Recombinant saccharomyces cerevisiae strain with high yield of asiatic acid and construction method

Abstract

The invention discloses a recombinant saccharomyces cerevisiae strain with high yield of asiatic acid and a construction method of the recombinant saccharomyces cerevisiae strain. The method comprises the following steps: integrating a C-2alpha hydroxylated cytochrome P450 oxidase gene AmCYP716C53 from avicennia marina and a C-23 hydroxylated cytochrome P450 oxidase gene CaCYP716E19 from centella asiatica to a chromosome 911b site of a chassis strain ZY35 by utilizing a CRISPR-Cas9 gene editing technology, so as to obtain ZSA01; then, genes MCH5, FLX1, RIB1, FMN1 and FAD1 are integrated to the 1014a site of the ZSA01, and ZSA02 is obtained; finally, the AmCYP716C53 is integrated to the HMX1 site of the ZSA02, and an engineering strain ZSA03 is constructed. Fermentation results show that ZSA03 reaches 1g / L in a 5L fermentation tank, and the ZSA03 has high-efficiency synthesis and industrial application potential.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and relates to a recombinant strain for producing asiatic acid, a construction method thereof and a biological production method of asiatic acid. BACKGROUND

[0002] Asiatic acid is a natural pentacyclic triterpenoid compound, which is widely present in the Umbelliferae plant asiatic acid. Asiatic acid has various biological activities such as anti-inflammatory, antioxidant, promoting wound healing, neuroprotective, anti-tumor and anti-anxiety, and is widely used in traditional medicine and food fields. However, the traditional plant extraction method not only consumes time and energy, and has low efficiency, but also is affected by season and climate, resulting in unstable product quality and limited production scale.

[0003] Microbial synthesis of asiatic acid is a potential sustainable alternative method. Asiatic acid is synthesized from ursolic acid by two consecutive cytochrome P450 enzymes, but the reported P450 enzymes have poor specificity and low activity, and the lack of cofactor supply also affects the electron transfer and catalytic efficiency of P450 enzymes, resulting in low level of microbial synthesis of asiatic acid. More sophisticated and effective metabolic engineering strategies and synthetic biology technologies are needed to improve the ability of microorganisms to produce asiatic acid. SUMMARY

[0004] The purpose of the present application is to overcome the deficiencies of the prior art, and to provide a recombinant Saccharomyces cerevisiae strain for high-yield production of asiatic acid.

[0005] The second purpose of the present application is to provide a construction method of a recombinant Saccharomyces cerevisiae strain for high-yield production of asiatic acid.

[0006] The third purpose of the present application is to provide an application of a recombinant Saccharomyces cerevisiae strain for high-yield production of asiatic acid in the fermentation preparation of asiatic acid.

[0007] The technical solution of the present application is summarized as follows: A construction method of a recombinant Saccharomyces cerevisiae for high-yield production of asiatic acid, comprising the following steps: (1) prepare the yeast ZY35 competent cells, and use the CRISPR-Cas9 editing technology to integrate the 911b upstream homologous arm, the 911b downstream homologous arm, the gene expression cassette TEF1 promoter-AmCYP716C53-ENO2 terminator and PGK1 promoter-CaCYP716E19-GPM1 terminator and the gRNA-911b plasmid into the 911b site of the yeast ZY35 chromosome, culture on the SD-URA solid plate for 2-3 days, extract the genome of the single colony and perform colony PCR verification, culture the correct strain in the YPD medium and discard the gRNA-911b plasmid, and obtain the recombinant yeast ZSA01; the AmCYP716C53 is a codon-optimized nucleic acid sequence, and is shown in SEQ ID NO. 2; the CaCYP716E19 is a codon-optimized nucleic acid sequence, and is shown in SEQ ID NO. 4; (2) prepare the recombinant yeast ZSA01 competent cells, and use the CRISPR-Cas9 editing technology to integrate the 1014a upstream homologous arm, the 1014a downstream homologous arm, the gene expression cassette TDH3 promoter-MCH5-ACS1 terminator, HXT7 promoter-FLX1-ENO2 terminator, TEF1 promoter-RIB1-GPM1 terminator, GAL1 promoter-FMN1-GPM1 terminator, GAL10 promoter-FAD1-FBA1 terminator and the gRNA-1014a plasmid into the 1014a site of the recombinant yeast ZSA01 chromosome, culture on the SD-URA solid plate for 2-3 days, extract the genome of the single colony and perform colony PCR verification, culture the correct strain in the YPD medium and discard the gRNA-1014a plasmid, and obtain the recombinant yeast ZSA02; (3) prepare the recombinant yeast ZSA02 competent cells, and use the CRISPR-Cas9 editing technology to integrate the HMX1 upstream homologous arm, the HMX1 downstream homologous arm, the gene expression cassette TEF1 promoter-AmCYP716C53-ENO2 and the gRNA-HMX1 plasmid into the HMX1 site of the recombinant yeast ZSA02, culture on the SD-URA solid plate for 2-3 days, extract the genome of the single colony and perform colony PCR verification, culture the correct strain in the YPD medium and discard the gRNA-HMX1 plasmid, and obtain the recombinant yeast ZSA03.

