Metabolic Engineering and Systems Biology Laboratory, Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE, USA.
Ginkgo Bioworks, Boston, MA, USA.
Nat Protoc. 2019 Oct;14(10):2856-2877. doi: 10.1038/s41596-019-0204-0. Epub 2019 Aug 30.
Precise quantification of metabolic pathway fluxes in biological systems is of major importance in guiding efforts in metabolic engineering, biotechnology, microbiology, human health, and cell culture. C metabolic flux analysis (C-MFA) is the predominant technique used for determining intracellular fluxes. Here, we present a protocol for C-MFA that incorporates recent advances in parallel labeling experiments, isotopic labeling measurements, and statistical analysis, as well as best practices developed through decades of experience. Experimental design to ensure that fluxes are estimated with the highest precision is an integral part of the protocol. The protocol is based on growing microbes in two (or more) parallel cultures with C-labeled glucose tracers, followed by gas chromatography-mass spectrometry (GC-MS) measurements of isotopic labeling of protein-bound amino acids, glycogen-bound glucose, and RNA-bound ribose. Fluxes are then estimated using software for C-MFA, such as Metran, followed by comprehensive statistical analysis to determine the goodness of fit and calculate confidence intervals of fluxes. The presented protocol can be completed in 4 d and quantifies metabolic fluxes with a standard deviation of ≤2%, a substantial improvement over previous implementations. The presented protocol is exemplified using an Escherichia coli ΔtpiA case study with full supporting data, providing a hands-on opportunity to step through a complex troubleshooting scenario. Although applications to prokaryotic microbial systems are emphasized, this protocol can be easily adjusted for application to eukaryotic organisms.
精确量化生物系统中的代谢途径通量对于指导代谢工程、生物技术、微生物学、人类健康和细胞培养等领域的努力至关重要。C 代谢通量分析（C-MFA）是用于确定细胞内通量的主要技术。在这里，我们介绍了一种 C-MFA 方案，该方案结合了平行标记实验、同位素标记测量和统计分析的最新进展，以及通过几十年经验发展的最佳实践。确保通量以最高精度估计的实验设计是该方案的一个组成部分。该方案基于在两个（或更多）平行培养物中用 C 标记的葡萄糖示踪剂培养微生物，然后进行气相色谱-质谱（GC-MS）测量蛋白质结合氨基酸、糖原结合葡萄糖和 RNA 结合核糖的同位素标记。然后使用 C-MFA 软件（如 Metran）估计通量，然后进行全面的统计分析以确定拟合度并计算通量的置信区间。该方案可以在 4 天内完成，并以≤2%的标准偏差定量代谢通量，比以前的实施有了很大的改进。该方案通过大肠杆菌ΔtpiA 案例研究进行了说明，并提供了完整的支持数据，为解决复杂故障排除情况提供了实践机会。尽管强调了该方案在原核微生物系统中的应用，但可以轻松调整该方案以应用于真核生物。
