Evaluating non-oxidative glycolysis using 13 C metabolic flux analysis

Non-oxidative glycolysis (NOG) converts glucose to acetyl-CoA without emitting CO 2 during Escherichia coli bioproduction. Although 13 C-metabolic flux analysis ( 13 C-MFA) has the potential to evaluate NOG flux distribution in vivo , the precision of these estimates depends on the labeling patterns of glucose tracers. Because E. coli has multiple native glycolysis pathways, selecting the optimal glucose tracer is important for precise estimates. This study used simulations to evaluate the performance of 18 commercially available glucose tracers in precisely predicting NOG flux. We artificially generated flux distributions of central metabolic pathways, including three types of NOG pathways, and obtained 13 C labeling patterns of amino acids metabolized from each glucose tracer. Using these data, we then calculated 95% confidence intervals (CIs) for NOG pathway fluxes. Comparing the sum of CI widths from three NOG fluxes showed that estimation with [1,3- 13 C]-glucose yielded the narrowest CIs. Several tracers, including [2,3- 13 C]glucose, were capable of determining fluxes with high precision, but [1- 13 C]glucose was not one of them, despite often being used for estimating glycolytic fluxes. We then experimentally evaluated [2,3- 13 C]- and [1- 13 C]glucose as tracers in E. coli strains with and without the expression of phosphoketolase, which is required for NOG pathways to function. The results suggested that [1- 13 C]glucose could not detect NOG fluxes, whereas [2,3- 13 C]glucose estimated the fluxes of all three NOG pathways with narrow CIs.