Abstract
A novel approach to C-13 metabolic flux analysis (MFA) is presented
using cytosolic metabolite pool sizes and their C-13 labeling data from
an isotopically non-stationary C-13 labeling experiment (INST-CLE). The
procedure is demonstrated with an E. coli wild type strain grown at fed
batch conditions. The intra cellular labeling dynamics are excited by a
sudden step increase of the C-13 portion in the substrate feed. Due to
unchanged saturation of the substrate uptake system, the metabolic
fluxes remain constant during the following sampling time period of only
16 s, in which 20 samples are taken by an automated rapid sampling
device immediately stopping metabolism by methanol quenching. Subsequent
cell disruptive sample preparation and LC-MS/MS enabled simultaneous
determination of pool sizes and mass isotopomers of intra cellular
metabolites requiring detection limits in the nM range. Based on this
data the new computational flux analysis tool 13CFLUX/INST is used to
determine the intra cellular fluxes based on a complex carbon labeling
network model. The measured data is in good agreement with the model
predictions, thus proving the applicability of the new isotopically
non-stationary C-13 metabolic flux analysis (INST-C-13-MFA) concept.
Moreover, it is shown that significant new information with respect to
flux identifiability, non-measurable pool sizes, data consistency, or
large storage pools can be taken from the novel kind of experimental
data. This offers new insight into the biological operation of the
metabolic network in vivo. (c) 2006 Elsevier B.V. All rights reserved.
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