Engineered yeast capable of producing mogrol, siamenoside i, mogroside IIIX, mogroside IVA, and / or mogroside v and use thereof

Abstract

The present invention relates to an engineered yeast capable of producing mogrol, siamenoside I, mogroside IIIX, mogroside IVA, and / or mogroside V and the use thereof. The present invention provides an engineered yeast capable of producing mogrol, siamenoside I, mogroside IIIX, mogroside IVA, and / or mogroside V. By optimizing and modifying chassis cells, a metabolic pathway of microorganisms is changed, or an expression level of related genes is regulated, thereby enhancing the synthesis efficiency and yield of target products, while improving the production efficiency.

Description

Yeast engineering bacteria producing mogroside, simenoside I, mogroside IIIX, mogroside IVA and / or mogroside V and their applications

[0001] This application claims priority to the Chinese patent application filed with the Patent Office of China on April 2, 2024, with application number 202410393819.2 and invention name “Yeast engineered bacteria producing mogroside, simenoside I and / or mogroside V and their applications”, the entire contents of which are incorporated herein by reference.

[0002] This application claims priority to the Chinese patent application filed with the Patent Office of China on April 2, 2024, with application number 202410393817.3 and invention name “Yeast engineered bacteria producing mogroside, simenoside I and / or mogroside V and their applications”, the entire contents of which are incorporated herein by reference.

[0003] This application claims priority to the Chinese patent application filed with the Patent Office of China on April 2, 2024, with application number 202410393824.3 and invention name “Yeast engineered bacteria producing mogroside, simenoside I and / or mogroside V and their applications”, the entire contents of which are incorporated herein by reference. Technical Field

[0004] The present invention relates to the field of microbial engineering, and in particular to yeast engineering bacteria producing mogroside, simenoside I and / or mogroside V and applications thereof. Background Art

[0005] Monk fruit is a plant of the Cucurbitaceae family, and its fruit has been used as a traditional herbal medicine and contains various bioactive components.

[0006] Mogrol and mogroside are the most commonly used active ingredients in Momordica grosvenori.

[0007] Among them, mogroside and its derivative mogroside are a type of cucurbitane-type triterpene glycoside, among which mogroside is a cucurbitane-type triterpene glycoside, and its aglycone is mogroside. The structure of mogroside is shown in the figure below:

[0008] In recent years, the pharmacological effects of mogrol have attracted widespread attention. Several studies have shown that mogrol can inhibit the ERK1 / 2 and STAT3 signaling pathways, while reducing CREB activity and activating AMPK. It has antioxidant, anti-inflammatory, and hypoglycemic potential, and has shown significant improvements in spatial cognition impairment and learning and memory impairment caused by Alzheimer's disease and ischemic dementia. In vitro cell experiments have shown that mogrol induces G0 / G1 cell cycle arrest in a dose-dependent manner and exhibits potent anti-tumor cell proliferation activity.

[0009] Mogroside is a general term for a variety of mogrosides isolated and identified from Momordica grosvenori. The basic structure is as follows:

[0010] Mogrosides share the same mogroside triterpene core, and the aglycone mogroside is glycosylated with different numbers of glucose moieties to form a variety of mogroside compounds. Different numbers of glucose moieties and the way they are linked produce different mogrosides, such as mogroside IIE, mogroside V, mogroside IV, and simonoside I. These mogrosides vary greatly in taste, for example, mogroside IIE is the main saponin component of immature Momordica grosvenori and tastes extremely bitter. As the Momordica grosvenori grows, it is successively transformed into mogroside II1, mogroside IV, and finally becomes the pentaglycosylated mogroside V with an extremely sweet taste.

[0011] Mogroside V is about 300-400 times sweeter than sucrose. It not only has antitussive, expectorant, and antioxidant effects, but can also be used as a sugar substitute. Compared to artificial saccharin, it is more natural, healthier, and has no side effects. It is a good additive for diabetics and obese people. The structure of Mogroside V is shown below:

[0012] Traditionally, mogroside and mogroside are obtained through water extraction from the fruit of the monk fruit, followed by concentration and separation. Typically, the fruit is chopped or crushed, soaked in water or other solvents, and then filtered, concentrated, separated, and purified through further purification steps to obtain high-purity mogroside and mogroside. This method preserves the natural properties of the product, but the content of mogroside and mogroside in monk fruit is extremely low, typically only around 1%. The cultivation of monk fruit also presents numerous difficulties, making large-scale production difficult and costly. This makes it difficult to achieve mass production through plant extraction, limiting its widespread application. Alternatively, mogroside can be produced through acid / base hydrolysis using mogroside as a raw material. However, obtaining mogroside is also extremely difficult, and the cost can even exceed that of mogroside. Furthermore, the hydrolysis reaction is relatively harsh, resulting in poor stability and difficulty in obtaining a stable final product. In contrast, biosynthesis allows for lower costs and large-scale production of mogroside and mogroside, offering greater controllability and stable products.

[0013] Transcriptomic and metabolomic analysis is an effective strategy for elucidating the biosynthetic pathways of plant natural products. In 2016, Israeli researchers Itkin et al., based on transcriptomic and genomic data from Luo Han Guo (Sheng Guo), achieved a complete delineation of the biosynthetic pathway for mogroside V. In the initial stage of the de novo synthesis of mogrosides from glucose, glucose undergoes glycolysis from pyruvate to acetyl-CoA. Acetyl-CoA then converts this into 2,3-oxidosqualene via the MVA pathway. This is further metabolized to disaccharides of tetrahydroxycucurbitadienol, which is then oxidized to mogroside alcohol. Mogroside alcohol undergoes branched glycosylation to produce sweet-tasting mogrosides IV and V. Figure 1 shows a schematic diagram of the biosynthetic pathway for mogroside V, from Sun Zemin's "Progress in Synthetic Biology of Mogroside V." This diagram provides a basic understanding of the biosynthetic pathway for mogroside V. This application also optimizes and improves this synthetic pathway.

[0014] Using glucose as a starting substrate, de novo microbial synthesis of mogroside or mogroside allows for precise control of synthesis steps and conditions, resulting in the desired product with high purity, safety, and environmental friendliness, a feat unattainable through chemical synthesis. However, de novo microbial synthesis requires long metabolic pathways, a complex network of competing pathways, and high demands on the stability and metabolic capacity of the chassis cells. Therefore, the chassis cells need to be modified to produce specific compounds or increase the yield of specific metabolites.

[0015] With the advancement of synthetic biology research and a deeper understanding of the biosynthetic mechanisms of mogroside molecules in Momordica grosvenori, the use of microorganisms to synthesize mogrosides has become a new approach for large-scale production. In the biosynthesis of mogrosides, UDPG (uridine diphosphate glucose) plays a key role, providing glucose molecules to triterpenoids. For example, UDP-glucosyltransferases (UGTs) use UDPG to attach glucose groups at the C3 and C24 positions of the triterpenoid core of mogrosides, forming mogroside V and imparting its sweetness and biological activity. However, insufficient UDPG supply has become a major bottleneck limiting synthetic throughput. In synthetic biology, the high consumption rate of UDPG and the limited natural supply pathways hinder the efficient glucosylation process, significantly affecting the synthesis efficiency and yield of mogrosides. The main drawbacks of existing mogroside biosynthesis technologies include: 1) insufficient UDPG donor supply, resulting in low synthesis efficiency and limiting synthetic throughput; and 2) room for improvement in the efficiency and specificity of UGT enzymes. Summary of the Invention

[0016] In view of this, the present invention provides an engineered yeast strain for producing mogroside, simonoside I, and / or mogroside V. By optimizing and modifying the chassis cells, the metabolic pathways of the microorganisms are altered or the expression levels of related genes are regulated, thereby increasing the synthesis efficiency and yield of the target products and improving production efficiency. In addition, the present invention also enhances the utilization rate of UDPG and the overall efficiency of biosynthesis by strengthening the microorganism's UDPG pathway and optimizing UGT enzyme activity, thereby achieving efficient and sustainable production of mogrosides.

[0017] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0018] In a first aspect, the present invention provides the use of any one of the following or a combination thereof in the preparation of mogroside, simenoside I and / or mogroside V;

[0019] (I) Overexpression of tHMG1 and IDI1 (308a), the key rate-limiting genes for terpenoid synthesis in the MVA pathway;

[0020] (II), mutant gene upc2-1(416d) that overexpresses the global transcription factor UPC2;

[0021] (III), overexpression of genes for acetaldehyde dehydrogenase ALD6 (1622b), pyruvate decarboxylase PDC1 (1021b), and acetyl-CoA synthetase ACS1 (911b);

[0022] (IV), knocking out the isocitrate dehydrogenase IDH1 gene;

[0023] (V) Overexpression of the citrate lyase ACL gene of Rhodosporidium toruloides;

[0024] (VI) Overexpression of the gene for alcohol dehydrogenase ADH2 (208a) from the rice base rot pathogen.

[0025] In some specific embodiments of the present invention, any one of (I) to (VI) or a combination thereof enhances the MVA pathway and / or the acetyl-CoA pathway.

[0026] In some specific embodiments of the present invention, any one of the following or a combination thereof further comprises:

[0027] (VII), integration into SgCDS, SgEPH3, CYP87D18, and / or SgCPR1;

[0028] (VIII), integrated into UGT720-269-1; and / or

[0029] (IX), integrated into UGT94-289-3.

[0030] In a second aspect, the present invention further provides a gene expression cassette comprising any one or a combination of the following:

[0031] (I) Overexpression of tHMG1 and IDI1 (308a), key rate-limiting genes for terpenoid synthesis in the MVA pathway;

[0032] (II), overexpression of the mutant gene upc2-1(416d) of the global transcription factor UPC2;

[0033] (III), overexpressed genes for acetaldehyde dehydrogenase ALD6 (1622b), pyruvate decarboxylase PDC1 (1021b), and acetyl-CoA synthetase ACS1 (911b);

[0034] (IV), genes excluding isocitrate dehydrogenase IDH1;

[0035] (V) Overexpression of the citrate lyase ACL gene of Rhodosporidium toruloides;

[0036] (VI) Overexpression of the rice base rot pathogen alcohol dehydrogenase ADH2 (208a) gene.

