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.
As the main bioactive constituents of Panax species, ginsenosides possess a wide range of notable medicinal effects such as anti-cancer, anti-oxidative, antiaging, anti-inflammatory, anti-apoptotic and neuroprotective activities. However, the increasing medical demand for ginsenosides cannot be met due to the limited resource of Panax species and the low contents of ginsenosides. In recent years, biotechnological approaches have been utilized to increase the production of ginsenosides by regulating the key enzymes of ginsenoside biosynthesis, while synthetic biology strategies have been adopted to produce ginsenosides by introducing these genes into yeast. This review summarizes the latest research progress on cloning and functional characterization of key genes dedicated to the production of ginsenosides, which not only lays the foundation for their application in plant engineering, but also provides the building blocks for the production of ginsenosides by synthetic biology.
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