodotorula glutinis.

16. The engineered yeast for de novo synthesis of mogroside according to any one of claims 10-14, characterized in that, The engineered yeast is Saccharomyces cerevisiae, and in its genome, the expression cassette P of the cucurbitadienol synthase gene is inserted at the 1622b locus tef1 -cds-T cyc1 ; Insert the epoxide hydrolase gene expression cassette P at the 911b site tef1 -eph3-T cyc1 ; Insert the cytochrome P450 enzyme-encoding gene CYP and the cytochrome P450 enzyme reductase gene expression cassette T at the 106a site tdh3 -cyp87d18-P gal1 / 10 -atcpr2-T cyc1 ; Insert the glycosyltransferase-encoding gene expression cassette T at the 308a site adh1 -ugt74ac1-P gal1 / 10 -ugtms1-T cyc1 ; Insert the expression cassette P of the truncated 3-hydroxy-3-methylglutaryl coenzyme A reductase gene at the gal80 locus tef1 -tHMG1-T hmg1 。 17. Use of the engineered yeast for de novo synthesis of mogroside according to any one of claims 9-16 in the biological fermentation production of mogroside.

18. The method for fermenting and producing mogroside by the engineered yeast for de novo synthesis of mogroside according to claim 14, characterized in that, Take the engineered yeast strain and culture it in a liquid YPD medium on a shaker at 30 °C and 220 r / min for 18 h to obtain a fermentation seed liquid. Transfer the fermentation seed liquid into a fermentation medium at an inoculation rate of 3% and culture it at 30 °C and 220 r / min for 96 h.

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