[0037] In some specific embodiments of the present invention, the gene expression cassette further comprises:

[0038] (VII), SgCDS, SgEPH3, CYP87D18, and / or SgCPR1;

[0039] (VIII), UGT720-269-1; and / or

[0040] (IX), UGT94-289-3.

[0041] In a third aspect, the present invention further provides the use of any one of the following or a combination thereof in the preparation of mogroside, simenoside I and / or mogroside V;

[0042] (I) downregulating the expression of the endogenous gene ERG7;

[0043] (II), knockout of the gene encoding the GAL4 inhibitory protein GAL80;

[0044] (III) Overexpression of squalene epoxidase SQE (GAL80) from Momordica grosvenori, cytochrome b5 (RsCyb5,511b) from Rubus sweetleaf, or yeast phospholipid biosynthesis regulatory gene INO2 (ER size regulator, replacement promoter).

[0045] In some specific embodiments of the present invention, any one of (I) to (III) or a combination thereof reduces the expression of the competing pathway and expands the space of the endoplasmic reticulum;

[0046] Preferably, the expression of the weakened competition pathway comprises:

[0047] Reduce the consumption of 2,3-epoxysqualene;

[0048] This allows the expression of the GAL gene to be freed from the control of the more costly galactose and to respond to the glucose concentration in the culture medium.

[0049] In some specific embodiments of the present invention, any one of the following or a combination thereof further comprises:

[0050] (IV), integration into SgCDS, SgEPH3, CYP87D18, and / or SgCPR1;

[0051] (V), integrated into UGT720-269-1; and / or

[0052] (VI), integrated into UGT94-289-3.

[0053] In a fourth aspect, the present invention further provides a gene expression cassette comprising any one or a combination of the following:

[0054] (I), downregulated endogenous gene ERG7;

[0055] (II), excluding the gene encoding the GAL4 inhibitory protein GAL80;

[0056] (III) Overexpression of squalene epoxidase SQE (GAL80) from Momordica grosvenori and cytochrome b5 (RsCyb5,511b) from Rubus sweetleaf interacted with the yeast phospholipid biosynthesis regulatory gene INO2 (ER size regulator, replacement promoter).

[0057] In some specific embodiments of the present invention, the gene expression cassette further comprises:

[0058] (IV), SgCDS, SgEPH3, CYP87D18, and / or SgCPR1;

[0059] (V), UGT720-269-1; and / or

[0060] (VI), UGT94-289-3.

[0061] In a fifth aspect, the present invention provides the use of any one of the following or a combination thereof in the preparation of mogroside, simenoside I and / or mogroside V;

[0062] (I) Screen and replace CPR genes, selecting CPR1, CPR2, and CPR3;

[0063] The CPR1 sequence is shown as SEQ ID No. 92;

[0064] The CPR2 sequence is shown as SEQ ID No. 93;

[0065] The CPR3 sequence is shown in SEQ ID No. 94;

[0066] (II) Increase the copy number of P450 enzyme CYP87D18 to 2-4 copies;

[0067] (III) reducing the expression intensity of CPR by replacing the relatively weak constitutive promoter;

[0068] The relatively weak constitutive promoter includes: PGI1p, HXT7p or PGK1p.

[0069] In some specific embodiments of the present invention, any one of (I) to (III) or a combination thereof improves the catalytic efficiency of cytochrome P450 (CYP450).

[0070] In some specific embodiments of the present invention, any one of the following or a combination thereof further comprises:

[0071] (IV), integration into SgCDS, SgEPH3, CYP87D18, and / or SgCPR1;

[0072] (V), integrated into UGT720-269-1; and / or

[0073] (VI), integrated into UGT94-289-3.

[0074] In some specific embodiments of the present invention, any one of the following or a combination thereof further comprises:

[0075] (I) downregulating the expression of the endogenous gene ERG7;

[0076] (II), knockout of the gene encoding the GAL4 inhibitory protein GAL80;

[0077] (III) Overexpression of squalene epoxidase SQE (GAL80) from Momordica grosvenori, cytochrome b5 (RsCyb5,511b) from Rubus sweetleaf, or yeast phospholipid biosynthesis regulatory gene INO2 (ER size regulator, replacement promoter).

[0078] In some specific embodiments of the present invention, any one of the following or a combination thereof further comprises:

[0079] (i) Overexpression of tHMG1 and IDI1 (308a), key rate-limiting genes in the biosynthesis of terpenoids in the MVA pathway;

[0080] (ii), upc2-1(416d), a mutant gene that overexpresses the global transcription factor UPC2;

[0081] (iii) overexpression of genes for acetaldehyde dehydrogenase ALD6 (1622b), pyruvate decarboxylase PDC1 (1021b), and acetyl-CoA synthetase ACS1 (911b);

[0082] (iv) knocking out the isocitrate dehydrogenase IDH1 gene;

[0083] (v) Overexpression of the citrate lyase ACL gene of Rhodosporidium toruloides;

[0084] (vi) Overexpression of the gene for alcohol dehydrogenase ADH2 (208a) from the rice base rot pathogen.

[0085] In a sixth aspect, the present invention provides a gene expression cassette comprising any one or a combination of the following:

[0086] (I), CPR1, CPR2, CPR3;

[0087] The CPR1 sequence is shown as SEQ ID No. 92;

[0088] The CPR2 sequence is shown as SEQ ID No. 93;

[0089] The CPR3 sequence is shown in SEQ ID No. 94;

[0090] (II), P450 enzyme CYP87D18;

[0091] (III), relatively weak constitutive promoter;

[0092] The relatively weak constitutive promoter includes: PGI1p, HXT7p or PGK1p.

[0093] In some specific embodiments of the present invention, the gene expression cassette further comprises:

[0094] (IV), SgCDS, SgEPH3, CYP87D18, and / or SgCPR1;

[0095] (V), UGT720-269-1; and / or

[0096] (VI), UGT94-289-3.

[0097] In some specific embodiments of the present invention, the gene expression cassette further comprises:

[0098] (I), downregulated endogenous gene ERG7;

[0099] (II), excluding the gene encoding the GAL4 inhibitory protein GAL80;

[0100] (III) Overexpression of squalene epoxidase SQE (GAL80) from Momordica grosvenori and cytochrome b5 (RsCyb5,511b) from Rubus sweetleaf interacted with the yeast phospholipid biosynthesis regulatory gene INO2 (ER size regulator, replacement promoter).

[0101] In some specific embodiments of the present invention, the gene expression cassette further comprises:

[0102] (i) Overexpression of tHMG1 and IDI1 (308a), key rate-limiting genes for terpenoid synthesis in the MVA pathway;

[0103] (ii), upc2-1(416d), a mutant gene of the overexpressed global transcription factor UPC2;

[0104] (iii) overexpressed genes for acetaldehyde dehydrogenase ALD6 (1622b), pyruvate decarboxylase PDC1 (1021b), and acetyl-CoA synthetase ACS1 (911b);

[0105] (iv) genes excluding isocitrate dehydrogenase IDH1;

[0106] (v) Overexpression of the citrate lyase ACL gene of Rhodosporidium toruloides;

[0107] (vi) Overexpression of the rice base rot pathogen alcohol dehydrogenase ADH2 (208a) gene.

[0108] In a seventh aspect, the present invention provides a construct comprising the gene expression cassette.

[0109] In an eighth aspect, the present invention provides a host comprising any one of the following:

[0110] (i), the gene expression cassette;

[0111] (ii) the construct.

[0112] In some specific embodiments of the present invention, the host includes but is not limited to yeast.

[0113] In some embodiments of the present invention, the host comprises Saccharomyces cerevisiae.

[0114] In a ninth aspect, the present invention further provides a method for constructing an engineered yeast, comprising the following steps:

[0115] Screen and replace CPR genes, using artificially synthesized CPR1, CPR2, and CPR3.

[0116] Increase the copy number of the P450 enzyme CYP87D18 to 2-4 copies.

[0117] By replacing the relatively weak constitutive promoter, the expression intensity of CPR is reduced.

[0118] In some specific embodiments of the present invention, the method for constructing an engineered yeast strain further comprises:

[0119] integration into SgCDS, SgEPH3, CYP87D18, and / or SgCPR1;

[0120] Incorporated into UGT720-269-1; and / or

[0121] Integrated into UGT94-289-3.

[0122] In some specific embodiments of the present invention, the method for constructing an engineered yeast strain further comprises:

[0123] IPP and DMAPP generate squalene under the action of endogenous ERG20 and ERG9, and then are catalyzed by ERG1 to generate 2,3;22,23-diepoxysqualene.

[0124] Downregulate the expression of the endogenous gene ERG7;

[0125] Knockout of the gene encoding the GAL4 inhibitory protein GAL80;

[0126] Overexpression of squalene epoxidase SQE (GAL80) from Momordica grosvenori, cytochrome b5 (RsCyb5,511b) from Rubus sweetleaf, and yeast phospholipid biosynthesis regulatory gene INO2 (ER size regulator, replacement promoter) expands the space of the endoplasmic reticulum.

[0127] In some specific embodiments of the present invention, the method for constructing an engineered yeast strain further comprises:

[0128] Overexpression of tHMG1 and IDI1 (308a), key rate-limiting genes for terpenoid biosynthesis in the MVA pathway;

[0129] upc2-1(416d), a mutant gene that overexpresses the global transcription factor UPC2;

[0130] Overexpression of acetaldehyde dehydrogenase ALD6 (1622b), pyruvate decarboxylase PDC1 (1021b), and acetyl-CoA synthetase ACS1 (911b);

[0131] Knockout of isocitrate dehydrogenase IDH1;

[0132] Overexpression of citrate lyase ACL from Rhodosporidium toruloides;

[0133] Overexpression of alcohol dehydrogenase ADH2 (208a) from Pathogen Pathogen Pathogen.

[0134] In a tenth aspect, the present invention provides the use of any one of the following or a combination thereof in the preparation of mogroside, simenoside I and / or mogroside V;

[0135] (i), the gene expression cassette;

[0136] (ii), the construct;

[0137] (iii) the host.

