Engineering UDP-Glycosyltransferase UGTPg29 for the Efficient Synthesis of Ginsenoside Rg3 from Protopanaxadiol

Rare ginsenosides Rg3 and Rh2, which exhibit diverse pharmacological effects, are derivatives of protopanaxadiol (PPD). UDP-glycosyltransferases, such as the M315F variant of Bs-YjiC (Bs-YjiCm) from Bacillus subtilis and UGTPg29 from Panax ginseng, can efficiently convert PPD into Rh2 and Rh2 into Rg3, respectively. In the present study, the N178I mutation of Bs-YjiCm was introduced, resulting in an increase in Rh2 production. UDP-glycosyltransferase UGTPg29 was then engineered to improve its robustness through semi-rational design. The variant R91M/D184M/A287V/A342L, which indicated desirable stability and activity, was utilized in coupling with the N178I variant of Bs-YjiCm and sucrose synthase AtSuSy from Arabidopsis thaliana to set up a "one-pot" three-enzyme reaction for the biosynthesis of Rg3. The influential factors, including the ratio and concentration of UDP-glycosyltransferases, pH, and the concentrations of UDP, sucrose, and DMSO, were optimized. On this basis, a fed-batch strategy was adopted to achieve a Rg3 yield as high as 12.38 mM (9.72 g/L) with a final yield of 68.78% within 24 h. This work may provide promising UDP-glycosyltransferase candidates for ginsenoside biosynthesis.