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. <em>Clostridium thermocellum</em> 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.糖酵解（Glycolysis）是一类古老、分布广泛且高度保守的代谢途径，可将葡萄糖转化为丙酮酸。在经典糖酵解途径中，磷酸果糖激酶（PFK）反应在调控途径通量方面发挥着关键作用。热纤梭菌（*Clostridium thermocellum*）拥有非典型糖酵解途径，其PFK反应以焦磷酸（PPi）替代三磷酸腺苷（ATP）作为磷酸供体。PPi依赖型PFK反应的热力学驱动力减弱，使得整个途径更趋近于热力学平衡，此前研究预测该变化会限制产物滴度。本研究将PPi-PFK反应替换为ATP依赖型PFK反应。实验证实，该局部改变与热力学预测相符：1,6-二磷酸果糖与6-磷酸果糖的比值升高，且该反应的反向通量（通过¹³C标记测定）有所降低。然而，发酵产物的最终滴度与分布并未发生改变，这表明PPi-PFK反应的热力学约束并非限制产物滴度的唯一因素。