Genetic investigation of hydrogenases in Thermoanaerobacterium thermosaccharolyticum suggests that redox balance via hydrogen cycling enables high ethanol yield

Abstract

Thermoanaerobacterium thermosaccharolyticum is an anaerobic and thermophilic bacterium that has been genetically engineered for ethanol production at very high yields. However, the underlying reactions responsible for electron flow, redox equilibrium, and how they relate to ethanol production in this microbe are not fully elucidated. Therefore, we performed a series of genetic manipulations to investigate the contribution of hydrogenase genes to high ethanol yield, generating evidence for the importance of hydrogen-reacting enzymes in ethanol production. Our results indicate that a high ethanol yield, >85% of the theoretical maximum, only occurs when the hfsD, hydAB, and nfnAB genes are all present together, while the hfsB gene is absent. We propose that the products of these three gene clusters facilitate an NADPH-generating reaction via hydrogen cycling, with a stoichiometry comparable with a canonical ferredoxin:NADP+ oxidoreductase (FNOR; EC 1.18.1.2) reaction. The hypothesized mechanism provides a balance of nicotinamide cofactors and facilitates ferredoxin recycling, leading to progress in optimizing the energy conversion of biomass-derived sugars to ethanol.