[0138] In the eleventh aspect, the present invention provides a method for preparing mogroside, simenoside I and / or mogroside V:

[0139] integrating the gene expression cassette;

[0140] transforming the construct;

[0141] fermenting and culturing the host;

[0142] The fermentation broth was collected and purified.

[0143] Furthermore, the biosynthesis of mogroside involves multiple steps, among which UDPG plays a key role as a glycosyl donor in the glycosylation reaction. An insufficient supply of UDPG is the primary factor limiting the synthesis of glycoside products. The present invention provides a fully synthetic strain with an enhanced UDPG pathway, which can enhance the synthesis flux of mogrosides, improving synthesis efficiency and yield. The main technical solution is to enhance the UDPG pathway, increase the supply of UDPG, and thus increase the synthesis flux, using glucose as a starting material to fully synthesize simanoside I, mogroside IIIX, mogroside IVA, and / or mogroside V.

[0144] The specific technical means are:

[0145] Knockout of the G1P metabolic enzyme gene GAL7;

[0146] Overexpression of the UDPG synthesis pathway gene HXK (increased copy number, regulated by the PGAL1 promoter);

[0147] Overexpression of PGM1 (increased copy number, regulated by the PGAL10 promoter);

[0148] Overexpression of PGM2 (increased copy number, regulated by the PGAL10 promoter);

[0149] Overexpression of UGP1 (regulated by the PGAL1 promoter); and / or

[0150] Integrate UGT720-269-1 into the sap155b locus; and / or

[0151] UGT94-289-3 was integrated into the His3b site.

[0152] In a twelfth aspect, the present invention further provides the use of any one of the following or a combination thereof in the preparation of mogroside, simenoside I, mogroside III X, mogroside IVA and / or mogroside V;

[0153] Screen and replace CPR genes, using CPR1, CPR2, and CPR3;

[0154] The CPR1 sequence is shown as SEQ ID No. 92;

[0155] The CPR2 sequence is shown as SEQ ID No. 93;

[0156] The CPR3 sequence is shown as SEQ ID No. 94;

[0157] Increase the number of copies of the P450 enzyme CYP87D18 to 2-4 copies;

[0158] By replacing the relatively weak constitutive promoter, the expression intensity of CPR is reduced;

[0159] The relatively weak constitutive promoter includes: PGI1p, HXT7p or PGK1p;

[0160] integration into SgCDS, SgEPH3, CYP87D18, and / or SgCPR1;

[0161] Integrated into UGT720-269-1;

[0162] Incorporated into UGT94-289-3; and

[0163] (I), knocking out the G1P metabolic enzyme gene GAL7; and / or

[0164] (II), overexpression of UDPG synthesis pathway gene HXK1; and / or

[0165] (III), overexpression of PGM1; and / or

[0166] (IV), overexpression of PGM2; and / or

[0167] (V) Overexpression of UGP1.

[0168] In some specific embodiments of the present invention, it further comprises:

[0169] (VI), the UGT720-269-1 is integrated into the sap155b site; and / or

[0170] (VII) The UGT94-289-3 is integrated into the His3b site.

[0171] In some embodiments of the present invention, the use comprises improving the catalytic efficiency of cytochrome P450 (CYP450).

[0172] In a thirteenth aspect, the present invention further provides a gene expression cassette comprising any one or a combination of the following:

[0173] CPR1, CPR2, CPR3;

[0174] The CPR1 sequence is shown as SEQ ID No. 92;

[0175] The CPR2 sequence is shown as SEQ ID No. 93;

[0176] The CPR3 sequence is shown in SEQ ID No. 94;

[0177] P450 enzyme CYP87D18;

[0178] Relatively weak constitutive promoter;

[0179] The relatively weak constitutive promoter includes: PGI1p, HXT7p or PGK1p;

[0180] SgCDS, SgEPH3, CYP87D18, and / or SgCPR1;

[0181] UGT720-269-1;

[0182] UGT94-289-3;

[0183] (I) knocking out the G1P metabolic enzyme gene GAL7; and / or

[0184] (II), overexpression of UDPG synthesis pathway gene HXK1; and / or

[0185] (III), overexpression of PGM1; and / or

[0186] (IV), overexpression of PGM2; and / or

[0187] (V) Overexpression of UGP1.

[0188] In some specific embodiments of the present invention, the gene expression cassette further comprises:

[0189] (VI), the UGT720-269-1 is integrated into the sap155b site; and / or

[0190] (VII) The UGT94-289-3 is integrated into the His3b site.

[0191] In a fourteenth aspect, the present invention also provides a construct comprising the gene expression cassette.

[0192] In a fifteenth aspect, the present invention further provides a host comprising any one of the following:

[0193] (i), the gene expression cassette;

[0194] (ii) the construct.

[0195] In some embodiments of the present invention, the host includes but is not limited to yeast.

[0196] In a sixteenth aspect, the present invention further provides the use of any one of the following or a combination thereof in the preparation of mogroside, simenoside I, mogroside IIIX, mogroside IVA and / or mogroside V;

[0197] (i), the gene expression cassette;

[0198] (ii), the construct;

[0199] (iii) the host.

[0200] In a seventeenth aspect, the present invention further provides a method for preparing mogroside, simenoside I, mogroside III X, mogroside IV A and / or mogroside V, comprising:

[0201] integrating the gene expression cassette;

[0202] transforming the construct;

[0203] fermenting and culturing the host;

[0204] The fermentation broth was collected and purified.

[0205] The present invention provides an engineered yeast strain for producing mogroside, simanoside I, mogroside IIIX, mogroside IVA and / or mogroside V. By optimizing and transforming chassis cells, the metabolic pathways of the microorganisms are changed or the expression levels of related genes are regulated, thereby increasing the synthesis efficiency and yield of the target products and improving production efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0206] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for describing the embodiments or the prior art.

[0207] FIG1 is a schematic diagram showing the biosynthetic pathway of mogroside V;

[0208] Figure 2 shows the CYP87D18 conversion rate in Example 1;

[0209] FIG3 shows the yield of mogrosanol in Example 10;

[0210] FIG4 shows the yields of mogroside I-A1 and mogroside II E in Example 10;

[0211] FIG5 shows the yields of mogroside IIIX, mogroside IVA, simenoside I, and mogroside V in Example 10. DETAILED DESCRIPTION

[0212] The present invention discloses an engineered yeast strain producing mogroside, simenoside I, mogroside IIIX, mogroside IVA, and / or mogroside V, and its applications. Those skilled in the art may refer to the contents herein and appropriately modify the process parameters to achieve the desired results. It should be noted that all similar substitutions and modifications obvious to those skilled in the art are considered encompassed by the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel will readily be able to modify, alter, and combine the methods and applications described herein without departing from the content, spirit, and scope of the present invention to implement and apply the technology of the present invention.

[0213] The metabolic pathway of de novo synthesis is long, with many steps and a complex metabolic network. The metabolic capacity of yeast itself can only produce trace amounts of non-specific compounds, which requires precise regulation and optimization to change the metabolic pathway of the microorganism or regulate the expression level of related genes, thereby increasing the synthesis efficiency and yield of the target product and improving production efficiency.

[0214] The main technical solution of the present invention is: chassis modification, which increases the supply of precursors, weakens competitive pathways, and screens for high gene expression by strengthening chassis cells, thereby achieving the purpose of increasing the yield of target products.

[0215] More specifically, in Module 1, the supply of precursors is increased by strengthening the MVA and acetyl-CoA pathways, thereby increasing the yield of the target product. Second, in Module 2, the metabolism of biosynthetic intermediates is reduced by weakening the expression of competing pathways, thereby increasing the yield of the target product. Third, in Module 2, the activity of oxidoreductases (CPRs) is enhanced, thereby increasing the efficient expression of cytochrome P450 (CYP450), thereby increasing the yield of the target product.

[0216] Regarding strategies to strengthen the MVA pathway and acetyl-CoA pathway to increase the supply of precursors, the specific technical means are:

[0217] Overexpression of tHMG1 and IDI1 (308a), key rate-limiting genes for terpenoid biosynthesis in the MVA pathway;

[0218] Overexpression of the mutant gene upc2-1(416d) of the global transcription factor UPC2 directly or indirectly upregulates the transcription efficiency of MVA pathway genes;

[0219] Overexpression of acetaldehyde dehydrogenase ALD6 (1622b), pyruvate decarboxylase PDC1 (1021b), acetyl-CoA synthetase ACS1 (911b), knockout of isocitrate dehydrogenase IDH1, overexpression of citrate lyase ACL from Rhodosporidium toruloides, and overexpression of alcohol dehydrogenase ADH2 (208a) from basidiomyces oryzae to increase the supply of acetyl-CoA;

[0220] To reduce the expression of competing pathways, the specific technical means are:

[0221] Down-regulate the expression of the endogenous gene ERG7 and reduce the consumption of 2,3-epoxysqualene;

[0222] Knocking out the gene encoding the GAL80 inhibitory protein of GAL4 frees GAL gene expression from the more costly galactose control and instead responds to the glucose concentration in the culture medium;

[0223] Overexpression of squalene epoxidase SQE (GAL80) from Momordica grosvenori; overexpression of cytochrome b5 (RsCyb5,511b) from Rubus bistorta; overexpression of yeast phospholipid biosynthesis regulatory gene INO2 (ER size regulator, replacement promoter) to expand the space of the endoplasmic reticulum.

[0224] To improve the catalytic efficiency of cytochrome P450 (CYP450), the specific technical means are:

[0225] Screen and replace CPR genes, using artificially synthesized CPR1, CPR2, and CPR3.

[0226] Increase the copy number of the P450 enzyme CYP87D18 to 2-4 copies.

[0227] By replacing the relatively weak constitutive promoter, the expression intensity of CPR is reduced.

[0228] In the yeast engineering bacteria for producing mogroside, simenoside I and / or mogroside V and applications thereof provided by the present invention, the raw materials and reagents used can all be purchased from the market.

[0229] Chassis cells: yeast cells.

