Saccharomyces cerevisiae genetically engineered bacterium for efficiently synthesizing mogroside V in multi-dimensional cytoplasm adaptability and construction method of saccharomyces cerevisiae genetically engineered bacterium

Abstract

The invention discloses a saccharomyces cerevisiae genetically engineered bacterium for efficiently synthesizing mogroside V through multi-dimensional cytoplasm adaptability and a construction method of the saccharomyces cerevisiae genetically engineered bacterium. The construction method of the saccharomyces cerevisiae genetically engineered bacterium comprises the following steps: integrating truncated 3-hydroxy-3-methylglutaryl coenzyme A reductase tHMG1, pentenyl pyrophosphate isomerase IDI1, squalene synthetase ERG9, epoxy squalene cyclization enzyme ERG1, choline kinase ScCK, isopentenyl phosphokinase AtIPK, phosphoketolase BbPK, phosphotransacetylase PTA and dihydroxyacetone hydrolase EcHAD at a site of a saccharomyces cerevisiae genome GAL80, and carrying out enzymolysis, so as to obtain the saccharomyces cerevisiae genetically engineered bacterium. The invention relates to a preparation method of a cytochrome P450 enzyme, which comprises the following steps of: preparing a cycloepoxide hydrolase SgEPH3, a cytochrome P450 enzyme CYP87D18 and a cytochrome P450 enzyme reductase AtCPR1; an ABC efflux protein PDR11, a glycosyl transferase UGTMG1, a sucrose synthase Susy and a glycosyl transferase SgUGT94-289-3 are integrated at a site Exg1 of a saccharomyces cerevisiae genome. The saccharomyces cerevisiae genetically engineered bacterium constructed by the invention realizes a higher level of mogroside V yield synthesized from the beginning in microorganisms, and has important industrial application potential.

Description

Technical Field

[0001] The present invention relates to a genetically engineered yeast strain for efficiently synthesizing mogroside V with multi-dimensional cytoplasmic adaptability and a construction method thereof, and belongs to the field of bioengineering. Background Art

[0002] A high-sugar diet contributes to a range of modern health problems, including tooth decay, obesity, diabetes, metabolic syndrome, and cardiovascular disease. Plant-derived mogrosides, known for their high sweetness, low calorie content, safety, fat-reducing properties, and resistance to tooth decay, have become a new generation of sweeteners, attracting increasing attention.

[0003] Mogroside V is the main source of mogroside sweetness. At one ten-thousandth of a part, its sweetness is 425 times that of 5% sucrose. It also exhibits numerous pharmacological activities, including antitussive and expectorant, anticancer, antioxidant, and blood sugar regulation, making it a new generation of functional sweetener being actively developed both domestically and internationally. However, the optimal cultivation area for monk fruit is narrow, primarily distributed in Guilin, and biological resources are limited. Furthermore, the content of mogroside V in the whole fruit is only 0.8% to 1.3%, making it difficult to purify complex analogues. Large-scale production is therefore impossible if it is extracted from monk fruit.

[0004] Therefore, using advanced synthetic biology technology to construct a microbial cell factory for the de novo synthesis of mogroside V, using glucose or other intermediate metabolites as raw materials to synthesize and separate high-purity mogroside V, and achieving low-cost, large-scale production will become a key technology to subvert traditional monk fruit extraction production methods, and can make an important contribution to meeting the "explosive" growth of global demand for natural non-sugar sweeteners.

[0005] Mogroside V is a cytoplasmic triterpenoid saponin. The whole genome of Momordica grosvenori was sequenced in 2016, and the biosynthetic pathway of mogroside V was fully elucidated. Saccharomyces cerevisiae is a safe eukaryotic model microorganism with a clear genetic background and mature genetic modification techniques. It also possesses a mevalonate (MVA) pathway for the biosynthesis of terpenes. IPP and DMAPP are sequentially synthesized into the triterpene precursor squalene, catalyzed by farnesyl pyrophosphate synthase ERG20 and squalene synthase ERG9. Squalene is then oxidized to diepoxysqualene (2,3;22,23-diepoxysqualene) by squalene epoxidase ERG1.

