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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 gETOH/gGluc 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. [ABSTRACT FROM AUTHOR]

Background: Caldicellulosiruptor bescii is a thermophilic cellulolytic bacterium that efficiently deconstructs lignocellulosic biomass into sugars, which subsequently can be fermented into alcohols, such as ethanol, and other products. Deconstruction of complex substrates by C. bescii involves a myriad of highly abundant, substrate-specific extracellular solute binding proteins (ESBPs) and carbohydrate-active enzymes (CAZymes) containing carbohydrate-binding modules (CBMs). Mass spectrometry-based proteomics was employed to investigate how these substrate recognition proteins and enzymes vary as a function of lignocellulosic substrates. Results: Proteomic analysis revealed several key extracellular proteins that respond specifically to either C5 or C6 mono- and polysaccharides. These include proteins of unknown functions (PUFs), ESBPs, and CAZymes. ESBPs that were previously shown to interact more efficiently with hemicellulose and pectin were detected in high abundance during growth on complex C5 substrates, such as switchgrass and xylan. Some proteins, such as Athe_0614 and Athe_2368, whose functions are not well defined were predicted to be involved in xylan utilization and ABC transport and were significantly more abundant in complex and C5 substrates, respectively. The proteins encoded by the entire glucan degradation locus (GDL; Athe_1857, 1859, 1860, 1865, 1867, and 1866) were highly abundant under all growth conditions, particularly when C. bescii was grown on cellobiose, switchgrass, or xylan. In contrast, the glycoside hydrolases Athe_0609 (Pullulanase) and 0610, which both possess CBM20 and a starch binding domain, appear preferential to C5/complex substrate deconstruction. Some PUFs, such as Athe_2463 and 2464, were detected as highly abundant when grown on C5 substrates (xylan and xylose), also suggesting C5-substrate specificity. Conclusions: This study reveals the protein membership of the C. bescii secretome and demonstrates its plasticity based on the complexity (mono-/disaccharides vs. polysaccharides) and type of carbon (C5 vs. C6) available to the microorganism. The presence or increased abundance of extracellular proteins as a response to specific substrates helps to further elucidate C. bescii’s utilization and conversion of lignocellulosic biomass to biofuel and other valuable products. This includes improved characterization of extracellular proteins that lack discrete functional roles and are poorly/not annotated. [ABSTRACT FROM AUTHOR]