[0230] Preferred: Saccharomyces cerevisiae, Yarrowia lipolytica, Kluyveromyces lactis, Ogataea polymorpha. Preferred: Saccharomyces cerevisiae CEN.PK2-1C. This application uses Saccharomyces cerevisiae CEN.PK2-1C (from the China Microbial Culture Bank, No. Bio-116324) as the starting strain for construction and modification.

[0231] Table 1. Primer information

[0232] Table 2. Main information of strains

[0233] Table 3. Primer information

[0234] Table 4. Strain information and verification results

[0235] Table 5. Strain information

[0236] Table 6. Primer information

[0237] Table 7

[0238] Table 8. Strain information

[0239] Table 9. Primer information

[0240] Sequence information:

[0241] tHMG1 (NCBI Accession: NP_013636.1)

[0242] IDI1 (NCBI Accession: QHB12144.1)

[0243] CYP87D18 > ON352538.1 Synthetic construct cytochrome P450 - dependent monooxygenase (cyp87d18) gene (NCBI Accession: ON352538.1)

[0244] CPR1 synthetic sequence (as shown in SEQ ID No. 92)

[0245] CPR2 synthetic sequence (as shown in SEQ ID No. 93)

[0246] CPR3 synthetic sequence (as shown in SEQ ID No. 94)

[0247] CaCPR1 > MK737062.1 Camptotheca acuminata cytochrome p450 reductase (CPR1)

[0248] mRNA (NCBI Accession: MK737062.1)

[0249] SgCPR1 > MH886510.1 Siraitia grosvenorii NADPH:cytochrome P450 reductase (CPR1)

[0250] mRNA (NCBI Accession: MH886510.1)

[0251] SgCDS from Siraitia grosvenorii (NCBI Accession: ON352536.1)

[0252] SgEPH3 from Siraitia grosvenorii (NCBI Accession: ON352537.1)

[0253] CYP87D18 from Siraitia grosvenorii (NCBI Accession: ON352538.1)

[0254] UGT720-269-1 from Siraitia grosvenorii (as shown in SEQ ID No. 95)

[0255] UGT94-289-3 from Siraitia grosvenorii (as shown in SEQ ID No. 96)

[0256] GAL7 (shown as SEQ ID No. 97)

[0257] HXK1 (shown in SEQ ID No. 98)

[0258] PGM1 (as shown in SEQ ID No. 99)

[0259] PGM2 (as shown in SEQ ID No. 100)

[0260] UGP1 (shown in SEQ ID No. 101)

[0261] DPP1 (shown in SEQ ID No. 102)

[0262] The raw materials and reagents used in the technical solution provided by the present invention can be purchased from the market.

[0263] The present invention will be further described below in conjunction with the embodiments:

[0264] Example 1 Construction of MOG001 strain

[0265] Overexpression of tHMG1 and IDI1

[0266] 1) The target gene was integrated into the Saccharomyces cerevisiae genome using the GTR-CRISPR-Cas9 gene editing system. A plasmid carrying the gene expressing the Cas9 protein and a sgRNA sequence targeting the corresponding site was constructed for targeted gene integration. First, the gRNA sequence for the 308a integration site was predicted using the Zhang Lab's CRISPOR (http: / / crispor.tefor.net / ) online website, and a gRNA sequence with high efficiency and no off-target effects was selected. Primers containing the gRNA sequence were designed (see Table 1). PCR amplification was performed using the plasmid pScURA as a template to obtain a gene fragment containing the corresponding gRNA and the selection marker Ura. The DNA fragment was then ligated with the pCas9 plasmid using the Golden Gate Assembly kit to generate the knockout plasmid pCas9-308a. Donor construction primers were designed (see Table 1 ). The DonorDNA gene fragment contained 50 bp of upstream and downstream homology arms, the promoter, and the inserted gene sequence. DonorDNA was obtained by OE-PCR: up-GAL1p-tHMG1-GAL10p-IDI1-down;

[0267] 2) The up-GAL1p-tHMG1-GAL10p-IDI1-down and pCas9-308a plasmids were co-transformed into S. cerevisiae CEN.PK2-1C competent cells by electroporation. The PGAL-target gene expression cassette was integrated using the homologous recombination ability of S. cerevisiae. The cells were plated on SC-URA plates and incubated at 30°C for 2-3 days. Positive clones were screened by colony PCR and verified by sequencing. Single clones confirmed by PCR were re-streaked onto SC plates containing 5-FOA (5-fluoroorotic acid) and incubated at 30°C to eliminate the Cas9 plasmid. Single clones that grew on SC plates (containing 5-FOA) were streaked onto YPD and SC-URA plates and incubated at 30°C. Those that grew on YPD but not SC-URA were identified as gene-edited strains. Single clones were selected and plated onto YPD liquid medium, incubated at 30°C for 24 hours, and then stored in 20% glycerol to obtain the base strain MOG001. The strain information is shown in Table 2.

[0268] Example 2 Construction of interM-01 and interM-02 strains

[0269] 1. The same method as steps 1)-2) of "1. Overexpression of tHMG1 and IDI1 in Example 1" was used. The primers involved are listed in Table 9.

[0270] The difference is that: step 2) is to co-transform the knockout plasmid pCas9-1309a and donor DNA: up-GAL1p-SgCDS-down into MOG001 chassis cells to obtain strain interM-01, and preserve the strain for future use (20% glycerol).

[0271] 2. The same method as steps 1)-2) of "Overexpression of tHMG1 and IDI1 in Example 1" was used. The primers involved are listed in Table 9. The difference is that in step 2), the knockout plasmid pCas9-106a and the donor DNA: up-GAL10p-SgEPH3-down were co-transformed into interM-01 to obtain strain interM-02, which was stored for future use (20% glycerol).

[0272] Example 3 Screening and Verification of CPR Genes

[0273] 1. Strain Construction

[0274] (1). Full gene synthesis of CYP87D18, CPR1, CPR2, CPR3, CaCPR1, and SgCPR1 was performed, and recombinant expression plasmids were constructed using pESC-HIS as a vector.

[0275] Primers were designed (Table 3). First, they were used to amplify the CYP87D18 fragment carrying the homology arms and the linearized pESC-HIS vector fragment. Following the kit instructions, the DNA fragment and the linearized vector fragment were subjected to homologous recombination at 50°C for 1 hour. The cells were then transformed into E. coli DH5α competent cells, plated with Amp-resistant plates, and incubated overnight at 37°C. Positive colonies were screened by colony PCR. Positive colonies were selected and plated in 5 mL of LB medium containing Amp for verification by sequencing. The sequenced plasmid pESC-HIS-CYP87D18 (MCS1) was then isolated and used for future use.

[0276] (2) Use the corresponding primers to amplify the CPR1 fragment carrying the homology arms and the linearized pESC-HIS-CYP87D18 (MCS1) vector fragment. The recombination reaction is the same as "(1)". Screen the positive clones by colony PCR, pick the positive clones and place them in 5 mL of LB medium containing Amp, verify by sequencing, and extract the plasmid pESC-HIS-CYP87D18-CPR1 with the correct sequencing.

[0277] The construction process of CPR2 was the same as that of “(2)”, and the plasmid pESC-HIS-CYP87D18-CPR2 with correct sequencing was extracted.

[0278] The construction process of CPR3 was the same as that of “(2)”, and the plasmid pESC-HIS-CYP87D18-CPR3 with correct sequencing was extracted.

[0279] The construction process of CaCPR1 was the same as that of “(2)”, and the plasmid pESC-HIS-CYP87D18-CaCPR1 with correct sequencing was extracted.

[0280] The construction process of SgCPR1 was the same as that of “(2)”, and the plasmid pESC-HIS-CYP87D18-SgCPR1 with correct sequencing was extracted.

[0281] (3) Use electroporation to transform the recombinant expression plasmid pESC-HIS-CYP87D18-CPR1 into Saccharomyces cerevisiae CEN.PK2-1C competent cells, spread on SC-HIS plates, and culture at 30°C for 2-3 days. Screen positive clones by colony PCR, inoculate them into 10 mL of SC-HIS liquid medium, and culture at 30°C, 230 rpm for 24 hours. Store strain MRO001.

[0282] In the same way, pESC-HIS-CYP87D18-CPR2 was transformed into Saccharomyces cerevisiae CEN.PK2-1C to obtain strain MRO002;

[0283] pESC-HIS-CYP87D18-CPR3 was transformed into S. cerevisiae CEN.PK2-1C to obtain strain MRO003;

[0284] pESC-HIS-CYP87D18-CaCPR1 was transformed into S. cerevisiae CEN.PK2-1C to obtain strain MRO004;

[0285] pESC-HIS-CYP87D18-SgCPR1 was transformed into Saccharomyces cerevisiae CEN.PK2-1C to obtain strain MRO005.

[0286] The strain information is shown in Table 4.

[0287] 2. Verification experiment:

[0288] Inoculate 1 mL of each of the above bacterial cultures into 50 mL of fresh SC-HIS liquid medium and incubate at 30°C and 230 rpm until growth reaches the plateau phase. Centrifuge at 3,000 rpm for 5 minutes at room temperature, discard the supernatant, and collect the Saccharomyces cerevisiae cells. Resuspend and wash the cells in approximately 50 mL of sterile water, centrifuge at 3,000 rpm for 5 minutes at room temperature, and collect the cells. Repeat this step three times. Resuspend the cells in approximately 2 mL of YPGal liquid medium containing 2% galactose and add them to 100 mL of YPGal liquid medium containing 2% galactose. Induce the cells at 28°C and 230 rpm for 24 hours. After induction, centrifuge the culture at 3,000 rpm and 4°C for 5 minutes to collect the cells. Resuspend the cells in Tris-HCl buffer (50 mM, pH 8.0) and wash them three times. Then, resuspend each gram of cells in 3 mL of buffer. Add glass beads and shake to disrupt the yeast cells. After stabilization, collect the cell lysate.

