Recombinant bacteria producing dammarenediol-ii glycoside and application thereof

Abstract

The application discloses a method for producing dammaradien-diol-II glycoside 3beta-O-Glc-DM and a construction method of a recombinant bacterium, the recombinant bacterium obtained by the method, and application of the recombinant bacterium in preparation of 3beta-O-Glc-DM.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to a method for constructing a recombinant bacterium that produces dammarenediol-II glycoside 3β-O-Glc-DM, the recombinant bacterium obtained by the above method, and the use of the above recombinant bacterium in the preparation of dammarenediol-II glycoside 3β-O-Glc-DM. Background Technology

[0002] Ginseng (Panax ginseng C.A. Meyer) is a traditional and precious medicinal herb with various pharmacological activities, including anti-cancer, anti-aging, anti-diabetic, anti-hypertensive, immunomodulatory, and neuroprotective effects. Ginsenosides are the main bioactive components of ginseng. To date, more than 150 natural ginsenosides have been isolated and identified from plants of the Panax genus. The structural and functional diversity of ginsenosides depends on the aglycone structure and the type, number, and position of the glycosyl ligands. Based on the different aglycone skeletons, they can be divided into dammarane-type tetracyclic triterpenoid saponins and oleanane-type pentacyclic triterpenoid saponins. Dammarane-type saponins constitute the vast majority of ginsenosides, and they are further divided into protopanaxadiol (PPD) type and protopanaxtriol (PPT) type ginsenosides. PPD-type ginsenosides are synthesized through glycosylation of PPD at C3-OH and / or C20-OH, while PPT-type ginsenosides are synthesized through glycosylation of PPT at C6-OH and / or C20-OH. Furthermore, the differences in the position and number of hydroxyl and glycosyl groups lead to the diversity of bioactivity of ginsenosides.

[0003] It has been reported that the cytotoxic activity of dammarane-type ginsenosides is negatively correlated with the number of hydroxyl groups in their aglycones. However, triterpenoid saponins based on dammarene diol-II (DM) have never been isolated from ginseng plants. DM, as a direct precursor of PPD, has fewer hydroxyl groups than PPD and PPT, possessing only two hydroxyl groups at C3 and C20 positions. Therefore, it is speculated that C3-glycosylated DM and C20-glycosylated DM may have higher cytotoxic activity than PPD-type and PPT-type ginsenosides. In vitro pharmacological activity assays showed that 3β-O-Glc-DM inhibited the growth of multiple colon cancer cell lines; in vivo pharmacological evaluations showed that, whether used alone or in combination with 5-FU, 3β-O-Glc-DM significantly inhibited the growth of C26 colon cancer xenograft tumors compared to the control groups Rg3 and Compound K.

[0004] In recent years, researchers have cloned and identified several UDP-glycosyltransferase (UGT) genes from ginseng plants. Among them, PgUGT74AE2, derived from ginseng plants, can selectively catalyze the glycosylation of PPD and Compound K at C3-OH, generating Rh2 and F2, respectively. Research on UGT-related aspects of ginsenoside biosynthesis lays the foundation for the production of natural or non-natural ginsenosides through metabolic engineering.

[0005] This invention cloned genes encoding dammarene diol-II synthase (DS) and PgUGT74AE2 from ginseng. PgUGT74AE2 was heterologously expressed in *Escherichia coli* BL21(DE3), and DM glycoside 3β-O-Glc-DM was obtained via in vitro enzymatic reaction. By introducing codon-optimized DS and PgUGT74AE2 genes into *Saccharomyces cerevisiae* with a knockout hexokinase 2 gene, a biosynthetic pathway for 3β-O-Glc-DM was constructed using yeast endogenous terpene biosynthetic genes. The DS and PgUGT74AE2 genes were integrated into the yeast genome using the CRISPR / Cas9 system. Several key enzymes upstream of the 3β-O-Glc-DM biosynthetic pathway were overexpressed, competitive branching metabolic pathways were downregulated, and transcription activator HAC1 was overexpressed to optimize the biosynthetic pathway of the recombinant strain, thereby increasing the yield of 3β-O-Glc-DM. This study provides an efficient method for producing 3β-O-Glc-DM, which can provide candidate compounds for new drug research. Summary of the Invention

[0006] The inventors have discovered that in recombinant bacteria that produce DM, the yield of DM is significantly increased when dammarene diol-II synthase (DS) is expressed in fusion with green fluorescent protein (GFP).

[0007] The inventors also discovered that ginsenoside glycosyltransferase PgUGT74AE2 can selectively catalyze the C3-OH of DM to generate 3β-O-Glc-DM.

[0008] Furthermore, the inventors discovered that knocking out hexokinase 2, a key enzyme in the glycolysis pathway, can adjust the metabolic flux of the glycolysis pathway, thereby increasing the yield of DM in recombinant bacteria.

[0009] To obtain a recombinant bacterium producing 3β-O-Glc-DM and a method for constructing such a strain, the present invention provides in the following paragraphs:

[0010] [1] A method for constructing recombinant bacteria, the method comprising the following steps: knocking out the hexokinase 2 gene in Saccharomyces cerevisiae and introducing into the Saccharomyces cerevisiae the gene expression cassette encoding the fusion protein of DS and GFP and the gene expression cassette encoding ginseng PgUGT74AE2.

[0011] [2] According to the method described in [1], the method further includes the following step: increasing the activity of 3-hydroxy-3-methylglutaryl-CoA reductase in the brewing yeast.

[0012] [3] According to any one of [1] or [2], the method further includes one or more of the following:

[0013] To enhance the activity of isopentenyl pyrophosphate isomerase IDI1 in Saccharomyces cerevisiae;

[0014] To increase the activity of farnesyl pyrophosphate synthase ERG20 in Saccharomyces cerevisiae;

[0015] Increase the activity of squalene monooxygenase ERG1 in Saccharomyces cerevisiae;

[0016] Increase the activity of squalene synthase ERG9 in Saccharomyces cerevisiae;

[0017] Reduces the activity of lanosterol synthase ERG7 in Saccharomyces cerevisiae;

[0018] Increase the level of the molecular chaperone BiP in Saccharomyces cerevisiae;

[0019] Increase the level of transcription factor HAC1 in Saccharomyces cerevisiae; or

[0020] Increase the level of disulfide isomerase PDI1 in Saccharomyces cerevisiae.

[0021] [4] According to any of [1]-[3], wherein the gene expression cassette encoding the DS-GFP fusion protein contains the gene encoding the DS-GFP fusion protein shown in SEQ ID NO:1.

[0022] [5] The method according to any one of [1]-[4], wherein the gene expression cassette encoding PgUGT74AE2 contains the gene encoding PgUGT74AE2 shown in SEQ ID NO:2.

[0023] [6] According to the method described in [2], the activity of 3-hydroxy-3-methylglutary-CoA reductase in the brewer's yeast is increased by introducing the tHMG1 expression cassette encoding the 3-hydroxy-3-methylglutary-CoA reductase gene into the brewer's yeast.

[0024] [7] According to any of the methods described in [3]-[6], wherein,

[0025] The activity of isopentenyl pyrophosphate isomerase IDI1 in Saccharomyces cerevisiae was enhanced by introducing the gene expression cassette encoding isopentenyl pyrophosphate isomerase IDI1 into Saccharomyces cerevisiae.

[0026] The activity of farnesyl pyrophosphate synthase ERG20 in Saccharomyces cerevisiae was enhanced by introducing a gene expression cassette encoding farnesyl pyrophosphate synthase ERG20 into Saccharomyces cerevisiae.

[0027] The activity of squalene monooxygenase ERG1 in Saccharomyces cerevisiae was increased by introducing a gene expression cassette encoding squalene monooxygenase ERG1 into Saccharomyces cerevisiae.

[0028] The activity of squalene synthase ERG9 in Saccharomyces cerevisiae was enhanced by introducing an expression cassette encoding the squalene synthase ERG9 gene into Saccharomyces cerevisiae.

[0029] The activity of lanosterol synthase ERG7 in Saccharomyces cerevisiae was reduced by introducing an expression cassette containing the antisense fragment of lanosterol synthase ERG7 into Saccharomyces cerevisiae.

[0030] The level of molecular chaperone BiP in Saccharomyces cerevisiae was increased by introducing a gene expression cassette encoding the molecular chaperone BiP into Saccharomyces cerevisiae.

[0031] The increase in HAC1 levels in Saccharomyces cerevisiae was achieved by introducing a gene expression cassette encoding HAC1 into Saccharomyces cerevisiae; or

[0032] The level of disulfide isomerase PDI1 in Saccharomyces cerevisiae was increased by introducing a gene expression cassette encoding disulfide isomerase PDI1 into Saccharomyces cerevisiae.

[0033] [8] The method according to [7] is characterized in that:

[0034] The nucleotide sequence encoding IDI1 is the sequence shown in SEQ ID NO:4;

[0035] The nucleotide sequence encoding ERG20 is the sequence shown in SEQ ID NO:5;

[0036] The nucleotide sequence encoding ERG1 is the sequence shown in SEQ ID NO:6;

[0037] The nucleotide sequence encoding ERG9 is the sequence shown in SEQ ID NO:7;

[0038] The nucleotide sequence encoding the ERG7 antisense fragment is the sequence shown in SEQ ID NO:8;

[0039] The nucleotide sequence encoding BiP is the sequence shown in SEQ ID NO:8;

[0040] The nucleotide sequence encoding HAC1 is the sequence shown in SEQ ID NO:10; or

[0041] The nucleotide sequence encoding PDI1 is shown in SEQ ID NO:11.

[0042] [9] According to any of the methods in [1]-[8], wherein the expression cassette is integrated into the Saccharomyces cerevisiae genome, preferably using CRISPR / Cas9 for the integration of the expression cassette.

[0043]

[10] Recombinant bacteria obtained by any of the methods described in [1]-[9].

[0044]

[11] Application of the recombinant bacteria described in [9] in the production of 3β-O-Glc-DM.

[0045]

[12] A method for producing 3β-O-Glc-DM, the method comprising fermenting a recombinant strain as described in [9] to obtain 3β-O-Glc-DM. Invention Details

[0047] The first aspect of the present invention is to provide a method for constructing recombinant bacteria, the method comprising the following steps: knocking out the hexokinase 2 gene in Saccharomyces cerevisiae, and introducing into the Saccharomyces cerevisiae a gene expression cassette encoding a fusion protein of dammarene diol-II synthase and GFP (hereinafter referred to as DS-GFP) and a gene expression cassette encoding ginsenoside transferase PgUGT74AE2.

[0048] In this invention, dammarene glycol-II synthase can be ginseng-derived dammarene glycol-II synthase. In a preferred embodiment, the ginseng dammarene glycol-II synthase gene ds (No. AB265170.1) can be used.

[0049] Green fluorescent protein (GFP) is a fluorescent protein isolated from the Victoria jellyfish. This protein emits green fluorescence when excited by blue light in the 450nm-490nm range, making it an ideal reporter molecule. Numerous studies have fused target proteins with GFP for expression, observing green fluorescence to determine subcellular localization of the target protein and explore its biological functions. In this invention, the GFP-encoding gene can be any polynucleotide capable of encoding GFP.

[0050] In this invention, GFP can be fused to the C-terminus of dammarenediol-II synthase.

