Adaptive rewiring of NADPH electron flow reveals escape routes from redox-balance-based growth coupling in Escherichia coli.

Redox-balance-based growth coupling links oxidoreductase activity to cellular fitness and can enable high-throughput enzyme selection and strain evolution. However, because growth reflects the fitness of the whole cell, metabolic adaptation can circumvent the intended selective pressure. Here, we investigated the evolutionary stability of an aerobic NADPH redox-balance selection strain of Escherichia coli engineered to accumulate NADPH during growth on glucose. Adaptive evolution first selected mutations predicted to relax cofactor specificity in NADH-dependent quinone oxidoreductases, allowing NADPH-derived reducing equivalents to enter respiratory electron flow. Removing major NADH-to-quinone oxidoreductases did not eliminate the capacity for evolutionary escape; in one successfully evolving lineage, adaptive evolution uncovered a latent lactate-mediated route for electron transfer into the quinone pool. These results reveal strong selective pressure to reconnect soluble nicotinamide redox metabolism with energy-conserving respiration and demonstrate that E. coli can access multiple pathways for restoring electron flux between these systems. Overall this work outlines design principles for aerobic, growth-coupled strains which rely upon redox balance, illustrating that successful designs must limit respiratory rewiring while preserving sufficient carbon and energy flux to support growth and pathway selection.