Stationary versus non-stationary (13)C-MFA: a comparison using a consistent dataset.
Besides the well-established (13)C-metabolic flux analysis ((13)C-MFA) which characterizes a cell's fluxome in a metabolic and isotopic stationary state a current area of research is isotopically non-stationary MFA. Non-stationary (13)C-MFA uses short-time isotopic transient data instead of long-time isotopic equilibrium data and thus is capable to resolve fluxes within much shorter labeling experiments. However, a comparison of both methods with data from one single experiment has not been made so far. In order to create a consistent database for directly comparing both methods a (13)C-labeling experiment in a fed-batch cultivation with a Corynebacterium glutamicum lysine producer was carried out. During the experiment the substrate glucose was switched from unlabeled to a specifically labeled glucose mixture which was immediately traced by fast sampling and metabolite quenching. The time course of labeling enrichments in intracellular metabolites until isotopic stationarity was monitored by LC-MS/MS. The resulting dataset was evaluated using the classical as well as the isotopic non-stationary MFA approach. The results show that not only the obtained relative data, i.e. intracellular flux distributions, but also the more informative quantitative fluxome data significantly depend on the combination of the measurements and the underlying modeling approach used for data integration. Taking further criteria on the experimental and computational part into consideration, the current limitations of both methods are demonstrated and possible pitfalls are concluded.
Noack
Stephan
S
Institut für Biotechnologie, Forschungszentrum Jülich, Leo-Brandt-Straße, Jülich, NRW, Germany. k.noeh@fz-juelich.de
Nöh
Katharina
K
Moch
Matthias
M
Oldiges
Marco
M
Wiechert
Wolfgang
W
D057486:Carbon Cycle; D002247:Carbon Isotopes / Q000032:analysis*; D048230:Corynebacterium glutamicum / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D007553:Isotope Labeling / Q000379:methods; D008239:Lysine / Q000096:biosynthesis
D002247:Carbon Isotopes; D005947:Glucose; D008239:Lysine