Production of a bioactive unnatural ginsenoside by metabolically engineered yeasts based on a new UDP-glycosyltransferase from Bacillus subtilis

Ginsenosides are the main bioactive constituents of Panax species, which are biosynthesized by glycosylation at C3-OH and/or C20-OH of protopanaxadiol (PPD), C6-OH and/or C20-OH of protopanaxatriol (PPT). The C12-glycosylated ginsenosides have scarcely been identified from Panax species. The C12-glycosylated ginsenosides produced from PPD by chemical semi-synthesis have been reported to exhibit higher cytotoxicity than the natural ginsenosides. However, the chemical semi-synthesis approach is not practical due to its complexity and high cost. In our study, a new UDP-glycosyltransferase UGT109A1 was identified from Bacillus subtilis. This enzyme transferred a glucose moiety to C3-OH and C20-OH of dammarenediol-II (DM), C3-OH and C12-OH of PPD and PPT respectively to produce the unnatural ginsenosides 3β-O-Glc-DM, 3β,20S-Di-O-Glc-DM, 3β,12β-Di-O-Glc-PPD and 3β,12β-Di-O-Glc-PPT. Among these unnatural ginsenosides, 3β,12β-Di-O-Glc-PPT is a new compound which has never been reported before. The anti-cancer activities of these unnatural ginsenosides were evaluated in vitro and in vivo. 3β,12β-Di-O-Glc-PPD exhibited higher anti-lung cancer activity than Rg3, which is the most active natural ginsenoside against lung cancer. Finally, we constructed metabolically engineered yeasts to produce 3β,12β-Di-O-Glc-PPD by introducing the genes encoding B. subtilis UGT109A1, Panax ginseng dammarenediol-II synthase (DS), P. ginseng cytochrome P450-type protopanaxadiol synthase (PPDS) together with Arabidopsis thaliana NADPH-cytochrome P450 reductase (ATR1) into Saccharomyces cerevisiae INVSc1. The yield of 3β,12β-Di-O-Glc-PPD was increased from 6.17mg/L to 9.05mg/L by overexpressing tHMG1. Thus, this study has established an alternative route to produce the unnatural ginsenoside 3β,12β-Di-O-Glc-PPD by synthetic biology strategies, which provides a promising candidate for anti-cancer drug discovery.