[0008] The above-mentioned construction method constructs a recombinant yeast strain with high yield of asiatic acid.

[0009] The above-mentioned recombinant yeast strain with high yield of asiatic acid is used for fermentation preparation of asiatic acid.

[0010] Advantages of this invention: Experiments have shown that the recombinant brewing yeast of the present invention can produce asiatic acid by fermentation. After 96 h of shake-flask fermentation, 339 mg / L of asiatic acid is produced. The yield of asiatic acid in a 5 L bioreactor is as high as 1 g / L. Detailed Implementation

[0011] The present invention will be further described below with reference to specific embodiments.

[0012] The present invention utilizes the previously constructed strain of *Saccharomyces cerevisiae* ZY35 (genotypes: GAL1 promoter-CrMAS-ADH1 terminator, GAL1 promoter-CrOAS-(GGGS)2-CrCPR1-ADH1 terminator, GAL1 promoter-EfMVAS-ADH1 terminator, GAL10 promoter-EfMVAE-CYC1 terminator, TPI1 promoter-ERG8-ERG8 terminator, ENO2 promoter-ERG12-ERG...). 12 terminators, GPD1 starter-IDI1-PRM9 terminator, GPM1 starter-MVD1-IDP1 terminator, HXT7 starter-ERG9-TPGI terminator, ADH1 starter-ERG20-TPI1 terminator, GAL1 starter-ERG1-ENO2 terminator, GAL1 starter-CrMAS-ADH1 terminator, GAL1 starter-CRMAS-ADH1 terminator, ERG7P::ERG7P -UBI4-DEGRON(F: K3K15), ΔSSM4, GAL1 promoter-CkPTA-GPM1 terminator, GAL10 promoter-BbFPK-FBA1 terminator, GAL1 promoter-ALD6-GPM1 terminator, GAL10 promoter-SeACSL641P-FBA1 terminator, TPI1 promoter-MPC1-MPC1 terminator, FBA1 promoter-MPC3-MPC3 terminator, TEF1 promoter-YHM2-YHM2 terminator, PGK1 promoter-CTP1-CTP1 terminator, HXT7 promoter-RtCIT1-ENO2 terminator, GAL1 promoter-AnACLA-GPM1 terminator, GAL10 promoter-AnACLB-FBA1 terminator, Zhu, Y., Yan, X., Li, W et al., 2025. Modularmetabolic engineering of Saccharomyces cerevisiae for enhanced production of ursolic acid. Journal of Agricultural and Food Chemistry73(6), 3580-3590 (starting strain was commercially available CEN-PK2-1C). Gene expression cassettes and genome integration fragments were constructed using overlap extension PCR. Competent cells were transformed with lithium acetate, and the fragments were integrated into specific chromosomal sites using CRISPR-Cas9 gene editing technology.

[0013] Preparation of Saccharomyces cerevisiae ZY35 competent cells Streaking yeast ZY35 frozen bacteria on YPD plates for 2-3 days, pick single colonies and culture them in YPD liquid medium for 18 hours, then inoculate at a 2% ratio to activate for 4-5 hours until the bacterial culture reaches OD. 600 The cells were collected by centrifugation at 3600 rpm for 5 min with a pH of 0.8-0.9. The cells were washed twice with sterile double-distilled water and then centrifuged again to obtain the competent cells of Saccharomyces cerevisiae ZY35.

[0014] The preparation of recombinant Saccharomyces cerevisiae ZSA01 competent cells and recombinant Saccharomyces cerevisiae ZSA02 competent cells was carried out with reference to the preparation of Saccharomyces cerevisiae ZY35 competent cells.

