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Metabolic flux analysis at ultra short time scale: Isotopically non-stationary 13C labeling experiments

Paper ID Volume ID Publish Year Pages File Format Full-Text
25464 43575 2007 19 PDF Available
Title
Metabolic flux analysis at ultra short time scale: Isotopically non-stationary 13C labeling experiments
Abstract

A novel approach to 13C metabolic flux analysis (MFA) is presented using cytosolic metabolite pool sizes and their 13C labeling data from an isotopically non-stationary 13C 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 13C 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 13C metabolic flux analysis (INST-13C-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.

Keywords
13PG, 1,3-diphosphateglycerate; 2PG, 2-phosphoglycerate; 3PG, 3-phosphoglycerate; 6PG, gluconate-6-phosphate; AcCoA, acetyl-CoA; AcN, cis-aconitate; AKG, α-keto-glutarate, 2-oxoglutarate; Asp, aspartate; Cit, citrate; DHAP, dihydroxy-acetone-phosphate; E4
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Metabolic flux analysis at ultra short time scale: Isotopically non-stationary 13C labeling experiments
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 129, Issue 2, 30 April 2007, Pages 249–267
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us