[0051] In this invention, GFP can be directly linked to dammarenediol-II synthase, or a spacer sequence, for example, 2 to 40 amino acids, preferably 5 to 20 amino acids, can exist between GFP and dammarenediol-II synthase. In a preferred embodiment of this invention, GFP can be fused to the C-terminus of dammarenediol-II synthase.

[0052] In a preferred embodiment, the gene encoding DS-GFP is prepared by the sequence shown in SEQ ID NO:1 (which is described in Liang Huichao et al., Study on expression, localization and function of ginseng dammarene diol-II synthase in Saccharomyces cerevisiae, Acta Pharmaceutica Sinica 2016, 51(6): 998-1003, which is incorporated herein by reference in its entirety).

[0053] In one embodiment of the present invention, the DS-GFP expression cassette further specifically includes the promoter TEF1, the coding gene for DS-GFP, and the terminator CYC1.

[0054] In a preferred embodiment of the present invention, an optimized gene sequence PgUGT74AE2 (SEQ ID NO:2) is synthesized based on the cDNA sequence information of ginseng glycosyltransferase PgUGT74AE2 (No. JX898529.1) and the codon preference of Saccharomyces cerevisiae.

[0055] In one embodiment of the present invention, the ginseng glycosyltransferase PgUGT74AE2 encoding gene expression cassette further specifically includes promoter TDH3, PgUGT74AE2 encoding gene, and terminator ADH2.

[0056] In this invention, the hexokinase 2 gene in *Saccharomyces cerevisiae* is knocked out using methods known in the art. For example, homologous recombination is used to knock out the hexokinase 2 gene HXK2.

[0057] In this invention, diploid and haploid Saccharomyces cerevisiae mutants with HXK2 gene deletion can be used, with haploid Saccharomyces cerevisiae mutants being preferred.

[0058] In a further embodiment of the present invention, the method further includes the step of: increasing the activity of 3-hydroxy-3-methylglutaryl-CoA reductase in the brewer's yeast.

[0059] 3-Hydroxy-3-methylglutaryl-CoA reductase (HMGR) is the first key enzyme in the mevalonate metabolic pathway, catalyzing the conversion of 3-hydroxy-3-methylglutaryl-CoA to mevalonate. This reaction is also the first rate-limiting step in the ginsenoside biosynthesis pathway. HMGR contains an N-terminal transmembrane domain and a C-terminal catalytic domain, which play localization and catalytic roles, respectively. Overexpression of HMGR in cells leads to feedback inhibition of the mevalonate metabolic pathway; that is, downstream products catalyzed by HMGR activate HMGR anchored to the endoplasmic reticulum membrane to enter the degradation pathway. Therefore, the localization function of its transmembrane domain plays an important role in the HMGR degradation pathway. In view of this, by removing the transmembrane domain, truncating the HMGR gene, and overexpressing it, the feedback inhibition of the mevalonate metabolic pathway can be effectively reduced, thereby promoting the biosynthesis of downstream products. In *Saccharomyces cerevisiae*, the HMGR pathway of the mevalonate pathway has two members, Hmg1p and Hmg2p, encoded by genes HMG1 and HMG2, with Hmg1p, encoded by HMG1, playing a major role. The cDNA sequence (No. NM_001182434.1) of the gene tHMG1, which encodes the HMGR catalytic domain, is shown in SEQ ID NO:3.

[0060] In this invention, overexpression of the HMGR catalytic domain increases the supply of the upstream precursor 2,3-oxidized squalene while avoiding feedback inhibition caused by the accumulation of downstream products, ultimately resulting in a significant increase in the DM content in the recombinant bacteria.

[0061] Therefore, in a preferred embodiment of the present invention, the method for increasing the activity level of 3-hydroxy-3-methyl-glutaryl-CoA reductase can be to introduce the tHMG1 expression cassette, which encodes the 3-hydroxy-3-methyl-glutaryl-CoA reductase gene.

[0062] In one embodiment of the present invention, the 3-hydroxy-3-methyl-glutaryl-CoA reductase encoding gene expression cassette further specifically includes promoter PGK1, tHMG1, and terminator ADH1.

[0063] In the method of the present invention, the method further includes one or more of the following:

[0064] To enhance the activity of isopentenyl pyrophosphate isomerase IDI1 in Saccharomyces cerevisiae;

[0065] To increase the activity of farnesyl pyrophosphate synthase ERG20 in Saccharomyces cerevisiae;

[0066] Increase the activity of squalene monooxygenase ERG1 in Saccharomyces cerevisiae;

[0067] Increase the activity of squalene synthase ERG9 in Saccharomyces cerevisiae;

[0068] Reduces the activity of lanosterol synthase ERG7 in Saccharomyces cerevisiae;

[0069] Increase the level of the molecular chaperone BiP in Saccharomyces cerevisiae;

[0070] Increase the level of transcription factor HAC1 in Saccharomyces cerevisiae; or

[0071] Increase the level of disulfide isomerase PDI1 in Saccharomyces cerevisiae.

[0072] In a specific implementation, the activity of isopentenyl pyrophosphate isomerase IDI1 in Saccharomyces cerevisiae is enhanced by introducing the gene expression cassette encoding isopentenyl pyrophosphate isomerase IDI1 into Saccharomyces cerevisiae.

[0073] The activity of farnesyl pyrophosphate synthase ERG20 in Saccharomyces cerevisiae was enhanced by introducing the gene expression cassette encoding farnesyl pyrophosphate synthase ERG20 into Saccharomyces cerevisiae.

[0074] The activity of squalene monooxygenase ERG1 in Saccharomyces cerevisiae was enhanced by introducing the gene expression cassette encoding squalene monooxygenase ERG1 into Saccharomyces cerevisiae.

[0075] The activity of squalene synthase ERG9 in Saccharomyces cerevisiae was enhanced by introducing the gene expression cassette encoding squalene synthase ERG9 into Saccharomyces cerevisiae.

[0076] The activity of lanosterol synthase ERG7 in Saccharomyces cerevisiae was reduced by introducing an expression cassette containing the antisense fragment of lanosterol synthase ERG7 into Saccharomyces cerevisiae.

[0077] The level of molecular chaperone BiP in Saccharomyces cerevisiae was increased by introducing a gene expression cassette encoding BiP into Saccharomyces cerevisiae.

[0078] The level of transcription factor HAC1 in Saccharomyces cerevisiae was increased by introducing a gene expression cassette encoding HAC1 into the yeast; or

[0079] The level of disulfide isomerase PDI1 in Saccharomyces cerevisiae was increased by introducing a gene expression cassette encoding disulfide isomerase PDI1 into Saccharomyces cerevisiae.

[0080] In one embodiment, the gene expression cassette encoding isopentenyl pyrophosphate isomerase IDI1 may include promoter TDH3, the gene encoding isopentenyl pyrophosphate isomerase IDI1, and terminator TPI1.

[0081] In one embodiment, the nucleotide sequence encoding IDI1 is the sequence shown in SEQ ID NO:4.

[0082] In one embodiment, the gene expression cassette encoding farnesyl pyrophosphate synthase ERG20 may include a promoter PGK1, the gene encoding farnesyl pyrophosphate synthase ERG20, and a terminator ADH1.

[0083] In one embodiment, the nucleotide sequence encoding ERG20 is the sequence shown in SEQ ID NO:5.

[0084] In one embodiment, the gene expression cassette encoding squalene monooxygenase ERG1 may include a promoter PGK1, a gene encoding squalene monooxygenase ERG1, and a terminator ADH1.

[0085] In one embodiment, the nucleotide sequence encoding ERG1 is the sequence shown in SEQ ID NO:6.

[0086] In one embodiment, the cassette encoding the squalene synthase ERG9 may include the promoter TEF1, the gene encoding squalene synthase ERG9, and the terminator TPI1.

[0087] In one embodiment, the nucleotide sequence encoding ERG9 is the sequence shown in SEQ ID NO:7.

[0088] In one embodiment, the gene expression cassette encoding the lanosterol synthase ERG7 antisense fragment may include the promoter TEF1, the gene encoding the lanosterol synthase ERG7 antisense fragment, and the terminator CYC1.

[0089] In one embodiment, the nucleotide sequence encoding the ERG7 antisense fragment is the sequence shown in SEQ ID NO:8.

[0090] In one embodiment, the gene expression cassette encoding the molecular chaperone BiP may include the promoter TEF1, the gene encoding the molecular chaperone BiP, and the terminator CYC1.

[0091] In one embodiment, the nucleotide sequence encoding BiP is the sequence shown in SEQ ID NO:9.

[0092] In one embodiment, the gene expression cassette encoding the transcription factor HAC1 may include the promoter TEF1, the gene encoding the transcription factor HAC1, and the terminator CYC1.

[0093] In one embodiment, the nucleotide sequence encoding HAC1 is the sequence shown in SEQ ID NO:10.

[0094] In one embodiment, the gene expression cassette encoding the disulfide isomerase PDI1 may include a promoter TEF1, a gene encoding the disulfide isomerase PDI1, and a terminator CYC1.

[0095] In one embodiment, the nucleotide sequence encoding PDI1 is the sequence shown in SEQ ID NO:11.

[0096] In embodiments of the present invention, the expression cassettes described above can be individually integrated into the genome of a *Saccharomyces cerevisiae* cell. Alternatively, the expression cassettes can be interconnected and integrated into the genome of a *Saccharomyces cerevisiae* cell. For example, all expression cassettes can be tandemly linked and integrated into the genome of a *Saccharomyces cerevisiae* cell. Alternatively, the expression cassettes can be constructed into multiple expression modules and then integrated into the genome of a *Saccharomyces cerevisiae* cell. In the present invention, an expression module refers to two or more expression cassettes that are operatively connected. In the present invention, expression cassettes and / or expression modules can be integrated into one or more sites. In the present invention, CRISPR / Cas9 is preferably used for the integration of expression cassettes and / or expression modules.

[0097] In preferred embodiments, the gene expression cassettes encoding the fusion protein of dammarene diol-II synthase and GFP, the gene expression cassettes encoding ginsenoside transferase PgUGT74AE2, and the gene expression cassettes encoding 3-hydroxy-3-methylglutaryl-CoA reductase can be integrated into the δ1 site of the *Saccharomyces cerevisiae* genome. The gene expression cassettes encoding isopentenyl pyrophosphate isomerase IDI1, farnesyl pyrophosphate synthase ERG20, squalene monooxygenase ERG1, squalene synthase ERG9, and the antisense fragment of lanosterol synthase ERG7 can be integrated into the δ4 site of the *Saccharomyces cerevisiae* genome. The gene expression cassettes encoding the molecular chaperone BiP, the transcription factor HAC1, and the disulfide bond isomerase PDI1 can be integrated into the *Saccharomyces cerevisiae* genomic rDNA site.

[0098] In a preferred embodiment, the gene expression cassettes encoding the fusion protein of dammarene diol-II synthase and GFP, the gene expression cassettes encoding ginsenoside transferase PgUGT74AE2 and 3-hydroxy-3-methylglutaryl-CoA reductase can be further constructed into expression modules and integrated into the δ1 site of the *Saccharomyces cerevisiae* genome. The gene expression cassettes encoding the isopentenyl pyrophosphate isomerase IDI1, farnesyl pyrophosphate synthase ERG20, squalene monooxygenase ERG1, squalene synthase ERG9, and the antisense fragment of lanosterol synthase ERG7 can be further constructed into expression modules and integrated into the δ4 site of the *Saccharomyces cerevisiae* genome. The gene expression cassettes encoding the molecular chaperone BiP, the transcription factor HAC1, and the disulfide bond isomerase PDI1 can be constructed into expression modules and integrated into the *Saccharomyces cerevisiae* genomic rDNA site.

