초록
<P><B>Background</B></P><P>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<SUB>2</SUB>, and amino acids that compete with ethanol production for carbon and electrons. Elimination of H<SUB>2</SUB> production could redirect carbon flux towards ethanol production by making more electrons available for acetyl coenzyme A reduction to ethanol.</P><P><B>Results</B></P><P>H<SUB>2</SUB> 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<SUB>2</SUB> production in <I>∆hydG∆ech</I> was undetectable, and the ethanol yield nearly doubled to 64% of the maximum theoretical yield. Genomic analysis of <I>∆hydG</I> 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, <I>∆hydG</I> and <I>∆hydG∆ech</I> are not more ethanol tolerant than the wild type, illustrating the complicated interactions between redox balancing and ethanol tolerance in <I>C. thermocellum</I>.</P><P><B>Conclusions</B></P><P>The dramatic increase in ethanol production suggests that targeting protein post-translational modification is a promising new approach for simultaneous inactivation of multiple enzymes.</P><P><B>Electronic supplementary material</B></P><P>The online version of this article (doi:10.1186/s13068-015-0204-4) contains supplementary material, which is available to authorized users.</P>