(291f) In silico and in vitro Analysis of Energy Conservation and Bifurcating Enzymes in Clostridium Thermocellum

Clostridium thermocellum str. DSM1313 is a fast growing (poly)saccharide fermenter that produces H₂, ethanol, acetate, formate, and lactate as major byproducts, making this organism of interest to consolidated bioprocessing. The metabolic capabilities of this organism have been extensively studied, particularly to understand and optimize the bioconversion of sugars to biomass and/or byproducts. Less is known about the energy conserving pathways, specifically the role enzymatically catalyzed electron bifurcating (BF-) transhydrogenase and BF-hydrogenase reactions play in electron flux and energy generation. The objectives of this study were to, 1) characterize growth, byproduct production, and redox poise of C. thermocellum cultured under low or high H₂ partial pressures (pH₂); 2) quantify the thermodynamic limits of reactions catalyzed by enzymes implicated in electron flux; and 3) identify molecular components and design principles which are necessary for enhanced electron-mediated energy conservation. Growth characterization, byproduct production, and redox poise (i.e., [NAD(P)H]/[NAD(P)⁺]) were determined by culturing C. thermocellum on cellobiose and in the presence of either Ar or H₂ headspace (1 bar). Classic thermodynamic modeling of redox reactions associated with electron flow were constrained by in vivo measurements to predict catalytic bias and reaction direction under defined pH₂ conditions. Stoichiometric metabolic networking, specifically Elementary Flux Mode Analysis (EFMA) and Flux Balance Analysis (FBA), was used to integrate growth data, genomics, and thermodynamic analysis to model energy and byproduct yields under simulated conditions. C. thermocellum exhibited extensive metabolic redundancy (e.g., pyruvate metabolism, hydrogenases, transhydrogenases), which enabled multiple pathways to accomplish redox balance. Redox tuning of ferredoxin (Fd) midpoint potentials was found to be the most sensitive parameter for determining the equilibria of Fd-dependent reactions, and although the in vivo ranges of [NAD(P)H]/[NAD(P)+] did not vary enough to significantly effect reaction equilibria, these measured ratios enabled the prediction of Fd midpoint potentials associated with the BF-NADH dependent NADPH:Fd transhydrogenase (NfnI) and the energy conserving Rnf complex. Finally, the electron bifurcating [FeFe] hydrogenase reactions were predicted to improve byproduct and ATP yields per mol of substrate oxidized with the highest ATP yields requiring concomitant evolution of H₂. Production of H₂ was also required for metabolic strategies associated with acclimation to low pH stress. The results of this study revealed the importance of electron bifurcating reactions in electron-mediated energy conservation of C. thermocellum and identified important control points that can be targeted for metabolic engineering and optimization.