FAD依赖的葡萄糖脱氢酶多拷贝毕赤酵母菌株的构建及高效表达  

Construction of Multicopy Strains and High Expression of FAD-Dependent Glucose Dehydrogenase in Pichia pastoris

作　　者：董聪 王玥 罗同阳 高庆华 王庆庆 DONG Cong;WANG Yue;LUO Tong-yang;GAO Qing-hua;WANG Qing-qing(Hebei Res.Inst.of Microbiol.Co LTD,Baoding 071051)

机构地区：[1]河北省微生物研究所有限公司,河北保定071051

出　　处：《微生物学杂志》2023年第4期18-25,共8页Journal of Microbiology

基　　金：河北省科学院科技计划项目(20202);河北省科学院高层次人才培养与资助项目(2021G27);河北省科学院高层次人才培养与资助项目(2022G16)。

摘　　要：通过体外多拷贝构建实现FAD依赖的葡萄糖脱氢酶(FAD-GDH)在毕赤酵母(Pichia pastoris)X33菌株中的高效表达。将前期构建的经密码子偏好性优化FAD-GDH基因插入到pPICZαA质粒中,通过同尾酶法酶切酶连构建含1~4个表达盒的重组表达质粒,分别电转至毕赤酵母X33中,成功筛选到各种重组菌株。qRT-PCR测定结果表明,载体所含的表达盒数目与嵌合进毕赤酵母基因组中的GDH基因拷贝数之间存在正相关关系。重组菌在试管水平用甲醇诱导72 h,酶活达到最高,其中4拷贝的转化子表达水平最高;选择1拷贝和4拷贝转化子进行10 L发酵罐扩大培养,1拷贝菌株诱导108 h酶活达到最高697.125 U/mL,4拷贝菌株诱导132 h酶活达到最高1063.279 U/mL,比1拷贝酶活提高52.52%。结果表明通过增加目的基因拷贝数策略有助于提高FAD-GDH的表达量,为其进一步扩大生产提供参考。The coexpression strains harboring different copies FAD-dependent glucose dehydrogenase gene(FADGDH)were obtained successfully by multi-copy construction in vitro.Pre-constructed FAD-GDH gene optimized by codon bias inserted into the pPICZαA plasmid.The expression vectors containing 1-4 expression cassettes were constructed by enzyme digestion and transferred into Pichia pastoris X33 strain to obtain the FAD-GDH recombinant strains.The results based on the real-time quantitative PCR(qRT-PCR)showed that there existed a positive correlation between the expression cassette number and the gene copy number.The recombinant strains were induced with methanol at the test tube level for 72 h,and the enzyme activity reached the highest level;the expression level of fourcopy transformants was the highest.1-copy and 4-copy transformants for expansion in 10 L fermenter were selected,108 h after induction culture,enzyme activity of 1-copy recombinant strain reached 697.125 U/mL;132 h after induction culture,enzyme activity of 4-copy recombinant strain reached 1063.279 U/mL,52.52%higher than 1-copy.The results showed that the strategy of increasing the copy number of the target gene was conducive to increase the expression of FAD-GDH,which provided a theoretical and technical foundation for its further production expansion.

关 键 词：FAD依赖的葡萄糖脱氢酶 多拷贝 重组菌株 高效表达 毕赤酵母 

分 类 号：Q935[生物学—微生物学]