Production of a Novel Protopanaxatriol-Type Ginsenoside by Yeast Cell Factories

Ginsenosides, the main active compounds in Panax species, are glycosides of protopanaxadiol (PPD) or protopanaxatriol (PPT). PPT-type ginsenosides have unique pharmacological activities on the central nervous system and cardiovascular system. As an unnatural ginsenoside, 3,12-Di-O-beta-D-glucopyranosyl-dammar-24-ene-3 beta,6 alpha,12 beta,20S-tetraol (3 beta,12 beta-Di-O-Glc-PPT) can be synthesized through enzymatic reactions but is limited by the expensive substrates and low catalytic efficiency. In the present study, we successfully produced 3 beta,12 beta-Di-O-Glc-PPT in Saccharomyces cerevisiae with a titer of 7.0 mg/L by expressing protopanaxatriol synthase (PPTS) from Panax ginseng and UGT109A1 from Bacillus subtilis in PPD-producing yeast. Then, we modified this engineered strain by replacing UGT109A1 with its mutant UGT109A1-K73A, overexpressing the cytochrome P450 reductase ATR2 from Arabidopsis thaliana and the key enzymes of UDP-glucose biosynthesis to increase the production of 3 beta,12 beta-Di-O-Glc-PPT, although these strategies did not show any positive effect on the yield of 3 beta,12 beta-Di-O-Glc-PPT. However, the unnatural ginsenoside 3 beta,12 beta-Di-O-Glc-PPT was produced in this study by constructing its biosynthetic pathway in yeast. To the best of our knowledge, this is the first report of producing 3 beta,12 beta-Di-O-Glc-PPT through yeast cell factories. Our work provides a viable route for the production of 3 beta,12 beta-Di-O-Glc-PPT, which lays a foundation for drug research and development.