Metabolic flux analysis in Escherichia coli by integrating isotopic dynamic and isotopic stationary 13C labeling data
✍ Scribed by Jochen Schaub; Klaus Mauch; Matthias Reuss
- Publisher
- John Wiley and Sons
- Year
- 2008
- Tongue
- English
- Weight
- 272 KB
- Volume
- 99
- Category
- Article
- ISSN
- 0006-3592
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✦ Synopsis
Abstract
The novel concept of isotopic dynamic ^13^C metabolic flux analysis (ID‐^13^C MFA) enables integrated analysis of isotopomer data from isotopic transient and/or isotopic stationary phase of a ^13^C labeling experiment, short‐time experiments, and an extended range of applications of ^13^C MFA. In the presented work, an experimental and computational framework consisting of short‐time ^13^C labeling, an integrated rapid sampling procedure, a LC‐MS analytical method, numerical integration of the system of isotopomer differential equations, and estimation of metabolic fluxes was developed and applied to determine intracellular fluxes in glycolysis, pentose phosphate pathway (PPP), and citric acid cycle (TCA) in Escherichia coli grown in aerobic, glucose‐limited chemostat culture at a dilution rate of D = 0.10 h^−1^. Intracellular steady state concentrations were quantified for 12 metabolic intermediates. A total of 90 LC‐MS mass isotopomers were quantified at sampling times t = 0, 91, 226, 346, 589 s and at isotopic stationary conditions. Isotopic stationarity was reached within 10 min in glycolytic and PPP metabolites. Consistent flux solutions were obtained by ID‐^13^C MFA using isotopic dynamic and isotopic stationary ^13^C labeling data and by isotopic stationary ^13^C MFA (IS‐^13^C MFA) using solely isotopic stationary data. It is demonstrated that integration of dynamic ^13^C labeling data increases the sensitivity of flux estimation, particularly at the glucose‐6‐phosphate branch point. The identified split ratio between glycolysis and PPP was 55%:44%. These results were confirmed by IS‐^13^C MFA additionally using labeling data in proteinogenic amino acids (GC‐MS) obtained after 5 h from sampled biomass. Biotechnol. Bioeng. 2008;99: 1170–1185. © 2007 Wiley Periodicals, Inc.