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

Abstract

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.

Keywords: 13C-labeling experiment; 13C-metabolic flux analysis; Computer simulation; Design of experiment; Escherichia coli.

Fig. 1

Procedure for computational simulation of ¹³C-MFA experiments. Step 1: The intracellular flux distribution (a) and the consumption and production rate (b) data were obtained from the literature. Step 2: The composition of carbon source was arbitrarily selected (c). Step 3: The theoretical MIDs were calculated and Gaussian noise at 1% levels was added to produce artificially measured MID data (d). Step 4: The metabolic flux distribution (e) and those 95% confidence intervals (f) were estimated. Step 5: An accuracy score S_i (g) was determined for each reaction i. Step 6: The sum of S_i of all reactions, S_sum, was calculated (h).

Fig. 2

Experimentally determined metabolic flux distributions used in this study. Flux values (red numbers) are normalized to a glucose uptake rate of 100. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Heatmap representations of the precision scores S_i and S_sum levels estimated by the computer simulation of ¹³C-MFA using 66 mixtures of non-labeled, [1-¹³C], and [U-¹³C]glucose as carbon sources. (a) Comparison of S_sum levels determined for the five metabolic flux distributions. (b) Precision scores S_i of each reaction determined for the metabolic flux distribution A (a continuous culture of E. coli MG1655). The red and blue colors in the boxes represent larger (better precision) and smaller (poorer precision) S_sum levels of flux estimation, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Heatmap representations of the precision scores S_i and S_sum levels estimated by the computer simulation of ¹³C-MFA using all the possible patterns of ¹³C-labeled glucose as carbon source. All results are shown in Supplementary data S2 and S3. (a) Comparison of S_sum levels determined for the five metabolic flux distributions. (b) Precision scores S_i of each reactions determined for the five metabolic flux distributions using [1,2-¹³C] and [1,2,4-¹³C]glucose. The red and blue colors represent larger (better precision) and smaller (poorer precision) S_sum levels of flux estimation, respectively. The precision scores of non-labeled, [1-¹³C], and [U-¹³C]glucose at ratios of 0:8:2 and 4:1:5 are also shown for comparison. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

¹³C-metabolic flux analysis of batch cultivated E. coli MG1655. (a and b) The metabolic flux distributions estimated from the labeling experiment using mixtures of non-labeled, [1-¹³C], and [U-¹³C]glucose at 0:8:2 (a) and 4:1:5 (b), respectively. (c and d) The 95% confidence intervals of estimated flux levels of the reactions in glycolysis, the anaplerotic and the glyoxylate pathways. Flux values are normalized to a glucose uptake rate of 100.