, Volume 16, Issue 3, pp 419-437

Re-examination of metabolic fluxes in Escherichia coli during anaerobic fermentation of glucose using 13C labeling experiments and 2-dimensional nuclear magnetic resonance (NMR) spectroscopy

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Improved design of metabolic flux estimation using mixed label 13C labeling experiments and identifiability analysis motivated re-examination of metabolic fluxes during anaerobic fermentation in the Escherichia coli. Comprehensive metabolic flux maps were determined by using a mixture of differently labeled glucose and compared to conventional flux maps obtained using extracellular measurements and comprehensive metabolic flux maps obtained using only U-13C glucose as the substrate. As expected, conventional flux analysis performs poorly in comparison to 13C-MFA, especially in the Embden-Meyerhof-Parnas (EMP) and pentose phosphate (PP) pathways. Identifiability analysis indicated and experiments confirmed that a mixture of 10% U-l3C glucose, 25% 1-13C glucose, and 65% naturally labeled glucose significantly improved the statistical quality of all calculated fluxes in the PP pathway, the EMP pathway, the anaplerotic reactions, and the tricarboxylic acid cycle. Modifying the network topology for the presence and absence of the Entner-Doudoroff pathway and the glyoxylate shunt did not affect the value or quality of estimated fluxes significantly. Extracellular measurement of formate production was necessary for the accurate estimation of the fluxes around the formate node.