Background: The native ability of <
i>
Clostridium thermocellum<
/i>
to rapidly consume cellulose and produce ethanol makes it a leading candidate for a consolidated bioprocessing (CBP) biofuel production strategy. <
i>
C. thermocellum<
/i>
also synthesizes lactate, formate, acetate, H-2, and amino acids that compete with ethanol production for carbon and electrons. Elimination of H-2 production could redirect carbon flux towards ethanol production by making more electrons available for acetyl coenzyme A reduction to ethanol. Results: H-2 production in <
i>
C. thermocellum<
/i>
is encoded by four hydrogenases. Rather than delete each individually, we targeted hydrogenase maturase gene <
i>
hydG<
/i>
, involved in converting the three [FeFe] hydrogenase apoenzymes into holoenzymes. Further deletion of the [NiFe] hydrogenase (<
i>
ech<
/i>
) resulted in a mutant that functionally lacks all four hydrogenases. H-2 production in ?hydG?ech was undetectable, and the ethanol yield nearly doubled to 64% of the maximum theoretical yield. Genomic analysis of ?hydG revealed a mutation in <
i>
adhE<
/i>
, resulting in a strain with both NADH- and NADPH-dependent alcohol dehydrogenase activities. While this same <
i>
adhE<
/i>
mutation was found in ethanol-tolerant <
i>
C. thermocellum<
/i>
strain E50C, ?hydG and ?hydG?ech are not more ethanol tolerant than the wild type, illustrating the complicated interactions between redox balancing and ethanol tolerance in <
i>
C. thermocellum<
/i>
. Conclusions: Finally, the dramatic increase in ethanol production suggests that targeting protein post-translational modification is a promising new approach for simultaneous inactivation of multiple enzymes.