[0015] Construction of gRNA-911b plasmid: using pCas9-URA plasmid (Xu, H., Han, M., Zhou, S.). et al. Chromosome drives via CRISPR-Cas9 in yeast. Nat Commu Using 11, 4344 (2020) as a template, and primers 1-F (SEQ ID NO. 5) and 2-R (SEQ ID NO. 6) as primers, circular plasmid PCR was performed. The plasmid was digested with DpnI at 37℃ for 20 min and transformed into *E. coli* (11, 4344 (2020)). DH5α The single clone was sequenced, and the plasmid that was correctly sequenced was named gRNA-911b.

[0016] Construction of gRNA-1014a plasmid: Using pCas9-URA plasmid as a template, and primers 3-F (SEQ ID NO.7) and 4-R (SEQ ID NO.8) as primers, circular plasmid PCR was performed. The plasmid was then digested with DpnI at 37℃ for 20 min and transformed into *E. coli*. DH5α The single clone was sequenced, and the plasmid that was correctly sequenced was named gRNA-1014a.

[0017] Construction of gRNA-HMX1 plasmid: Using pCas9-URA plasmid as a template, and primers 5-F (SEQ ID NO. 9) and 6-R (SEQ ID NO. 10) as primers, circular plasmid PCR was performed, followed by transformation into Escherichia coli after digestion with DpnI at 37℃ for 20 min. DH5 α The single clone was sequenced, and the plasmid that was correctly sequenced was named gRNA-HMX1.

[0018] White bone soil ( Avicennia marina The amino acid sequence of the C-2α-hydroxylated cytochrome P450 oxidase AmCYP716C53 from [source name missing] is shown in SEQ ID NO.1, and its codon-optimized nucleic acid sequence from *Saccharomyces cerevisiae* is shown in SEQ ID NO.2. Centella asiatica ( Centella asiatica The amino acid sequence of C-23 hydroxylated cytochrome P450 oxidase CaCYP716E19 from the source is shown in SEQ ID NO.3, and its codon-optimized nucleic acid sequence from Saccharomyces cerevisiae is shown in SEQ ID NO.4.

[0019] Example 1 A method for constructing a recombinant brewing yeast that produces high levels of asiatic acid includes the following steps: (1) Prepare competent cells of Saccharomyces cerevisiae ZY35, and use CRISPR-Cas9 editing technology to co-transfer the upstream homologous arm of 911b, the downstream homologous arm of 911b, the gene expression cassette TEF1 promoter-AmCYP716C53-ENO2 terminator and PGK1 promoter-CaCYP716E19-GPM1 terminator, and gRNA-911b plasmid into the 911b site of chromosome 911b of Saccharomyces cerevisiae ZY35. The cells were plated on SD-URA solid plates and cultured for 2-3 days. The genome of the grown single clones was extracted and colony PCR was performed for verification. The verified strains were cultured in YPD medium and the gRNA-911b plasmid was discarded to obtain recombinant Saccharomyces cerevisiae ZSA01. The AmCYP716C53 is a codon-optimized nucleic acid sequence, shown in SEQ ID NO.2. The CaCYP716E19 is a codon-optimized nucleic acid sequence, shown in SEQ ID NO.4. Preparation of the upstream homologous arm of 911b: Using the genome of Saccharomyces cerevisiae ZY35 as a template, PCR amplification was performed using primers 27-F (SEQ ID NO.31) and 28-R (SEQ ID NO.32) to obtain the upstream homologous arm of 911b; Preparation of the downstream homologous arm of 911b: Using the genome of Saccharomyces cerevisiae ZY35 as a template, PCR amplification was performed using primers 29-F (SEQ ID NO.33) and 30-R (SEQ ID NO.34) to obtain the downstream homologous arm of 911b; Preparation of the TEF1 promoter: Using the genome of Saccharomyces cerevisiae ZY35 as a template, the TEF1 promoter was obtained by PCR amplification using primers 7-F (SEQ ID NO. 11) and 8-R (SEQ ID NO. 12); Preparation of the ENO2 terminator: Using the genome of Saccharomyces cerevisiae ZY35 as a template, PCR amplification was performed using primers 9-F (SEQ ID NO. 13) and 10-R (SEQ ID NO. 14) to obtain the ENO2 terminator; Preparation of the PGK1 promoter: Using the genome of Saccharomyces cerevisiae ZY35 as a template, the PGK1 promoter was obtained by PCR amplification using primers 11-F (SEQ ID NO. 15) and 12-R (SEQ ID NO. 16); Preparation of the GPM1 terminator: Using the genome of Saccharomyces cerevisiae ZY35 as a template, the GPM1 terminator was obtained by PCR amplification using primers 13-F (SEQ ID NO. 17) and 14-R (SEQ ID NO. 18). Promoter, gene, and terminator fragments were fused in vitro using overlap extension PCR to obtain the corresponding gene expression cassette.

