Simultaneous Achievement of High Ethanol Yield and Titer in Clostridium Thermocellum

Background: Biofuel production from plant cell walls offers the potential for sustainable and economically attractive alternatives to petroleum-based products. Fuels from cellulosic biomass are particularly promising, but would benefit from lower processing costs. Clostridium thermocellum can rapidly solubilize and ferment cellulosic biomass, making it a promising candidate microorganism for consolidated bioprocessing for biofuel production, but increases in product yield and titer are still needed. Results: Here, we started with an engineered C. thermocellum strain where the central metabolic pathways to products other than ethanol had been deleted. After two stages of adaptive evolution, an evolved strain was selected with improved yield and titer. On chemically defined medium with crystalline cellulose as substrate, the evolved strain produced 22.4 +/- 1.4 g/L ethanol from 60 g/L cellulose. The resulting yield was about 0.39 g(ETOH)/g(Gluc eq), which is 75 % of the maximum theoretical yield. Genome resequencing, proteomics, and biochemical analysis were used to examine differences between the original and evolved strains. Conclusions: A two step selection method successfully improved the ethanol yield and the titer. This evolved strain has the highest ethanol yield and titer reported to date for C. thermocellum, and is an important step in the development of this microbe for industrial applications