Integrated 13C-metabolic flux analysis of 14 parallel labeling experiments in Escherichia coli

The use of parallel labeling experiments for ¹³C metabolic flux analysis (¹³C-MFA) has emerged in recent years as the new gold standard in fluxomics. The methodology has been termed COMPLETE-MFA, short for complementary parallel labeling experiments technique for metabolic flux analysis. In this contribution, we have tested the limits of COMPLETE-MFA by demonstrating integrated analysis of 14 parallel labeling experiments with Escherichia coli. An effort on such a massive scale has never been attempted before. In addition to several widely used isotopic tracers such as [1,2-¹³C]glucose and mixtures of [1-¹³C]glucose and [U-¹³C]glucose, four novel tracers were applied in this study: [2,3-¹³C]glucose, [4,5,6-¹³C]glucose, [2,3,4,5,6-¹³C]glucose and a mixture of [1-¹³C]glucose and [4,5,6-¹³C]glucose. This allowed us for the first time to compare the performance of a large number of isotopic tracers. Overall, there was no single best tracer for the entire E. coli metabolic network model. Tracers that produced well-resolved fluxes in the upper part of metabolism (glycolysis and pentose phosphate pathways) showed poor performance for fluxes in the lower part of metabolism (TCA cycle and anaplerotic reactions), and vice versa. The best tracer for upper metabolism was 80% [1-¹³C]glucose+20% [U-¹³C]glucose, while [4,5,6-¹³C]glucose and [5-¹³C]glucose both produced optimal flux resolution in the lower part of metabolism. COMPLETE-MFA improved both flux precision and flux observability, i.e. more independent fluxes were resolved with smaller confidence intervals, especially exchange fluxes. Overall, this study demonstrates that COMPLETE-MFA is a powerful approach for improving flux measurements and that this methodology should be considered in future studies that require very high flux resolution.