过表达HAC1提高葡萄糖氧化酶在毕赤酵母中分泌表达  

Overexpression of HAC1 enhances the expression of glucose oxidase in Pichia pastoris

作　　者：王庆庆 高庆华 罗同阳 董聪 王玥 刘小娜 唐兆宏 贾振华 WANG Qingqing;GAO Qinghua;LUO Tongyang;DONG Cong;WANG Yue;LIU Xiaona;TANG Zhaohong;JIA Zhenhua(Hebei Research Institute of Microbiology Co.,Ltd,Baoding 071051;Baoding Xian′erkang Biological Engineering Co.,Ltd,Baoding 071051;Institute of Biology,Hebei Academy of Sciences,Shijiazhuang 050051,China)

机构地区：[1]河北省微生物研究所有限公司,河北保定071051 [2]保定鲜尔康生物工程有限责任公司,河北保定071051 [3]河北省科学院生物研究所,河北石家庄050051

出　　处：《生物技术》2024年第5期537-542,共6页Biotechnology

基　　金：河北省科学院高层次人才培养与资助项目(2022G14;2023G23);河北省科学院科技计划项目(19203)

摘　　要：[目的]旨在提高重组葡萄糖氧化酶(GOD)在毕赤酵母中的表达。[方法]将构建的表达载体pPICZB-HAC1、pPICZB-Hrd1、pPIC3.5K-Ubc1线性化后,电击转化青霉葡萄糖氧化酶毕赤酵母感受态细胞,用含有50μg/mL Zeocin或200μg/mL G418的YPD平板筛选阳性转化子。阳性转化子进行试管诱导培养,筛选得到1株高产重组菌;在此基础上进行10 L发酵罐培养时,外源添加氯化血红素来提高葡萄糖氧化酶在毕赤酵母中的表达量。[结果]在试管水平筛选过表达转录激活因子HAC1、下游分子伴侣Hrd1和Ubc1的重组菌株,相比出发菌株酶活分别提高了56.46%、43.51%和48.02%,其中过表达HAC1重组青霉葡萄糖氧化酶菌株在10 L发酵罐酶活达到1308 U/mL。[结论]通过过表达HAC1或者分子伴侣可以辅助蛋白正确折叠,其中过表达HAC1菌株表达的葡萄糖氧化酶酶活最高,将酶活提高了86%。[Objective]To increase the expression of glucose oxidase in Pichia pastoris.[Method]The constructed expression vectors pPICZB-HAC1,pPICZB-Hrd1 and pPIC3.5K-Ubc1 were linearized and integrated into the genome of Penicillium glucose oxidase receptor cells of Pichia pastoris by electroporation.The recombinant yeasts were screened with 50μg/mL zeocin or 200μg/mL G418.After the transformants were fermented in testtube and a high-yield recombinant strain was obtained.On this basis,the expression of glucose oxidase in Pichia pastoris was increased by exogenous addition of hemin in 10 L fermentor.[Result]The enzyme activity of recombinant strains overexpressing transcriptional activator HAC1,downstream chaperone Hrd1 and Ubc1 was increased by 56.46%,43.51%and 48.02%,respectively,compared with that of the original strains.Among them,the enzyme activity of recombinant Penicillium glucose oxidase strain overexpressing HAC1 reached 1308 U/mL in 10 L fermenter.[Conclusion]Overexpression of HAC1 or chaperones can help the protein to fold correctly,and the overexpression of HAC1 strains has the highest expression of glucose oxidase enzyme activity,increasing the enzyme activity by 86%.

关 键 词：毕赤酵母 葡萄糖氧化酶 未折叠蛋白响应 HAC1基因 分子伴侣 氯化血红素 异源表达 分泌蛋白 

分 类 号：Q789[生物学—分子生物学]