ABSTRACTThermoanaerobacterium saccharolyticumandClostridium thermocellumare 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 theadhEgene inC. thermocellumandT. saccharolyticum. Deletion ofadhEreduced ethanol production by >95% in bothT. saccharolyticumandC. thermocellum, confirming thatadhEis necessary for ethanol formation in both organisms. In bothadhEdeletion 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, theadhEdeletion 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. saccharolyticumparent strain but did not increase activity in theadhEdeletion strain, suggesting that ALDH activity was inhibited. InC. thermocellum, theadhEdeletion strain lost >90% of ALDH and ADH activity in cell extracts. TheC. thermocellumadhEdeletion 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.IMPORTANCEThermoanaerobacterium saccharolyticumandClostridium thermocellumare 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 ofadhEreduced ethanol production by >95% in bothT. saccharolyticumandC. thermocellum, confirming thatadhEis necessary for ethanol formation in both organisms. In strains withoutadhE, we note changes in biochemical activity, product formation, and growth.
The ethanol pathway from Thermoanaerobacterium saccharolyticum improves ethanol production in Clostridium thermocellum
