title: The bifunctional alcohol and aldehyde dehydrogenase gene, adhE, is necessary for ethanol production in Clostridium thermocellum and Thermoanaerobacterium saccharolyticum.
abstract: Thermoanaerobacterium saccharolyticum and Clostridium thermocellum are anaerobic thermophilic bacteria being investigated for their ability to produce biofuels from plant biomass. The bifunctional alcohol and aldehyde dehydrogenase gene, adhE, is present in these bacteria and has been known to be important for ethanol formation in other anaerobic alcohol producers. This study explores the inactivation of the adhE gene in C. thermocellum and T. saccharolyticum. Deletion of adhE reduced ethanol production by >95% in both T. saccharolyticum and C. thermocellum, confirming that adhE is necessary for ethanol formation in both organisms. In both adhE deletion strains, fermentation products shifted from ethanol to lactate production and resulted in lower cell density and longer time to reach maximal cell density. In T. saccharolyticum, the adhE deletion strain lost >85% of alcohol dehydrogenase (ADH) activity. Aldehyde dehydrogenase (ALDH) activity did not appear to be affected, although ALDH activity was low in cell extracts. Adding ubiquinone-0 to the ALDH assay increased activity in the T. saccharolyticum parent strain but did not increase activity in the adhE deletion strain, suggesting that ALDH activity was inhibited. In C. thermocellum, the adhE deletion strain lost >90% of ALDH and ADH activity in cell extracts. The C. thermocellum adhE deletion strain contained a point mutation in the lactate dehydrogenase gene, which appears to deregulate its activation by fructose 1,6-bisphosphate, leading to constitutive activation of lactate dehydrogenase.
  
  
  OBJECTIVE
  Thermoanaerobacterium saccharolyticum and Clostridium thermocellum are bacteria that have been investigated for their ability to produce biofuels from plant biomass. They have been engineered to produce higher yields of ethanol, yet questions remain about the enzymes responsible for ethanol formation in these bacteria. The genomes of these bacteria encode multiple predicted aldehyde and alcohol dehydrogenases which could be responsible for alcohol formation. This study explores the inactivation of adhE, a gene encoding a bifunctional alcohol and aldehyde dehydrogenase. Deletion of adhE reduced ethanol production by >95% in both T. saccharolyticum and C. thermocellum, confirming that adhE is necessary for ethanol formation in both organisms. In strains without adhE, we note changes in biochemical activity, product formation, and growth.
authors: Department of Biological Sciences, Dartmouth College, Hanover, New Hampshire, USA BioEnergy Science Center, Oak Ridge, Tennessee, USA.
authors: Jonathan
authors: J
authors: Lo
authors: Department of Biological Sciences, Dartmouth College, Hanover, New Hampshire, USA BioEnergy Science Center, Oak Ridge, Tennessee, USA.
authors: Tianyong
authors: T
authors: Zheng
authors: Thayer School of Engineering, Dartmouth College, Hanover, New Hampshire, USA BioEnergy Science Center, Oak Ridge, Tennessee, USA.
authors: Shuen
authors: S
authors: Hon
authors: Thayer School of Engineering, Dartmouth College, Hanover, New Hampshire, USA BioEnergy Science Center, Oak Ridge, Tennessee, USA.
authors: Daniel G
authors: DG
authors: Olson
authors: Department of Biological Sciences, Dartmouth College, Hanover, New Hampshire, USA Thayer School of Engineering, Dartmouth College, Hanover, New Hampshire, USA BioEnergy Science Center, Oak Ridge, Tennessee, USA Lee.R.Lynd@Dartmouth.edu.
authors: Lee R
authors: LR
authors: Lynd
mesh_terms: D000426:Alcohol Dehydrogenase / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D000445:Aldehyde Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D048013:Acetivibrio thermocellus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D000431:Ethanol / Q000378:metabolism*; D005285:Fermentation; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D055785:Gene Knockdown Techniques; D008969:Molecular Sequence Data; D009154:Mutation; D046950:Thermoanaerobacterium / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism
chemical_list: D000431:Ethanol; D000426:Alcohol Dehydrogenase; D000445:Aldehyde Oxidoreductases