异源表达【FeFe】氢酶的基因工程大肠杆菌的构建  

Engineering of Escherichia coli for convenient expression of[FeFe]-hydrogenase

作　　者：于瑞嵩 宗文明[2,3] 周志华[2] 

机构地区：[1]上海市农业遗传育种重点实验室,上海市农业科学院畜牧兽医研究所,上海201106 [2]中国科学院合成生物学重点实验室,中国科学院上海生命科学研究院植物生理生态研究所,上海200032 [3]安徽农业大学工学院,合肥230036

出　　处：《微生物学报》2011年第11期1468-1475,共8页Acta Microbiologica Sinica

基　　金：国家自然科学基金(3107009830770271);中国科学院方向性项目(KSCX2-EW-G-13-1)~~

摘　　要：【目的】探讨一种构建异源表达【FeFe】氢酶的重组大肠杆菌的新方法。【方法】通过同源重组,依次将来源于丙酮丁醇梭菌中促进【FeFe】氢酶成熟的3个辅助基因hydE、hydF和hydG分别整合到大肠杆菌BW2513-10(缺失氢酶基因)的丙酮酸甲酸脱氢酶(ybiW)、乳酸脱氢酶(ldh)和乙醇脱氢酶(adhE)编码基因位点上。在此基础上进一步将含有来源于丁酸梭菌的氢酶基因的表达载体转化上述重组菌,并对转化子的氢酶活性进行分析。【结果】PCR和RT-PCR的检测结果表明,3个辅助基因都整合到BW 2513-10基因组相应位点并能够正常转录;丁酸梭菌【FeFe】氢酶的活性表达表明,组成型表达3个辅助因子能够促进【FeFe】氢酶的成熟,并获得了异源表达【FeFe】氢酶的重组大肠杆菌菌株BW23113-15。【结论】利用同源重组整合至染色体特定部位的方法可以实现【FeFe】氢酶辅助基因的组成型表达,简化了【FeFe】氢酶在大肠杆菌中异源表达的程序。该系统也将为不同氢酶的活性筛选及在大肠杆菌中构建新的产氢代谢途径奠定基础。[Objective] A new method used to heterologously express [FeFe]-hydrogenase in Escherichia coli was investigated in our present study.[Methods] By homologous recombination,three assistant genes（hydE、hydF and hydG） for hydrogenase were integrated into the chromosome of E.coli BW 25113-10,in which all hydrogenase genes were inactivated.A hydrogenase structural gene hydA from Clostridium butyricum was used to test the hydrogenase maturation ability of the recombined E.coli.BW 25113-13 [Results] The corrected integration of the three assistant genes was confirmed by PCR,and RT-PCR results indicated that the three accessory genes were transcripted in the recombinant.The active expression of hydA indicated that the constitutively expressed accessory proteins could assist the maturation of the [FeFe]-hydrogenases.[Conclusions] A simplified [FeFe]-hydrogenase expression recombinant E.coli BW25113-13 was constructed.It would lay foundations for the functional screening of [FeFe]-hydrogenases and the construction of novel hydrogen producing pathways in E.coli.

关 键 词：【FeFe】氢酶 异源表达 重组大肠杆菌 

分 类 号：Q78[生物学—分子生物学]