[0099] In a further preferred embodiment, the present invention constructs a CRISPR / Cas9 system based on the δ1 site of the Saccharomyces cerevisiae genome. By utilizing the Cas9 endonuclease in this system to mediate double-strand breaks, the engineered yeast strain of the present invention is obtained through homologous recombination mechanism.

[0100] The brewing yeast of the present invention can be any brewing yeast available in the art. For example, commercially available brewing yeasts such as INVSC1, BY4742, YPH499, or W303-1B are available.

[0101] The gene expression cassettes described in this invention are integrated into the genome of Saccharomyces cerevisiae; or, these gene expression cassettes exist in Saccharomyces cerevisiae cells in the form of plasmids.

[0102] In some embodiments, the plasmid vector is selected from pESC-HIS, pESC-URA, pESC-TRP, and pESC-TRP (Invitrogen, USA).

[0103] The dammarene diol-II synthase, GFP, glycosyltransferase PgUGT74AE2, 3-hydroxy-3-methylglutaryl-CoA reductase, isopentenyl pyrophosphate isomerase, farnesyl pyrophosphate synthase, squalene monooxygenase, squalene synthase, lanosterol synthase, molecular chaperone BiP, transcription factor HAC1, or disulfide isomerase PDI1 in this invention may be:

[0104] (a) Naturally occurring wild-type enzymes;

[0105] (b) A polypeptide of a wild-type enzyme formed by substitution, deletion or addition of one or more amino acid residues, or by the addition of a signal peptide sequence, and possessing the corresponding activity.

[0106] (c) A polypeptide whose sequence contains the polypeptide sequence described in (a) or (b);

[0107] (d) A polypeptide whose amino acid sequence is ≥85% or ≥90% (preferably ≥95%) identical to that of the wild-type enzyme and which has wild-type enzyme activity.

[0108] In this invention, the coding gene used may be a natural polynucleotide sequence (e.g., cDNA sequence, genomic sequence, or RNA, etc.) encoding any one of (a)-(d) above, or a degenerate variant thereof. As used herein, "degenerate variant" refers to a polynucleotide sequence that encodes any one of (a)-(d) above, but differs from the natural polynucleotide sequence. Codon-optimized DNA sequences are preferred. Polynucleotides can generally be obtained by PCR amplification, recombination, or artificial synthesis.

[0109] Exogenous gene expression cassettes can be integrated into the genome of Saccharomyces cerevisiae for expression, or they can be expressed in plasmid form outside the genome.

[0110] The free carrier of Saccharomyces cerevisiae can be a commercially available carrier or any carrier with the same function. For example, the free carrier can be the pESC series carriers, including pESC-HIS, pESC-URA, pESC-TRP and pESC-TRP; pYES2; or pAUR123 (Invitrogen, USA).

[0111] The main method for integrating exogenous genes into the yeast genome is homologous recombination. Homologous recombination involves amplifying the upstream and downstream sequences of the integration site as upstream or downstream homologous arms, constructing a target gene expression cassette (containing a promoter, target gene, and terminator), typically with a selection marker gene also present in the upstream homologous arm, and then linking these components in the order of upstream homologous arm, gene expression cassette, and downstream homologous arm to form a fragment suitable for homologous recombination. This fragment is then introduced into *Saccharomyces cerevisiae*, and positive transformants are selected based on the selection markers to obtain integrative recombinant *Saccharomyces cerevisiae*.

[0112] Integration sites in the Saccharomyces cerevisiae genome can be selected from the following sites: δ site, 1-10 random locations among multiple δ genes on the Saccharomyces cerevisiae chromosome; rDNA site, 1-10 random locations among multiple ribosomal genes on the Saccharomyces cerevisiae chromosome; HIS3 site, the location of the HIS3 gene in the histidine biosynthesis pathway on the Saccharomyces cerevisiae chromosome; or Trp1 site, the location of the Trp1 gene in the tryptophan biosynthesis pathway on the Saccharomyces cerevisiae chromosome.

[0113] The available selection markers for Saccharomyces cerevisiae gene integration can be any selection marker known to those skilled in the art, as long as the selection markers used for integrating different fragments into the same Saccharomyces cerevisiae strain are different from each other. Common selection markers include auxotrophic selection markers and resistance selection markers. Among them, auxotrophic selection markers can be selected from LEU, HIS, URA, or TRP. Resistance selection markers can be G418 or HYG.

[0114] The promoter can be any promoter that can be used in Saccharomyces cerevisiae. For example, the promoter can be selected from the group consisting of the following promoters: pPGK, pADH1, pTDH3, pTEF2, pPDC1, and pTPI1. The terminator can be any terminator that can be used in Saccharomyces cerevisiae. For example, the terminator can be selected from the group consisting of the following terminators: PGK1t, ADH1t, and FBA1t.

[0115] Homologous recombinant fragments or recombinant plasmids are introduced into *Saccharomyces cerevisiae* using methods known in the art. The transformation of *Saccharomyces cerevisiae* can be achieved using various transformation methods known to those skilled in the art, such as electroconversion and lithium acetate chemical conversion.

[0116] The activity level of ergosterol synthase in Saccharomyces cerevisiae can be reduced by decreasing the expression level of the ergosterol synthase gene erg7 or by reducing the activity of the ergosterol synthase protein.

[0117] In the method for constructing the recombinant strain of the present invention, any method known to those skilled in the art can be used to reduce the expression level of the target gene (e.g., the erg7 gene) or reduce the activity of ergosterol synthase (including inactivating the target gene). Such methods include, but are not limited to, gene knockout, site-directed mutagenesis, or RNA interference (RNAi).

[0118] In the embodiments of the present invention relating to RNAi, there is no particular limitation on the method of achieving RNAi. Various RNAi technologies known to those skilled in the art can be used. For example, the transcription or translation of a target gene (e.g., the erg7 gene) can be blocked by using small interfering RNA (siRNA), antisense nucleic acid, microRNA, etc., thereby causing a decrease in the expression level of the target gene.

[0119] A second aspect of the present invention is to provide recombinant bacteria prepared by the method described in the first aspect of the present invention.

[0120] The third aspect of the present invention is the application of the recombinant bacteria described in the second aspect in the production of 3β-O-Glc-DM.

[0121] A fourth aspect of the present invention provides a method for producing 3β-O-Glc-DM, the method comprising fermenting the recombinant bacteria described in the second aspect to obtain 3β-O-Glc-DM.

[0122] In this invention, the fermentation of the recombinant bacteria can be carried out according to various known methods in the art.

[0123] Beneficial technical effects

[0124] This invention obtained a recombinant strain producing dammarenediol-II glycoside 3β-O-Glc-DM by transferring the dammarenediol-II synthase gene and the ginseng glycosyltransferase PgUGT74AE2 gene into *Saccharomyces cerevisiae* with the hexokinase HXK2 gene knocked out. Based on this, CRISPR / Cas9 technology was used to promote the integration of exogenous genes into the *Saccharomyces cerevisiae* genome, overexpressing upstream key enzymes isopentenyl pyrophosphate isomerase, farnesyl pyrophosphate synthase, squalene monooxygenase, and squalene synthase, downregulating lanosterol synthase expression using antisense technology, and overexpressing transcription factor HAC1, further increasing the yield of 3β-O-Glc-DM in the engineered strain. This invention is the first to obtain a high-yield engineered strain producing the rare ginsenoside 3β-O-Glc-DM, laying the foundation for its large-scale production. Attached Figure Description

[0125] Figure 1 A schematic diagram of the construction of the knockout element LoxP-KanMX-LoxP is shown.

[0126] Figure 2 The electrophoresis diagrams show the amplification of the left and right homologous arms of the HXK2 (hexokinase 2) gene, a primary metabolism-related gene in Saccharomyces cerevisiae. 1: Left homologous arm of HXK2; 2: Right homologous arm of HXK2.

[0127] Figure 3 The image shows an amplified electrophoresis diagram of the KanMX gene expression cassette.

[0128] Figure 4 The electrophoresis results of the knockout element LoxP-KanMX-LoxP are shown. Among them, 1: HXK2 gene knockout element...

[0129] Figure 5 A schematic diagram of diagnostic PCR primers for gene knockout strain verification is shown.

[0130] Figure 6The electrophoresis verification results of the HXK2 gene knockout strain are shown. Primers for lanes 1 and 5: HXK2-1F / KanMX-R; primers for lanes 2 and 6: KanMX-F / HXK2-2R; primers for lanes 3 and 7: HXK2-1F / HXK2-2R; primers for lanes 4 and 8: HXK2-YF / YR. Y-ΔHXK2: YPH499 with the HXK2 gene knocked out; WT: YPH499 genome.

[0131] Figure 7 The electrophoresis results of integrated modules I and II are shown.

[0132] Figure 8 The electrophoresis results for integrated modules IV, V, and VI are shown.

[0133] Figure 9 The electrophoresis results for integrated modules VII, VIII, and IX are shown.

[0134] Figure 10 A schematic diagram of the construction of each integrated module is shown.

[0135] Figure 11 The Cas9 expression box element P is shown. TEF1 -Cas9-T CYC1 (1) and fusion element gRNA-P TEF1 -Cas9-T CYC1 (2) Electrophoresis results.

[0136] Figure 12 The standard curve for the standard 3β-O-Glc-DM is shown.

[0137] Figure 13 The HPLC detection results of 3β-O-Glc-DM in recombinant bacteria are shown.

[0138] Figure 14 The LC-MS detection results of 3β-O-Glc-DM in recombinant bacteria are shown.

[0139] Figure 15 The genotype of recombinant strain Y1C and the screening results of high-yielding strains are shown. Figure 15 A shows the genotype of recombinant strain Y1C; Figure 15 B shows the yield of 3β-O-Glc-DM from recombinant strains Y1C-1 to Y1C-20.

[0140] Figure 16 The genotype of recombinant strain Y1CS and the screening results of high-yielding strains are shown. Figure 16 A shows the genotype of the recombinant strain Y1CS; Figure 16B shows the yield of 3β-O-Glc-DM from recombinant strains Y1CS-1 to Y1CS-20.

[0141] Figure 17 The genotypes of recombinant bacteria Y1CSB, Y1CSH, and Y1CSP, as well as the screening results of high-yielding strains, are shown. Figure 17 A shows the genotypes of recombinant bacteria Y1CSB, Y1CSH, and Y1CSP; Figure 17 B shows the yield of 3β-O-Glc-DM from recombinant strains Y1CSB-1 to Y1CSB-10, Y1CSH-1 to Y1CSH-10, and Y1CSP-1 to Y1CSP-10.

[0142] Figure 18 The results of shake-flask culture yield of recombinant yeast producing 3β-O-Glc-DM are shown.

[0143] Figure 19 The results of high-density fermentation yield detection for recombinant yeast producing 3β-O-Glc-DM are shown. Detailed Implementation

[0144] The following describes exemplary embodiments of the present invention. It should be understood by those skilled in the art that these embodiments do not limit the specific implementation of the invention, but should be understood to include all variations, equivalents, or substitutions within the spirit and scope of the invention. Various modifications and other embodiments are within the capabilities of those skilled in the art and are contemplated to fall within the scope of the present invention.

