体外多酶级联反应体系高效合成莱鲍迪苷M

A Multiple-enzyme Cascade Reaction Strategy in Vitro for Efficient Synthesis of Rebaudioside M

为建立体外多酶级联反应体系高效合成莱鲍迪苷M(Rebaudioside M,RM),该文分别构建了含有甜叶菊来源的糖基转移酶UGT76G1和水稻来源的糖基转移酶EUGT11以及拟南芥来源的蔗糖合成酶SUS1的重组大肠杆菌,将甜菊苷经一锅法催化合成RM。为提升该体系中限速酶EUGT11的酶活,从而提升整个体外多酶级联反应体系的RM产量,本文将该酶表达质粒的T7启动子核心区域串联,并比较一重、二重、三重和四重启动子启动转录时该酶活性,当三重启动子启动转录时,重组酶EUGT11的酶活力最高,是一重启动子启动转录时的2.13倍,在酶活力配比为SUS1:UGT76G1:EUGT11/3 copies of T7=1:2:6.39的体外多酶级联反应体系中,RM产量为3.79 mmol/L,较酶活力配比为SUS1:UGT76G1:EUGT11=1:2:3的体外多酶级联反应体系中,RM的产量提升了2.35倍。该文成功建立了高效合成RM的体外多酶级联反应体系,为此后工业化酶法合成RM提供理论和数据支持。

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.