کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
31568 44819 2012 9 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Parallel labeling experiments with [U-13C]glucose validate E. coli metabolic network model for 13C metabolic flux analysis
موضوعات مرتبط
مهندسی و علوم پایه مهندسی شیمی بیو مهندسی (مهندسی زیستی)
پیش نمایش صفحه اول مقاله
Parallel labeling experiments with [U-13C]glucose validate E. coli metabolic network model for 13C metabolic flux analysis
چکیده انگلیسی

13C-metabolic flux analysis (MFA) is a widely used method for measuring intracellular metabolic fluxes in living cells. 13C MFA relies on several key assumptions: (1) the assumed metabolic network model is complete, in that it accounts for all significant enzymatic and transport reactions; (2) 13C-labeling measurements are accurate and precise; and (3) enzymes and transporters do not discriminate between 12C- and 13C-labeled metabolites. In this study, we tested these inherent assumptions of 13C MFA for wild-type E. coli by parallel labeling experiments with [U-13C]glucose as tracer. Cells were grown in six parallel cultures in custom-constructed mini-bioreactors, starting from the same inoculum, on medium containing different mixtures of natural glucose and fully labeled [U-13C]glucose, ranging from 0% to 100% [U-13C]glucose. Macroscopic growth characteristics of E. coli showed no observable kinetic isotope effect. The cells grew equally well on natural glucose, 100% [U-13C]glucose, and mixtures thereof. 13C MFA was then used to determine intracellular metabolic fluxes for several metabolic network models: an initial network model from literature; and extended network models that accounted for potential dilution effects of isotopic labeling. The initial network model did not give statistically acceptable fits and produced inconsistent flux results for the parallel labeling experiments. In contrast, an extended network model that accounted for dilution of intracellular CO2 by exchange with extracellular CO2 produced statistically acceptable fits, and the estimated metabolic fluxes were consistent for the parallel cultures. This study illustrates the importance of model validation for 13C MFA. We show that an incomplete network model can produce statistically unacceptable fits, as determined by a chi-square test for goodness-of-fit, and return biased metabolic fluxes. The validated metabolic network model for E. coli from this study can be used in future investigations for unbiased metabolic flux measurements.


► E. coli was grown in six parallel cultures in mini-bioreactors at 10-mL.
► Steady-state isotopic labeling experiments were performed using 0–100% [U-13C]glucose.
► Off-gas data and growth profiles did not provide evidence for kinetic isotope effect.
► An initial network model from literature produced biased metabolic fluxes and poor fits.
► The validated network model accounted for dilution of intracellular CO2 by inlet air.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Metabolic Engineering - Volume 14, Issue 5, September 2012, Pages 533–541
نویسندگان
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