摘要Both natural ginsenoside F2 and unnatural ginsenoside 3β,20S-Di-O-Glc-DM were reported to exhibit anti-tumor activity.Traditional approaches for producing them rely on direct extraction from Panax ginseng,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 3β,20S-Di-O-Glc-DM in Saccharomyces cerevisiae via the CRISPR/Cas9 system.The titers of F2 and 3β,20S-Di-O-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 3β,20S-Di-O-Glc-DM exhibited anti-pancreatic cancer activity and the activity of 3β,20S-Di-O-Glc-DM was even better.Furthermore,the titer of 3β,20S-Di-O-Glc-DM reached 2.6 g/L by fed-batch fermen-tation in a 3 L bioreactor.To our knowledge,this is the first report on demonstrating the anti-pancreatic cancer activity of F2 and 3β,20S-Di-O-Glc-DM,and achieving their de novo biosynthesis by the engineered yeasts.Our work presents an alternative approach to produce F2 and 3β,20S-Di-O-Glc-DM from renewable biomass,which lays a foundation for drug research and development.