[0006] Jiangnan University disclosed in CN117887599A a method for constructing an engineered yeast for synthesizing mogrosides from scratch. By genetic engineering methods, exogenous genes were integrated into the yeast, including the cucurbitadienol synthase encoding gene CDS from Momordica grosvenori, the epoxide hydrolase encoding gene EPH3, the cytochrome P450 enzyme encoding gene CYP87D18, the glycosyltransferase encoding genes UGT74AC1 and UGTMS1, and the cytochrome P450 enzyme reductase encoding gene AtCPR2 from Arabidopsis thaliana. CYP87D18 was expressed on a high-copy plasmid and expressed by using a strong endogenous promoter of yeast. The method of adjusting the gene copy number was used to enhance the expression of the yeast endogenous truncated 3-hydroxy-3-methylglutaryl-CoA reductase gene HMG1, hydroxymethylglutaryl-CoA synthase gene ERG13, squalene synthase ERG9 and squalene epoxidase gene ERG1. In addition, the lanosterol synthase gene ERG7 was knocked out, thereby constructing an engineered yeast that synthesizes mogroside from scratch. The fermentation results showed that the fermentation products of the engineered yeast included a mogroside mixture consisting of mogroside IIE, mogroside IIIX, mogroside IVA, simenoside I and mogroside V, among which the yield of mogroside V was 5.6 mg / L.

[0007] Sichuan Yingjia Hesheng Technology Co., Ltd. disclosed in CN118147262A and CN118147263A a yeast engineered bacterium producing mogroside, simenoside I and / or mogroside V and its application, wherein CN118147262A weakened the competition pathway by replacing the endogenous promoter method to down-regulate the lanosterol synthase gene ERG7 and up-regulating the phospholipid biosynthesis regulatory gene INO2, and integrated exogenous genes into the yeast genome on the basis of knocking out the GAL4 inhibitory protein GAL80 encoding gene, including the squalene epoxidase gene GAL80 from mogroside, the cucurbitadienol synthase encoding gene CDS, An engineered yeast capable of synthesizing mogroside was constructed by using the epoxide hydrolase encoding gene EPH3, the cytochrome P450 enzyme encoding gene CYP87D18, the cytochrome P450 enzyme reductase encoding gene CRR1, the glycosyltransferase encoding genes UGT720-269-1 and UGT94-289-3, and the cytochrome b5 gene from Rubus serrata. The fermentation results showed that the fermentation products of the engineered yeast included a mogroside mixture consisting of mogroside I-AI, mogroside IIE, mogroside IIIX, mogroside IVA, simenoside I, and mogroside V, with the yield of mogroside V being 5.57 mg / L. CN118147263A reduces the expression intensity of CRR by using an endogenous constitutive weak promoter and increases the expression amount of CYP87D18 derived from Momordica grosvenori by increasing the copy number. Then, exogenous genes, including the cucurbitadienol synthase encoding gene CDS, the epoxide hydrolase encoding gene EPH3 and the cytochrome P450 enzyme reductase encoding gene CRR1, derived from Momordica grosvenori, are integrated into the yeast genome to construct an engineered yeast capable of synthesizing mogroside. The fermentation results show that the fermentation product of the engineered yeast includes a mogroside mixture consisting of mogroside I-AI, mogroside IIE, mogroside IIIX, mogroside IVA, simenoside I and mogroside V, wherein the yield of mogroside V reaches 22.07 mg / L.

[0008] Although there have been reports on the de novo synthesis of mogroside V using Saccharomyces cerevisiae, no related strains have been used in industrial production. The reasons may be as follows: (1) Acetyl CoA produced by sugar metabolism is mainly generated in mitochondria and mainly used in the tricarboxylic acid cycle, and the acetyl CoA flux to the MVA pathway is insufficient; (2) In addition to part of the squalene in yeast flowing to the ergosterol pathway to synthesize lanosterol, most of it is stored in non-polar lipid droplets, resulting in a low flux of squalene to the mogroside V synthesis pathway; (3) The cucurbitacin in the mogroside V synthesis pathway is not sufficient. The styrofolate synthase SgCDS, epoxide hydrolase SgEPH3, cytochrome P450 enzyme CYP87D18 and cytochrome P450 enzyme reductase are located in different subcellular regions and have low self-activity. The synthesis of mogroside V is low after heterologous expression. (4) The specificity of mogroside glycosyltransferase is low. In addition to synthesizing a small amount of mogroside V, it also synthesizes many other mogrosides such as mogroside I-AI, mogroside IIE, mogroside IIIX, mogroside IVA, and simenoside I, which makes it difficult to purify and separate the fermentation product and the product quality is low. Therefore, multi-dimensional enhancement of the exogenous mogroside V synthesis pathway and the adaptability of its key enzymes to the yeast host metabolic network and intracellular microenvironment is helpful to improve the synthesis of mogroside V. Summary of the Invention

[0009] The technical problem to be solved by the present invention is to provide a genetically engineered yeast strain of Saccharomyces cerevisiae that can efficiently synthesize mogroside V with multi-dimensional cytoplasmic adaptability and a construction method thereof.

