Increasing ethanol production in Clostridium thermocellum by optimizing electron transfer

Biofuels derived from plant cell walls offers the potential to be a sustainable and economically attractive alternative to petroleum-based products.  Fuels from cellulosic biomass are particularly promising, but would benefit from lower processing costs. Clostridium thermocellum is a thermophilic bacterium that can rapidly solubilize and ferment cellulosic biomass. These two features make it a good candidate microorganism for consolidated bioprocessing for biofuel production. However commercialization is limited by low titer and low yield of ethanol. Engineered strains of Thermoanaerobacterium saccharolyticum, another thermophilic (although non-cellulolytic) bacterium, have been shown to produce over 50 g/L ethanol at near-theoretical yield. An important difference between these organisms is their method of electron transfer from ferredoxin to nicotinamide cofactors (FNOR activity). In vitro enzyme assay shown that Thermoanaerobacterium saccharolyticum has a strong capacity to transfer the electron from ferredoxin to both NAD⁺ and NADP⁺.  By contrast, in C. thermocellum the activity of this reaction was much lower and limited to NAD⁺. In this work, we have identified the main FNOR enzymes in Thermoanaerobacterium saccharolyticum and heterologously expressed them in Clostridium thermocellum. With the help of these FNOR enzymes, ethanol titer and yield were increased.