Combining protein and metabolic engineering to achieve green biosynthesis of 12 beta-O-Glc-PPD in Saccharomyces cerevisiae

12-O-beta-d-Glucopyranosyl-dammar-24-ene-3 beta,12 beta,20S-triol (12 beta-O-Glc-PPD) is a promising candidate for the development of anti-lung cancer drugs. Currently it can only be obtained by chemical semi-synthesis from protopanaxadiol (PPD) as a precursor, which leads to high cost and environmental unfriendliness. Synthetic biology has been acknowledged as a green and economical approach for the production of bioactive products. Herein, we first optimized the PPD-producing chassis to increase the precursor PPD supply. Then, the catalytic efficiency of UGT109A1 from Bacillus subtilis was improved through protein engineering to produce 3,12-Di-O-beta-d-glucopyranosyl-dammar-24-ene-3 beta,12 beta,20S-triol (3 beta,12 beta-Di-O-Glc-PPD) more effectively. Next, a high-efficiency beta-glycosidase Bgy2 was identified from Lactobacillus brevis and used to hydrolyze the C3 glucosyl moiety of 3 beta,12 beta-Di-O-Glc-PPD to produce 12 beta-O-Glc-PPD. Finally, combining protein and metabolic engineering led to de novo biosynthesis of 12 beta-O-Glc-PPD in Saccharomyces cerevisiae with a titer of 38.6 mg L-1. This study utilizes a green and sustainable biotechnology to produce 12 beta-O-Glc-PPD, which lays the foundation for its industrial production.