甾醇C-24甲基转移酶基因在酿酒酵母中的内源表达及工程菌麦角甾醇的合成

Endogenesis Expression of Sterol C-24 Methyltransferase Increases Ergosterol Biosynthesis in Saccharomyces cerevisiae

摘要:

通过高保真PCR克隆到含酿酒酵母甾醇C-24甲基转移酶基因编码序列及终止子序列的DNA片段ERG6, 以大肠杆菌-酿酒酵母穿梭质粒YEp352为载体, 磷酸甘油酸激酶基因PGK1启动子为上游调控元件构建了酵母菌表达质粒pPERG6。通过同源重组, 以铜离子螯合蛋白基因CUP1替换染色体上ERG6基因内部序列获得ERG6破坏菌株YS58-erg6, 其中麦角甾醇的合成被阻断, 同时细胞的生长也受到明显抑制。表达质粒pPERG6转化破坏菌株YS58-erg6后, 不但使细胞恢复了合成麦角甾醇的能力, 细胞生物量也得到明显提高, 这说明表达质粒上的ERG6基因得到了功能性的表达。分别用载体质粒YEp352和表达质粒pPERG6转化酿酒酵母单倍体菌株YS58, 获得对照菌株YS58(YEp352)和重组菌株YS58(pPERG6)。重组菌株YS58(pPERG6) 生物量和麦角甾醇含量分别是对照菌YS58(YEp352)的1.23和1.32倍。可见甾醇C-24甲基转移酶基因的高表达可以增强酵母细胞麦角甾醇的合成能力。

Abstract:

Ergosterol is economically important as the precursor of vitamin D2 and the main material to produce steroid drugs. The biosynthesis of sterols in yeast is complex. ERG6 gene encodes the sterol C-24 methyltransferase catalyzing the fifteenth reaction in ergosterol biosynthesis. In this study, ERG6 gene was cloned from Saccharomyces cerevisiae YSF-20 by PCR. To express ERG6 gene properly in S. cerevisiae, expression plasmid pPERG6 containing ERG6 under the control of PGK1 promoter was constructed and then introduced into S. cerevisiae YS58 to generate recombinant strain YS58(pPERG6). Results of ERG6 disruption and complementation showed that the ERG6 gene on plasmid pPERG6 expressed functionally in S. cerevisiae. The ergosterol content in recombinant strains YS58(pPERG6) was 1.32 times of that in the control strain YS58(YEp352), and at the same time, the biomass of the recombinant strain was 1.23 times of that of the control strain. Results in this study showed that the internal expression of ERG6 gene enhanced the ergosterol biosynthesis in yeast.