毕赤酵母己糖运载体HXT1基因缺失菌株的构建

Construction of Hexose Transporter-like HXT1 Deletion Mutant in Pichia pastoris

摘要:

葡萄糖在酵母胞内的转运是依靠己糖运载体家族成员完成的。目前在酿酒酵母中已鉴定了18个己糖运载体基因, 各自功能也已得到深入的研究。然而在作为优良蛋白表达系统的毕赤酵母中, 目前国内外尚未有此类基因的相关报道。本文基于酵母同源重组率高的特性, 以G418作为抗性筛选标记, 分别在其5￠和3￠端连接待缺失毕赤酵母基因HXT1开放阅读框ORF的上下游各200 bp作为同源重组区, 电转毕赤酵母GS115感受态, 通过不同浓度G418抗性平板筛选, 最终得到了HXT1基因缺失的毕赤酵母菌株GS115ΔHXT1。通过与野生株比较发现, HXT1基因缺失株的生长状况及葡萄糖利用都有所下降。

Abstract:

Glucose was transported by the large number of hexose transporters in yeast cells. There were 18 hexose transporter genes had been identified in Saccharomyces cerevisiae. However, as an excellent expression system, there was no information of these genes had been reported in Pichia pastoris. Based on high homologous recombination efficiency in yeast, we chose G418 resistance for screening, 200 bp were cloned from the up and down sequences of HXT1 ORF respectively, then ligated to the 5￠ and 3￠ end of G418 resistance gene for recombination. After electroporation of GS115 spheroplast and screened through different G418 concentration plates, finally we obtained one HXT1 gene deletion mutant named GS115ΔHXT1. The growth rate and glucose consumption of this mutant were both lower than the wide type.