Pyrophosphate-Free Glycolysis in Clostridium thermocellum Increases Both Thermodynamic Driving Force and Ethanol Titers

Background Clostridium thermocellum is a promising candidate for production of cellulosic biofuels, however its final product titer is too low for commercial application, and this may be due to thermodynamic limitations in glycolysis. Previous studies in this organism have revealed a metabolic bottleneck at the phosphofructokinase (PFK) reaction in glycolysis. In the wild type organism, this reaction uses pyrophosphate (PPi) as an energy cofactor, which is thermodynamically less favorable compared reactions that use ATP as a cofactor. Previously we showed that replacing the PPi-linked PFK reaction with an ATP-linked reaction increased the thermodynamic driving force of glycolysis, but only had a local effect on intracellular metabolite concentrations, and did not affect final ethanol titer. Results In this study, we substituted PPi- pfk with ATP- pfk , deleted the other PPi-requiring glycolytic gene pyruvate:phosphate dikinase ( ppdk ), and expressed a soluble pyrophosphatase ( PPase ) and pyruvate kinase ( pyk ) genes to engineer PPi-free glycolysis in C. thermocellum . We demonstrated a decrease in the reversibility of the PFK reaction, higher levels of lower glycolysis metabolites, and an increase in ethanol titer by an average of 38% (from 15.1 g/L to 21.0 g/L) by using PPi-free glycolysis. Conclusions By engineering PPi-free glycolysis in C. thermocellum , we achieved an increase in ethanol production. These results demonstrate that optimizing the thermodynamic landscape through metabolic engineering can enhance product titers. While further increases in ethanol titers are necessary for commercial application, this work represents a significant step toward engineering glycolysis in C. thermocellum to increase ethanol titers.