Abstract:To efficiently synthesize rebaudioside M (RM) by multiple-enzyme cascade reaction method, a recombinant E. coli containing UDP-glucosyltransferase UGT76G1 from Stevia rebaudiana, UDP-glucosyltransferase EUGT11 from Oryza sativa, and the sucrose synthase SUS1 from Arabidopsis thaliana was constructed respectively. To improve the catalytic efficiency of the rate-limiting enzyme, here, the EUGT11 single-promoter expression plasmid was modified by using multiple-promoter. When the triple-promoter initiated transcription, the enzyme activity of the recombinase EUGT11 was the highest, which was 2.13 times than that of single-promoter. When it participated in the in vitro multiple-enzyme cascade reaction system, under the condition that the enzyme ratio was SUS1: UGT76G1: EUGT11/3 copies of T7=1: 2: 6.39, the yield of RM was 3.79 mmol/L, which was 2.35 times higher than before. The in vitro multiple-enzyme cascade reaction system for the efficient synthesis of RM was successfully established. In summary, this study improved the strategy of enzymatic synthesis of RM and provided theoretical data support for industrial production.
