title: Mutant alcohol dehydrogenase leads to improved ethanol tolerance in Clostridium thermocellum.
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 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.
authors.affiliation: Biosciences Division and BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA. brownsd@ornl.gov
authors.forename: Steven D
authors.initials: SD
authors.lastname: Brown
authors.forename: Adam M
authors.initials: AM
authors.lastname: Guss
authors.forename: Tatiana V
authors.initials: TV
authors.lastname: Karpinets
authors.forename: Jerry M
authors.initials: JM
authors.lastname: Parks
authors.forename: Nikolai
authors.initials: N
authors.lastname: Smolin
authors.forename: Shihui
authors.initials: S
authors.lastname: Yang
authors.forename: Miriam L
authors.initials: ML
authors.lastname: Land
authors.forename: Dawn M
authors.initials: DM
authors.lastname: Klingeman
authors.forename: Ashwini
authors.initials: A
authors.lastname: Bhandiwad
authors.forename: Miguel
authors.initials: M
authors.lastname: Rodriguez
authors.forename: Babu
authors.initials: B
authors.lastname: Raman
authors.forename: Xiongjun
authors.initials: X
authors.lastname: Shao
authors.forename: Jonathan R
authors.initials: JR
authors.lastname: Mielenz
authors.forename: Jeremy C
authors.initials: JC
authors.lastname: Smith
authors.forename: Martin
authors.initials: M
authors.lastname: Keller
authors.forename: Lee R
authors.initials: LR
authors.lastname: Lynd
mesh_terms: D000426:Alcohol Dehydrogenase / Q000235:genetics*; D000445:Aldehyde Oxidoreductases; D048013:Acetivibrio thermocellus / Q000201:enzymology / Q000235:genetics* / Q000502:physiology; D004361:Drug Tolerance / Q000235:genetics*; D000431:Ethanol / Q000378:metabolism*; D009154:Mutation; D009243:NAD; D009249:NADP
chemical_list: D009243:NAD; D000431:Ethanol; D009249:NADP; D000426:Alcohol Dehydrogenase; D000445:Aldehyde Oxidoreductases; C027014:acetaldehyde dehydrogenase (acylating)