National Renewable Energy Laboratory, Golden, CO, USA.
Methods Mol Biol. 2020;2096:179-196. doi: 10.1007/978-1-0716-0195-2_14.
Metabolic flux analysis represents an essential perspective to understand cellular physiology and offers quantitative information to guide pathway engineering. A valuable approach for experimental elucidation of metabolic flux is dynamic flux analysis, which estimates the relative or absolute flow rates through a series of metabolic intermediates in a given pathway. It is based on kinetic isotope labeling experiments, liquid chromatography-mass spectrometry (LC-MS), and computational analysis that relate kinetic isotope trajectories of metabolites to pathway activity. Herein, we illustrate the mathematic principles underlying the dynamic flux analysis and mainly focus on describing the experimental procedures for data generation. This protocol is exemplified using cyanobacterial metabolism as an example, for which reliable labeling data for central carbon metabolites can be acquired quantitatively. This protocol is applicable to other microbial systems as well and can be readily adapted to address different metabolic processes.
代谢通量分析代表了理解细胞生理学的一个重要视角，并提供定量信息来指导途径工程。实验阐明代谢通量的一种有价值的方法是动态通量分析，它估计给定途径中一系列代谢中间产物的相对或绝对流速。它基于动力学同位素标记实验、液相色谱-质谱（LC-MS）和计算分析，将代谢物的动力学同位素轨迹与途径活性联系起来。在这里，我们说明了动态通量分析的数学原理，并主要集中描述用于生成数据的实验程序。该方案以蓝细菌代谢为例进行了说明，对于该方案，可以定量地获得中心碳代谢物的可靠标记数据。该方案也适用于其他微生物系统，并且可以很容易地适应不同的代谢过程。
