1-41: Mutant Clostridium thermocellum alcohol dehydrogenase confers improved ethanol tolerance

Clostridium thermocellum is a thermophilic, obligate anaerobic Gram-positive bacterium that is a candidate microorganism for converting cellulosic biomass into ethanol via consolidated bioprocessing. Ethanol intolerance is a major stumbling block and has generally been thought to be a complex and likely a multigenic trait for which complex gene interactions come into play. Here, we identify single nucleotide changes (SNPs) associated with an ethanol tolerant mutant, and show ethanol tolerance is conferred by a single mutated bifunctional acetaldehyde-CoA/alcohol dehydrogenase gene (adhE). A mutation in AdhE lies within the NADH binding pocket of the alcohol dehydrogenase (ADH) domain, suggesting that specificity for NADH binding may be altered. Indeed, biochemical assays confirm a complete loss of NADH-dependent activity with concomitant acquisition of NADPH-dependent activity, which likely affects electron flow in the mutant strain. The simplicity of the ethanol-tolerant genetic phenotype revealed here enhances perspectives for rational engineering of mutant microbial strains for cellulosic ethanol production.