Mutant alcohol dehydrogenase leads to improved ethanol tolerance in Clostridium thermocellum

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 108; no. 33; pp. 13752 - 13757
• Main Authors: Brown, Steven D., Guss, Adam M., Karpinets, Tatiana V., Parks, Jerry M., Smolin, Nikolai, Yang, Shihui, Land, Miriam L., Klingeman, Dawn M., Bhandiwad, Ashwini, Rodriguez, Miguel, Raman, Babu, Shao, Xiongjun, Mielenz, Jonathan R., Smith, Jeremy C., Keller, Martin, Lynd, Lee R.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 16-08-2011; National Acad Sciences
• Subjects: 09 BIOMASS FUELS; ALCOHOL DEHYDROGENASE; Alcohol Dehydrogenase - genetics; Alcohols; Aldehyde Oxidoreductases; Anaerobic microorganisms; BESC Clostridium thermocellum ethanol; Biofuels; Biological Sciences; BIOMASS; CELLULOSIC ETHANOL; CLOSTRIDIUM THERMOCELLUM; Clostridium thermocellum - enzymology; Clostridium thermocellum - genetics; Clostridium thermocellum - physiology; Cofactors; Dehydrogenases; Drug tolerance; Drug Tolerance - genetics; ECONOMICS; ELECTRONS; ENZYMES; ETHANOL; Ethanol - metabolism; Ethanol production; GENES; Genetic mutation; GENETICS; Genomes; Genotype & phenotype; Gram-positive bacteria; HYPOTHESIS; Intolerance; METRICS; MICROORGANISMS; MUTANTS; Mutation; NAD; NADP; OXIDOREDUCTASES; PHENOTYPE; PRODUCTION; SPECIFICITY; STRAINS; Thermophilic microorganisms; TOLERANCE
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1102444108

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.