In situ monitoring of intracellular NADP+/NADPH state: Engineered E. coli redox biosensor for carbon metabolism and toxicant detection

Continuous monitoring of the intracellular NADP⁺/NADPH redox state in living bacteria remains a major challenge in the ability to track and optimize the redox-dependent process during bioproduction and environmental monitoring. Continuous monitoring of the intracellular redox state can be achieved using a bacterial redox sensor like soxRS. The current study developed a whole-cell redox biosensor using the soxR:psoxS sensor fused to the lacZ reporter. This biosensor construct was developed for Escherichia coli and senses shifts in the intracellular NADP⁺/NADPH ratio, resulting in the differential expression of β-galactosidase (lacZ). The continuous and non-destructive monitoring of the cellular redox state was modulated by constitutively expressing lactose permease (lacY), enabling the direct uptake of reporter substrates, o-nitrophenyl-β-galactoside (ONPG) and p-aminophenyl β-D-galactopyranoside (PAPG). This study validated the performance of the redox sensor strain by cultivating it on various carbon sources, such as acetate, glucose, xylose, and glycerol, and observed intracellular redox changes through a colorimetric response. In addition, the NADPH assays were conducted to correlate the biosensor color changes quantitatively with the intracellular NADP⁺/NADPH levels. Finally, the redox biosensor application was extended to detect the organic pollutant triclosan using screen-printed electrodes. This study provides a robust platform for intracellular NADP⁺/NADPH redox monitoring with applications in environmental and microbial fermentation.