NADH and NAD+ are essential cofactors involved in numerous cellular oxidation-reduction reactions. They play critical roles in energy metabolism, redox homeostasis, and signaling processes. The intracellular NADH/NAD+ ratio serves as a key indicator reflecting redox state and metabolic efficiency, particularly in engineered cells containing synthetic pathways involving redox reactions. However, precise real-time monitoring and regulation of in vivo redox ratio remain challenging due to its instant variation and limitations of existing analytical methods. In this study, we engineered a transcription factor-based biosensor capable of dynamically responding to intracellular NADH/NAD+ ratio fluctuations in Escherichia coli. Firstly, we systematically evaluated multiple redox-responsive components and selected the most effective candidates to construct an initial biosensor system. Subsequently, we characterized its response curves under gradient intracellular redox states. To overcome the performance limitations of the regulator, we conducted site-directed mutagenesis to enhance sensitivity and broaden detection range, obtaining variants with improved detection windows. We further demonstrated the utility of the biosensor for identifying redox imbalances and metabolic bottlenecks in synthetic pathways. In addition, incorporating the biosensor into dynamic regulation circuits enabled real-time redox balancing and bioproduction titer improvement. Overall, this work provides a robust biosensor element to enrich the metabolic engineering toolbox and presented a novel strategy for dynamic monitoring and regulation based on the cellular redox level.
