Identification and engineering of a sucrose synthase from Stevia rebaudiana for glycosylation applications

Sucrose synthase (SuSy) is a unique glycosyltransferase that can be utilized in the production of nucleoside monosaccharides, such as diphosphate (UDP)-glucose, which serve as essential sugar donors for the glycosylation reactions catalyzed by UDP-dependent glycosyltransferases (UGTs). The selection of an appropriate SuSy coupled with a UGT is crucial for achieving the efficient synthesis of glycoside products. In this study, three candidate SuSy genes were identified from the transcriptome sequencing of Stevia rebaudiana, among which SrSUS1 was found to be expressed and active in Escherichia coli. The optimal temperature and pH for SrSUS1 were determined to be 55°C and pH 7.0, respectively. A variant SrSUS1_{T49A/L90P/V104E} was generated based on a consensus sequence strategy, exhibiting a 3.6-fold increase in activity and the enhanced affinity for sucrose (K_m = 52.32 mM), as well as the improved thermal stability and catalytic efficiency. By coupling SrSUS1_{T49A/L90P/V104E} with glycosyltransferase UGTAn85_Q23E/N65D or UGT76G4, respectively, the production of 163.32 mM (43.63 g/L) of 2-phenylethyl-β-D-pyranoside and 72.29 mM (93.35 g/L) of rebaudioside M was achieved within 24 h in one-pot, two-enzyme fed-batch reactions. This study provides new insights into plant-derived SuSys and presents a promising biocatalyst for industrial glycosylation applications.