[0010] The method for constructing a genetically engineered yeast strain of Saccharomyces cerevisiae that can efficiently synthesize mogroside V with multi-dimensional cytoplasmic adaptability to solve the above technical problems is as follows:

[0011] (1) Integrate truncated 3-hydroxy-3-methylglutaryl-CoA reductase tHMG1, prenyl pyrophosphate isomerase IDI1, squalene synthase ERG9, epoxysqualene cyclase ERG1, choline kinase ScCK, isopentenyl phosphate kinase AtIPK, phosphoketolase BbPK, phosphotransacetylase PTA, dihydroxyacetone hydrolase EcHAD, rhamnose isomerase PsLRhI, cucurbitadienol synthase SgCDS, epoxyepoxide hydrolase SgEPH3, cytochrome P450 enzyme CYP87D18, and cytochrome P450 enzyme reductase AtCPR1 into the GAL80 locus of the Saccharomyces cerevisiae genome;

[0012] (2) The ABC efflux protein PDR11, glycosyltransferase UGTMG1, sucrose synthase Susy and glycosyltransferase SgUGT94-289-3 were integrated into the Exg1 locus of the Saccharomyces cerevisiae genome.

[0013] In the step (1):

[0014] The HMG1 (NCBI Sequence ID: NP_013636.1) N-terminal truncation of 527 amino acids tHMG1, prenyl pyrophosphate isomerase IDI1 (NCBI Sequence ID: NP_015208.1), choline kinase ScCK (NCBI Sequence ID: NP_013234.1), isopentenyl phosphate kinase AtIPK (NCBI Sequence ID: NP_173986.2), phosphoketolase BbPK (NCBI Sequence ID: WP_026645831.1), phosphotransacetylase PTA (NCBI Sequence ID: CCJ44813.1), dihydroxyacetone hydrolase EcHAD (NCBI Sequence ID: WP_314879630.1), and rhamnose isomerase PsLRhI (NCBI Sequence ID: BAD14073.1).

[0015] The amino and carboxyl ends of the epoxysqualene cyclase ERG1 (NCBI Sequence ID: NP_011691.1) are respectively connected to the erythromycin polyketide synthase polypeptide interaction tag D3 via a flexible linker (GGGGS) 3. N DD (nucleotide sequence shown in SEQ ID NO. 1) and polypeptide interaction tag RIAD (nucleotide sequence shown in SEQ ID NO. 2) are fused.

[0016] Squalene synthase ERG9 (NCBI Sequence ID: NP_012060.1) amino terminus interacts with erythromycin polyketide synthase peptide D2 C DD (nucleotide sequence shown in SEQ ID NO.3) fusion.

[0017] The amino and carboxyl ends of the cucurbitacinol synthase SgCDS (NCBI Sequence ID: K7NBZ9.1) are respectively connected to the erythromycin polyketide synthase polypeptide interaction tag D5 via a flexible linker (GGGGS) 3. N DD (nucleotide sequence shown in SEQ ID NO.4) and polypeptide interaction tag RIDD (nucleotide sequence shown in SEQ ID NO.5) are fused.

[0018] The amino and carboxyl ends of the epoxide hydrolase SgEPH3 (NCBI Sequence ID: P0DO70.1) are respectively connected to the chlortetracycline polyketide synthase polypeptide interaction tag A2 through a flexible linker (GGGGS) 3. N DD (nucleotide sequence shown in SEQ ID NO.6) and erythromycin polyketide synthase polypeptide interaction tag D4C DD (nucleotide sequence shown in SEQ ID NO.7) fusion.

[0019] The amino terminus of the cytochrome P450 enzyme CYP87D18 (NCBI Sequence ID: K7NBR2.1) interacts with the tacrolimus polyketide synthase polypeptide via a flexible linker (GGGGS) 3. N DD (nucleotide sequence as SEQ ID NO.8) fusion.

[0020] 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.9) fusion.

[0021] In the step (2):

[0022] The carboxyl terminus of the ABC efflux protein PDR11 (NCBI Sequence ID: NP_012252.1) is connected to the tacrolimus polyketide synthase polypeptide interaction tag F4 via a flexible linker (GGGGS) 3 C DD (nucleotide sequence such as SEQ ID NO.10) fusion.

