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

Abstract

12-O-β-D-Glucopyranosyl-dammar-24-ene-3β,12β,20S-triol (12β-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-β-D-glucopyranosyl-dammar-24-ene-3β,12β,20S-triol (3β,12β-Di-O-Glc-PPD) more effectively. Next, a high-efficiency β-glycosidase Bgy2 was identified from Lactobacillus brevis and used to hydrolyze the C3 glucosyl moiety of 3β,12β-Di-O-Glc-PPD to produce 12β-O-Glc-PPD. Finally, combining protein and metabolic engineering led to de novo biosynthesis of 12β-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 12β-O-Glc-PPD, which lays the foundation for its industrial production.