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.

鉴定代谢途径中的限速步骤是代谢工程中增强途径流动的重要挑战。尽管特定的酶活性（V _max）为鉴定提供了有价值的线索，但是由于每种酶需要不同的测定规程，因此耗时且难以测量途径中的多种酶。在本研究中，我们提出了一种通过动力学模型同时通过体外实验确定中间浓度随时间变化的方法来同时确定多种酶的V _max值的方法。为了证明这种方法，在大肠杆菌中进行了9次糖酵解反应，将6磷酸葡萄糖（G6P）转化为丙酮酸被认为是。在含有G6P和辅因子的反应混合物中，通过添加从固定相细胞获得的粗细胞提取物来开始糖酵解。使用动力学模型对V _max值进行了优化，以最大程度地减少了实测和模拟时程之间的差异。使用具有估计的V _max值的动力学模型进行代谢控制分析，结果表明果糖二磷酸醛缩酶（FBA）是糖酵解上部的限速步骤。在反应混合物中加入FBA成功地增加了体外的糖酵解通量。此外，体内，在稳定期，FBA过表达菌株的比葡萄糖消耗率比对照菌株高1.4倍。这些结果证实，FBA是固定相下糖酵解的限速步骤。此方法可为动力学模型的代谢控制分析提供途径中多种酶的V _max值。