Metabolic engineering has evolved to the point of fulfilling the dream of having our industrial chemicals produced renewably. Carboxylic acids (e.g., short chain fatty acids (SCFAs) such as octanoic acid (C8)) are one such chemical intermediate that can be produced with Escherichia coli engineered with a short chain thioesterase. However, C8 is toxic to E. coli growth at concentrations greater than 10 mM. To design a better host strain for the production of large amount of those SCFAs, metabolic flux analysis of E. coli grown under C8 stress was performed. When E. coli MG1655 was grown in M9 minimal media supplemented with 35 mM C8 under aerobic conditions, cell growth was inhibited by 55.1% ± 10.0% (mean ± standard deviation, n=4) compared to control (without C8). To study fluxes in central carbon metabolism, 10 g/L of a mixture of labeled and unlabeled glucose (25% uniform label glucose, 25% 1-label glucose and 50% natural glucose) was used as the sole carbon source for bacterial growth. Proteinogenic amino acid isotopomers were measured by 2D (13C, 1H) NMR. Notable differences of several amino acid isotopomer enrichments were observed between control condition and C8 stress condition, suggesting the precursor nodes of these amino acids in metabolic pathways are responding to the stress. More specifically, the difference suggested that the flux distribution among tricarboxylic acid (TCA) cycle, pyruvate node and α-ketoglutarate node and oxaloacetate node was changed. Under C8 stress, acetate production flux increased by ~50% and the flux from phosphoenolpyruvate to pyruvate increased by ~50%, while the TCA cycle pathway, the malic enzyme pathway, and the pentose phosphate pathway decreased by 25%, 90%, and 20% respectively. Downregulation of the TCA cycle can occur if an excess of NADH occurs, due to feedback inhibition of isocitrate synthase. Since our flux data shows that less NADH is produced via central carbon metabolism under C8 stress, an excess of NADH could occur if C8 stress inhibits regeneration of NAD+ from NADH, via membrane disruption. Our transcriptome and proteomic data suggest that carboxylic acids may negatively impact the function and/or integrity of the cell membrane. Flux data combined with transcriptomic and proteomic analysis allow the development of hypotheses in context with the mechanism of C8 toxicity, which in turn helps to engineer strains with increased tolerance of C8.
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