[0023] The carboxyl terminus and amino terminus of the glycosyltransferase UGTMG1 (NCBI Sequence ID: 6L8W_A) are fused to the Aga1p (NCBI Sequence ID: NP_014442.1) and Aga2p (amino acid sequence shown in SEQ ID NO.11) subunits of α-agglutinin, respectively.

[0024] The carboxyl terminus and amino terminus of the sucrose synthase Susy (NCBI Sequence ID: WP_011381564.1) were fused to the Aga1p and Aga2p subunits of α-agglutinin, respectively.

[0025] The glycosyltransferase SgUGT94-289-3 V148M / G152A (NCBI Sequence ID: 8HJO_A) The carboxyl terminus and amino terminus are fused to the Aga1p and Aga2p subunits of α-agglutinin, respectively.

[0026] The synthesis of mogroside V is carried out in two different regions, wherein the precursor mogroside alcohol is synthesized in the cytoplasm, and mogroside V is synthesized outside the cell wall.

[0027] The precursor mogroside is transported from the cytoplasm to the extracellular space via the ABC efflux protein PDR11.

[0028] The epoxysqualene cyclase ERG1, cucurbitadienol synthase SgCDS, epoxyepoxide hydrolase SgEPH3, cytochrome P450 enzyme CYP87D18 and cytochrome P450 enzyme reductase AtCPR1 form a linear directional multi-enzyme complex in the cell membrane matrix side through a polypeptide interaction tag and an ABC efflux protein PDR11.

[0029] The epoxysqualene cyclase ERG1 and the cucurbitadienol synthase SgCDS construct a three-enzyme complex in the cytoplasm in the form of one ERG1 molecule and two SgCDS molecules through the peptide interaction tags RIAD and RIDD.

[0030] The glycosyltransferase UGTMG1, sucrose synthase Susy and glycosyltransferase SgUGT94-289-3 are displayed on the surface of yeast cell wall through the α-lectin system.

[0031] The mogroside V is synthesized by the glycosyltransferase UGTMG1, sucrose synthase Susy and glycosyltransferase SgUGT94-289-3 displayed on the surface of yeast cell walls to catalyze the synthesis of extracellular mogroside alcohol.

[0032] The uridine diphosphate glucose required for the synthesis of mogroside V is provided by sucrose in the culture medium catalyzed by sucrose synthase Susy displayed on the surface of yeast cell walls.

[0033] 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.12).

[0034] The present invention also provides a genetically engineered yeast strain of Saccharomyces cerevisiae that can efficiently synthesize mogroside V with multi-dimensional cytoplasmic adaptability, which is specifically constructed using the above-mentioned construction method.

[0035] The present invention also provides a method for producing mogroside V, which comprises fermenting and producing mogroside V using the engineered yeast Saccharomyces cerevisiae provided by the present invention.

[0036] In the present invention, when fermenting to produce mogroside V, YPD medium supplemented with 10 mM sucrose, 10 mM uridine diphosphate (UDP), 21 mM 3-methyl-3-butene-1-ol and 9 mM 3-methyl-2-butene-1-ol is used as the fermentation medium.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] Mogroside V (MG-V) is a triterpenoid. Acetyl CoA, isopentenyl diphosphate (IPP), dimethylallyl diphosphate (DMAPP), squalene, and mogroside alcohol are all key precursors for MG-V biosynthesis. This study increases intracellular acetyl CoA supply by constructing a heterologous isopentenol utilization pathway (IUP). Furthermore, by constructing a heterologous phosphoketolase pathway and overexpressing key enzymes of the native mevalonate (MVA) pathway, the synthesis of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) is enhanced.

[0039] Squalene epoxidase ERG1 is a key enzyme at the junction of MG-V synthesis and ergosterol synthesis. The present invention uses a multi-enzyme complex directed assembly strategy to form a three-enzyme complex with ERG1 and the cucurbitadienol synthase encoding gene SgCDS at a molecular ratio of 1:2, pulling squalene towards the synthesis of mogrosantheol. In addition, a multi-enzyme complex directed assembly strategy is used to assemble ERG1 with cucurbitadienol synthase, epoxide hydrolase, cytochrome P450 enzyme, and cytochrome P450 enzyme reductase to comprehensively increase the yield of mogrosantheol.

