体外偶联UDP-糖基转移酶与蔗糖合成酶高效催化合成莱鲍迪苷A

Efficient Synthesis of Rebaudioside A Catalyzed by in vitro Coupling UDP-Glycosyltransferase and Sucrose Synthase

本研究构建了UDP-糖基转移酶UGT76G1基因的重组毕赤酵母菌株GS115/pPIC9K-UGT76G1和绿豆来源的蔗糖合成酶(mbSUS)基因的重组毕赤酵母菌株GS115/pPIC9K/pPICZA-mbSUS。通过甲醇诱导产酶,制备UDP-糖基转移酶UGT76G1和蔗糖合成酶,并构建生物催化级联反应体系,生物催化甜菊糖苷(Stevioside,ST)合成莱鲍迪苷A(rebaudioside A,RA),有效实现了级联反应体系中尿苷二磷酸葡萄糖(UDPG)的循环利用。本研究通过反应条件优化,发现级联反应中的限速酶是糖基转移酶UGT76G1,添加酶活比例为U_((UGT76G1)):U_((mbSUS))=6:1为合成莱鲍迪苷A的最佳酶活比例,在pH7.0,UDP浓度1mM,蔗糖浓度50mM,MgCl_2浓度3 mM的反应条件下,10 mM ST转化合成8.20±0.11 mM RA,RA的产率达到82.91%,与在大肠杆菌表达系统中相比,极大缩短了催化反应时间。利用体外偶联UDP-糖基转移酶与蔗糖合成酶催化合成RA为酶法高效生物合成RA及其产业化应用提供技术支持。

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 MgCl23 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.