Enhancing PgUGT activity and thermostability via mutation site screening aided by molecular docking structure and...

Rebaudioside D (Reb D) is a natural high-intensity, zero-calorie sweetener with a superior taste profile compared to stevioside and rebaudioside A (Reb A), creating high consumer demand. In this study, the glycosyltransferase PgUGT_M0 was engineered via rational design and computational strategies to enhance the biocatalytic production of Reb D. A combinatorial mutant, PgUGT_M2 (M0-M87H/I146F/H149W/Y164W/I169F/N178Y/A342L), was constructed, exhibiting a 15.4-fold increase in enzymatic activity and a 33.5-fold extension in half-life. Molecular dynamics simulations attributed this enhanced thermostability to improved cavity filling, increased hydrophobicity, and strengthened hydrogen bonding networks. Furthermore, a dual-enzyme cascade system coupling PgUGT_M2 with sucrose synthase mbSUS was established inP. pastoris. Under optimized fed-batch conditions at 50 °C, a Reb D titer of 223.3 g/L (89.9% conversion) was achieved. Following the optimization of cell disruption and pH control, the process was scaled up to a 1.5 L enzymatic reactor, yielding 166.1 g/L Reb D with a 91.3% conversion rate within 20 h. This study presents an integrated approach-spanning enzyme engineering to process intensification-marking a critical advancement toward the industrial-scale production of Reb D.