[0040] In order to reduce the effect of MG-V on yeast cell growth, the present invention utilizes the ABC efflux pump PDR11 to transport mogrosanol to the extracellular space to synthesize MG-V. In addition, through polypeptide interaction, the present invention forms a linear multi-enzyme complex with cucurbitadienol synthase, epoxide hydrolase, cytochrome P450 enzyme, cytochrome P450 enzyme reductase, and PDR11, thereby integrating mogrosanol synthesis and efflux, and reducing the effect of mogrosanol on yeast cell growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 Schematic diagram of integration into the GAL80 locus of the Saccharomyces cerevisiae genome.

[0042] Figure 2 Schematic diagram of integration into the Exg1 locus of the Saccharomyces cerevisiae genome. DETAILED DESCRIPTION

[0043] 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.

[0044] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0045] Unless otherwise specified, the test materials used in the following examples were purchased from conventional biochemical reagent stores.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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:

[0050] Table 1 List of artificial genes

[0051]

[0052]

[0053] Example 2 Backbone plasmid construction

[0054] The information of the backbone plasmids involved in the construction of the following examples is shown in Table 2.

[0055] Table 2 Backbone plasmid list

[0056] Plasmid name Basic Information pM350 Contains the SmaI-TDH1t-SacII-AscI-pGAL1,10-PacI-SbfI-GPM1t-StuI backbone pM351 Contains the SmaI-ADH1t-SacII-AscI-pGAL1,10-PacI-SbfI-ADH2t-StuI backbone pM352 Contains the SmaI-TDH2t-SacII-AscI-pGAL1,10-PacI-SbfI-TDH3t-StuI backbone pM353 Contains the SmaI-CYC1t-SacII-AscI-pGAL1,10-PacI-SbfI-TEF1t-StuI backbone pM354 Contains the SmaI-TPI1t-SacII-AscI-pGAL1,10-PacI-SbfI-FBA1t-StuI backbone pM355 Contains the SmaI-ACT1t-SacII-AscI-pGAL1,10-PacI-SbfI-PGI1t-StuI backbone pM356 Contains the PmeI-pGAL1-PacI-SbfI-ENO1t-StuI backbone pM357 Contains the PmeI-pGAL1-PacI-SbfI-FBA1t-StuI backbone

[0057] (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.

[0058] 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.

[0059] 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.

[0060] Table 3 Primer list

[0061]

[0062]

[0063]

[0064] (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.

[0065] 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.

[0066] (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.

[0067] 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.

[0068] 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.

[0069] Similarly, plasmids pM351, pM352, pM353, pM354, pM355, pM356, and pM357 were constructed.

[0070] Example 2 Expression cassette plasmid construction

[0071] The plasmid information for constructing the expression cassettes involved in the following examples is shown in Table 4.

[0072] Table 4 Expression cassette plasmids

[0073]

[0074]

[0075] (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.

[0076] 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.

[0077] 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.

[0078] (2) Take D5 in the second column of Table 5 N Overlap extension PCR (OE-PCR) was performed using the DD fragment, SgCDS fragment, and RIDD fragment as templates. The OE-PCR amplification product was recovered by gel cutting to obtain D5. N DD-SgCDS-RIDD fragment.

[0079] 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.

[0080] 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.

[0081] (3) Double digestion of D5 with restriction endonucleases AscI and SacII N The DD-SgCDS-RIDD fragment and pM355 plasmid were purified by PCR product purification kit to obtain D5 NDD-SgCDS-RIDD-AscI:SacII sticky-end fragment and pM355-AscI:SacII linear vector.

[0082] 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.

[0083] (4) Use T4 DNA ligase to ligate D5 N The DD-SgCDS-RIDD-AscI:SacII sticky end fragment was connected to the pM355-AscI:SacII linear vector. The T4 DNA ligase reaction system 10μL is: 10×T4 DNA Ligase Buffer 1μL, D5 N 30 ng of the DD-SgCDS-RIDD-AscI:SacII sticky-end fragment and pM355-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.

[0084] (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.

[0085] Using bacteria as templates, primers ACT1t-f1 (5'-ctgaaaagcgatgaagagatgat-3') and pGAL1-r1 (5'-gacgaggacgcacggaggagagt-3') were used for verification to obtain the recombinant plasmid pM355-D5. N DD-SgCDS-RIDD. PCR verification system 25μL: 2×SanTaq PCR Mix 12.5μL, drop solution 1μL, ACT1t-f1 and pGAL1-r1 1μL each, distilled water 11.5μL. N DD-SgCDS-RIDD was sequenced, and the sequencing results showed that the recombinant plasmid contained SmaI-ACT1t-SacII-RIDD-SgCDS-D5 NDD-AscI-pGAL1,10-PacI-SbfI-PGI1t-StuI expression cassette.

