Acetate fluxes in Escherichia coli are determined by the thermodynamic control of the Pta-AckA pathway

Abstract

Escherichia coli excretes acetate upon growth on fermentable sugars, but the regulation of this production remains elusive. Acetate excretion on excess glucose is thought to be an irreversible process. However, dynamic 13C-metabolic flux analysis revealed a strong bidirectional exchange of acetate between E. coli and its environment. The Pta-AckA pathway was found to be central for both flux directions, while alternative routes (Acs or PoxB) play virtually no role in glucose consumption. Kinetic modelling of the Pta-AckA pathway predicted that its flux is thermodynamically controlled by the extracellular acetate concentration in vivo. Experimental validations confirmed that acetate production can be reduced and even reversed depending solely on its extracellular concentration. Consistently, the Pta-AckA pathway can rapidly switch from acetate production to consumption. Contrary to current knowledge, E. coli is thus able to co-consume glucose and acetate under glucose excess. These metabolic capabilities were confirmed on other glycolytic substrates which support the growth of E. coli in the gut. These findings highlight the dual role of the Pta-AckA pathway in acetate production and consumption during growth on glycolytic substrates, uncover a novel regulatory mechanism that controls its flux in vivo, and significantly expand the metabolic capabilities of E. coli.

Enhanced production of heterologous proteins by a synthetic microbial community: Conditions and trade-offs

“…When the glucose is almost completely depleted (∼ 4 h), cells continue growth on acetate at a lower rate. As shown in the same plot, the simulations are in good agreement with the experimental data of Enjalbert et al [13]. The capability to reproduce diauxic growth depends on the inclusion of a regulatory term for CCR [34,47] in Eq.…”

“…The fact that these experiments have been carried out with different E. coli strains and in different conditions carries the risk of obtaining parameter values that are mutually inconsistent. In order to avoid this problem, we have calibrated the model as much as possible against a single, recently published data set for an E. coli wild-type strain grown in batch in minimal medium with different concentrations of glucose and acetate [13], that is, for conditions in which D = 0 h −1 and Y h = 0. Only the values for the biomass degradation constant k deg , the threshold for acetate overflow l, and the half-maximal saturation constants K g and K a were taken from the literature.…”

“…Concentrations in the bioreactor are denoted by G for glucose, A for acetate, H for heterologous protein, and B for autocatalytic biomass. For clarity, regulatory interactions due to growth inhibition by acetate and carbon catabolite repression have been omitted from the figure. (B) and (C) Experimental data from [13] used to identify the parameter values of the system of Eqs 1-6. The data include measurements of the growth rate µ + , the glucose uptake rate r g+ up , the (net) acetate uptake rate r a+ up , and the acetate overflow rate r a+ over during exponential growth of an E. coli wild-type strain in batch conditions in minimal medium with glucose as the sole carbon source (B left panel), acetate as the sole carbon source (B right panel) and in glucose with increasing concentrations of acetate added (C).…”

“…The possibility of the cell to continue to take up glucose at a reduced rate, through other, non-specific systems, is advantageous for our purpose, since it allows the cleaner to grow faster than on acetate alone and thus enlarges the coexistence range in continuous-mode cultivation (see below). Overexpressing acs has been shown to increase the growth rate of E. [13] (dots). (B) Effect of lower growth yield Y g , accounting for the use of an energy-dissipating LacY mutant, on the onset of acetate overflow, indicated by the overflow rate (black curves) reached at steady state in a bioreactor operating in continuous mode, with G in = 20 g L −1 .…”

Trehalose protects Escherichia coli against carbon stress manifested by protein acetylation and aggregation

“…This is consistent with the earlier studies demonstrating that the Δ otsA mutant exhibited an approximately twofold higher level of UDP‐glucose than the WT strain (Böhringer et al , ) and with our previous study showing that early stationary phase Δ otsA cells accumulated more glycogen than WT cells (Kuczyńska‐Wiśnik et al , ). In the absence of trehalose synthesis in Δ otsA cells, the excess glucose‐6‐phosphate could be converted to pyruvate and then to acetyl‐CoA and AcP (Enjalbert et al , ) that is engaged in nonenzymatic lysine acetylation, which is the dominant form of acetylation in E. coli cultures in stationary phase (Kuhn et al , ; Wolfe, ). Slightly enhanced level of acetyl lysines (AcK) was immunodetected in the Δ otsA extract as compared to that in the WT extract (Fig.…”