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

[Image: see text] Isoprene is a valuable platform chemical, which is produced by engineered microorganisms, albeit in low quantities. The amount of isoprene produced is usually measured by gas chromatography, which can be time-consuming and expensive. Alternatively, biosensors have evolved as a powerful tool for real-time high-throughput screening and monitoring of product synthesis. The AraC-pBAD-inducible system has been widely studied, evolved, and engineered to develop biosensors for small molecules. In our preliminary studies, the AraC-pBAD system was mildly induced at higher isoprene concentrations when arabinose was also available. Hence, in the present study, we designed and constructed a synthetic biosensor based on the AraC-pBAD system, wherein the ligand-binding domain of AraC was replaced with IsoA. On introducing this chimeric AraC-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 isoprene and arabinose. The biosensor sensitivity and dynamic range were further enhanced by removing operator sequences and by substituting the native promoter (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 essential for the functioning of our biosensor, while AcIa provides enhanced sensitivity and specificity for isoprene. However, insights into how AraC-AcIa interacts and the possible working mechanism remain to be explored. This study provides a prototype for developing chimeric AraC-based biosensors with proteins devoid of known dimerizing domains and opens a new avenue for further study and exploration.