[0020] (2) Prepare recombinant Saccharomyces cerevisiae ZSA01 competent cells, and use CRISPR-Cas9 editing technology to integrate the upstream homologous arm of 1014a, the downstream homologous arm of 1014a, the gene expression cassette TDH3 promoter-MCH5-ACS1 terminator, HXT7 promoter-FLX1-ENO2 terminator, TEF1 promoter-RIB1-GPM1 terminator, GAL1 promoter-FMN1-GPM1 terminator, GAL10 promoter-FAD1-FBA1 terminator and gRNA-1014a plasmid into the 1014a site of chromosome of recombinant Saccharomyces cerevisiae ZSA01. Spread on SD-URA solid plates and culture for 2-3 days. Extract the genome of the grown single clones and perform colony PCR verification. The correct strains are cultured in YPD medium and the gRNA-1014a plasmid is discarded to obtain recombinant Saccharomyces cerevisiae ZSA02. MCH5 The nucleic acid sequence, GenBank number NC_001147.6; FLX1 The nucleic acid sequence, GenBank number NC_001141.2; RIB1 The nucleic acid sequence, GenBank number NC_001134.8; FMN1 The nucleic acid sequence, GenBank number NC_001136.9; FAD1 The nucleic acid sequence, GenBank number NC_001136.10; Preparation of the upstream homologous arm of 1014a: Using the genome of Saccharomyces cerevisiae ZY35 as a template, PCR amplification was performed using primers 31-F (SEQ ID NO. 35) and 32-R (SEQ ID NO. 36) to obtain the upstream homologous arm of 1014a; Preparation of the downstream homologous arm of 1014a: Using the genome of Saccharomyces cerevisiae ZY35 as a template, PCR amplification was performed using primers 33-F (SEQ ID NO.37) and 34-R (SEQ ID NO.38) to obtain the downstream homologous arm of 1014a; Preparation of the TDH3 promoter: Using the genome of Saccharomyces cerevisiae ZY35 as a template, the TDH3 promoter was obtained by PCR amplification using primers 15-F (SEQ ID NO. 19) and 16-R (SEQ ID NO. 20); Preparation of the ACS1 terminator: Using the genome of Saccharomyces cerevisiae ZY35 as a template, the ACS1 terminator was obtained by PCR amplification using primers 17-F (SEQ ID NO. 21) and 18-R (SEQ ID NO. 22). Preparation of the HXT7 promoter: Using the genome of Saccharomyces cerevisiae ZY35 as a template, the HXT7 promoter was obtained by PCR amplification using primers 19-F (SEQ ID NO. 23) and 20-R (SEQ ID NO. 24); Preparation of the GAL1 promoter: Using the genome of Saccharomyces cerevisiae ZY35 as a template, the GAL1 promoter was obtained by PCR amplification using primers 21-F (SEQ ID NO. 25) and 22-R (SEQ ID NO. 26); Preparation of the GAL10 promoter: Using the genome of Saccharomyces cerevisiae ZY35 as a template, the GAL10 promoter was obtained by PCR amplification using primers 23-F (SEQ ID NO. 27) and 24-R (SEQ ID NO. 28); Preparation of the FBA1 terminator: Using the genome of Saccharomyces cerevisiae ZY35 as a template, PCR amplification was performed using primers 25-F (SEQ ID NO. 29) and 26-R (SEQ ID NO. 30) to obtain the FBA1 terminator; Promoter, gene, and terminator fragments were fused in vitro using overlap extension PCR to obtain the corresponding gene expression cassette.

