Metabolic engineering of yeasts for green and sustainable production of bioactive ginsenosides F2 and 3,20-Di--Glc-DM

Both natural ginsenoside F2 and unnatural ginsenoside 3,20-Di--Glc-DM were reported to exhibit anti-tumor activity. Traditional approaches for producing them rely on direct extraction from , enzymatic catalysis or chemical synthesis, all of which result in low yield and high cost. Metabolic engineering of microbes has been recognized as a green and sustainable biotechnology to produce natural and unnatural products. Hence we engineered the complete biosynthetic pathways of F2 and 3,20-Di--Glc-DM in the CRISPR/Cas9 system. The titers of F2 and 3,20-Di--Glc-DM were increased from 1.2 to 21.0 mg/L and from 82.0 to 346.1 mg/L at shake flask level, respectively, by multistep metabolic engineering strategies. Additionally, pharmacological evaluation showed that both F2 and 3,20-Di--Glc-DM exhibited anti-pancreatic cancer activity and the activity of 3,20-Di--Glc-DM was even better. Furthermore, the titer of 3,20-Di--Glc-DM reached 2.6 g/L by fed-batch fermentation in a 3 L bioreactor. To our knowledge, this is the first report on demonstrating the anti-pancreatic cancer activity of F2 and 3,20-Di--Glc-DM, and achieving their biosynthesis by the engineered yeasts. Our work presents an alternative approach to produce F2 and 3,20-Di--Glc-DM from renewable biomass, which lays a foundation for drug research and development.