[0145] Unless otherwise stated, the experimental methods used below are conventional methods well known to those skilled in the art, and can be performed using standard procedures described in the following works: Sambrook et al., *Molecular Cloning: A Laboratory Manual* (3rd edition) (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2001); Davis et al., *Basic Methods in Molecular Biology* (Elsevier Science Publishing, Inc., New York, USA, 1995); and Juan S. Bonifacino et al., *Current Protocols in Cell Biology* (John Wiley and Sons, Inc.).

[0146] Example

[0147] The invention can be better understood with the help of the following embodiments, which are only used to illustrate the invention and should not be construed as limiting the invention.

[0148] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0149] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0150] Example 1. Construction of the yeast gene knockout element LoxP-KanMX-LoxP

[0151] Gene knockout elements consist of portions homologous to the 5′ and 3′ untranslated regions of the target gene at both ends, with the kanamycin resistance gene (KanMX) in the middle serving as a reporter gene for screening gene deletion mutants. Using plasmid pUC6 as a template, the Kan resistance gene expression cassette (kanMX) is amplified using primers KAN-F / R. Overlap extension PCR (OE-PCR) is then used to fuse the left and right homologous arms of the target gene with the Kan resistance gene expression cassette (kanMX) to obtain the target gene knockout element. The LoxP-KanMX-LoxP knockout element is obtained as follows... Figure 1 As shown.

[0152] The nucleotide sequence (NC_001139.9) of HXK2 (hexokinase 2), a gene related to primary metabolism in *Saccharomyces cerevisiae*, was obtained from GenBank Nucleotide (http: / / www.ncbi.nlm.nih.gov / nuccore / ). Primers were designed based on the sequence information (Table 1). HXK2-1F / R and 2F / R were used as upstream and downstream primers, respectively. Using *Saccharomyces cerevisiae* INVSC1 genomic DNA as a template, the left and right homologous arms of the above-mentioned target genes were amplified using high-fidelity enzymes (sequences SEQ ID NO:12 and SEQ ID NO:13, respectively). Figure 2 The band size is approximately 360–400 bp.

[0153] Table 1 Primer sequences for gene knockout elements

[0154]

[0155] Primers were designed based on the pUC6 sequence information (Table 2). Using this plasmid as a template, the Kan resistance expression cassette (kanMX) (SEQ ID NO:14) was amplified using primers KAN-F and KAN-R in Table 2. Figure 3The target band was 1613 bp in size, and the aminoglycoside phosphotransferase expressed by it could inactivate kanamycin, thus serving as a resistance marker for screening gene knockout bacteria.

[0156] Table 2 Primer sequences for cloning the resistance gene

[0157]

[0158] The PCR amplification system is as follows (50 μL):

[0159]

[0160] The PCR amplification conditions are as follows:

[0161] 98℃, 30s;

[0162] 98℃, 10s; 55-60℃, 30s; 72℃, 1kb / 15-30s; 30 cycles;

[0163] 72℃, 5 min;

[0164] 4℃, ∞;

[0165] The PCR products were detected by 1.0% agarose gel electrophoresis.

[0166] The left and right homologous arms of the target gene and the resistance gene expression cassette were recovered and amplified using OE-PCR with primers CF / CR to obtain the target gene knockout element.

[0167] OE-PCR reaction system (50uL):

[0168]

[0169] OE-PCR amplification conditions:

[0170] 95℃, 30s;

[0171] 95℃, 10s; 50-60℃, 30s; 72℃, 1kb / 15-30s; 10 cycles;

[0172] 95℃, 10s; 55℃, 30s; 72℃, 1kb / 15-30s; 20 cycles;

[0173] 72℃, 5 min;

[0174] 4℃, ∞;

[0175] Electrophoresis results showed that the actual size of the fusion fragment was consistent with the theoretical value. Figure 4 ).

[0176] 4 μL of OE-PCR product was ligated into 1 μL of the cloning vector pEASY-Blunt simple vector and transformed into Trans1-T1 competent cells. Positive transformants were screened, plasmids were extracted, and the plasmids were sequenced to obtain the full sequence of the target gene knockout element LoxP-KanMX-LoxP. The successfully constructed plasmid was named pEASY-HXK2.

[0177] Example 2: Transformation and Screening of Saccharomyces cerevisiae

[0178] The yeast gene knockout element LoxP-KanMX-LoxP was transformed into competent *Saccharomyces cerevisiae* YPH499 cells. The following transfection mixture was added sequentially using the LiAC / ssDNA / PEG yeast transfection method:

[0179]

[0180]

[0181] A control was prepared by replacing the target gene knockout element with 50 μL ddH2O. An appropriate amount of competent *Saccharomyces cerevisiae* YPH499 cells were added to the above system for yeast transformation. After transformation, the cell slurries from the experimental and control groups were spread onto solid culture plates containing antibiotic G418 (0.3 mg / ml) using a glass rod and cultured at 30°C for 2 days until transformants appeared. (Note: After adding PEG3350, the cell slurry should be thoroughly resuspended; ssDNA needs to be pre-treated at 100°C for 10 min to denature, and then immediately cooled on ice).

[0182] Transformants with good growth were selected from G418 resistant plates. Genomic DNA was extracted from these transformants and used as templates. Corresponding diagnostic PCR primers HXK2-1F / HXK2-2R, HXK2-1F / MA, MB / HXK2-2R, and HXK2-YF / HXK2-YR (Tables 1 and 3) were selected to screen for gene knockout-positive transformants. The diagnostic PCR primer design is as follows: Figure 5 As shown.

[0183] Table 3 Diagnostic PCR Primer Sequences

[0184]

[0185] Positive transformants with gene knockout were screened using diagnostic PCR. 5 μL of each diagnostic PCR sample was analyzed by agarose gel electrophoresis. The results showed that diploid and haploid *Saccharomyces cerevisiae* mutants with the HXK2 gene deletion were successfully screened using homologous recombination. Figure 6 ).

[0186] like Figure 6 As shown, using a single colony of *Saccharomyces cerevisiae* YPH499 transformed with the HXK2 knockout element and its corresponding wild-type yeast genomic DNA as templates, PCR diagnostic experiments were performed using three pairs of diagnostic primers: HXK2-1F / KanMX-R, KanMX-F / HXK2-2R, and HXK2-1F / 2R. Compared with the control group, the transformed group showed bands consistent with the theoretical size of the knockout element, indicating that the knockout element had undergone homologous recombination, replacing the target gene HXK2 on the genome. Simultaneously, when verified using the gene's own primer HXK2-YF / YR, the transformed group did not show the corresponding band of the target gene, indicating that the approximately 1.0 kb blurred band in the transformed group was non-specific amplification rather than diploid gene knockout. Based on these results, it is demonstrated that the knockout element successfully replaced the target gene on the *Saccharomyces cerevisiae* genome using the homologous recombination mechanism, resulting in a *Saccharomyces cerevisiae* HXK2 gene-deficient strain, named Y-ΔHXK2.

[0187] The Saccharomyces cerevisiae gene-deficient strains constructed in this embodiment are shown in Table 4.

[0188] Table 4. Gene-deficient Saccharomyces cerevisiae strains

[0189]

[0190] To investigate the growth status of *Saccharomyces cerevisiae* after gene knockout in different culture media, the time-growth curve of Y-ΔHXK2 was determined. The genetically modified *Saccharomyces cerevisiae* and wild-type *Saccharomyces cerevisiae* were inoculated into 2 mL LYPD liquid medium and cultured at 30°C, 220 rpm, and shaking for 14 h to induce the late logarithmic growth phase, which was then used as the seed culture. The seed culture was then divided into two groups based on the initial OD values. 600 The yeast was inoculated at a ratio of 0.4 in 30 mL of LYPD and YPG liquid medium, with three replicates. The cultures were incubated at 30 °C with shaking at 220 rpm. The absorbance (OD) of the gene-deficient *Saccharomyces cerevisiae* cultures obtained in different media was measured at 600 nm using a UV spectrophotometer at 4 h, 8 h, 12 h, and 16 h. The results showed that the growth rate of the knockout strain Y-ΔHXK2 was slightly higher than that of the control strain YPH499 in both media, indicating that knocking out the HXK2 gene did not affect the normal growth of YPH499.

[0191] Example 3: Construction of Gene Expression Cassettes

[0192] Construction of expression cassettes for genes DS-GFP, PgUGT74AE2, and tHMG1

[0193] According to the GenBank registration information, the cDNA (No. AB265170.1) sequence information of the dammarene diol-II synthase gene DS from ginseng was obtained. Based on the codon preference of Saccharomyces cerevisiae, the optimized DS gene and DS-GFP sequence (SEQ ID NO:1) were synthesized to obtain the plasmid pESC-HIS-DS-GFP (prepared according to the method described in Liang Huichao et al., Expression, localization and function of ginseng dammarene diol-II synthase in Saccharomyces cerevisiae, Acta Pharmaceutica Sinica 2016, 51(6): 998-1003, which is incorporated herein by reference in its entirety).

[0194] Using plasmid pESC-HIS-DS-GFP as a template, the synDS-GFP gene (3036 bp) was amplified by PCR using primers DS-TEF1-F and DS-CYC1-R (Table 5). Using *Saccharomyces cerevisiae* INVSC1 genomic DNA as a template, the *Saccharomyces cerevisiae* promoter TEF1 (430 bp, SEQ ID NO:15) and terminator CYC1 (189 bp, SEQ ID NO:16) sequence fragments were amplified using overlapping extension primers A-TEF1-delta1-F / TEF1-DS-R and CYC1-DS-F / CYC1-PGK1-R (Table 5), respectively. Using OE-PCR with primers A-TEF1-delta1-F and CYC1-PGK1-R (Table 5), the synDS-GFP gene expression cassette element was fused to obtain P. TEF1 -synDS-GFP-T CYC1 .

[0195] Based on GenBank registration information, the cDNA sequence information of the glycosyltransferase gene PgUGT74AE2 (No. JX898529.1) derived from ginseng was obtained. Based on the codon preference of Saccharomyces cerevisiae, the optimized gene sequence PgUGT74AE2 (SEQ ID NO:2) was synthesized, and the plasmid pUC57-PgUGT74AE2 was obtained.

[0196] Using plasmid pUC57-PgUGT74AE2 as a template, the synPgUGT74AE2 gene (1356 bp) was amplified using primers UGT74AE2-TDH3-F and UGT74AE2-ADH2-R (Table 5). Using Saccharomyces cerevisiae INVSC1 genomic DNA as a template, the Saccharomyces cerevisiae promoter TDH3 (800 bp, SEQ ID NO:17) and terminator ADH2 (566 bp, SEQ ID NO:18) sequence fragments were amplified using primers TDH3-ADH1-F / TDH3-UGTPg1-R and ADH2-UGTPg1-F / ADH2-HIS-R (Table 5), respectively. OE-PCR was performed using primers TDH3-ADH1-F and ADH2-HIS-R (Table 5) to fuse the synDS-GFP gene expression cassette element: P. TDH3 -synPgUGT74AE2-T ADH2 .

[0197] Primers tHMG1-PGK1-F / tHMG1-ADH1-R were designed based on the cDNA sequence (No. NM_001182434.1) of the Saccharomyces cerevisiae 3-hydroxy-3-methylglutaryl-CoA reductase gene HMG1 registered in GenBank.

