Abstract: Previously, the glycosyltransferase mutant TW (S129T/F168W) was obtained to improve the production of salidroside. In this research, a cascade reaction comprising glycosyltransferase mutant TW and sucrose synthaseAtSuSy for salidroside synthesis was constructed. The cascade reaction conditions including temperature, pH, uridine diphosphate (UDP), sucrose, tyrosol, metal ions and two-enzyme ratio were optimized. Furthermore, the fed-batch cascade reaction was carried out under the optimal conditions to achieve the regeneration of uridine diphosphate-glucose (UDPG) and promote salidroside synthesis. Optimal conditions for salidroside synthesis were determined as: temperature of 35 ℃, pH 8.0, UDP concentration of 0.5 mmol/L, sucrose concentration of 1 mol/L, tyrosol concentration of 5 mmol/L, Ca2+ concentration of 5 mmol/L, and TW∶AtSuSy enzyme activity ratio of 0.5∶6. In a 10 mL reaction system, the yield of salidroside reached 10.3 g/L by cascade reaction using fed-batch strategy. Moreover, the possible molecular mechanism responsible for salidroside synthesis was also investigated by molecular docking, demonstrating that effective hydrogen bonds contributed to the efficient synthesis of salidroside. This study establishes a foundation for efficient salidroside preparation in industrial application in the future.

Key words: glycosyltransferase, salidroside, two-enzyme coupling, UDPG regeneration, fed-batch