Exploring the Strategy of Fusing Sucrose Synthase to Glycosyltransferase UGT76G1 in Enzymatic Biotransformation

Abstract

Uridine diphosphate glycosyltransferases (UGTs) as fine catalysts of glycosylation are increasingly used in the synthesis of natural products. Sucrose synthase (SuSy) is recognized as a powerful tool for in situ regenerating sugar donors for the UGT-catalyzed reaction. It is crucial to select the appropriate SuSy for cooperation with UGT in a suitable way. In the present study, eukaryotic SuSy from Arabidopsisthaliana (AtSUS1) helped stevia glycosyltransferase UGT76G1 achieve the complete conversion of stevioside (30 g/L) into rebaudioside A (RebA). Position of the individual transcription units containing the genes encoding AtSUS1 and UGT76G1 in the expression plasmid has an effect, but less than that of the fusion order of these genes on RebA yield. Fusion of the C-terminal of AtSUS1 and the N-terminal of UGT76G1 with rigid linkers are conducive to maintaining enzyme activities. When the same fusion strategy was applied to a L637M-T640V double mutant of prokaryotic SuSy from Acidithiobacillus caldus (AcSuSym), 18.8 ± 0.6 g/L RebA (a yield of 78.2%) was accumulated in the reaction mixture catalyzed by the fusion protein Acm-R3-76G1 (the C-terminal of AcSuSym and the N-terminal of UGT76G1 were linked with (EAAAK)3). This work would hopefully reveal the potential of UGT-SuSy fusion in improving the cascade enzymatic glycosylation.

Keywords

• glycosyltransferase UGT76G1
• rebaudioside A
• stevioside
• <i>Stevia rebaudiana</i>
• sucrose synthase