[0198] Using *Saccharomyces cerevisiae* INVSC1 genomic DNA as a template, the tHMG1 gene (1634 bp, SEQ ID NO:3) encoding the HMGR catalytic domain was amplified using primers tHMG1-PGK1-F and tHMG1-ADH1-R (Table 5). Using *Saccharomyces cerevisiae* INVSC1 genomic DNA as a template, the promoter PGK1 (750 bp, SEQ ID NO:19) and terminator ADH1 (158 bp, SEQ ID NO:20) sequence fragments were amplified using primers PGK1-CYC1-F / PGK1-tHMG1-R and ADH1-tHMG1-F / ADH1-R (Table 5), respectively. OE-PCR was performed using primers PGK1-CYC1-F and ADH1-R to fuse the tHMG1 gene expression cassette element: P. PGK1 -tHMG1-T ADH1 .

[0199] Table 5 Primer sequences for gene cloning

[0200]

[0201]

[0202] 4 μL of OE-PCR product was ligated into 1 μL of the cloning vector pEASY-Blunt-Zero vector and transformed into Trans1-T1 competent cells. Positive transformants were screened, and plasmids were extracted and named pEASY-DS-GFP, pEASY-PgUGT74AE2, and pEASY-tHMG1, respectively. Sequencing results of the target fragment were consistent with the theoretical sequence.

[0203] Construction of expression cassettes for genes IDI1, ERG20, ERG1, ERG9, and ERG7

[0204] Primers IDI1-TDH3-F and IDI1-TPI1-R were designed based on the DNA sequence of the Saccharomyces cerevisiae isopentenyl pyrophosphate isomerase IDI1 gene (No. NC_001148.4) registered in GenBank (Table 6).

[0205] Using *Saccharomyces cerevisiae* YPH499 genomic DNA as a template, the IDI1 gene (867 bp, SEQ ID NO:4) was amplified by PCR using primers IDI1-TDH3-F and IDI1-TPI1-R (Table 6). Using *Saccharomyces cerevisiae* YPH499 genomic DNA as a template, the promoter TDH3 (800 bp, SEQ ID NO:17) and terminator TPI1 (422 bp, SEQ ID NO:21) sequence fragments were amplified by using overlapping extension primers TDH3-TY4-1-F / TDH3-IDI1-R and TPI1-IDI1-F / TPI1-PGK1-R, respectively. OE-PCR was then performed using primers TDH3-TY4-1-F and TPI1-PGK1-R to fuse the IDI1 gene expression cassette element: P. TDH3 -IDI1-T TPI1 .

[0206] Primers ERG20-PGK1-F / ERG20-ADH1-R were designed based on the DNA sequence of the Saccharomyces cerevisiae farnesyl pyrophosphate synthase gene ERG20 (No. NC_001142.9) registered in GenBank (Table 6).

[0207] Using *Saccharomyces cerevisiae* YPH499 genomic DNA as a template, the ERG20 gene (1059 bp, SEQ ID NO: 5) was amplified by PCR using primers ERG20-PGK1-F and ERG20-ADH1-R (Table 6). Using *Saccharomyces cerevisiae* YPH499 genomic DNA as a template, the promoter PGK1 (750 bp, SEQ ID NO: 19) and terminator ADH1 (158 bp, SEQ ID NO: 20) sequence fragments were amplified by PCR using overlapping extension primers PGK1-TPI1-F / PGK1-ERG20-R and ADH1-ERG20-F / ADH1-TEF1-R (Table 6), respectively. OE-PCR was then performed using primers PGK1-TPI1-F and ADH1-TEF1-R to fuse the ERG20 gene expression cassette element: P. PGK1 -ERG20-T ADH1 .

[0208] Primers ERG1-PGK1-F / ERG1-ADH1-R were designed based on the DNA sequence of the Saccharin monooxygenase gene ERG1 registered in GenBank (No. NC_001139.9) (Table 6).

[0209] Using *Saccharomyces cerevisiae* YPH499 genomic DNA as a template, the ERG1 gene (1491 bp, SEQ ID NO: 6) was amplified by PCR using primers ERG1-PGK1-F / ERG1-ADH1-R (Table 6). Using *Saccharomyces cerevisiae* YPH499 genomic DNA as a template, the promoter PGK1 (750 bp, SEQ ID NO: 19) and terminator ADH1 (158 bp, SEQ ID NO: 20) sequence fragments were amplified by PCR using overlapping extension primers PGK1-CYC1-F / PGK1-ERG1-R and ADH1-ERG1-F / ADH1-TEF1-R (Table 6), respectively. Using OE-PCR with primers PGK1-CYC1-F and ADH1-TEF1-R, the ERG1 gene expression cassette element was fused to obtain P. PGK1 -ERG1-T ADH1 .

[0210] Primers ERG9-TEF1-F / ERG9-CYC1-R were designed based on the DNA sequence of the Saccharin synthase gene ERG9 registered in GenBank (No. NC_001140.6) (Table 6).

[0211] Using *Saccharomyces cerevisiae* YPH499 genomic DNA as a template, the ERG9 gene (1335 bp, SEQ ID NO:7) was amplified by PCR using primers ERG9-TEF1-F / ERG9-CYC1-R (Table 6). Using *Saccharomyces cerevisiae* YPH499 genomic DNA as a template, the promoter TEF1 (430 bp, SEQ ID NO:15) and terminator CYC1 (189 bp, SEQ ID NO:16) sequence fragments were amplified by PCR using overlapping extension primers TEF1-ADH1-F / TEF1-ERG9-R and CYC1-ERG9-F / CYC1-PGK1-R (Table 6), respectively. Using OE-PCR with primers TEF1-ADH1-F and CYC1-PGK1-R, the ERG9 gene expression cassette element was fused: P TEF1 -ERG9-T CYC1 .

[0212] With plasmid pESC-URA-ERG7 - Using the template (Wang Qinghua et al., Inhibiting the expression of lanosterol synthase gene in Saccharomyces cerevisiae using antisense RNA technology, Acta Pharmaceutica Sinica 2015, 50(1): 118-122, which is incorporated herein by reference in its entirety), the long fragment of the ERG7 antisense gene (2247bp, SEQ ID NO:8) was amplified using the primers ERG7-TEF1-F / ERG7-CYC1-R in Table 6; using Saccharomyces cerevisiae genomic DNA as a template, the promoter TEF1 (430bp, SEQ ID NO:15) and terminator CYC1 (189bp, SEQ ID NO:16) sequence fragments of Saccharomyces cerevisiae were amplified using the primers TEF1-ADH1-F / TEF1-ERG7-R and CYC1-ERG7-F / CYC1-LEU-R listed in Table 6, respectively. OE-PCR was performed using primers TEF1-ADH1-F and CYC1-LEU-R to fuse the ERG7 antisense gene expression cassette element: P TEF1 -ERG7 - -T CYC1 .

[0213] Table 6 Primer sequences for gene cloning

[0214]

[0215]

[0216] 4 μL of OE-PCR product was ligated into 1 μL of the cloning vector pEASY-Blunt-Zero and transformed into Trans1-T1 competent cells. Positive transformants were screened, and plasmids were extracted and named pEASY-IDI1, pEASY-ERG1, pEASY-ERG20, pEASY-ERG9, and pEASY-ERG7, respectively. Sequencing confirmed that the target fragment sequence was consistent with the expected sequence.

[0217] Construction of expression cassettes for genes BiP, HAC1, and PDI1

[0218] Primers BIP-TEF1-F and BIP-CYC1-R were designed based on the DNA sequence of the Saccharomyces cerevisiae molecular chaperone BiP gene (No. NC_001142.9) registered in GenBank (Table 7).

[0219] Using *Saccharomyces cerevisiae* YPH499 genomic DNA as a template, the BiP gene (2049 bp, SEQ ID NO: 9) was amplified by PCR using primers BIP-TEF1-F and BIP-CYC1-R (Table 7). Using *Saccharomyces cerevisiae* genomic DNA as a template, the promoter TEF1 (430 bp, SEQ ID NO: 15) and terminator CYC1 (189 bp, SEQ ID NO: 16) sequence fragments were amplified by PCR using overlapping extension primers GJ-F / TEF1-BIP-R and CYC1-BIP-F / GJ-R (Table 7), respectively. OE-PCR was then performed using primers GJ-F and GJ-R to fuse the BiP gene expression cassette element: P. TEF1 -BiP-T CYC1 .

[0220] Primers HAC1-TEF1-F and HAC1-CYC1-R were designed based on the DNA sequence of the Saccharomyces cerevisiae transcription factor HAC1 gene (No. NC_001138.5) registered in GenBank (Table 7).

[0221] Using *Saccharomyces cerevisiae* YPH499 genomic DNA as a template, the HAC1 gene (717 bp, SEQ ID NO: 10) was amplified by PCR using primers HAC1-TEF1-F and HAC1-CYC1-R (Table 7). Using *Saccharomyces cerevisiae* YPH499 genomic DNA as a template, the promoter TEF1 (430 bp, SEQ ID NO: 15) and terminator CYC1 (189 bp, SEQ ID NO: 16) sequence fragments were amplified by PCR using overlapping extension primers GJ-F / TEF1-HAC1-R and CYC1-HAC1-F / GJ-R (Table 7), respectively. Using OE-PCR with primers GJ-F and GJ-R, the BiP gene expression cassette element was fused: P TEF1 -HAC1-T CYC1 .

[0222] Primers PDI1-TEF1-F and HAC1-CYC1-R were designed based on the DNA sequence (No. NC_001135.5) of the Saccharomyces cerevisiae molecular chaperone (disulfide isomerase) PDI1 gene registered in GenBank (Table 7).

[0223] Using *Saccharomyces cerevisiae* YPH499 genomic DNA as a template, the PDI1 gene (1569 bp, SEQ ID NO: 11) was amplified by PCR using primers PDI1-TEF1-F and PDI1-CYC1-R (Table 7). Using *Saccharomyces cerevisiae* YPH499 genomic DNA as a template, the promoter TEF1 (430 bp, SEQ ID NO: 15) and terminator CYC1 (189 bp, SEQ ID NO: 16) sequence fragments were amplified by PCR using overlapping extension primers GJ-F / TEF1-PDI1-R and CYC1-PDI1-F / GJ-R, respectively. Using OE-PCR with primers GJ-F and GJ-R, the PDI1 gene expression cassette element was fused to obtain P... TEF1 -PDI1-T CYC1 .

[0224] Table 7 Primer sequences for gene cloning and plasmid construction

[0225]

[0226] 4 μL of OE-PCR product was ligated into 1 μL of the cloning vector pEASY-Blunt-Zero vector and transformed into Trans1-T1 competent cells. Positive transformants were screened, and plasmids were extracted and named pEASY-BiP, pEASY-HAC1, and pEASY-PDI1, respectively. Sequencing confirmed that the target fragment sequence was consistent with the expected sequence.

