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pH 7.0，UDP浓度1 mM，蔗糖浓度50 mM，MgCl2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 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.

国家重点研发计划项目（2018YFA0901700）；国家自然科学基金资助项目（31871739）