Engineering electron metabolism to increase ethanol production in Clostridium thermocellum

The NfnAB (NADH-dependent reduced ferredoxin: NADP⁺ oxidoreductase) and Rnf (ion-translocating reduced ferredoxin: NAD⁺ oxidoreductase) complexes are thought to catalyze electron transfer between reduced ferredoxin and NAD(P)⁺. Efficient electron flux is critical for engineering fuel production pathways, but little is known about the relative importance of these enzymes in vivo. In this study we investigate the importance of the NfnAB and Rnf complexes in Clostridium thermocellum for growth on cellobiose and Avicel using gene deletion, enzyme assays, and fermentation product analysis. The NfnAB complex does not seem to play a major role in metabolism, since deletion of nfnAB genes had little effect on the distribution of fermentation products. By contrast, the Rnf complex appears to play an important role in ethanol formation. Deletion of rnf genes resulted in a decrease in ethanol formation. Overexpression of rnf genes resulted in an increase in ethanol production of about 30%, but only in strains where the hydG hydrogenase maturation gene was also deleted.