[0086] (6) Take A2 in the second column of Table 5 N DD fragment, SgEPH3 fragment and D4 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 A2 N DD-SgEPH3-D4 C DD fragment.

[0087] (7) Double digest A2 with restriction endonucleases PacI and SbfI N DD-SgEPH3-D4 C DD fragment and pM355-D5 N DD-SgCDS-RIDD plasmids were purified using a PCR product purification kit to obtain A2 N DD-SgEPH3-D4 C DD-PacI:SbfI sticky-end fragment and pM355-PacI:SbfI linear vector.

[0088] (8) Use T4 DNA ligase to ligate A2 N DD-SgEPH3-D4 C The DD-PacI:SbfI sticky-end fragment was ligated to the pM355-PacI:SbfI linear vector.

[0089] (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.

[0090] Using bacteria as templates and primers pGAL1-f1 (5'-caaccataggatgataatgcgat-3') and PGI1t-r1 (5'-cacaataaagtcttcacgacgctaa-3') for verification, the recombinant plasmid pM355-C6 was obtained. The PCR verification system (25 μL) consisted of: 12.5 μL of 2× SanTaq PCR Mix, 1 μL of liquid, 1 μL each of pGAL1-f1 and PGI1t-r1, and 11.5 μL of distilled water. The recombinant plasmid was sequenced, and the sequencing results showed that the recombinant plasmid contained SmaI-ACT1t-SacII-RIDD-SgCDS-D5. N DD-AscI-pGAL1,10-PacI-A2 N DD-SgEPH3-D4 C DD-SbfI-PGI1t-StuI expression cassette.

[0091] Similarly, the plasmids listed in Table 4 were obtained.

[0092] Table 5 Primer list

[0093]

[0094]

[0095] Example 3 Construction of p426-GAL80-Exg1 gRNA dual-target plasmid

[0096] (1) Using KOD-Plus high-fidelity enzyme and p426 plasmid as templates, primer pair 56, primer pair 57, primer pair 58, and primer pair 59 shown in Table 6 were used for PCR amplification, and the corresponding PCR amplification products were recovered by gel cutting to obtain GAL80 gRNA-1, GAL80 gRNA-2, Exg1 gRNA-1, and Exg1 gRNA-2 fragments.

[0097] (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.

[0098] (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.

[0099] 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.

[0100] (4) Using KOD-Plus high-fidelity enzyme, p426-GAL80 gRNA plasmid and p426-Exg1 gRNA plasmid as templates, PCR amplification was performed using primer pair 60 and primer pair 61 shown in Table 6, and the corresponding PCR amplification products were recovered by gel excision. The corresponding PCR amplification products were recovered by gel excision to obtain GAL80 gRNA-3 and Exg1 gRNA-3 fragments, and the GAL80 gRNA-3 and Exg1 gRNA-3 fragments were homologously recombined using Seamless cloning Master Mix. OP10 competent cells were transformed and PCR verification was performed using primers gRNA-F and gRNA-R to obtain p426-GAL80-Exg1 gRNA recombinant plasmid. The sequencing results of the recombinant plasmid showed that the recombinant plasmid contained GAL80 gRNA (5'-gtcggtctcaacgcagccaa-3') and Exg1 gRNA (5'-gaacaaattgaatggaagaa-3').

[0101] Table 6 Primer list

[0102]

[0103] Example 4 Construction of recombinant yeast

[0104] (1) Using the plasmids shown in column 1 of Table 7 as templates, PCR amplification was performed using the primers shown in columns 3 and 4 of Table 7, and the corresponding PCR amplification products were recovered to obtain the screening markers Leu2 and His3 fragments and the modules C-M7 and C-M3'.

[0105] Table 7 Primer list

[0106]

[0107] (2) The plasmids shown in column 2 of Table 8 were double-digested with the restriction endonucleases shown in column 3 of Table 8, and the double-digested products of the sizes shown in column 4 of Table 8 were recovered from the gel, thereby obtaining modules C-M1, C-M2, C-M3, C-M4, C-M5, C-M6, C-M1′, and C-M2′.

