AraC-Based Biosensor for the Detection of Isoprene in E. coli

Isoprene is a valuable platform chemical, which is producedbyengineered microorganisms, albeit in low quantities. The amount ofisoprene produced is usually measured by gas chromatography, whichcan be time-consuming and expensive. Alternatively, biosensors haveevolved as a powerful tool for real-time high-throughput screeningand monitoring of product synthesis. The AraC-pBAD-inducible systemhas been widely studied, evolved, and engineered to develop biosensorsfor small molecules. In our preliminary studies, the AraC-pBAD systemwas mildly induced at higher isoprene concentrations when arabinosewas also available. Hence, in the present study, we designed and constructeda synthetic biosensor based on the AraC-pBAD system, wherein the ligand-bindingdomain of AraC was replaced with IsoA. On introducing this chimericAraC-IsoA (AcIa) transcription factor with the native P-BAD promoter system regulating rfp gene expression,fluorescence output was observed only when wild-type Escherichia coli cells were induced with both isopreneand arabinose. The biosensor sensitivity and dynamic range were furtherenhanced by removing operator sequences and by substituting the nativepromoter (P-AraC) with the strong tac promoter (P-tac). The chimeric sensor did not work in AraC knockout strains; however,functionality was restored by reintroducing AraC. Hence, AraC is essentialfor the functioning of our biosensor, while AcIa provides enhancedsensitivity and specificity for isoprene. However, insights into howAraC-AcIa interacts and the possible working mechanism remain to beexplored. This study provides a prototype for developing chimericAraC-based biosensors with proteins devoid of known dimerizing domainsand opens a new avenue for further study and exploration.