Type II ganoderic acids (GAs) produced by the traditional medicinal mushroom Ganoderma are a group of triterpenoids with superior biological activities. However, challenges in the genetic manipulation of the native producer, low level of accumulation in the farmed mushroom, the vulnerabilities of the farming-based supply chain, and the elusive biosynthetic pathway have hindered the efficient production of type II GAs. Here, we assemble the genome of type II GAs accumulating G. lucidum accession, screen cytochrome P450 enzymes (CYPs) identified from G. lucidum in baker’s yeast, identify key missing CYPs involved in type II GAs biosynthesis, and investigate the catalytic reaction sequence of a promiscuous CYP. Then, we engineer baker’s yeast for bioproduciton of GA-Y (3) and GA-Jb (4) and achieve their production at higher level than those from the farmed mushroom. Our findings facilitate the further deconvolution of the complex GA biosynthetic network and the development of microbial cell factories for producing GAs at commercial scale.
Efficient bioproduction of triterpenoids is gaining increasing interest because of their significant biological applications; however, the secretion and bioproduction of triterpenoids are hindered by untapped genetic determinants. In our previous study, we observed that different engineered Saccharomyces cerevisiae strains exhibit different abilities for secreting the antitumor triterpenoid ganoderic acid 3-hydroxy-lanosta-8,24-dien-26-oic acid (GA-HLDOA). In the present study, we performed comparative proteomics analyses of the engineered strains and identified two genes, encoding an aspartic protease, YPS3, and a cell wall glucanase, SCW10, as the most effective determinants that enhance the secretion of GA-HLDOA. Compared with this control strain, strain BJ5464-r demonstrated an overexpression of YPS3 and SCW10 resulting in 3.9-fold and 4.7-fold higher secretion of GA-HLDOA, respectively, and these increases were accompanied by an increase in cell permeability. Moreover, compared with the YPS3-overexpressing strain, the SCW10-overexpressing strain had a thinner outer mannan layer. Our findings offer valuable insights into designing microbial cell factories for the efficient secretion of triterpenoids.
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