On the bioenergetic balance between biomass degradation and biofuel production: Insights from Clostridium thermocellum

Clostridium thermocellum is a consolidated bioprocessing bacterium which exhibits fast growth on crystalline cellulose and is capable of converting cellulose to biofuels (e.g., ethanol, hydrogen, and isobutanol). Several metabolic engineering strategies have approached but not yet met industrially relevant yields and titers due to complex, highly branched overflow metabolism. Therefore, a predictive model of C. thermocellum metabolism would be beneficial to direct future metabolic engineering efforts. In this work, we constructed a new genome scale model (GEM) for C. thermocellum DSM 1313 and trained the model with multiple experimental datasets for model-guided metabolic engineering. This model contains the first implementation of a dynamic cellulosome component and allows exploration of energetic constraints binding cellulose degradation, cell growth, and biofuel production. Using the GEM, we determined the energy cost associated with cellulosome production and secretion to be 2-fold higher than typical protein synthesis. Using experimental data on multiple carbon sources (cellobiose vs. cellulose) as well as multiple genotypes (disrupting hydrogen and/or acetate production), we were able to quantify effects of those perturbations on redox cofactor turnover and robustness of the overall metabolic network. Further, we used data from high-substrate loading experiments to dynamically study how metabolism changes throughout an industrially relevant batch condition to infer regulatory mechanisms. Finally, we explored the sensitivity of C. thermocellum metabolism to cellulosome expression perturbations on cell growth and fermentation products. These analyses elucidate the bioenergetic landscape regulating how C. thermocellum metabolism proceeds through a batch fermentation, and guide strategies for enhanced conversion of biomass to biofuels.