[0021] (3) Prepare recombinant Saccharomyces cerevisiae ZSA02 competent cells, and use CRISPR-Cas9 editing technology to integrate the upstream homologous arm of HMX1, the downstream homologous arm of HMX1, the gene expression cassette TEF1 promoter-AmCYP716C53-ENO2 and gRNA-HMX1 plasmid into the HMX1 site of recombinant Saccharomyces cerevisiae ZSA02. Spread on SD-URA solid plates and culture for 2-3 days. Extract the genome of the grown single clones and perform colony PCR verification. The verified strains are cultured in YPD medium and the gRNA-HMX1 plasmid is discarded to obtain recombinant Saccharomyces cerevisiae ZSA03.

[0022] Preparation of the upstream homologous arm of HMX1: Using the genome of Saccharomyces cerevisiae ZY35 as a template, primers 35-F (SEQ ID NO.39) and 36-R (SEQ ID NO.40) were used for PCR amplification to obtain the upstream homologous arm of HMX1; Preparation of the downstream homologous arm of HMX1: Using the genome of Saccharomyces cerevisiae ZY35 as a template, PCR amplification was performed using primers 37-F (SEQ ID NO.41) and 38-R (SEQ ID NO.42) to obtain the downstream homologous arm of HMX1; Promoter, gene, and terminator fragments were fused in vitro using overlap extension PCR to obtain the corresponding gene expression cassette.

[0023] Example 2 Fermentation and yield detection of recombinant brewing yeast Seed preparation: Streaking the frozen strain onto YPD solid medium and activating it twice. Pick single clones and inoculate them into YPD liquid medium, and culture at 30℃ and 220 rpm until the logarithmic growth phase.

[0024] Shake-flask fermentation: Seeds in the logarithmic growth phase were inoculated at a ratio of 5% into shake-flask YPD liquid medium and fermented at 30℃ and 220 rpm. After 96 h of fermentation, 2 mL of the fermentation broth was transferred to a centrifuge tube and centrifuged at 7500 rpm for 10 min. The supernatant was removed, and the centrifuged cells were mixed with an appropriate amount of steel balls and methanol. The cells were then disrupted using a high-throughput grinder. The disrupted cells were centrifuged at 7500 rpm for 10 min, and the organic phase was collected and filtered through a 0.22 μm organic filter membrane. The results were then analyzed by liquid chromatography.

[0025] The asiatic acid yield of ZSA01 was 55 mg / L, that of ZSA02 was 219 mg / L, and that of ZSA03 was 339 mg / L.

[0026] Fermentation in a fermenter: Seeds in the logarithmic growth phase were inoculated at a ratio of 10% into a 5 L fermenter containing 2 L of fermentation medium. The fermenter rotation speed was maintained between 200-800 rpm, dissolved oxygen level not lower than 30%, temperature around 30℃, pH around 5.5, and aeration rate of 3.0-6.0 vvm. When glucose in the medium was depleted, glucose was added again after 24 h to maintain a glucose concentration of 1.0-5.0 g / L. After 48 h, anhydrous ethanol was added to maintain an ethanol content of 1.0-5.0 g / L. After 132 h of fermentation, the fermentation broth was transferred to centrifuge tubes and centrifuged at 7500 rpm for 10 min. A suitable amount of steel balls and methanol were added, and the cells were disrupted using a high-throughput grinder. The disrupted bacterial solution was centrifuged at 7500 rpm for 10 min, and the organic phase was collected and filtered through a 0.22 μm organic filter membrane. The processed sample could be directly analyzed by liquid chromatography. The liquid chromatography system used was an Agilent 1260 HPLC system with a photodiode array detector and an Ultimate® XS-C18 column (4.6 × 250 mm, 5 μm). The mobile phase consisted of a mixture of solvent A (acetonitrile), solvent B (methanol), and solvent C (0.5% aqueous ammonium acetate) in a ratio of 67:12:21 (v / v / v). 10 μL of the prepared sample was injected into the HPLC system, and isocratic elution was performed at a flow rate of 1 mL / min for 27 min. The column temperature was maintained at 30 °C, and detection was performed at a wavelength of 210 nm.

[0027] ZSA03 has a asiatic acid yield of 1 g / L.

[0028] YPD liquid medium: 20 g / L glucose, 20 g / L peptone and 5 g / L yeast extract. YPD solid medium is supplemented with 1.5-2.0% (w / v) agar powder.

