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
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摘要: 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。Abstract: In this study, a new expression vector p ESCD which contains the two constitutive promoters, p PGK1 and p TEF1 that replaced the original induced promoter GAL1 / GAL10 in p ESC- Leu, was designed and constructed.The synthetic glycosyltransferase UGT76G1 coding gene was inserted into the restriction sites, Sal I and Xho I of p ESCD to construct the recombinant plasmid p ESCD- 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 24 h 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, 288 mg / L RA was produced after reaction for 15 h using the whole- cell catalyst, which was 5 times higher than that of thecontrol group.