Redox-guided dynamic regulation for enhanced bioproduction using engineered NADH/NAD⁺ biosensors.

Efficient microbial production requires coordinated control of carbon flux and intracellular redox balance. However, NADH/NAD+ imbalance often constrains pathway performance, particularly when the NADH/NAD+-dependent synthetic pathways are introduced. Here, a S. aureus transcription factor Rex-based NADH/NAD+ biosensor was engineered and applied for redox-guided optimization of microbial production in Escherichia coli. Intracellular redox states were systematically tuned using NADH-consuming and NADH-generating enzymes, enabling quantitative characterization of the biosensor response. The optimized biosensor exhibited a 4.37-fold dynamic range in response to decreased NADH/NAD+ ratios. Rational mutagenesis of the Rex DNA-binding domain generated the A47Y variant, Rexm, which expanded the operational range and enabled a 3.14-fold response to increased NADH/NAD+ ratios. Rexm monitored redox dynamics during NADH-dependent 3-hydroxybutyrate biosynthesis and captured metabolic transitions during fermentation. Furthermore, the biosensor was integrated into a bifunctional dynamic regulation circuit that activated the non-oxidative glycolysis pathway while repressing glycolysis according to intracellular redox status. This redox-guided circuit balanced NADH supply and carbon flux allocation, increasing mevalonate titer by 28% to 6.23 g/L. Overall, the engineered Rex-based biosensor provides a tunable platform that enables intracellular redox sensing and dynamic regulation for enhanced production of value-added chemicals.