Strong synthetic stationary phase promoter-based gene expression system for Escherichia coli

Abstract

The Gram-negative bacterium Escherichia coli has been the work horse for recombinant protein production since the past several years. However, most of the gene expression systems used either require expensive inducers or exhibit low strength. In the present study, we have generated a strong promoter by repeated rounds of random mutagenesis in a stationary phase promoter isolated from Gordonia sp. IITR100. The promoter activity increased 16-fold as compared to the wild-type promoter. The resultant synthetic promoter showed β-galactosidase activities of ~16,000 Miller units which is comparable to the strong T7 promoter ~13,000 Miller units. The amount of LacZ produced by the synthetic promoter was found to be active for several days in stationary phase. The advantage of this synthetic promoter over T7 promoter includes its stationary phase auto-inducibility thereby saving the cost of addition of inducers. Expression of GFP_uv was observed in all the cells of E. coli due to the absence of requirement of inducer. A general-purpose vector containing the synthetic promoter with an MCS ready for use has been developed in the study. It has also been used to demonstrate the production of two heterologous proteins.