Biosynthesis of Active Ginsenoside Aglycone Analogues Using Two Oxidases Mined from Mucor spinosus

The pharmacological activity of protopanaxadiol (PPD) can be enhanced by oxidative modification. Compared with chemical synthesis and biotransformation, synthetic biology offers a more efficient and eco-friendly approach for modifying PPD. In this study, we identified two oxidases from Mucor spinosus. MsSDR3 oxidizes C3-OH of both dammarenediol-II and PPD to a ketone, while MsCYP3 oxidizes C12-OH of PPD to a ketone and hydroxylates PPD at C7β, C15α, and C11β positions. Combining MsSDR3/MsCYP3 with the enzymes involved in ginsenoside biosynthesis, we achieved de novo biosynthesis of seven ginsenoside aglycone analogues in Saccharomyces cerevisiae. Pharmacological evaluation indicated that 12-oxo-15α-hydroxy-protopanaxadiol (p3) showed higher anticolon cancer activity, and 7β-hydroxy-protopanaxadiol (p6) exhibited not only higher anticolon, antigastric, antiliver, antilung, and antipancreatic cancer activities but also higher cardioprotective activity than PPD. Our work establishes a green and sustainable platform for producing active ginsenoside aglycone analogues, paving the way for the development of drugs and functional foods.