[0108] Table 8 Plasmid list

[0109] Module Plasmid name Endonuclease name Size (bp) C-M1 pM350-CP1 SmaI and StuI 4358 C-M2 pM351-CP2 SmaI and StuI 3389 C-M3 pM352-CP3 SmaI and StuI 4305 C-M4 pM353-CP4 SmaI and StuI 6514 C-M5 pM354-CP5 SmaI and StuI 4053 C-M6 pM355-CP6 SmaI and StuI 4631 C-M1 pM357-CP1 PmeI and StuI 5518 C-M2 pM355-CP2 SmaI and StuI 8891

[0110] (3) Prepare Saccharomyces cerevisiae competent cells according to the instructions of the yeast competent cell preparation kit. To Saccharomyces cerevisiae competent cells, 0.1 μg of module M1, 0.1 μg of module M2, 0.1 μg of module M3, 0.1 μg of module M4, 0.1 μg of module M5, 0.1 μg of module M6, 0.1 μg of module M7, 0.1 μg of module M1`, 0.1 μg of module M2`, 0.1 μg of module M3`, 0.1 μg of module M4`, and 0.1 μg of module M5`, 0.2 μg of plasmid p426-GAL80-Exg1 gRNA, 0.2 μg of plasmid p414-Cas9, 0.1 μg of fragment Leu2, and 0.1 μg of fragment His3 were added. After 2.7 kV electroporation, 1 mL of mol / L sorbitol solution was added, and the cells were revived at 30°C for 1 hour. The cells were then plated onto screening medium to obtain several single clones, which were cultured at 30°C for at least 36 hours. The screening medium consisted of: 0.8% SD-His-Leu-Trp-Ura, 2% glucose and 2% agar powder.

[0111] (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, M7 and the screening marker Leu2 were correctly inserted into the GAL80 site of the yeast genome, and M1', M2', M3', M4', M5' and the screening marker were correctly inserted into the Exg1 site of the yeast genome.

[0112] Table 9 Primer list

[0113]

[0114]

[0115] Example 6: Fermentation of engineered yeast Saccharomyces cerevisiae and detection of mogroside V

[0116] (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.

[0117] (2) Fermentation culture: The seed culture was inoculated at a 1% to 5% inoculum into YPD liquid medium supplemented with 10 mM sucrose, 10 mM uridine diphosphate (UDP), 21 mM 3-methyl-3-butene-1-ol, and 9 mM 3-methyl-2-butene-1-ol. The culture was carried out at 30°C and 220 rpm for 96 h, and the fermentation broth was collected.

[0118] (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.

[0119] (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 38.2 mg / L.

[0120] Although the present invention discloses the preferred embodiments as described above, it is not intended to limit the present invention. Any technician familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for constructing a genetically engineered yeast strain of Saccharomyces cerevisiae that can efficiently synthesize mogroside V with multi-dimensional cytoplasmic adaptability, characterized by: The following steps are involved: (1) Integrate truncated 3-hydroxy-3-methylglutaryl-CoA reductase tHMG1, prenyl pyrophosphate isomerase IDI1, squalene synthase ERG9, epoxysqualene cyclase ERG1, choline kinase ScCK, isopentenyl phosphate kinase AtIPK, phosphoketolase BbPK, phosphotransacetylase PTA, dihydroxyacetone hydrolase EcHAD, rhamnose isomerase PsLRhI, cucurbitadienol synthase SgCDS, epoxyepoxide hydrolase SgEPH3, cytochrome P450 enzyme CYP87D18, and cytochrome P450 enzyme reductase AtCPR1 into the GAL80 locus of the Saccharomyces cerevisiae genome; (2) The ABC efflux protein PDR11, glycosyltransferase UGTMG1, sucrose synthase Susy and glycosyltransferase SgUGT94-289-3 were integrated into the Exg1 locus of the Saccharomyces cerevisiae genome.

