Direct Construction and Screening of the Expression Vector of Anaerobic Clostridium in Escherichia coli

Abstract

The complex procedures of molecular biological experiments with anaerobic microbes, such as reconstruction of metabolic pathway and expression of the recombinant gene, would introduce oxygen easily, which may result in death of the anaerobic microorganisms. This study was aimed at the problem that the process of construction and screening of shuttle vector in Clostridium would lethally exposed strains to oxygen. The possibility of initializing gene transcription by the thiolase promoter in Escherichia coli was analyzed using endoglucanase gene Cphy3367 as a reporter gene. Compared with control strain, recombinant strains containing pSOS95-Cphy3367 plasmid grew more slowly and had a biomass reduction. The results of real-time quantitative PCR and activity detection of recombinant enzyme showed that the thiolase promoter in E. coli initiated the expression of gene Cphy3367. The initiation of gene Cphy3367 peaked at 48 h incubation at 37 ℃. In conclusion,the thiolase promoter can be recognized by host cell and the gene can be translated into protein in E. coli. The anaerobic Clostridium shuttle vector containing the thiolase promoter can be actively screened in E. coli, thereby reduce complexity and difficulty of the anaerobic operation.