Increasing the thermodynamic driving force of the phosphofructokinase reaction in Clostridium thermocellum

Glycolysis is an ancient, widespread, and highly conserved metabolic pathway that converts glucose into pyruvate. In the canonical pathway, the phosphofructokinase (PFK) reaction plays an important role in controlling flux through the pathway. Clostridium thermocellum has an atypical glycolysis and uses pyrophosphate (PPi) instead of ATP as the phosphate donor for the PFK reaction. The reduced thermodynamic driving force of the PPi-PFK reaction shifts the entire pathway closer to thermodynamic equilibrium, which has been predicted to limit product titers. In this work, we replace the PPi-PFK reaction with an ATP-PFK reaction. We demonstrate that the local changes are consistent with thermodynamic predictions: the ratio of fructose 1,6-bisphosphate to fructose-6-phosphate increases, and the reverse flux through the reaction (determined by ¹³C labeling) decreases. The final titer and distribution of fermentation products, however, do not change, demonstrating that the thermodynamic constraints of the PPi-PFK reaction are not the sole factor limiting product titer.