Clostridium thermocellum is an increasingly well-studied organism with considerable advantages for consolidated bioprocessing towards ethanol production. A potential limitation to increased ethanol production rates by C. thermocellum is the low thermodynamic driving force along its glycolytic pathway leading to increased carbon efficiency at the expense of rate of throughput. Here, a genome-scale resource balance analysis (RBA) model of C. thermocellum, ctxRBA791, is reconstructed based on a recently published stoichiometric model (iCTH669), global proteomics, and 13C MFA datasets to analyze proteome allocation, its metabolic burden, and how protein capacity limits metabolism. Glycolytic and fermentation enzyme concentrations were modeled with high predictive accuracy. The metabolic burden associated with the formation of the cellulosome grown on multiple carbon substrates was assessed, with the overall effect found to be relatively small, given the small size of the complex (consistently less that 2% of the total estimated proteome). Previously constructed strains LL1210 and LL1570 were simulated and assessed for ethanol yield, along with strain redesign strategies involving single enzyme substitutions. All explored strains achieved higher ethanol yields compared to wild-type, with strain LL1210 strain reaching the highest predicted yield (76.5% of theoretical max). Model ctxRBA791 provides a framework for assessing the effect of genome changes on the proteome allocation and cost and analyzing potential strain designs of C. thermocellum for ethanol production or other bioprocessing applications.
