过表达KAR2基因对毕赤酵母产米黑根毛霉脂肪酶的影响

黄金金 孙梦雪 王一洲 赵庆伊 张馨 张恺倪

摘要 Kar2p是分子伴侣蛋白，可与新生肽结合，促进新生肽进入内质网。为研究其对米黑根毛霉脂肪酶 （RML） 在毕赤酵母中表达的影响，通过PCR方法从毕赤酵母菌株中克隆到 KAR2 基因，并用毕赤酵母胞内表达载体pPIC3.5K构建过表达质粒 KAR2 -pPIC3.5K。用电转化的方法，将 KAR2 -pPIC3.5K二次转化到含4-拷 贝rml基 因的毕赤酵母重组菌株4pRML-X33中，获得能同时过表达RML和KAR2p的二次转化子4pRML-X33- KAR2 。摇瓶发酵发现，过表达 KAR2 基因对菌株生长无明显影响，但RML胞外酶活降低。采用RT-qPCR方法分析菌 中rml 及UPR相关基因的转录水平，发现 KAR2 基因转录水平上调了3.7倍，虽然仅引起与UPR相关的 HAC1 基因转录水平的下调，但却 使rml的 表达下调43%。说明 KAR2 过表达导致RML产量降低是降低了 rml 的mRNA量，而不是增加内质网中蛋白折叠的压力。

关键词 米黑根毛霉脂肪酶; KAR2 ;过表达;毕赤酵母;UPR

中图分类号 Q78 文献标识码 A

文章编号 0517-6611（2020）14-0103-05

doi：10.3969/j.issn.0517-6611.2020.14.028

Abstract Kar2p is a molecular chaperone that binds to new peptides and facilitates their entry into the endoplasmic reticulum （ER）.In order to study its effect on the expression of Rhizomucor miehei lipase （RML） in Pichia pastoris，KAR2 gene was cloned from Pichia pastoris gDNA by PCR method.And it was ligated to vector pPIC3.5K to obtained the intracellular overexpression plasmid KAR2 -pPIC3.5K. KAR2 -pPIC3.5K was then transformed into Pichia pastoris recombinant strain 4pRML-X33 which containing 4 copies of rml gene used electroporation transformation method.The twice transformant 4pRML-X33- KAR2 that could overexpress both RML and KAR2p was obtained.Shaking flask fermentation found that overexpression of KAR2 gene had no significant effect on strain growth，but the extracellular enzyme activity of RML decreased.Then rml mRNA level and transcription level of UPR-related genes were analyzed used RT-qPCR method.Results found that the transcription level of KAR2 increased 3.7 times，and only caused downregulation of the transcription level of the HAC1 gene associated with UPR，while rml mRNA decreased 43%.Those results suggested that the decrease in RML production after overexpression of KAR2 was due to the reduction of rml mRNA，rather than increased the protein folding pressure in ER.

Key words Rhizomucor miehei lipase; KAR2 ;Overexpression; Pichia pastoris; UPR

巴斯德畢赤酵母 （Pichia pastoris） 作为异源基因的表达体系已有近30年的历史，该表达体系成功表达了多种饲料酶、食品酶等，目前已发展成生产异源蛋白质最重要的工业宿主之一[1]。巴斯德毕赤酵母具有其他蛋白表达宿主不可替代的优势：严格的醇氧化酶启动子调控机制;基因操作方法简单;工业发酵过程成熟;内源蛋白分泌少;可对异源蛋白进行翻译后修饰等[2]。2009年，巴斯德毕赤酵母完整的基因组序列被公布[3]。据统计，有超过5 000种异源蛋白在巴斯德毕赤酵母表达体系中成功表达[4]。目前在该表达体系中报道的异源蛋白产量最高的是里氏木霉纤维素酶，产量高达18 g/L[5]。……