Identification of a rate-limiting step in a metabolic pathway using the kinetic model and in vitro experiment

Identification of the rate-limiting step in a metabolic pathway is an important challenge in metabolic engineering for enhancing pathway flow. Although specific enzyme activities (V_max) provide valuable clues for the identification, it is time-consuming and difficult to measure multiple enzymes in the pathway because different assay protocols are required for each enzyme. In the present study, we propose a method to simultaneously determine the V_max values of multiple enzymes using a kinetic model with a time course of the intermediate concentrations through an in vitro experiment. To demonstrate this method, nine glycolysis reactions for converting glucose-6-phosphate (G6P) to pyruvate in Escherichia coli were considered. In a reaction mixture containing G6P and cofactors, glycolysis was initiated by adding a crude cell extract obtained from stationary phase cells. The V_max values were optimized to minimize the difference between the measured and simulated time-courses using a kinetic model. Metabolic control analysis using the kinetic model with the estimated V_max values revealed that fructose bisphosphate aldolase (FBA) was the rate-limiting step in the upper part of glycolysis. The addition of FBA in the reaction mixture successfully increased the glycolytic flux in vitro. Furthermore, in vivo, the specific glucose consumption rate of an FBA overexpression strain was 1.4 times higher than that of the control strain during the stationary phase. These results confirmed that FBA was the rate-limiting step in glycolysis under the stationary phase. This approach provides V_max values of multiple enzymes in a pathway for metabolic control analysis with a kinetic model.