Yu Ruisong, Zong Wenming, Zhou Zhihua
Shanghai Key Laboratory of Agricultural Genetics and Breeding, Institute of Animal Science and Veterinary Medicine, Shanghai Academy of Agricultural Sciences, Shanghai 201106, China.
Wei Sheng Wu Xue Bao. 2011 Nov 4;51(11):1468-75.
A new method used to heterologously express [FeFe]-hydrogenase in Escherichia coli was investigated in our present study.
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.
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.
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.
本研究探究了一种用于在大肠杆菌中异源表达[FeFe]-氢化酶的新方法。
通过同源重组，将氢化酶的三个辅助基因（hydE、hydF和hydG）整合到大肠杆菌BW 25113-10的染色体中，该菌株中所有氢化酶基因均已失活。使用来自丁酸梭菌的氢化酶结构基因hydA来测试重组大肠杆菌BW 25113-13的氢化酶成熟能力。
通过PCR确认了三个辅助基因的正确整合，RT-PCR结果表明这三个辅助基因在重组体中被转录。hydA的活性表达表明组成型表达的辅助蛋白可以协助[FeFe]-氢化酶的成熟。
构建了一种简化的[FeFe]-氢化酶表达重组大肠杆菌BW25113-13。这将为[FeFe]-氢化酶的功能筛选以及在大肠杆菌中构建新型产氢途径奠定基础。
