Mutant alcohol dehydrogenase leads to improved ethanol tolerance in Clostridium thermocellum

Steven D. Brown, Adam M. Guss, Tatiana V. Karpinets, Jerry M. Parks, Nikolai Smolin, Shihui Yang, Miriam L. Land, Dawn M. Klingeman, Ashwini Bhandiwad, Miguel Rodriguez, Babu Raman, Xiongjun Shao, Jonathan R. Mielenz, Jeremy C. Smith, Martin Keller, Lee R. Lynd

Research output: Contribution to journalArticlepeer-review

154 Scopus citations

Abstract

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 ethanoltolerant 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.

Original languageEnglish
Pages (from-to)13752-13757
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume108
Issue number33
DOIs
StatePublished - Aug 16 2011

Keywords

  • 454
  • Bioenergy
  • Genomics
  • Inhibitor
  • Resequencing

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