S. D Brown, A. M Guss, T. V Karpinets, J. M Parks, N. Smolin, S. Yang, M. L Land, D. M Klingeman, A. Bhandiwand, M. Rodriguez, B. Raman, X. Shao, J. R Mielenz, J. C Smith, M. Keller, and L. R Lynd (2011)
Mutant Alcohol Dehydrogenase Leads to Improved Ethanol Tolerance in Clostridium thermocellum
Proceedings of the National Academy of Sciences (U.S.A.), 108(33):13752-13757.
Clostridium thermocellum is a thermophilic, obligately anaerobic, Gram-positive bacterium that is a candidate microorganism for converting cellulosic biomass into ethanol through consolidated bioprocessing. Ethanol intolerance is an important metric in terms of process economics, and tolerance has often been described as a complex and likely multigenic trait for which complex gene interactions come into play. Here, we resequence the genome of an ethanol-tolerant mutant, show that the tolerant phenotype is primarily due to a mutated bifunctional acetaldehyde-CoA/alcohol dehydrogenase gene (adhE), hypothesize based on structural analysis that cofactor specificity may be affected, and confirm this hypothesis using enzyme assays. 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. The simplicity of the genetic basis for the ethanol-tolerant phenotype observed here informs rational engineering of mutant microbial strains for cellulosic ethanol production.