2. The method for constructing a genetically engineered yeast strain of Saccharomyces cerevisiae for efficiently synthesizing mogroside V with multi-dimensional cytoplasmic adaptability according to claim 1, characterized in that: In the step (1): The HMG1 N-terminus is truncated with 527 amino acids, tHMG1, prenyl pyrophosphate isomerase IDI1, choline kinase ScCK, isopentenyl phosphate kinase AtIPK, phosphoketolase BbPK, phosphotransacetylase PTA, dihydroxyacetone hydrolase EcHAD, and rhamnose isomerase PsLRhI; The amino and carboxyl ends of the epoxysqualene cyclase ERG1 are respectively connected to the erythromycin polyketide synthase polypeptide interaction tag D3 through a flexible linker (GGGGS) 3. N DD and peptide interaction tag RIAD fusion; Squalene synthase ERG9 amino terminus interacts with erythromycin polyketide synthase peptide tag D2 C DD fusion; The amino and carboxyl ends of the cucurbitacinol synthase SgCDS are respectively connected to the erythromycin polyketide synthase polypeptide interaction tag D5 through a flexible linker (GGGGS) 3. N DD and peptide interaction tag RIDD fusion; The amino and carboxyl ends of the cycloepoxide hydrolase SgEPH3 are respectively connected to the chlortetracycline polyketide synthase polypeptide interaction tag A2 through a flexible linker (GGGGS) 3. N DD and erythromycin polyketide synthase peptide interaction tag D4 C DD fusion; The amino terminus of the cytochrome P450 enzyme CYP87D18 interacts with the tacrolimus polyketide synthase polypeptide through 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 a genetically engineered yeast strain of Saccharomyces cerevisiae for efficiently synthesizing mogroside V with multi-dimensional cytoplasmic adaptability according to claim 1, characterized in that: In the step (2): The carboxyl end of the ABC efflux protein PDR11 interacts with the tacrolimus polyketide synthase polypeptide through a flexible linker (GGGGS) 3 and a tag F4. C DD fusion; The carboxyl terminus and amino terminus of the glycosyltransferase UGTMG1 are fused to the Aga1p and Aga2p subunits of α-agglutinin, respectively; The carboxyl terminus and amino terminus of the sucrose synthase Susy are fused to the Aga1p and Aga2p subunits of α-agglutinin respectively; The glycosyltransferase SgUGT94-289-3 V148M / G152A The carboxyl terminus and amino terminus were fused to the Aga1p and Aga2p subunits of α-agglutinin, respectively.

4. The method for constructing a genetically engineered yeast strain of Saccharomyces cerevisiae for efficiently synthesizing mogroside V with multi-dimensional cytoplasmic adaptability according to claim 1, characterized in that: The synthesis of mogroside V is carried out in two different regions, wherein the precursor mogroside alcohol is synthesized in the cytoplasm and transported from the cytoplasm to the extracellular space through the ABC efflux protein PDR11, and mogroside V is synthesized on the outside of the cell wall.

5. The method for constructing a genetically engineered yeast strain of Saccharomyces cerevisiae for efficiently synthesizing mogroside V with multi-dimensional cytoplasmic adaptability according to claim 1, characterized in that: The epoxysqualene cyclase ERG1, cucurbitadienol synthase SgCDS, epoxyepoxide hydrolase SgEPH3, cytochrome P450 enzyme CYP87D18 and cytochrome P450 enzyme reductase AtCPR1 form a linear directional multi-enzyme complex in the cell membrane matrix side through a polypeptide interaction tag and an ABC efflux protein PDR11.

6. The method for constructing a genetically engineered yeast strain of Saccharomyces cerevisiae for efficiently synthesizing mogroside V with multi-dimensional cytoplasmic adaptability according to claim 1, characterized in that: The epoxysqualene cyclase ERG1 and the cucurbitacadienol synthase SgCDS construct a three-enzyme complex in the cytoplasm in the form of one ERG1 molecule and two SgCDS molecules through the peptide interaction tags RIAD and RIDD.

7. The method for constructing a genetically engineered yeast strain of Saccharomyces cerevisiae for efficiently synthesizing mogroside V with multi-dimensional cytoplasmic adaptability according to claim 1, characterized in that: The glycosyltransferase UGTMG1, sucrose synthase Susy and glycosyltransferase SgUGT94-289-3 are displayed on the surface of yeast cell wall through an α-lectin system, and jointly catalyze the conversion of mogroside alcohol into mogroside V.

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

9. A genetically engineered yeast strain of Saccharomyces cerevisiae that efficiently synthesizes mogroside V with multi-dimensional cytoplasmic adaptability is characterized by: Constructed by the construction method according to any one of claims 1 to 8.

10. A method for producing mogroside V, characterized in that: The cerevisiae engineered bacteria according to claim 9 are used to ferment and produce mogroside V, using YPD medium supplemented with 10mM sucrose, 10mM uridine diphosphate, 21mM 3-methyl-3-butene-1-ol and 9mM 3-methyl-2-butene-1-ol as the fermentation medium.

Images