[0289] P450 catalytic activity assay: A 100 μL reaction system containing 50 mM Tris-HCl buffer (50 mM, pH 8.0), 1 mM 2,4,25-dihydroxycucurbitadienol, 5 mM NADPH, and an appropriate amount of cell disruption buffer was incubated at 30°C and 230 rpm for 2 h. The reaction was quenched by adding an equal volume of methanol and centrifuged at 12,000 rpm for 10 min. The supernatant was filtered through a 0.22 μm filter and analyzed by HPLC. Strain information and HPLC analysis results are shown in Table 10.

[0290] Table 10. CYP87D18 conversion rate

[0291] From the results in Table 10, it can be seen that the conversion rates of strains MRO001-003 using CPR1-3 are higher than those of strains CaCPR1 and SgCPR1, among which strain MRO002 using CPR2 has the best catalytic efficiency.

[0292] Example 4 Expression and Optimization of CPR Genes

[0293] 1. The application of CPR1-3 in the de novo synthesis of mogroside alcohol was verified in plasmid form. P450 and CPR1-3 were separately transferred into strain interM-02 in plasmid form to obtain strains M24-M26.

[0294] The specific process is as follows:

[0295] (1) Using pIYC04-HIS as a vector, construct the recombinant expression plasmid pIYC04-HIS-P PGK1-CYP87D18. The primers involved are listed in Table 3. The primers were used to amplify the CYP87D18 fragment carrying the homology arms and the linearized pIYC04-HIS vector fragment. According to the kit instructions, the DNA fragment and the linearized vector fragment were homologously recombined, reacted at 50°C for 1 hour, transformed into E. coli DH5α competent cells, and coated with Amp-resistant plates and cultured at 37°C overnight. Positive clones were screened by colony PCR. Positive clones were picked and plated in 5 mL of LB medium containing Amp for sequencing verification. The recombinant plasmid pIYC04-HIS-P was extracted and sequenced correctly. PGK1 -CYP87D18 spare.

[0296] (2) Use the corresponding primers to amplify the CPR1 fragment carrying the homology arms and the linearized pIYC04-HIS-P PGK1 -CYP87D18 vector fragment. The recombination reaction was the same as "(1)". The positive clones were screened by colony PCR, and the positive clones were picked and placed in 5 mL of LB medium containing Amp, and sequenced for verification to obtain pIYC04-HIS-P PGK1 -CYP87D18-P TEF1 -CPR1 recombinant plasmid is reserved.

[0297] The construction process of CPR2 was the same as that of (2), and the plasmid pIYC04-HIS-P PGK1 -CYP87D18-P TEF1 -CPR2.

[0298] The construction process of CPR3 was the same as that of (2), and the plasmid pIYC04-HIS-P PGK1 -CYP87D18-P TEF1 -CPR3.

[0299] (3) Using electroporation, the recombinant expression plasmid pIYC04-HIS-P PGK1 -CYP87D18-P TEF1 -CPR1 was transformed into interM-02 competent cells, plated on SC-HIS plates, and cultured at 30°C for 2-3 days. Positive colonies were screened by colony PCR and selected and inoculated into 10 mL of SC-HIS liquid medium. Incubate at 30°C, 230 rpm for 24 hours to preserve strain M24.

[0300] In the same way, pIYC04-HIS-P PGK1 -CYP87D18-P TEF1 -CPR2 was transformed into interM-02 to obtain strain M25;

[0301] In the same way, pIYC04-HIS-P PGK1-CYP87D18-P TEF1 -CPR3 was transformed into interM-02 to obtain strain M26.

[0302] The strain information is shown in Table 5.

[0303] Verification experiment: Detection of Mogrol

[0304] Glycerol culture was removed from a -80°C freezer, streaked onto SC-HIS plates, and incubated at 30°C for 2-3 days. A single colony was picked from the plate and incubated in 10 mL of SC-HIS medium at 30°C, 250 rpm, and cultured overnight. The seed solution was transferred to a 250 mL shake flask containing 25 mL of SC-HIS liquid medium at a 5% inoculum level and fermented at 30°C, 250 rpm, with three replicates per group. After 120 hours of fermentation, 5 mL of the fermentation broth was added to glass beads and shaken for 30 minutes to disrupt the cells. Extraction was then performed with an equal volume of ethyl acetate, and the organic phase was collected by centrifugation at 12,000 rpm for 5 minutes. This extraction was repeated three times. Finally, the organic phase was placed in a 70°C vacuum drying oven, the solvent was evaporated, and the solution was reconstituted with 200 μL of methanol (containing 1% formic acid) and analyzed by HPLC. The results are detailed in Example 9.

[0305] 2. Integrate CPR1-3 strains into chassis cells and optimize the promoters. The specific methods are as follows:

[0306] (1) Overexpression of CYP87D18 and CPR1:

[0307] 1) The target gene was integrated into the Saccharomyces cerevisiae genome using the GTR-CRISPR-Cas9 gene editing system. A plasmid carrying the gene expressing the Cas9 protein and a sgRNA sequence targeting the corresponding site was constructed for targeted gene integration. First, the gRNA sequence for the 308a integration site was predicted using the Zhang Lab's CRISPOR (http: / / crispor.tefor.net / ) online website, and a gRNA sequence with high efficiency and no off-target effects was selected. Primers containing the gRNA sequence were designed (see Table 1). PCR amplification was performed using the plasmid pScURA as a template to obtain a gene fragment containing the corresponding gRNA and the selection marker Ura. The DNA fragment was then ligated with the pCas9 plasmid using the Golden Gate Assembly kit to generate the knockout plasmid pCas9-308a. Donor construction primers were designed (see Table 1 ). The DonorDNA gene fragment contained 50 bp of upstream and downstream homology arms, the promoter, and the inserted gene sequence. DonorDNA was obtained by OE-PCR: up-GAL1p-tHMG1-GAL10p-IDI1-down;

[0308] 2) The knockout plasmid pCas9-720a and donor DNA: up-GAL1p-CYP87D18-GAL10p-CPR1-down were co-transformed into the strain interM-02, and the homologous recombination ability of Saccharomyces cerevisiae was used to achieve P GAL - Integration of the target gene expression cassette was performed by plating on SC-URA plates and incubating at 30°C for 2-3 days. Positive clones were screened by colony PCR and sequenced. Single clones confirmed by PCR were re-streaked onto SC plates containing 5-FOA (5-fluoroorotic acid) and incubated at 30°C to eliminate the Cas9 plasmid. Single clones growing on SC (containing 5-FOA) plates were streaked onto YPD and SC-URA plates and incubated at 30°C. The strains that grew on YPD plates but not on SC-URA plates were gene-edited. Single clones were selected and plated on YPD liquid medium. After incubation at 30°C for 24 hours, the strain was preserved (20% glycerol) to obtain strain M27.

[0309] The primers involved are listed in Table 6.

[0310] The same method as steps 1)-2) of "Overexpression of tHMG1 and IDI1 in Example 1" was used, and the primers involved are listed in Table 6. The difference is that in step 2), the knockout plasmid pCas9-720a and the donor DNA: up-GAL1p-CYP87D18-PGI1p-CPR1-down were co-transformed into the strain interM-02 to obtain strain M28, which was stored for future use (20% glycerol);

[0311] The same method as steps 1)-2) of "Overexpression of tHMG1 and IDI1 in Example 1" was used, and the primers involved are listed in Table 6. The difference is that in step 2), the knockout plasmid pCas9-720a and the donor DNA: up-GAL1p-CYP87D18-HXT7p-CPR1-down were co-transformed into the strain interM-02 to obtain strain M29, which was stored for future use (20% glycerol);

[0312] The same method as steps 1)-2) of "Overexpression of tHMG1 and IDI1 in Example 1" was used, and the primers involved are listed in Table 6. The difference is that in step 2), the knockout plasmid pCas9-720a and the donor DNA: up-GAL1p-CYP87D18-PGK1p-CPR1-down were co-transformed into the strain interM-02 to obtain strain M30, which was stored for future use (20% glycerol);

[0313] Using the same method, CYP87D18 and CPR2 and CPR3 regulated by different promoters were integrated into the genome of interM-02 chassis cells to construct strains M31-M38 that synthesize mogroside alcohol from scratch.

[0314] Please see Table 7 for strain information.

[0315] Verification experiment: Detection of Mogrol

[0316] Remove the glycerol stock from the -80°C freezer, streak it onto a YPD plate, and incubate it at 30°C for 2-3 days. Pick a single colony from the plate and incubate it in 10 mL of YPD medium at 30°C, 250 rpm, and incubate it overnight. Transfer the seed solution to a 5% inoculum into a 250 mL shake flask containing 25 mL of YPD liquid medium and ferment it at 30°C, 250 rpm, with three replicates per group. Ferment for 120 hours, then remove 5 mL of the fermentation broth, add glass beads, and shake to disrupt the cells for 30 minutes. Extract with an equal volume of ethyl acetate, centrifuge at 12,000 rpm for 5 minutes, and collect the organic phase. Repeat the extraction three times. Finally, place the organic phase in a 70°C vacuum drying oven, evaporate the solvent, reconstitute with 200 μL of methanol (containing 1% formic acid), and analyze by HPLC. For detailed results, see Example 6.

