用经密码子优化后合成的甜叶菊UDP糖基转移酶UGT76G1编码基因构建了表达该酶的重组酿酒酵母YPH499(pYES2-UGT)。建立了通过柠檬酸钠调节酿酒酵母细胞内由葡萄糖到尿苷二磷酸葡萄糖(UDPG)的代谢通量,用于催化甜菊苷合成莱鲍迪苷A的全细胞催化方法。优化后的催化体系为:甜菊苷1 g/L,葡萄糖20g/L,普郎尼克F68 10 g/L,MgCl2 6 g/L,柠檬酸钠15 g/L,pH 7.2,细胞密度200 g湿细胞/L反应液,在37℃下经72 h后转化生成莱鲍迪苷A 267.89 mg/L。

The synthetic UDP glycosyl-transferase UGT76G1 coding gene with modification was inserted into the vector pYES2 with the restriction site of EcoR I and Xho I to construct the recombinant Saccharomyces cerevisiae YPH499(pYES2–UGT) strain,which can express UDP glycosyl-transferase UGT76G1.A whole cell catalysis method for adjusting UDPG synthesis metabolic pathway of the yeast cell was established by providing glucose for carbon source and enhancing the UDPG flux with the substrate Stevioside.The optimal conditions for the whole cell catalysis system were as follows:1 g/L Stevioside,20 g/L glucose,10 g/L PluronicF68,6 g/L MgCl2,15 g/L citric acid sodium,pH 7.2,the cell density was 200 g wet cells /L reaction solution,the reaction temperature was 37 ℃,the reaction time was 72 h.Under the optimal conditions,the output of Rebaudioside A could reach 267.89 mg/L.