[0227] Example 4: Construction of the Genome Integration Module

[0228] Integration Module I δ1-1-P TEF1 -synDS-GFP-T CYC1 -P PGK1 -tHMG1-T ADH1 Construction

[0229] Using *Saccharomyces cerevisiae* INVSC1 genomic DNA as a template, the *Saccharomyces cerevisiae* genome Delta1(δ1) site sequence fragment (410 bp, SEQ ID NO: 22) was amplified using primers Delta1-2F and Delta2-1R listed in Table 8. This fragment was then ligated into the pEASY-Blunt-Simple vector to obtain the plasmid pEASY-INδ. Using plasmid pEASY-INδ as a template, the genomic integration site δ1-1 fragment was amplified using primers Delta1-2F and Delta1-TEF1-2R listed in Table 8. The δ1-1 fragment was then ligated into the pEASY-Blunt-Simple vector using OE-PCR with primers Delta1-2F and CYC1-PGK1-R listed in Table 8. TEF1 -synDSGFP-T CYC1 By fusing, the element δ1-1-P is obtained. TEF1 -synDS-GFP-T CYC1 .

[0230] Referring to the OE-PCR reaction system and conditions, using element δ1-1-P TEF1 -synDS-GFP-T CYC1 and P PGK1 -tHMG1-T ADH1 Using the primers Delta1-2F and ADH1-TDH3-R listed in Table 8 as templates, a second round of OE-PCR was performed to obtain the integrated module I δ1-1-P. TEF1 -synDS-GFP-T CYC1 -P PGK1 -tHMG1-T ADH1 4 μL of the second-round OE-PCR product was ligated into 1 μL of the cloning vector pEASY-Blunt-Zero vector and transformed into Trans1-T1 competent cells; positive transformants were screened, plasmids were extracted and sequenced for verification, and the recombinant plasmid was named pEASY-3. Gel electrophoresis and DNA sequencing results showed that the amplified fragment matched the theoretical sequence. Figure 7 ).

[0231] Integration Module II overlap-P TDH3 -synPgUGT74AE2-T ADH2Construction of -HIS-δ1-2

[0232] Using plasmid pEASY-3 as a template, a 455 bp fragment from the 3' end of the tHMGR gene expression cassette was amplified using primers B-400F and ADH1-TDH3-R listed in Table 8. This fragment was then used for OE-PCR. Following the OE-PCR reaction system and conditions, the fragment was amplified using element P. TDH3 -synPgUGT74AE2-T ADH2 Using the fragment amplified above as a template, OE-PCR was performed with primers B400-F and ADH2-HIS-R listed in Table 8 to obtain the overlap-P element. TDH3 -synPgUGT74AE2-T ADH2 .

[0233] Using plasmid pESC-HIS as a template, the HIS expression cassette sequence (1169 bp, SEQ ID NO: 23) of the resistance marker gene was amplified using primers HIS-ADH2-F and HIS-Delta2-R listed in Table 8. Using plasmid pEASY-INδ as a template, the δ1-2 fragment of the genome integration site was amplified using primers Delta2-HIS-2F and Delta2-1R listed in Table 8. Following the OE-PCR reaction system and conditions, the δ1-2 fragment and the HIS expression cassette sequence were fused using primers HIS-ADH2-F and Delta2-1R listed in Table 8 to obtain HIS-δ1-2.

[0234] Referring to the OE-PCR reaction system and conditions, the element overlap-P TDH3 -synPgUGT74AE2-T ADH2 Using HIS-δ1-2 as a template, a second round of OE-PCR was performed with primers B400-F and Delta2-1R listed in Table 8 to obtain the integration module II overlap-P. TDH3 -synPgUGT74AE2-T ADH2 -HIS-δ1-2. 4 μL of the second-round OE-PCR product was ligated into 1 μL of the cloning vector pEASY-Blunt-Zero vector and transformed into Trans1-T1 competent cells; positive transformants were screened, plasmids were extracted and sequenced for verification, and the recombinant plasmid was named pEASY-1. Gel electrophoresis and DNA sequencing results showed that the amplified fragment matched the theoretical sequence. Figure 7 ).

[0235] Integration Module Ⅳδ4-1-P TDH3 -IDI1-T TPI1 -P PGK1 -ERG20-T ADH1 Construction

[0236] Using the genomic DNA of *Saccharomyces cerevisiae* YPH499 as a template, the Delta4 (δ4) site sequence fragment (371 bp, SEQ ID NO: 24) of the *Saccharomyces cerevisiae* genome was amplified using primers TY4-F1 and TY4-R2 listed in Table 8. This fragment was then ligated into the pEASY-Blunt-Simple vector to obtain plasmid pEASY-TY4. Using plasmid pEASY-TY4 as a template, the δ4-1 genomic integration site fragment was amplified using primers TY4-F1 and TY4-1-TDH3-R listed in Table 8. The δ4-1 fragment was then ligated into the P… using OE-PCR with primers TY4-F1 and TPI1-PGK1-R listed in Table 8. TDH3 -IDI1-T TPI1 The components δ4-1-P were fused together to obtain the element. TDH3 -IDI1-T TPI1 .

[0237] Referring to the OE-PCR reaction system and conditions, using element δ4-1-P TDH3 -IDI1-T TPI1 and P PGK1 -ERG20-T ADH1 Using the template, a second round of OE-PCR was performed with primers TY4-F1 and ADH1-TEF1-R listed in Table 8 to obtain the integrated module IVδ4-1-P. TDH3 -IDI1-T TPI1 -P PGK1 -ERG20-T ADH1 4 μL of the second-round OE-PCR product was ligated into 1 μL of the cloning vector pEASY-Blunt-Zero vector and transformed into Trans1-T1 competent cells; positive transformants were screened, plasmids were extracted and sequenced for verification, and the recombinant plasmid was named pEASY-S28. Gel electrophoresis and DNA sequencing results showed that the amplified fragment matched the theoretical sequence. Figure 8 ).

[0238] Integration Module VIP PGK1 -ERG1-T ADH1 -P TEF1 -ERG7 - -T CYC1 Construction of -LEU-δ4-2

[0239] Using plasmid pESC-LEU as a template, the LEU2 expression cassette sequence (2178 bp, SEQ ID NO: 25) of the resistance marker gene was amplified using primers LEU-CYC1-F and LEU-TY4-2-R listed in Table 8. Using plasmid pEASY-TY4 as a template, the δ4-2 fragment of the genome integration site was amplified using primers TY4-2-LEU-F and TY4-2R listed in Table 8. Following the OE-PCR reaction system and conditions, the δ4-2 fragment and the LEU2 expression cassette sequence were fused using primers LEU-CYC1-F / TY4-2R to obtain LEU2-δ4-2.

[0240] Referring to the OE-PCR reaction system and conditions, with element P PGK1 -ERG1-T ADH1 P TEF1 -ERG7 - -T CYC1 Using LEU2-δ4-2 as a template, a second round of OE-PCR was performed with the primers PGK1-CYC1-F and TY4-2R listed in Table 8 to obtain the integration module VIP. PGK1 -ERG1-T ADH1 -P TEF1 -ERG7 - -T CYC1 -LEU2-δ4-2. 4 μL of the second-round OE-PCR product was ligated into 1 μL of the cloning vector pEASY-Blunt-Zero vector and transformed into Trans1-T1 competent cells; positive transformants were screened, plasmids were extracted and sequenced for verification, and the recombinant plasmid was named pEASY-S1319. Gel electrophoresis and DNA sequencing results showed that the amplified fragment matched the theoretical sequence. Figure 8 ).

[0241] Integration module V overlap-P TEF1 -ERG9-T CYC1 -overlap construction

[0242] Using plasmid pEASY-S28 as a template, a 515bp fragment from the 3' end of the ERG20 gene expression cassette was amplified using primers S28-400F / TEF1-ERG9-R listed in Table 8, serving as the first fragment for OE-PCR. Using plasmid pEASY-S1319 as a template, a 548bp fragment from the 5' end of the ERG1 gene expression cassette was amplified using primers PGK1-CYC1-F and S1319-400R listed in Table 8, serving as the second fragment for OE-PCR. Following the OE-PCR reaction system and conditions, element P... TEF1 -ERG9-T CYC1Using the two fragments obtained from the amplification above as templates, OE-PCR was performed using primers S28-400F and S1319-400R listed in Table 8 to fuse them into the integration module Voverlap-P. TEF1 -ERG9-T CYC1 -overlap. 4 μL of the second-round OE-PCR product was ligated into 1 μL of the cloning vector pEASY-Blunt-Zero vector and transformed into Trans1-T1 competent cells; positive transformants were screened, plasmids were extracted and sequenced for verification, and the recombinant plasmid was named pEASY-S813. Gel electrophoresis and DNA sequencing results showed that the amplified fragment matched the theoretical sequence. Figure 8 ).

[0243] Construction of recombinant vector prDNA-TRP

[0244] Using the genomic DNA of Saccharomyces cerevisiae YPH499 as a template, the genomic rDNA site sequence of Saccharomyces cerevisiae (1264bp, SEQ ID NO:26) was amplified using the primers rDNA1-MQWD-F and rDNA2-MQWD-R listed in Table 8, and then ligated into the pEASY-Blunt-Simple vector to obtain the plasmid pEASY-rDNA. Using plasmid pEASY-rDNA as a template, the genomic integration site rDNA-1 fragment was amplified using primers GJ-RDNA1-2U-F and rDNA1-MQWD-R listed in Table 8; the genomic integration site rDNA-2 fragment was amplified using primers rDNA2-TRP-F and GJ-RDNA2-PUC-R listed in Table 8; using plasmid pESC-TRP as a template, the resistance marker gene TRP expression cassette sequence (1365bp, SEQ ID NO:27) was amplified using primers TRP-MQWD-F and TRP-rDNA2-R listed in Table 8; and using plasmid pESC-TRP as a template, the plasmid backbone sequence was amplified using primers PUC-GJ-RDNA2-F and 2U-GJ-RDNA1-R listed in Table 8. Following the eFusion reaction system and conditions, the four fragments were seamlessly ligated; the ligation product was transformed into Trans1-T1 competent cells; positive transformants were screened, plasmids were extracted and sequenced for verification, and the recombinant plasmid was named plasmid prDNA-TRP.

[0245] Table 8 Primer sequences for the genome integration module

[0246]

[0247]

[0248] Integration module VII rDNA1-PTEF1 -BiP-T CYC1 Construction of -rDNA2

[0249] The plasmids prDNA-TRP and pEASY-BiP (obtained in Example 3) were double-digested using restriction endonucleases Sal I and Xho I; the digested target gene fragment and plasmid vector were recovered using a gel extraction kit; and the P gene fragment was ligated using T4 DNA ligase. TEF1 -BiP-T CYC1 The plasmid prDNA-TRP was ligated to obtain recombinant prDNA-TRP-BiP; the plasmid prDNA-TRP-BiP was then double-digested with restriction endonucleases BamHI and SacI to obtain the integration module VIIrDNA1-P. TEF1 -BiP-T CYC1 -rDNA2. Gel electrophoresis and DNA sequencing results showed that the amplified fragment matched the theoretical sequence. Figure 9 Integration module VIII rDNA1-P TEF1 -HAC1-T CYC1 Construction of -rDNA2

[0250] The plasmids prDNA-TRP and pEASY-HAC1 (obtained in Example 3) were double-digested using restriction endonucleases Sal I and Xho I; the digested target gene fragment and plasmid vector were recovered using a gel extraction kit; and the P DNA ligase was used to ligate the target gene fragment and plasmid vector. TEF1 -HAC1-T CYC1 The plasmid prDNA-TRP was ligated to obtain recombinant prDNA-TRP-HAC1; the plasmid prDNA-TRP-HAC1 was then double-digested with restriction endonucleases BamHI and SacI to obtain the integration module VIII rDNA1-P. TEF1 -HAC1-T CYC1 -rDNA2. Gel electrophoresis and DNA sequencing results showed that the amplified fragment matched the theoretical sequence. Figure 9 ).

