| Summary: | 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 <i>Arabidopsis</i><i>thaliana</i> (<i>At</i>SUS1) helped <i>stevia</i> 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 <i>At</i>SUS1 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 <i>C</i>-terminal of <i>At</i>SUS1 and the <i>N</i>-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 <i>Acidithiobacillus caldus</i> (<i>Ac</i>SuSym), 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 <i>C</i>-terminal of <i>Ac</i>SuSym and the <i>N</i>-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.
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