Elucidating Clostridium thermocellum central metabolism enables rational strain design towards enhanced ethanol production

Consolidated bioprocessing (CBP) is a potentially feasible route for sustainable production of bio-based fuels that condenses multiple steps of biomass degradation and sugar fermentation into a single step. Clostridium thermocellum is an anaerobic, thermophilic bacterium that is capable of degrading cellulosic biomass directly into ethanol. Despite its growing popularity, the central metabolism of C. thermocellum is still lacking in understanding, particularly with respect to cofactor specificity, regulatory effects around the pyruvate/ acetyl-CoA node, and the complex redox cofactor recycling system which can synthesize many undesired side products. A complete understanding of the central metabolism is necessary for any rational strain design efforts, and so a central metabolic model of C. thermocellum has been developed which correlates strongly with experimental data. Using elementary mode analysis, we investigate the range of phenotypes that C. thermocellum can reach with varying genotypes. This central metabolic model has shown its ability to encompass the phenotype displayed by a number of genetically modified strains of C. thermocellum and provide strong evidence for the presence of certain catabolic reactions which have been disputed previously. Further, we show the model’s rationale for why previous metabolic engineering strategies have low target ethanol yields and provide genetic engineering strategies for reaching the target yield. Finally, we incorporate experimental data to infer enzymatic reaction bottlenecks in the electron transport chain which is hindering ethanol production via CBP.