[0029] Fermentation medium: 40 g / L glucose, 50 g / L peptone, 1.2 g / L dipotassium hydrogen phosphate, 9 g / L potassium dihydrogen phosphate, 5.12 g / L magnesium sulfate heptahydrate, 3.5 g / L potassium sulfate, 0.28 g / L sodium sulfate, 1.0 g / L uracil, 2 mM 5-aminopropionylacetic acid, 10 mL trace metal element solution and 12 mL vitamin solution.

[0030] To prepare a 1L trace metal element solution, take 5.75 g zinc sulfate heptahydrate, 0.32 g manganese chloride tetrahydrate, 0.47 g cobalt chloride hexahydrate, 0.48 g sodium molybdate dihydrate, 2.9 g calcium chloride dihydrate, 2.8 g ferrous sulfate heptahydrate, and 80 mL / L 0.5 M ethylenediaminetetraacetic acid, and add water to 1L.

[0031] To prepare a 1L vitamin solution, take 0.05 g vitamin H, 1 g calcium pantothenate, 1 g niacin, 25 g inositol, 1 g ammonium sulfate hydrochloride, 1 g pyridoxal hydrochloride, and 0.2 g para-aminobenzoic acid, and add water to 1L. Although the present invention has been described above, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many modifications under the guidance of the present invention without departing from the spirit of the present invention, and these modifications are all within the protection scope of the present invention.

Claims

1. A method for constructing a recombinant brewing yeast that produces high levels of asiatic acid, characterized in that... Includes the following steps: (1) Prepare competent cells of Saccharomyces cerevisiae ZY35, and use CRISPR-Cas9 editing technology to co-transfer the upstream homologous arm of 911b, the downstream homologous arm of 911b, the gene expression cassette TEF1 promoter-AmCYP716C53-ENO2 terminator and PGK1 promoter-CaCYP716E19-GPM1 terminator, and gRNA-911b plasmid into the 911b site of chromosome 911b of Saccharomyces cerevisiae ZY35. The cells were plated on SD-URA solid plates and cultured for 2-3 days. The genome of the grown single clones was extracted and colony PCR was performed for verification. The correct strain was cultured in YPD medium and the gRNA-911b plasmid was discarded to obtain recombinant Saccharomyces cerevisiae ZSA01. The AmCYP716C53 is a codon-optimized nucleic acid sequence, shown in SEQ ID NO.

2. The CaCYP716E19 is a codon-optimized nucleic acid sequence, shown in SEQ ID NO.

4. (2) Prepare recombinant Saccharomyces cerevisiae ZSA01 competent cells, and use CRISPR-Cas9 editing technology to integrate the upstream homologous arm of 1014a, the downstream homologous arm of 1014a, the gene expression cassette TDH3 promoter-MCH5-ACS1 terminator, HXT7 promoter-FLX1-ENO2 terminator, TEF1 promoter-RIB1-GPM1 terminator, GAL1 promoter-FMN1-GPM1 terminator, GAL10 promoter-FAD1-FBA1 terminator and gRNA-1014a plasmid into the 1014a site of chromosome of recombinant Saccharomyces cerevisiae ZSA01. Spread on SD-URA solid plates and culture for 2-3 days. Extract the genome of the grown single clones and perform colony PCR verification. The correct strains are cultured in YPD medium and the gRNA-1014a plasmid is discarded to obtain recombinant Saccharomyces cerevisiae ZSA02. (3) Prepare recombinant Saccharomyces cerevisiae ZSA02 competent cells, and use CRISPR-Cas9 editing technology to integrate the upstream homologous arm of HMX1, the downstream homologous arm of HMX1, the gene expression cassette TEF1 promoter-AmCYP716C53-ENO2 and gRNA-HMX1 plasmid into the HMX1 site of recombinant Saccharomyces cerevisiae ZSA02. Spread on SD-URA solid plates and culture for 2-3 days. Extract the genome of the grown single clones and perform colony PCR verification. The verified strains are cultured in YPD medium and the gRNA-HMX1 plasmid is discarded to obtain recombinant Saccharomyces cerevisiae ZSA03.

2. A recombinant Saccharomyces cerevisiae strain that produces high levels of asiatic acid, constructed using the method of claim 1.

3. The application of the recombinant Saccharomyces cerevisiae strain with high asiatic acid production according to claim 2 for the fermentation preparation of asiatic acid.