Abstract:In this study, a UDP-glycosyltransferase UGT76G1 gene recombinant pichia pastoris strain GS115/pPIC9K-UGT76G1 and a sucrose synthase mbSUS gene recombinant Pichia pastoris strain GS115/ pPIC9K/pPICZA-mbSUS derived from mung bean were constructed. Methanol was used to induce the production of UDP-glycosyltransferase and sucrose synthase, and a biocatalytic cascade reaction system was constructed to catalyze the bioconversion of stevioside (ST) into rebaudioside A (RA), which realized the effective recycling of uridine diphosphate glucose (UDPG) in the cascade reaction system. Through optimization of reaction conditions, the rate-limiting enzyme in the cascade reaction was found to be the glycosyltransferase UGT76G1, with the optimal enzyme activity ratio, U(UGT76G1):U(mbSUS), as 6:1. Under the reaction conditions (pH 7.0, UDP 1 mM, sucrose 50 mM and MgCl2 3 mM), ST at 10 mM was converted to RA at 8.20±0.11 mM, with the RA yield as 82.91%. The catalytic reaction time was greatly shortened compared to the Escherichia coli expression system. The in vitro coupling of UDP-glycosyltransferase and sucrose synthetase can lead to effective catalysis for the efficient synthesis of RA, and this research provides technical support for RA enzymatic biosynthesis and associated industrial application.
