Ferredoxin:NAD<
sup>
+<
/sup>
oxidoreductase (NADH-FNOR) catalyzes the transfer of electrons from reduced ferredoxin to NAD<
sup>
+<
/sup>
. This enzyme has been hypothesized to be the main enzyme responsible for ferredoxin oxidization in the NADH-based ethanol pathway in <
i>
Thermoanaerobacterium saccharolyticum<
/i>
however, the corresponding gene has not yet been identified. Here, we identified the Tsac_1705 protein as a candidate FNOR based on the homology of its functional domains. We then confirmed its activity <
i>
in vitro<
/i>
with a ferredoxin-based FNOR assay. To determine its role in metabolism, the <
i>
tsac_1705<
/i>
gene was deleted in different strains of T. saccharolyticum. In wild-type <
i>
T. saccharolyticum<
/i>
, deletion of <
i>
tsac_1705<
/i>
resulted in a 75% loss of NADH-FNOR activity, which indicated that Tsac_1705 is the main NADH-FNOR in <
i>
T. saccharolyticum<
/i>
. When both NADH- and NADPH-linked FNOR genes were deleted, the ethanol titer decreased and the ratio of ethanol to acetate approached unity, indicative of the absence of FNOR activity. As a result, we tested the effect of heterologous expression of Tsac_1705 in Clostridium thermocellum and found improvements in both the titer and the yield of ethanol.