基于联合策略提高FAD依赖的葡萄糖脱氢酶的酵母表达  

Increasing the Expression of FAD-dependent Glucose Dehydrogenase by Recombinant Pichia pastoris Using a Combined Strategy

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

机构地区：[1]河北省微生物研究所有限公司,保定071051

出　　处：《生物技术通报》2023年第6期316-324,共9页Biotechnology Bulletin

基　　金：河北省科学院高层次人才培养与资助项目(2021G27,2022G16)。

摘　　要：为了提高了FAD依赖的葡萄糖脱氢酶在毕赤酵母中的表达量,本研究通过G418浓度梯度筛选,构建含有不同分子伴侣基因拷贝数共表达毕赤酵母重组菌株实现高效表达,优化摇瓶发酵条件。经G418浓度梯度筛选,得到1株高产重组菌,在此基础上分别构建了HAC1、Ero1、PDI三种分子伴侣1-4拷贝共表达重组菌株,RT-qPCR检测结果符合预期。试管水平酶活表明3拷贝PDI、3拷贝HAC1和2拷贝Ero1分别比1拷贝提高83%、77%和51%。总体比较而言,共表达3拷贝PDI的重组菌株酶活最高,比对照提高198%,优化摇瓶发酵条件表明培养基初始pH 7.0,摇瓶装液量50 mL,甲醇添加量1%,诱导温度28℃时,效果最佳。通过抗生素浓度筛选、增加分子伴侣的基因拷贝数策略可以提高FAD-GDH在毕赤酵母中的发酵产量。In order to increase the expression of FAD-dependent glucose dehydrogenase(FAD-GDH)in Pichia pastoris,the recombinant strains of co-expressing different copy numbers of molecular chaperone genes were constructed to achieve high-efficiency expression through the G418 concentration gradient screening,and the shake flask fermentation conditions were optimized.One high-yielding recombinant strain was obtained by G418 concentration gradient screening.On this basis,the recombinant strains co-expressing 1-4 copies of three molecular chaperones,HAC1,Ero1 and PDI,were constructed respectively,and the results of RT-qPCR were in line with expectations.The enzyme activity at the test tube level showed that 3 copies of PDI,3 copies of HAC1 and 2 copies of Ero1 increased by 83%,77%and 51%,respectively,compared with 1 copy.The recombinant strain with 3 copies of PDI had the highest enzyme activity,which was 198%higher than the control.The optimal conditions of the recombinant strain were as follows:initial pH 7.0,liquid loading volume 50 mL,the methanol addition amount 1%,and induction temperature 28℃.The efficient expression of FAD-GDH in P.pastoris is achieved by screening of antibiotic concentration and increasing the gene copy number of molecular chaperones.

关 键 词：FAD依赖的葡萄糖脱氢酶 毕赤酵母 抗生素梯度筛选 分子伴侣多拷贝 发酵条件 

分 类 号：TQ925[轻工技术与工程—发酵工程]