α-Amyrin, lupeol and germanicol are important pentacyclic triterpenoids, with excellent anti-inflammatory, anti-tumor, anti-virus activities and properties. To construct cell factories for the production of pentacyclic triterpenoids, genes encoding Catharanthus roseus α-amyrin synthase (CrαAS), Glycyrrhiza glabra lupeol synthase (GgLUS) and Bauhinia forficata germanicol synthase (BfGES) were separately integrated into the Saccharomyces cerevisiae chromosome using CRISPR/Cas9 technology, resulting in engineered strains S01, S02, and S03. After 48 h of fermentation in YPG medium, 50.7 mg/L α-amyrin, 45.8 mg/L lupeol and 17.6 mg/L germanicol were achieved with strain S01, S02, and S03, respectively. This demonstrated the successful construction of the S. cerevisiae cell factories for pentacyclic triterpenoids production. To increase the supply of precursor 2,3-oxidosqualene, the squalene epoxidase (ERG1), a key enzyme in the mevalonic acid (MVA) pathway, was further overexpressed in strains S01, S02, and S03. With the resulting recombinant strains S04, S05 and S06, the yield of α-amyrin, lupeol and germanicol increased to 96.3, 91.6, and 38.7 mg/L, respectively. Therefore, this study constructed triterpenoid-producing platforms, which provided a theoretical and technical basis for the construction of S.cerevisiae pentacyclic triterpenoid acids cell factories with potential for industrial applications.
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