[0317] Example 5 Expression of P450 enzyme CYP87D18

[0318] Increased copy number of the P450 enzyme CYP87D18

[0319] 1) The target gene was integrated into the Saccharomyces cerevisiae genome using the GTR-CRISPR-Cas9 gene editing system. A plasmid carrying the gene expressing the Cas9 protein and a sgRNA sequence targeting the corresponding site was constructed for targeted gene integration. First, the gRNA sequence for the 308a integration site was predicted using the Zhang Lab's CRISPOR (http: / / crispor.tefor.net / ) online website, and a gRNA sequence with high efficiency and no off-target effects was selected. Primers containing the gRNA sequence were designed (see Table 1). PCR amplification was performed using the plasmid pScURA as a template to obtain a gene fragment containing the corresponding gRNA and the selection marker Ura. The DNA fragment was then ligated with the pCas9 plasmid using the Golden Gate Assembly kit to generate the knockout plasmid pCas9-308a. Donor construction primers were designed (see Table 1 ). The DonorDNA gene fragment contained 50 bp of upstream and downstream homology arms, the promoter, and the inserted gene sequence. DonorDNA was obtained by OE-PCR: up-GAL1p-tHMG1-GAL10p-IDI1-down;

[0320] 2) The knockout plasmid pCas9-1114a and donor DNA: up-GAL1p-CYP87D18-down were co-transformed into strain M30, and the homologous recombination ability of Saccharomyces cerevisiae was used to achieve P GAL -Integration of the target gene expression cassette, apply SC-URA plates, and culture at 30°C for 2-3 days. Screen positive clones by colony PCR and sequence verification. The correct single clones verified by PCR are re-streaked on SC plates containing 5-FOA (5-fluoroorotic acid) and cultured at 30°C to eliminate the Cas9 plasmid. The single clones grown on SC (containing 5-FOA) plates are streaked on YPD and SC-URA plates respectively and cultured at 30°C. The bacteria that grow on YPD plates but not on SC-URA plates are gene-edited bacteria. Pick a single clone and culture it in YPD liquid medium. After culturing at 30°C for 24 hours, preserve the strain (20% glycerol) to obtain strain M39. The primers involved are listed in Table 6.

[0321] The same method as steps 1)-2) was used, and the primers involved are listed in Table 6. The difference is that in step 2), the knockout plasmid pCas9-1114a and the donor DNA: up-GAL1p-CYP87D18-GAL10p-CYP87D18-down were co-transformed into strain M30 to obtain strain M40, and the strain was preserved (20% glycerol) for future use;

[0322] The same method as steps 1)-2) was used, and the primers involved are listed in Table 6. The difference is that in step 2), the knockout plasmid pCas9-1114a and donorDNA: up-GAL1p-CYP87D18-down were co-transformed into strain M34 to obtain strain M41, which was preserved for future use (20% glycerol);

[0323] The same method as steps 1)-2) was used, and the primers involved are listed in Table 6. The difference is that in step 2), the knockout plasmid pCas9-1114a and the donor DNA: up-GAL1p-CYP87D18-GAL10p-CYP87D18-down were co-transformed into strain M34 to obtain strain M42, which was then preserved (20% glycerol) for future use;

[0324] The same method as steps 1)-2) was used, and the primers involved are listed in Table 6. The difference is that in step 2), the knockout plasmid pCas9-1114a and donorDNA:up-GAL1p-CYP87D18-down were co-transformed into strain M38 to obtain strain M43, and the strain was preserved for future use (20% glycerol);

[0325] The same method as steps 1)-2) was used, and the primers involved are listed in Table 6. The difference is that in step 2), the knockout plasmid pCas9-1114a and the donor DNA: up-GAL1p-CYP87D18-GAL10p-CYP87D18-down were co-transformed into strain M38 to obtain strain M44, which was then preserved (20% glycerol) for future use.

[0326] Verification experiment: Detection of Mogrol

[0327] Remove the glycerol stock from the -80°C freezer, streak it onto a YPD plate, and incubate at 30°C for 2-3 days. Pick a single colony from the plate and incubate it in 10 mL of YPD medium at 30°C, 250 rpm, and incubate overnight. Transfer the seed solution to a 5% inoculum into a 250 mL shake flask containing 25 mL of YPD liquid medium and ferment at 30°C, 250 rpm, with three replicates per group. Ferment for 120 hours, then remove 5 mL of the fermentation broth, add glass beads, and shake to disrupt the cells for 30 minutes. Extract with an equal volume of ethyl acetate, centrifuge at 12,000 rpm for 5 minutes, and collect the organic phase. Repeat the extraction three times. Finally, place the organic phase in a 70°C vacuum drying oven, evaporate the solvent, reconstitute with 200 μL of methanol (containing 1% formic acid), and analyze by HPLC. For detailed results, see Example 9.

[0328] Example 6 Construction of strain E for de novo synthesis of mogroside I-A1 and mogroside II

[0329] The enzymes required for the synthesis of mogroside I-A1 and mogroside II E were transferred into the previously constructed strains M27, M30, M31, M34, M35, M38, M40, M42, and M44 in the form of plasmids to obtain strains MGV06-MGV14 that synthesize mogroside I-A1 and mogroside II E from scratch.

[0330] The enzymes introduced are:

[0331] UGT720-269-1

[0332] Taking the MGV06 strain as an example, the specific construction process is as follows:

[0333] 1. Construction of UGT720-269-1 recombinant plasmid

[0334] Using pIYC04-HIS as a vector, the recombinant expression plasmid pIYC04-HIS-P PGK1-UGT720-269-1. The primers involved are listed in Table 9. The primers were used to amplify the UGT720-269-1 fragment carrying the homology arms and the linearized pIYC04-HIS vector fragment. According to the kit instructions, the DNA fragment and the linearized vector fragment were subjected to homologous recombination reaction. After reacting at 50°C for 1 hour, the DNA fragment was transformed into E. coli DH5α competent cells, and the cells were coated with Amp-resistant plates and cultured at 37°C overnight. Positive clones were screened by colony PCR. Positive clones were picked and plated in 5 mL of LB medium containing Amp. After sequencing verification, the recombinant expression plasmid pIYC04-HIS-P was extracted and sequenced correctly. PGK1 -UGT720-269-1 spare.

[0335] 2. Strain Construction

[0336] The recombinant expression plasmid pIYC04-HIS-P was transformed by electroporation. PGK1 Transform UGT720-269-1 into M27 competent cells, plate onto SC-HIS plates, and incubate at 30°C for 2-3 days. Screen positive colonies by colony PCR, pick a positive colony and inoculate it into 10 mL of SC-HIS liquid medium. Incubate at 30°C, 230 rpm for 24 hours. Strain MGV06 was stored.

[0337] Using the same method, expression plasmid pIYC04-HIS-P was transferred into competent cells M30, M31, M34, M35, M38, M40, M42, and M44, respectively. PGK1 -UGT720-269-1, and constructed strains MGV07-MGV14 that synthesized mogroside I-A1 and mogroside II E from scratch.

[0338] Experimental verification: Detection of mogroside I-A1 and mogroside II E

[0339] The glycerol culture was removed from the -80°C freezer, streaked onto an SC-HIS plate, and cultured at 30°C for 2-3 days; a single colony was picked from the plate and incubated in 10 mL of SC-HIS medium at 30°C, 250 rpm, and cultured overnight; the seed liquid was transferred to a 250 mL shake flask containing 25 mL of SC-HIS liquid medium at a 5% inoculum volume, and fermented at 30°C, 250 rpm, with 3 replicates per group; after 120 h of fermentation, 1 mL of fermentation broth was added to 1 mL of methanol, and the supernatant was collected by centrifugation at 12,000 rpm for 5 min, filtered, and then analyzed by HPLC. The specific results are shown in Example 9.

[0340] Example 8 Construction of strains for de novo synthesis of mogroside III X, mogroside IVA, simenoside I, and mogroside V

[0341] The enzymes required for the synthesis of mogroside III X, mogroside IVA, simenoside I and mogroside V were transferred into the previously constructed strains M27, M30, M31, M34, M35, M38, M40, M42 and M44 in the form of plasmids to obtain strains MGV20-MGV28 that synthesize mogroside III X, mogroside IVA, simenoside I and mogroside V de novo.

[0342] The enzymes introduced are:

[0343] UGT720-269-1

[0344] UGT94-289-3

[0345] Taking the MGV20 strain as an example, the specific construction process is as follows:

[0346] 1. Construction of UGT94-289-3 recombinant plasmid

[0347] The same method as step 1) of "1. UGT720-269-1 recombinant plasmid construction in Example 7" was used, and the primers involved are listed in Table 9. The difference is:

[0348] pIYC04-HIS-P PGK1 -UGT720-269-1 was used as plasmid, and the UGT94-289-3 fragment carrying the homology arm and the linearized vector fragment were amplified using primers to obtain pIYC04-HIS-P PGK1 -UGT720-269-1-P TEF1 -UGT94-289-3 recombinant expression plasmid is reserved.

[0349] 2. Strain Construction

[0350] The recombinant expression plasmid pIYC04-HIS-P was transformed by electroporation. PGK1 -UGT720-269-1-P TEF1 Transform UGT94-289-3 into M27 competent cells, plate onto SC-HIS plates, and incubate at 30°C for 2-3 days. Screen for positive colonies by colony PCR. Select a positive colony and inoculate it into 10 mL of SC-HIS liquid medium. Incubate at 30°C, 230 rpm, for 24 hours. Strain MGV15 was preserved.

[0351] Using the same method, expression plasmid pIYC04-HIS-P was transferred into competent cells M30, M31, M34, M35, M38, M40, M42, and M44, respectively. PGK1 -UGT720-269-1-P TEF1-UGT94-289-3, and constructed strains MGV21-MGV28 that synthesized mogroside III X, mogroside IVA, simenoside I, and mogroside V de novo.

[0352] Experimental verification: detection of mogroside III X, mogroside IVA, simenoside I and mogroside V

[0353] The glycerol culture was removed from the -80°C freezer, streaked onto an SC-HIS plate, and cultured at 30°C for 2-3 days; a single colony was picked from the plate and incubated in 10 mL of SC-HIS medium at 30°C, 250 rpm, and cultured overnight; the seed liquid was transferred to a 250 mL shake flask containing 25 mL of SC-HIS liquid medium at a 5% inoculum volume, and fermented at 30°C, 250 rpm, with 3 replicates per group; after 120 h of fermentation, 1 mL of fermentation broth was added to 1 mL of methanol, and the supernatant was collected by centrifugation at 12,000 rpm for 5 min, filtered, and then analyzed by HPLC. The specific results are shown in Example 9.

[0354] Example 9 Output of target product

[0355] 1. Mogrol production

[0356] Table 11

[0357] 2. Production of Mogroside I-A1 and Mogroside II E

[0358] Table 12

[0359] Table 13

[0360] 3. Production of mogroside III X, mogroside IVA, simenoside I and mogroside V

[0361] Table 14

[0362] Table 15

[0363] Table 16

[0364] Table 17

[0365] By screening and replacing the CPR gene, artificially synthesized CPR1, CPR2, and CPR3 were used to increase the copy number of the P450 enzyme CYP87D18 to 2-4 copies, and the relatively weak constitutive promoter was replaced, the expression intensity of CPR was reduced, the catalytic efficiency of cytochrome P450 (CYP450) was improved, and ultimately the product yield was promoted.

