Investigation of useful carbon tracers for 13C-metabolic flux analysis of Escherichia coli by considering five experimentally determined flux distributions

•Useful compositions of 13C-labeled glucose are investigated for 13C-MFA of E. coli.•Computer simulations revealed that [1,2-13C] was one of the best first choices.•Mixture of non-labeled, [1-13C] and [U-13C] at 0:8:2 was also suitable for 13C-MFA.•Mixture at 4:1:5 was specifically effective for estimation of glyoxylate pathway.•The wet 13C-MFA experiments of E. coli confirmed the findings.

The 13C-MFA experiments require an optimal design since the precision or confidence intervals of the estimated flux levels depends on factors such as the composition of 13C-labeled carbon sources, as well as the metabolic flux distribution of interest. In this study, useful compositions of 13C-labeled glucose for 13C-metabolic flux analysis (13C-MFA) of Escherichia coli are investigated using a computer simulation of the stable isotope labeling experiment. Following the generation of artificial mass spectra datasets of amino acid fragments using five literature-reported flux distributions of E. coli, the best fitted flux distribution and the 95% confidence interval were estimated by the 13C-MFA procedure. A comparison of the precision scores showed that [1, 2-13C]glucose and a mixture of [1-13C] and [U-13C]glucose at 8:2 are one of the best carbon sources for a precise estimation of flux levels of the pentose phosphate pathway, glycolysis and the TCA cycle. Although the precision scores of the anaplerotic and glyoxylate pathway reactions were affected by both the carbon source and flux distribution, it was also shown that the mixture of non-labeled, [1-13C], and [U-13C]glucose at 4:1:5 was specifically effective for the flux estimation of the glyoxylate pathway reaction. These findings were confirmed by wet 13C-MFA experiments.