Thermodynamic Analysis of Glycolysis in Clostridium thermocellum and Thermoanaerobacterium saccharolyticum Using C and H Tracers.

and are thermophilic anaerobic bacteria with complementary metabolic capabilities that utilize distinct glycolytic pathways for the conversion of cellulosic sugars to biofuels. We integrated quantitative metabolomics with H and C metabolic flux analysis to investigate the reversibility and thermodynamics of the central metabolic networks of these two microbes. We found that the glycolytic pathway in operates remarkably close to thermodynamic equilibrium, with an overall drop in Gibbs free energy 5-fold lower than that of or anaerobically grown The limited thermodynamic driving force of glycolysis in could be attributed in large part to the small free energy of the phosphofructokinase reaction producing fructose bisphosphate. The ethanol fermentation pathway was also substantially more reversible in than in These observations help explain the comparatively low ethanol titers of and suggest engineering interventions that can be used to increase its ethanol productivity and glycolytic rate. In addition to thermodynamic analysis, we used our isotope tracer data to reconstruct the central metabolic network, revealing exclusive use of the Embden-Meyerhof-Parnas (EMP) pathway for glycolysis, a bifurcated tricarboxylic acid (TCA) cycle, and a sedoheptulose bisphosphate bypass active within the pentose phosphate pathway. Thermodynamics constitutes a key determinant of flux and enzyme efficiency in metabolic networks. Here, we provide new insights into the divergent thermodynamics of the glycolytic pathways of and , two industrially relevant thermophilic bacteria whose metabolism still is not well understood. We report that while the glycolytic pathway in is as thermodynamically favorable as that found in model organisms, such as or , the glycolytic pathway of operates near equilibrium. The use of a near-equilibrium glycolytic pathway, with potentially increased ATP yield, by this cellulolytic microbe may represent an evolutionary adaptation to growth on cellulose, but it has the drawback of being highly susceptible to product feedback inhibition. The results of this study will facilitate future engineering of high-performance strains capable of transforming cellulosic biomass to biofuels at high yields and titers.