[0251] Integration module IX rDNA1-P TEF1 -PDI1-T CYC1 Construction of -rDNA2

[0252] The plasmids prDNA-TRP and pEASY-PDI1 (obtained in Example 3) were double-digested using restriction endonucleases Sal I and Xho I; the digested target gene fragment and plasmid vector were recovered using a gel extraction kit; and the P DNA ligase was used to ligate the target gene fragment and plasmid vector. TEF1 -PDI1-T CYC1The plasmid prDNA-TRP was ligated to obtain recombinant prDNA-TRP-PDI1; the plasmid prDNA-TRP-PDI1 was then double-digested with restriction endonucleases BamHI and SacI to obtain the integrated module IXrDNA1-P. TEF1 -PDI1-T CYC1 -rDNA2. Gel electrophoresis and DNA sequencing results showed that the amplified fragment matched the theoretical sequence. Figure 9 ).

[0253] The diagram of each integrated module is shown below. Figure 10 As shown.

[0254] The double enzyme digestion system and digestion conditions used are shown below:

[0255] Double enzyme digestion system (100 μL):

[0256]

[0257] Enzyme digestion overnight at 37°C.

[0258] The connection system and connection conditions used are as follows:

[0259] Connection system (20 μL):

[0260]

[0261]

[0262] At room temperature, after ligation for 30 min, the cells were immediately transformed into E. coli Trans1-T1 competent cells; positive transformants were screened and plasmids were extracted.

[0263] The eFusion connectivity architecture and connectivity conditions used are as follows:

[0264] Connection system (15μL):

[0265]

[0266] At room temperature, after ligation for 30 min, the cells were immediately transformed into E. coli Trans1-T1 competent cells; positive transformants were screened and plasmids were extracted.

[0267] Construction of Cas9 expression plasmid

[0268] A CRISPR-Cas9 expression system based on the δ1 site of the Saccharomyces cerevisiae genome was constructed using humanized Cas9 protein derived from Streptococcus pyogenes.

[0269] Using plasmid FM-1 (Zhang et al., 2016, Fungal Genet Biol, 86:47–57) as a template, PCR was performed using primers Cas9-TEF1-F / Cas9-ADH2-R listed in Table 9 to amplify the humanized Cas9 gene sequence (4272bp, SEQ ID NO:28) derived from Streptococcus pyogenes. Using the Saccharomyces cerevisiae genome YPH499 as a template, the promoter TEF1p (430bp, SEQ ID NO:15) and terminator ADH2t (566bp, SEQ ID NO:29) sequence fragments of Saccharomyces cerevisiae were amplified using primers TEF1-SUP4t-MSC-F / TEF1-Cas9-R and ADH2-Cas9-F / ADH2-pESC-R listed in Table 9, respectively. OE-PCR was performed using the primers TEF1-SUP4t-MSC-F and ADH2-pESC-R listed in Table 9 to obtain the Cas9 gene expression cassette element: P TEF1 -Cas9-T CYC1 Gel electrophoresis and DNA sequencing results showed that the amplified fragment matched the theoretical sequence. Figure 11 ).

[0270] A δ1 site-specific gRNA expression cassette sequence fragment (458 bp, SEQ ID NO:30) containing the RNA polymerase III nucleolar small RNA (snoRNA) promoter SNR52p and the yeast tRNA gene terminator SUP4t was artificially synthesized and ligated into plasmid pUC57, named pUC57-sgRNA. Using plasmid pUC57-sgRNA as a template, the δ1 site-specific gRNA expression cassette sequence fragment was amplified using primers listed in Table 9: SNR52p-MSC-pESC-F / SUP4t-MSC-TEF1-R. The plasmid backbone fragment was amplified using pESC-URA as a template with primers listed in Table 9: pESC-ADH2-F / pESC-SNR52P-MCS-R.

[0271] Referring to the OE-PCR reaction system and conditions, with element P TEF1 -Cas9-T CYC1 Using the gRNA expression cassette sequence as a template, a second round of OE-PCR was performed using the primers SNR52p-MSC-pESC-F and ADH2-pESC-R listed in Table 9 to fuse the DNA element gRNA-P. TEF1 -Cas9-T CYC1 Gel electrophoresis and DNA sequencing results showed that the amplified fragment matched the theoretical sequence. Figure 11 ).

[0272] Following the eFusion reaction system and conditions, DNA element gRNA-P TEF1 -Cas9-T CYC1 Seamless ligation was performed with the plasmid backbone fragment; the ligation product was transformed into Trans1-T1 competent cells; positive transformants were screened, plasmids were extracted and sequenced for verification, and the recombinant plasmid was named p-Cas9-δ.

[0273] Table 9 Primers for constructing CRISPR-Cas9 expression plasmids

[0274]

[0275] Table 10 shows the constructed recombinant plasmids.

[0276]

[0277]

[0278] Example 5: Transformation of Saccharomyces cerevisiae and Screening of Recombinant Strains

[0279] The LiAc / SS Carrier DNA / PEG transformation method was used to transform the integration module I δ1-1-P TEF1 -synDS-GFP-T CYC1 -P PGK1 -tHMG1-T ADH1 and integration module II overlap-P TDH3 -synPgUGT74AE2-T ADH2 -HIS-δ1-2 was transformed into the Saccharomyces cerevisiae mutant strain Y-△HXK2 to construct the recombinant yeast strain Y1 producing 3β-O-Glc-DM; simultaneously, the integration module I δ1-1-P was incorporated. TEF1 -synDS-GFP-T CYC1 -P PGK1 -tHMG1-T ADH1 Integration Module II overlap-P TDH3 -synPgUGT74AE2-T ADH2 The HIS-δ1-2 and Cas9 expression plasmid p-Cas9-δ were transformed into the Saccharomyces cerevisiae mutant strain Y-△HXK2 to construct the recombinant yeast strain Y1C. Positive transformants were screened on SD auxotrophic medium. Transformants were picked from SD plates, and genomic DNA was extracted as templates. PCR amplification was performed using appropriate specific primers to verify the correct introduction of the gene module.

[0280] Integrate module Ⅳδ4-1-P TDH3 -IDI1-T TPI1 -P PGK1 -ERG20-T ADH1Integration module Voverlap-P TEF1 -ERG9-T CYC1 -overlap and integration module VIP PGK1 -ERG1-T ADH1 -P TEF1 -ERG7 - -T CYC1 -LEU-δ4-2 was used to transform recombinant Saccharomyces cerevisiae Y1C to construct recombinant yeast Y1CS. Positive transformants were screened on SD auxotrophic medium. Transformants were picked from SD plates, and genomic DNA was extracted as templates. PCR amplification was performed using appropriate specific primers to verify the correct introduction of the gene module.

[0281] The integration module VII rDNA1-P was respectively TEF1 -BiP-T CYC1 -rDNA2, integration module VIII rDNA1-P TEF1 -HAC1-T CYC1 -rDNA2, integration module IXrDNA1-P TEF1 -PDI1-T CYC1 -rDNA2 was transformed into recombinant Saccharomyces cerevisiae Y1CS-6 to construct recombinant yeast strains Y1CSB, Y1CSH, and Y1CSP. Positive transformants were randomly screened on SD auxotrophic medium. Transformants were picked from SD plates, and genomic DNA was extracted as templates. PCR amplification was performed using appropriate specific primers to verify the correct introduction of the gene module.

[0282] Example 63: Plotting the Standard Curve of β-O-Glc-DM

[0283] Accurately weigh 5.0 mg of standard 3β-O-Glc-DM, dissolve it in methanol to prepare a 1.0 mg / mL stock solution, and prepare five standard solutions of 1.0 mg / mL, 0.5 mg / mL, 0.25 mg / mL, 0.125 mg / mL, and 0.0625 mg / mL respectively. Inject 10 μL of each standard solution under the following chromatographic conditions (HPLC conditions: Cosmosil C18 reversed-phase column, 4.6 × 150 mm, flow rate 1 mL / min, UV detection wavelength 203 nm, injection 10 μL; mobile phase conditions: 0 min, 58% ACN; 30 min, 58% ACN). Inject each sample three times. Plot a standard curve with the mean peak area as the ordinate and the sample concentration as the abscissa.

[0284] Plot a standard curve with the mean peak area on the ordinate and the sample concentration on the abscissa. Figure 12The linear regression equation for 3β-O-Glc-DM in the range of 0.0625–1.0 mg / mL is y = 4573.4x - 11.19, R0. 2 =0.9997.

[0285] Example 7: Validation and Optimization of Recombinant Bacteria Producing 3β-O-Glc-DM

[0286] The fermentation products of the recombinant bacteria were extracted and analyzed by HPLC and LC-MS. HPLC results showed that compounds with UV absorption and Rt values ​​consistent with those of the 3β-O-Glc-DM standard were present in both the recombinant bacteria and the culture medium extract. Figure 13 LC-MS results showed that the fragment ion peaks of this compound were consistent with those of the 3β-O-Glc-DM standard. Figure 14 ).

[0287] HPLC detection conditions: Cosmosil C18 reversed-phase column, 4.6 × 150 mm, flow rate 1 mL / min, UV detection wavelength 203 nm, injection 10 μL. Mobile phase conditions: 0 min, 58% ACN; 30 min, 58% ACN.

[0288] Twenty positive transformants from recombinant yeast strains Y1 and Y1C were randomly selected and cultured in 10 mL YPD medium at 30 °C and 200 rpm for 12 h; OD was measured. 600 (10-20), take an appropriate amount of culture medium and transfer it to 50 ml LYPD medium to allow it to reach its final OD. 600 The value was 0.2. Recombinant bacteria Y1 and Y1C were cultured at 30℃ and 220rpm for 3 days, and the bacterial cells were collected by centrifugation.

[0289] 1.0 g of recombinant dried bacteria was added to 100 mL of 70% ethanol, refluxed at 70 °C for 1 h, allowed to cool naturally, filtered to remove bacterial residue, evaporated to dryness under reduced pressure, dissolved in 100 mL of water, and extracted three times with 100 mL of water-saturated n-butanol, each time standing for 1 h. The extracts were combined and the n-butanol was evaporated to dryness, the product was dissolved in 2 mL of methanol, filtered through a 0.22 μm filter membrane, and 10 μL was injected for HPLC analysis. The 3β-O-Glc-DM content in the recombinant bacteria was determined according to the standard curve. The results showed that only two of the 20 transformants of recombinant strain Y1 produced 3β-O-Glc-DM; the yield of 3β-O-Glc-DM was significantly increased in the recombinant yeast strains obtained using CRISPR / Cas9 technology, with recombinant strain 19 showing the highest yield. The genotype and screening results of recombinant strain Y1C are shown in [reference needed]. Figure 15 .

[0290] Twenty positive transformants of recombinant yeast strain Y1CS were randomly selected for fermentation and product extraction. The 3β-O-Glc-DM content in the recombinant strains was determined by HPLC according to a standard curve. The results showed that overexpression of key upstream enzymes in the 3β-O-Glc-DM biosynthetic pathway further increased the 3β-O-Glc-DM yield in the recombinant strains, with strain 6 exhibiting the highest yield. The genotype and screening results of recombinant strain Y1CS are shown below. Figure 16 .

