The Bifunctional Alcohol and Aldehyde Dehydrogenase Gene, adhE , Is Necessary for Ethanol Production in Clostridium thermocellum and Thermoanaerobacterium saccharolyticum

Thermoanaerobacterium saccharolyticum andClostridium 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 theadhE gene inC. thermocellum andT. saccharolyticum . Deletion ofadhE reduced ethanol production by >95% in bothT. saccharolyticum andC. thermocellum , confirming thatadhE is necessary for ethanol formation in both organisms. In bothadhE deletion strains, fermentation products shifted from ethanol to lactate production and resulted in lower cell density and longer time to reach maximal cell density. InT. saccharolyticum , theadhE 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 theT. saccharolyticum parent strain but did not increase activity in theadhE deletion strain, suggesting that ALDH activity was inhibited. InC. thermocellum , theadhE deletion strain lost >90% of ALDH and ADH activity in cell extracts. TheC. 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.IMPORTANCE Thermoanaerobacterium saccharolyticum andClostridium 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 ofadhE , a gene encoding a bifunctional alcohol and aldehyde dehydrogenase. Deletion ofadhE reduced ethanol production by >95% in bothT. saccharolyticum andC. thermocellum , confirming thatadhE is necessary for ethanol formation in both organisms. In strains withoutadhE , we note changes in biochemical activity, product formation, and growth.