Mutants lacking global regulators, fis and arcA, in Escherichia coli enhanced growth fitness under acetate metabolism by pathway reprogramming.

Global regulatory transcription factors play a significant role in controlling microbial metabolism under genetic and environmental perturbations. A system-level effect of carbon sources such as acetate on microbial metabolism under disrupted global regulators has not been well established. Acetate is one of the major substrates available in various nutrient niches such as the mammalian gut and a keto diet. A substantial amount of acetate gets secreted in aerobic metabolism. Therefore, investigating the study on acetate metabolism is highly significant. It is known that the global regulators fis and arcA regulate acetate uptake genes in E. coli under glucose conditions. This study deciphered the growth and flux distribution of E. coli transcription regulatory knockouts Δfis, ΔarcA and double deletion mutant, ΔarcAΔfis under acetate using 13C-metabolic flux analysis (MFA), which has not been investigated before. We observed that the mutants exhibited an expeditious growth rate (~ 1.2-1.6-fold) with a proportionate increase in acetate uptake rates compared to the wild type. 13C-MFA displayed the distinct metabolic reprogramming of intracellular fluxes via the TCA cycle, anaplerotic pathway and gluconeogenesis, which conferred an advantage of a faster growth rate with better carbon usage in all the mutants. This resulted in higher metabolic fluxes through the TCA cycle (~ 18-90%), lower gluconeogenesis (~ 15-35%) and higher CO2 and ATP production with the proportional increase in growth rate. The study reveals a novel insight by stating the sub-optimality of the wild-type strain grown under acetate substrate aerobically. These mutant strains efficiently oxidize acetate, thus acting as potential candidates for the biosynthesis of isoprenoids, biofuels, vitamins and various pharmaceutical products.Key Points• Mutants exhibited a better balance between energy and precursor synthesis than WT.• Leveraged in the unravelling of regulatory control under various nutrient shifts.• Metabolic readjustment resulted in optimal biomass requirement and faster growth.