[0291] Ten positive transformants from recombinant yeast strains Y1CSB, Y1CSH, and Y1CSP were randomly selected for fermentation and product extraction. The 3β-O-Glc-DM content in each recombinant strain was determined by HPLC according to a standard curve. The results showed that overexpression of transcription factor HAC1 in the recombinant yeast strain Y1CSB significantly increased the yield of 3β-O-Glc-DM, with strain 3 exhibiting the highest yield. The genotypes and screening results of the recombinant strains Y1CSB, Y1CSH, and Y1CSP are shown below. Figure 17 .

[0292] Example 8: Yield Detection of Engineered Bacteria Producing 3β-O-Glc-DM in Shake Flask Culture

[0293] Recombinant yeast strains Y1, Y1C, Y1CS, and Y1CSH, which produce 3β-O-Glc-DM, were activated on YPD solid plates and cultured at 30°C and 200 rpm for 24 h. Single colonies were picked and inoculated into 10 ml of LYPD liquid medium and cultured at 30°C and 200 rpm for 12 h. Then, they were transferred to 100 ml of LYPD medium to allow the OD to terminate. 600 The value was 0.2. Recombinant yeast was cultured at 30℃ and 220rpm. 5mL of feed medium was added to the culture medium at 48h, 72h and 96h, respectively. The culture was continued until day 6. The cells and fermentation broth were collected by centrifugation, and the cells were cooled and dried.

[0294] Weigh 1.0 g of dried bacteria, add 100 mL of 70% ethanol, reflux at 70 °C for 1 h, cool naturally, filter to remove bacterial residue, evaporate the extract under reduced pressure to dryness, dissolve in 100 mL of water, and extract three times with 100 mL of water-saturated n-butanol, allowing to stand for 1 h each time. Combine the extracts and evaporate the n-butanol to dryness, dissolve the product in 2 mL of methanol, filter through a 0.22 μm filter membrane, and inject 10 μL for HPLC analysis. Determine the 3β-O-Glc-DM content in the recombinant bacteria according to the standard curve.

[0295] Take 100 mL of the supernatant from the centrifuged fermentation broth and extract it three times with 100 mL of water-saturated n-butanol, allowing it to stand for 1 h each time. Combine the extracts and evaporate the n-butanol to dryness. Dissolve the product in 2 mL of methanol, filter through a 0.22 μm filter membrane, and inject 10 μL for HPLC analysis. Determine the 3β-O-Glc-DM content in the fermentation broth according to the standard curve.

[0296] HPLC quantitative analysis revealed that the total 3β-O-Glc-DM yield of recombinant yeast Y1 was 14.8 mg / L. The total 3β-O-Glc-DM yield of recombinant yeast Y1C, obtained by CRISPR / Cas9 technology-mediated integration of exogenous genes into the yeast genome, was 115.3 mg / L, representing a 7.79-fold increase compared to Y1. Overexpression of key upstream enzymes in the 3β-O-Glc-DM biosynthesis pathway resulted in a total 3β-O-Glc-DM yield of 261.9 mg / L for recombinant yeast Y1CS, a 2.27-fold increase compared to Y1C. Overexpression of the transcription activator HAC1 in recombinant yeast resulted in a total 3β-O-Glc-DM yield of 414.8 mg / L for recombinant yeast Y1CSH, a 1.58-fold increase compared to Y1CS. Figure 18 ).

[0297] Example 9: Production of 3β-O-Glc-DM engineered bacteria by fed-exponential high-density fermentation

[0298] The engineered strain Y1CSH was activated on SD auxotrophic solid medium. A single colony was picked and inoculated into 100 ml of LYPD liquid medium for seed culture at 30°C and 220 rpm. The seed culture was then inoculated at a rate of 10% into a 3L fermenter containing 1 ml of LYPD fermentation medium (Shanghai Baoxing Bio-equipment Co., Ltd.). The fermentation temperature was 30°C, the aeration rate was 3 L / min, the dissolved oxygen (DO) was 30%, and the stirring rate was 300–900 rpm. The pH was maintained at 5.5 ± 0.2 using 5 M ammonia. After 20 hours of fermentation, an exponentially fed-batch culture medium was introduced. The medium contained 578 g / L glucose, 9 g / L KH₂PO₄, 5.12 g / L MgSO₄·7H₂O, 3.5 g / L K₂SO₄, 0.28 g / L Na₂SO₄, 2.1 g / L adenine, 2.5 g / L uracil, 5 g / L lysine, and 10 mL / L trace element solution (15 g EDTA, 10.2 g ZnSO₄·7H₂O, 0.5 g MnCl₂·4H₂O, 0.5 g CuSO₄, 0.86 g CoCl₂·6H₂O, 0.56 g Na₂MoO₄·2H₂O, 3.84 g CaCl₂·2H₂O, and 5.12 g MgSO₄·7H₂O). (Add FeSO4·7H2O to 1L of distilled water, filter and sterilize, store at 4℃) and a 12mL / L vitamin solution (0.05g biotin, 1g ubiquitin calcium, 1g niacin, 25g inositol, 1g thiamine hydrochloride, 1g pyridoxine hydrochloride and 0.2g aminobenzoic acid, add to 1L of distilled water, filter and sterilize, store at 4℃). Glucose concentration should be controlled below 1.0g / L, and ethanol concentration should not exceed 5.0g / L.

[0299] Samples were taken every 24 hours, and biomass and product content were measured. Biomass continued to increase until 96 hours later, then reached a plateau, with the fermentation broth OD at 168 hours. 600 The yield of β-O-Glc-DM reached a maximum of 1522 g / L at 144 h. Figure 19 In addition, during the fermentation process, mycelial blocks continuously accumulated on the walls of the fermenter. After 192 hours of fermentation, 2.49 g (dry weight, DCW) of mycelial blocks were collected from the tank walls, which contained 180 mg (72.3 mg / g DCW) of the product 3β-O-Glc-DM.

[0300] It will be apparent to those skilled in the art that various modifications and variations can be made to the method and recombinant bacteria of the present invention without departing from the spirit or scope of the invention. Therefore, the present invention covers such modifications and variations as long as they fall within the scope of the appended claims and their equivalents.

Claims

1. A method for constructing recombinant bacteria, the method comprising the following steps: knocking out the hexokinase 2 gene in *Saccharomyces cerevisiae*, introducing into the *Saccharomyces cerevisiae* a gene expression cassette of a fusion protein of dammarenediol-II synthase and GFP and a gene expression cassette of ginsenoside transferase PgUGT74AE2, and increasing the activity of 3-hydroxy-3-methylglutaryl-CoA reductase in the *Saccharomyces cerevisiae*, wherein, The ginseng glycosyltransferase PgUGT74AE2 encoding gene expression cassette contains the encoding gene for ginseng glycosyltransferase PgUGT74AE2 as shown in SEQ ID NO:2, wherein the dammarene diol-II synthase / GFP fusion protein encoding gene expression cassette contains the encoding gene for the dammarene diol-II synthase / GFP fusion protein as shown in SEQ ID NO:

1. Specifically, the activity of 3-hydroxy-3-methylglutaryl-CoA reductase in the *Saccharomyces cerevisiae* is enhanced by introducing a 3-hydroxy-3-methylglutaryl-CoA reductase-encoding gene expression cassette into the *Saccharomyces cerevisiae*. The 3-hydroxy-3-methylglutaryl-CoA reductase-encoding gene expression cassette contains the gene tHMG1 encoding the catalytic domain of the 3-hydroxy-3-methylglutaryl-CoA reductase shown in SEQ ID NO:

3. The expression cassette was integrated into the Saccharomyces cerevisiae genome using CRISPR / Cas9 technology.

2. The method according to claim 1, further comprising one or more of the following: To increase the activity of isopentenyl pyrophosphate isomerase IDI1 in the aforementioned Saccharomyces cerevisiae; To increase the activity of farnesyl pyrophosphate synthase ERG20 in the aforementioned Saccharomyces cerevisiae; To increase the activity of squalene monooxygenase ERG1 in the brewer's yeast; To increase the activity of squalene synthase ERG9 in the brewer's yeast; Reduce the activity of lanosterol synthase ERG7 in the brewer's yeast; Increase the level of the molecular chaperone BiP in the brewer's yeast; Increase the level of transcription factor HAC1 in the aforementioned Saccharomyces cerevisiae; or Increase the level of disulfide isomerase PDI1 in the brewer's yeast.

3. The method according to claim 2, wherein, The activity of isopentenyl pyrophosphate isomerase IDI1 in the *Saccharomyces cerevisiae* was enhanced by introducing the gene expression cassette encoding isopentenyl pyrophosphate isomerase IDI1 into the *Saccharomyces cerevisiae*. The activity of farnesyl pyrophosphate synthase ERG20 in the *Saccharomyces cerevisiae* was increased by introducing a gene expression cassette encoding farnesyl pyrophosphate synthase ERG20 into the *Saccharomyces cerevisiae*. The activity of squalene monooxygenase ERG1 in the *Saccharomyces cerevisiae* was increased by introducing a gene expression cassette encoding squalene monooxygenase ERG1 into the *Saccharomyces cerevisiae*. The activity of squalene synthase ERG9 in the *Saccharomyces cerevisiae* was enhanced by introducing an expression cassette encoding the squalene synthase ERG9 gene into the *Saccharomyces cerevisiae*. The activity of lanosterol synthase ERG7 in the yeast was reduced by introducing an expression cassette containing the antisense fragment of lanosterol synthase ERG7 into the yeast. The level of molecular chaperone BiP in the *Saccharomyces cerevisiae* was increased by introducing a gene expression cassette encoding the molecular chaperone BiP into the *Saccharomyces cerevisiae*. The level of transcription factor HAC1 in the *Saccharomyces cerevisiae* was increased by introducing a gene expression cassette encoding transcription factor HAC1 into the *Saccharomyces cerevisiae*. or The level of disulfide isomerase PDI1 in the *Saccharomyces cerevisiae* was increased by introducing a gene expression cassette encoding disulfide isomerase PDI1 into the *Saccharomyces cerevisiae*.

4. The method according to claim 3, wherein, The nucleotide sequence encoding IDI1 is the sequence shown in SEQ ID NO:4; The nucleotide sequence encoding ERG20 is the sequence shown in SEQ ID NO:5; The nucleotide sequence encoding ERG1 is the sequence shown in SEQ ID NO:6; The nucleotide sequence encoding ERG9 is the sequence shown in SEQ ID NO:7; The nucleotide sequence encoding the ERG7 antisense fragment is the sequence shown in SEQ ID NO:8; The nucleotide sequence encoding BiP is the sequence shown in SEQ ID NO:9; The nucleotide sequence encoding HAC1 is the sequence shown in SEQ ID NO:10; or The nucleotide sequence encoding PDI1 is shown in SEQ ID NO:

11.

5. The recombinant bacteria prepared by the method according to any one of claims 1-4.

6. The use of the recombinant bacteria according to claim 5 in the production of 3β-O-Glc-DM.

7. A method for producing 3β-O-Glc-DM, the method comprising fermenting the recombinant bacteria of claim 5 to obtain 3β-O-Glc-DM.

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