[0366] Example 10 Knockout of G1P Metabolizing Enzyme Gene GAL7

[0367] (1) The GTR-CRISPR-Cas9 gene editing system was used to integrate the target gene into the Saccharomyces cerevisiae genome, and a plasmid carrying a gene expressing Cas9 protein and a sgRNA sequence targeting the corresponding site was constructed for targeted gene integration.

[0368] First, the gRNA sequence for the GAL7 integration site was predicted using the website CRISPOR (http: / / crispor.tefor.net / ), and a gRNA sequence with high efficiency and no off-target effects was selected. Primers containing the gRNA sequence were designed (see Table 18), and PCR amplification was performed using the plasmid pScURA as a template to obtain a gene fragment containing the corresponding gRNA and the screening marker Ura. The DNA fragment was then ligated to the pCas9 plasmid using the Golden Gate Assembly kit to obtain the knockout plasmid pCas9-GAL7. Donor construction primers were designed (see Table 18). The Donor DNA gene fragment contained 50bp of gene sequences for the upstream and downstream homology arms. Donor DNA was obtained by OE-PCR: up-OverLap(GAL7)-down.

[0369] (2) Donor DNA:up-OverLap(GAL7)-down and pCas9-GAL7 plasmid were co-transformed into MGV27 competent cells by electroporation, and the target gene GAL7 was knocked out by utilizing the homologous recombination ability of Saccharomyces cerevisiae. The cells were coated on SC-URA plates and cultured at 30°C for 2-3 days.

[0370] Positive clones were screened by colony PCR and verified by sequencing. Single clones verified by PCR were re-streaked onto SC plates containing 5-FOA (5-fluoroorotic acid) and incubated at 30°C to eliminate the Cas9 plasmid. Single clones growing on SC plates (containing 5-FOA) were streaked onto YPD and SC-URA plates and incubated at 30°C. Those that grew on YPD but not SC-URA were gene-edited strains. Single clones were selected and plated onto YPD liquid medium. After incubation at 30°C for 24 hours, the strain was preserved (20% glycerol) to obtain strain MUDP001.

[0371] Table 18 Primer sequence table

[0372] Example 11 Overexpression of UDPG synthesis pathway gene HXK1

[0373] The same method as in Example 10 "Knockout of G1P metabolic enzyme gene GAL7" was used, and the primers involved are listed in Table 18. The differences are:

[0374] Step 2) The knockout plasmid pCas9-dpp1 and the donor DNA: up-GAL1p-HXK1-down were co-transformed into the MGV27 strain to obtain the chassis strain MUDP002, and the strain was preserved for future use (20% glycerol).

[0375] Step 2) The knockout plasmid pCas9-dpp1 and donor DNA: up-GAL1p-HXK1-down were co-transformed into the MUDP001 strain to obtain the chassis strain MUDP003, and the strain was preserved for future use (20% glycerol).

[0376] Example 12 Overexpression of PGM1

[0377] The same method as in Example 10 "Knockout of G1P metabolic enzyme gene GAL7" was used, and the primers involved are listed in Table 18. The differences are:

[0378] Step 2) The knockout plasmid pCas9-SAP155c and donor DNA: up-GAL1p-PGM1-down were co-transformed into the MGV27 strain to obtain the chassis strain MUDP004, and the strain was preserved for future use (20% glycerol).

[0379] Step 2) The knockout plasmid pCas9-SAP155c and donor DNA: up-GAL1p-PGM1-down were co-transformed into the MUDP003 strain to obtain the chassis strain MUDP005, and the strain was preserved for future use (20% glycerol).

[0380] Example 13 Overexpression of PGM2

[0381] The same method as in Example 10 "Knockout of G1P metabolic enzyme gene GAL7" was used, and the primers involved are listed in Table 18. The differences are:

[0382] Step 2) The knockout plasmid pCas9-1114a and donor DNA: up-GAL1p-PGM2-down were co-transformed into the MGV27 strain to obtain the chassis strain MUDP006, and the strain was preserved for future use (20% glycerol).

[0383] Step 2) The knockout plasmid pCas9-1114a and donor DNA: up-GAL1p-PGM2-down were co-transformed into the MUDP005 strain to obtain the chassis strain MUDP007, and the strain was preserved for future use (20% glycerol).

[0384] Example 14 Overexpression of UGP1

[0385] The same method as in Example 10 "Knockout of G1P metabolic enzyme gene GAL7" was used, and the primers involved are listed in Table 18. The differences are:

[0386] Step 2) The knockout plasmid pCas9-1014a and donor DNA: up-GAL1p-UGP1-down were co-transformed into the MGV27 strain to obtain the chassis strain MUDP008, and the strain was preserved for future use (20% glycerol).

[0387] Step 2) The knockout plasmid pCas9-1014a and donor DNA: up-GAL1p-UGP1-down were co-transformed into the MUDP007 strain to obtain the chassis strain MUDP009, and the strain was preserved for future use (20% glycerol).

[0388] Table 19 Basic information of strains

[0389] Example 15 Verification Experiment

[0390] The frozen strains MUDP001-MUDP009 were taken out of the -80℃ freezer, streaked on YPD plates, and cultured at 30℃ for 2-3 days; single colonies were picked from the plates and placed in 10mL YPD medium, cultured at 30℃, 250rpm, overnight; the seed liquid was transferred to a 250mL shake flask containing 25mL YPD liquid medium at a 5% inoculation rate, and fermented at 30℃, 250rpm, with 3 parallels per group; fermented for 120h, 5mL of fermentation liquid was taken, glass beads were added, and the cells were shaken for 30min, extracted with an equal volume of ethyl acetate, centrifuged at 12,000rpm for 5min, and the organic phase was collected. The extraction was repeated 3 times, and finally the organic phase was placed in a 70℃ vacuum drying oven, the solvent was evaporated, and it was re-dissolved with 200μL methanol (containing 1% formic acid) and detected by HPLC. The specific results are shown in Tables 20 to 23.

[0391] Table 20 III-X production

[0392] Table 21 IV-A yield

[0393] Table 22 SI production

[0394] Table 23 MV production

[0395] Example 16: Integration of UGT720-269-1 into the sap155b site and integration of UGT94-289-3 into the His3b site

[0396] 1. Construction of strains with UGT720-269-1 integrated into the sap155b locus

[0397] The chassis cells were the previously constructed M43 or MUDP009 (two groups of strains were constructed, and M43 was not enhanced by the introduction of the UDPG pathway), and strains MGV29 and MGV30 were constructed, respectively. The constructed strains were able to synthesize mogroside I-A1 and mogroside II E from scratch.

[0398] The same method as in Example 10 "Knockout of G1P metabolic enzyme gene GAL7" was used, and the primers involved are listed in Table 24. The differences are:

[0399] Step 2) The knockout plasmid pCas9-sap155b and the donor DNA: up-GAL1p-UGT720-269-1-down were co-transformed into the M43 strain to obtain the chassis strain MGV29, and the strain was preserved for later use (20% glycerol).

[0400] Step 2) The knockout plasmid pCas9-sap155b and donor DNA: up-GAL1p-UGT720-269-1-down were co-transformed into the MUDP009 strain to obtain the chassis strain MGV30, and the strain was preserved for future use (20% glycerol).

[0401] 2. Construction of strains with UGT94-289-3 integrated into the His3b site

[0402] The chassis cells were MGV29 or MGV30 constructed previously (two groups of strains were constructed, and MGV29 was not enhanced by the introduction of UDPG), and strains MGV31 and MGV32 were constructed respectively. The constructed strains were able to synthesize mogroside III X, mogroside IVA, simenoside I and mogroside V from scratch.

[0403] The same method as in Example 10 "Knockout of G1P metabolic enzyme gene GAL7" was used, and the primers involved are listed in Table 24. The differences are:

[0404] Step 2) The knockout plasmid pCas9-His3b and the donor DNA: up-GAL1p-UGT94-289-3-down were co-transformed into the MGV29 strain to obtain the chassis strain MGV31, and the strain was preserved for future use (20% glycerol).

[0405] Step 2) The knockout plasmid pCas9-His3b and the donor DNA: up-GAL1p-UGT94-289-3-down were co-transformed into the MGV30 strain to obtain the chassis strain MGV32, and the strain was preserved for future use (20% glycerol).

[0406] Table 24 Primer sequence table

[0407] Table 25 Basic information of strains

[0408] Example 17 Verification Experiment

[0409] The frozen strains MGV29-MGV32 were taken out of the -80°C freezer, streaked on YPD plates, and cultured at 30°C for 2-3 days; a single colony was picked from the plate and placed in 10 mL YPD medium, cultured at 30°C, 250 rpm, and overnight; the seed liquid was transferred to a 250 mL shake flask containing 25 mL YPD liquid medium at a 5% inoculation rate, and fermented at 30°C, 250 rpm, with 3 parallels per group; after 120 h of fermentation, 5 mL of fermentation liquid was taken, glass beads were added, and the cells were shaken for 30 minutes, extracted with an equal volume of ethyl acetate, and the organic phase was collected by centrifugation at 12,000 rpm for 5 minutes. The extraction was repeated 3 times, and finally the organic phase was placed in a 70°C vacuum drying oven, the solvent was evaporated, and after re-dissolving with 200 μL of methanol (containing 1% formic acid), HPLC detection was performed. The specific results are shown in Tables 26 to 31.

