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Metabolic Flux Analysis

Metabolic flux analysis (MFA) is widely used to probe fluxes in microbes. Fluxes describe what the cell does and, as such, provide an informative description of the overall cellular physiology. Monitoring flux changes in response to environmental and genetic perturbations provides key information about the distribution and control of metabolism that can be used for biotechnological and biomedical applications. Measuring intracellular fluxes is important because a typical coarse description of cell physiology that relies on just a few macroscopic variables such as growth rate, substrate uptake rate, and product accumulation is often not sufficient to understand intracellular network operations.

Physical behavior of cells is the integrated outcome of numerous cellular processes and emerges through the interaction of genes, proteins, and metabolites at multiple metabolic and regulatory levels. Metabolic fluxes constitute the final output of these interactions, and hence, experimental determination of these fluxes is crucial to observe, and eventually understand and manipulate the operation of biological networks

Metabolic flux analysis has emerged as a tool of great significance for metabolic engineering and clinical investigations. Metabolic fluxes are a fundamental determinant of cell physiology and a necessary parameter to quantify metabolic control within biochemical reaction networks. Fluxes describe the rate of metabolite interconversion in the context of the entire cell. As such, experimental determination of metabolic fluxes is crucial to observe, and eventually understand and manipulate the operation of biological networks.


Metabolic Flux Analysis

Fig 1. Metabolic flux analysis based on tracer experiments, GC-MS analysis, and EMU modeling


Fluxes are determined experimentally by stable-isotope tracer experiments, GC-MS analysis of labeling incorporation, and computational tools for isotopomer, or EMU balancing. One novel technology being developed in our lab is based on the use of multiple stable-isotope tracers, i.e. 13C, 2H and 18O, and quantitative analysis of labeling by tandem mass spectrometry, i.e. GC-MS/MS, for quantitative monitoring of network responses to genetic and exogenous perturbations. Such in vivo fluxes are then integrated with other genome-wide data to provide a global perspective on the integrated genetic and metabolic regulations that occur within cellular systems.


References

Antoniewicz MR, Kelleher JK, Stephanopoulos G.
Determination of confidence intervals of metabolic fluxes estimated from stable isotope measurements. Metab Eng 8(4): 324-337, 2006

Antoniewicz MR, Kelleher JK, Stephanopoulos G.
Elementary Metabolite Units (EMU): A novel framework for modeling isotopic distributions. Metab Eng 9(1): 68-86, 2007

Antoniewicz MR, Kraynie DF, Laffend LA, González-Lergier J, Kelleher JK, Stephanopoulos G.
Metabolic flux analysis in a nonstationary system: fed-batch fermentation of a high yielding strain of E. coli producing 1,3-propanediol. Metab Eng 9(3): 277-92, 2007

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