Thermodynamic analysis of the pathway for ethanol production from cellobiose in Clostridium thermocellum.

Clostridium thermocellum is a candidate for consolidated bioprocessing by carrying out both cellulose solubilization and fermentation. However, despite significant efforts the maximum ethanol titer achieved to date remains below industrially required targets. Several studies have analyzed the impact of increasing ethanol concentration on C. thermocellum's membrane properties, cofactor pool ratios, and altered enzyme regulation. In this study, we explore the extent to which thermodynamic equilibrium limits maximum ethanol titer. We used the max-min driving force (MDF) algorithm (Noor et al., 2014) to identify the range of allowable metabolite concentrations that maintain a negative free energy change for all reaction steps in the pathway from cellobiose to ethanol. To this end, we used a time-series metabolite concentration dataset to flag five reactions (phosphofructokinase (PFK), fructose bisphosphate aldolase (FBA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), aldehyde dehydrogenase (ALDH) and alcohol dehydrogenase (ADH)) which become thermodynamic bottlenecks under high external ethanol concentrations. Thermodynamic analysis was also deployed in a prospective mode to evaluate genetic interventions which can improve pathway thermodynamics by generating minimal set of reactions or elementary flux modes (EFMs) which possess unique genetic variations while ensuring mass and redox balance with ethanol production. MDF evaluation of all generated (336) EFMs indicated that, i) pyruvate phosphate dikinase (PPDK) has a higher pathway MDF than the malate shunt alternative due to limiting CO

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