[0410] Table 26 I-A1 production

[0411] Table 27 II-E production

[0412] Table 28 III-X production

[0413] Table 29 IV-A production

[0414] Table 30 SI production

[0415] Table 31 MV production

[0416] The above describes in detail the engineered yeast strains producing mogroside, simenoside I, and / or mogroside V, and their applications, provided by the present invention. This article uses specific examples to illustrate the principles and implementation methods of the present invention. The description of the above examples is intended only to facilitate understanding of the methods and core concepts of the present invention. It should be noted that those skilled in the art will be able to make various improvements and modifications to the present invention without departing from the principles of the present invention, and such improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. Use of any one of the following or a combination thereof in the preparation of mogroside, simenoside I and / or mogroside V; (I) Overexpression of tHMG1 and IDI1 (308a), the key rate-limiting genes for terpenoid synthesis in the MVA pathway; (II), mutant gene upc2-1(416d) that overexpresses the global transcription factor UPC2; (III), overexpression of genes for acetaldehyde dehydrogenase ALD6 (1622b), pyruvate decarboxylase PDC1 (1021b), and acetyl-CoA synthetase ACS1 (911b); (IV), knocking out the isocitrate dehydrogenase IDH1 gene; (V) Overexpression of the citrate lyase ACL gene of Rhodosporidium toruloides; (VI) Overexpression of the gene for alcohol dehydrogenase ADH2 (208a) from the rice base rot pathogen.

2. The use according to claim 1, characterized in that Any one of (I) to (VI) or a combination thereof strengthens the MVA pathway and / or the acetyl-CoA pathway.

3. The use according to claim 1, characterized in that Any one of the following items or a combination thereof also includes: (VII), integration into SgCDS, SgEPH3, CYP87D18, and / or SgCPR1; (VIII), integrated into UGT720-269-1; and / or (IX), integrated into UGT94-289-3.

4. A gene expression cassette, characterized in that Includes any one or a combination of the following: (I) Overexpression of tHMG1 and IDI1 (308a), key rate-limiting genes for terpenoid synthesis in the MVA pathway; (II), overexpression of the mutant gene upc2-1(416d) of the global transcription factor UPC2; (III), overexpressed genes for acetaldehyde dehydrogenase ALD6 (1622b), pyruvate decarboxylase PDC1 (1021b), and acetyl-CoA synthetase ACS1 (911b); (IV), genes excluding isocitrate dehydrogenase IDH1; (V) Overexpression of the citrate lyase ACL gene of Rhodosporidium toruloides; (VI) Overexpression of the rice base rot pathogen alcohol dehydrogenase ADH2 (208a) gene.

5. The gene expression cassette according to claim 4, wherein Also includes: (VII), SgCDS, SgEPH3, CYP87D18, and / or SgCPR1; (VIII), UGT720-269-1; and / or (IX), UGT94-289-3.

6. Use of any one of the following or a combination thereof in the preparation of mogroside, simenoside I and / or mogroside V; (I) downregulating the expression of the endogenous gene ERG7; (II), knockout of the gene encoding the GAL4 inhibitory protein GAL80; (III) Overexpression of squalene epoxidase SQE (GAL80) from Momordica grosvenori, cytochrome b5 (RsCyb5,511b) from Rubus sweetleaf, or yeast phospholipid biosynthesis regulatory gene INO2 (ER size regulator, replacement promoter).

7. The use according to claim 6, characterized in that Any one of (I) to (III) or a combination thereof weakens the expression of the competing pathway and expands the space of the endoplasmic reticulum; Preferably, the expression of the weakened competition pathway comprises: Reduce the consumption of 2,3-epoxysqualene; This allows the expression of the GAL gene to be freed from the control of the more costly galactose and to respond to the glucose concentration in the culture medium.

8. The use according to claim 6, characterized in that Any one of the following items or a combination thereof also includes: (IV), integration into SgCDS, SgEPH3, CYP87D18, and / or SgCPR1; (V), integrated into UGT720-269-1; and / or (VI), integrated into UGT94-289-3.

9. A gene expression cassette, characterized in that Includes any one or a combination of the following: (I), downregulated endogenous gene ERG7; (II), excluding the gene encoding the GAL4 inhibitory protein GAL80; (III) Overexpression of squalene epoxidase SQE (GAL80) from Momordica grosvenori and cytochrome b5 (RsCyb5,511b) from Rubus sweetleaf interacted with the yeast phospholipid biosynthesis regulatory gene INO2 (ER size regulator, replacement promoter).

10. The gene expression cassette according to claim 9, wherein Also includes: (IV), SgCDS, SgEPH3, CYP87D18, and / or SgCPR1; (V), UGT720-269-1; and / or (VI), UGT94-289-3.

11. Use of any one of the following or a combination thereof in the preparation of mogroside, simenoside I and / or mogroside V; (I) Screen and replace CPR genes, selecting CPR1, CPR2, and CPR3; The CPR1 sequence is shown as SEQ ID No. 92; The CPR2 sequence is shown as SEQ ID No. 93; The CPR3 sequence is shown as SEQ ID No. 94; (II) Increase the copy number of P450 enzyme CYP87D18 to 2-4 copies; (III) reducing the expression intensity of CPR by replacing the relatively weak constitutive promoter; The relatively weak constitutive promoters include: PGI1p, HXT7p, or PGK1p.

12. The use according to claim 11, characterized in that For example, any one of (I) to (VI) or a combination thereof improves the catalytic efficiency of cytochrome P450 (CYP450).

13. The use according to claim 11, characterized in that Any one of the following items or a combination thereof also includes: (IV), integration into SgCDS, SgEPH3, CYP87D18, and / or SgCPR1; (V), integrated into UGT720-269-1; and / or (VI), integrated into UGT94-289-3.

14. A gene expression cassette, characterized in that Includes any one or a combination of the following: (I), CPR1, CPR2, CPR3; The CPR1 sequence is shown as SEQ ID No. 92; The CPR2 sequence is shown as SEQ ID No. 93; The CPR3 sequence is shown as SEQ ID No. 94; (II), P450 enzyme CYP87D18; (III), relatively weak constitutive promoter; The relatively weak constitutive promoter includes: PGI1p, HXT7p or PGK1p.

15. The gene expression cassette according to claim 14, wherein Also includes: (IV), SgCDS, SgEPH3, CYP87D18, and / or SgCPR1; (V), UGT720-269-1; and / or (VI), UGT94-289-3.

16. A construct, characterized in that Comprising the gene expression cassette of claim 4, 5, 9, 10, 14 or 15.

17. A host, characterized in that Includes any of the following: (i) a gene expression cassette according to claim 4, 5, 9, 10, 14 or 15; (ii) The construct according to claim 16.

18. The host according to claim 17, wherein Including but not limited to yeast.

19. Use of any one of the following or a combination thereof in the preparation of mogroside, simenoside I and / or mogroside V; (i) a gene expression cassette according to claim 4, 5, 9, 10, 14 or 15; (ii) the construct of claim 16; (iii) The host according to claim 17 or 18.

20. A method for preparing mogroside, simenoside I and / or mogroside V, characterized in that: Integrating the gene expression cassette of claim 4, 5, 9, 10, 14 or 15; Transforming the construct of claim 16; Fermentation culture of the host according to claim 17 or 18; The fermentation broth was collected and purified.

21. Use of any one of the following or a combination thereof in the preparation of mogroside, simenoside I, mogroside IIIX, mogroside IVA and / or mogroside V; Screen and replace CPR genes, using CPR1, CPR2, and CPR3; The CPR1 sequence is shown as SEQ ID No. 92; The CPR2 sequence is shown as SEQ ID No. 93; The CPR3 sequence is shown as SEQ ID No. 94; Increase the number of copies of the P450 enzyme CYP87D18 to 2-4 copies; By replacing the relatively weak constitutive promoter, the expression intensity of CPR is reduced; The relatively weak constitutive promoters include: PGI1p, HXT7p, or PGK1p; integration into SgCDS, SgEPH3, CYP87D18, and / or SgCPR1; Integrated into UGT720-269-1; Incorporated into UGT94-289-3; and (I), knocking out the G1P metabolic enzyme gene GAL7; and / or (II), overexpression of UDPG synthesis pathway gene HXK1; and / or (III), overexpression of PGM1; and / or (IV), overexpression of PGM2; and / or (V) Overexpression of UGP1.

22. The use according to claim 21, characterized in that Also includes: (VI), integrating the UGT720-269-1 into the sap155b site; and / or (VII) integrating the UGT94-289-3 into the His3b site.

23. The use according to claim 21 or 22, characterized in that The applications include improving the catalytic efficiency of cytochrome P450 (CYP450).

24. A gene expression cassette, characterized in that Includes any one or a combination of the following: CPR1, CPR2, CPR3; The CPR1 sequence is shown as SEQ ID No. 92; The CPR2 sequence is shown as SEQ ID No. 93; The CPR3 sequence is shown as SEQ ID No. 94; P450 enzyme CYP87D18; Relatively weak constitutive promoter; The relatively weak constitutive promoter includes: PGI1p, HXT7p or PGK1p; SgCDS, SgEPH3, CYP87D18, and / or SgCPR1; UGT720-269-1; UGT94-289-3; (I) knocking out the G1P metabolic enzyme gene GAL7; and / or (II), overexpression of UDPG synthesis pathway gene HXK1; and / or (III), overexpression of PGM1; and / or (IV), overexpression of PGM2; and / or (V) Overexpression of UGP1.

25. The gene expression cassette according to claim 24, wherein Also includes: (VI), the UGT720-269-1 is integrated into the sap155b site; and / or (VII) The UGT94-289-3 is integrated into the His3b site.

26. A construct, characterized in that Comprising the gene expression cassette according to claim 24 or 25.

27. A host, characterized in that Includes any of the following: (i) The gene expression cassette according to claim 24 or 25; (ii) The construct according to claim 26.

28. The host according to claim 27, wherein Including but not limited to yeast.

29. Use of any one of the following or a combination thereof in the preparation of mogroside, simenoside I, mogroside IIIX, mogroside IVA and / or mogroside V; (i) The gene expression cassette according to claim 24 or 25; (ii) the construct of claim 26; (iii) The host according to claim 27 or 28.

30. A method for preparing mogroside, simanoside I, mogroside IIIX, mogroside IVA and / or mogroside V, characterized in that: Integrating the gene expression cassette according to claim 24 or 25; Transforming the construct of claim 26; Fermentation culture of the host according to claim 27 or 28; The fermentation broth was collected and purified.

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