酿酒酵母组成型表达甜叶菊糖基转移酶UGT76G1及相关性质研究

Constitutive expression and characteristics of glycosyltransferase UGT76G1 in Saccharomyces cerevisiae

本研究将酿酒酵母穿梭质粒p ESC-Leu的诱导型启动子GAL1和GAL10分别替换为PGK1和TEF1启动子,构建了组成型双启动子表达载体p ESCD,再将甜叶菊糖基转移酶UGT76G1的编码基因亚克隆到p ESCD的Sal I和Xho I酶切位点之间,构建了组成型表达UGT76G1的重组质粒p ESCD-UGT。将p ESCD-UGT转化到酿酒酵母YPH499中进行表达,结果确定该重组酵母在SD-L液体培养基培养24h达到稳定期,菌体培养8h蛋白表达量高,选用1%的甲苯作为重组菌全细胞催化的通透剂时,催化15h产生288mg/L的莱鲍迪甙A,其产量是对照组的5倍。

In this study, a new expression vector pESCD which contains the two constitutive promoters, pPGKt and pTEFt that replaced the original induced promoter GALl/GALl0 in pESC-Leu,was designed and constructed.The synthetic glycosyltransferase UGT76Gl coding gene was inserted into the restriction sites, Sail and Xho I of pESCD to construct the recombinant plasmid pESCD-UGT, which was then transformed into the Saccharomyces cerevisiae strain YPH499.The results confirmed that the recombinant cells grew into the stable phase when cultured for 24h and the highest expression of the recombinant protein was observed when cultured for 8h in SD-L.In case of 1 % methylbenzence as the cell permeating agent,288mg/L RA was produced after reaction for i5h using the whole-cell catalyst,which was 5 times higher than that of thecontrol group.