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

摘要:

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

Abstract:

Abstract:［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．