A distinct class of ferredoxin:NADP oxidoreductase enzymes driving thermophilic ethanol production.

Biofuel production from lignocellulosic biomass offers a transformative solution to reduce global fossil fuel dependency. Certain thermophilic anaerobes, including Clostridium thermocellum, show promise for renewable ethanol production due to their ability to break down plant material at high temperatures. However, achieving commercially viable ethanol yields has proven challenging despite extensive engineering efforts. Here, we characterized 27 ferredoxin:NADP oxidoreductase (Fnor) enzymes for their enzyme activity, nicotinamide cofactor specificity, thermotolerance, and functional expression in C. thermocellum. We identified a subset of 10 of these enzymes as a novel class of Fnor enzymes suited for metabolic pathways aimed at high-titer ethanol production. When expressed in engineered C. thermocellum, these enzymes increased ethanol production up to 2.2-fold. These findings establish a novel ethanol pathway and provide insights into physiological roles and biotechnological applications of this new class of Fnor enzymes.