毕赤酵母醇氧化酶1启动子突变体的分离与鉴定

Isolation and Identification of AOX1 Promoter Mutant in Pichia pastoris

目的:通过醇氧化酶1启动子突变,筛选毕赤酵母高水平表达菌株。方法:通过致错PCR构建醇氧化酶1启动子突变体,经酶切连接到改造过的质粒HSA-pPIC9上,转化毕赤酵母GS115感受态细胞,摇瓶培养表达筛选人血清白蛋白(HSA)高表达突变体菌株。结果:克隆测序结果表明,突变的醇氧化酶1启动子-910和-569位点处共2个碱基发生了T→C突变;获得一株高表达菌株,摇瓶中HSA的表达量由200 mg/L提高到335 mg/L。结论:通过醇氧化酶1启动子突变成功构建了HSA高表达菌株。

Objective： To achieve high expressive clone of Pichia pastoris through AOX1 promoter mutation. Methods： AOX1 promoter mutants were constructed by random mutagenesis PCR. After digested by enzyme, AOX1 promoter mutants were ligated to modificated HSA-pPIC9 plasmid and then transformed to P.pastoris GS115. A high human serum albumin （HSA） expressive clone was achieved through expressive selection. Results： The mutated AOX1 promoter sequence analysis showed that there were two T→C point mutations at position of -910 and -569. A high expressive clone was achieved and the expression level of HSA in conical flask increased from 200 mg/L to 335 mg/L. Conclusion： A high HSA expressive clone was achieved through AOX1 promoter mutation.