Replacing anaerobic fermentation with reverse β-oxidation enables growth-coupled evolution for enhanced fatty-alcohol production

Engineering robust fermentation hosts remains a barrier to scalable biomanufacturing. In this work we replace canonical mixed-acid fermentation in Escherichia coli with a redox‑balanced reverse β‑oxidation (rBOX) pathway that produces medium‑chain fatty alcohols and is bioenergetically equivalent to ethanol fermentation. This synthetic fermentation restores anaerobic growth, tightly coupling fitness to product formation and enabling adaptive laboratory evolution that selects for mutations increasing rBOX flux. Evolved strains increase maximum productivity fourfold (110 ± 0.15 mg/L/hr) and titer more than threefold up to 4.0 ± 0.28 g/L compared to the unevolved strain. Proteomic analyses of evolved strains indicate differential expression of enzymes at key metabolic nodes and activation of latent genes with functions overlapping those of rBOX enzymes. These results establish redox-balanced growth coupling as a powerful tool for strain improvement and demonstrate rBOX as a practical synthetic fermentation pathway.