Promoter Engineering Enables Control of Glucose-Responsive Gene Expression by Using a Transcriptional Regulator HexR

Abstract

In metabolic engineering, inducible promoters are important DNA elements that control bacterial cell’s physiology by expressing specific gene over the timing, location, and amount of a given gene product. However, typical chemical inducers such as IPTG cause toxicity to cell and make cost for bioproduction expensive. Using substrate-mediated induction is a solution for this issue and in this context, construction of HexR-mediated promoter is a promising tool for metabolic engineering. HexR is a regulator that represses glycolysis pathway of Pseudomonads dependent on concentration of 2-keto-deoxy-6-phosphogluconate (KDPG), an intermediate of Entner-Doudoroff pathway (EDP). Thus, in strains using EDP, HexR-based gene expression system can be used to control gene expression in the absence or the presence of glucose. In this study, a glucose-responsive hybrid promoter, PtachexO is constructed by introducing HexR binding site into a well-known tac promoter. Newly constructed PtachexO promoter is fused with gfp+ encoding green fluorescent protein and the gene expression from the promoter was analyzed in medium with or without glucose. PtachexO –gfp+ produced higher fluorescent signal when glucose exists, and it proved that introduction of HexR binding site can make promoter responsive to glucose.