Systems biology approach reveals that overflow metabolism of acetate in Escherichia coli is triggered by carbon catabolite repression of acetyl-CoA synthetase

Abstract

BackgroundThe biotechnology industry has extensively exploited Escherichia coli for producing recombinant proteins, biofuels etc. However, high growth rate aerobic E. coli cultivations are accompanied by acetate excretion i.e. overflow metabolism which is harmful as it inhibits growth, diverts valuable carbon from biomass formation and is detrimental for target product synthesis. Although overflow metabolism has been studied for decades, its regulation mechanisms still remain unclear.ResultsIn the current work, growth rate dependent acetate overflow metabolism of E. coli was continuously monitored using advanced continuous cultivation methods (A-stat and D-stat). The first step in acetate overflow switch (at μ = 0.27 ± 0.02 h-1) is the repression of acetyl-CoA synthethase (Acs) activity triggered by carbon catabolite repression resulting in decreased assimilation of acetate produced by phosphotransacetylase (Pta), and disruption of the PTA-ACS node. This was indicated by acetate synthesis pathways PTA-ACKA and POXB component expression down-regulation before the overflow switch at μ = 0.27 ± 0.02 h-1 with concurrent 5-fold stronger repression of acetate-consuming Acs. This in turn suggests insufficient Acs activity for consuming all the acetate produced by Pta, leading to disruption of the acetate cycling process in PTA-ACS node where constant acetyl phosphate or acetate regeneration is essential for E. coli chemotaxis, proteolysis, pathogenesis etc. regulation. In addition, two-substrate A-stat and D-stat experiments showed that acetate consumption capability of E. coli decreased drastically, just as Acs expression, before the start of overflow metabolism. The second step in overflow switch is the sharp decline in cAMP production at μ = 0.45 h-1 leading to total Acs inhibition and fast accumulation of acetate.ConclusionThis study is an example of how a systems biology approach allowed to propose a new regulation mechanism for overflow metabolism in E. coli shown by proteomic, transcriptomic and metabolomic levels coupled to two-phase acetate accumulation: acetate overflow metabolism in E. coli is triggered by Acs down-regulation resulting in decreased assimilation of acetic acid produced by Pta, and disruption of the PTA-ACS node.

Global metabolic network reorganization by adaptive mutations allows fast growth of Escherichia coli on glycerol

“…3d) is consistent with a previous study 26 that associated overflow with catabolite repression on glucose and decreased concentrations of acetyl-CoA synthetase and cyclic AMP (cAMP). In the present study, acetyl-CoA synthetase, the key protein for acetate utilization, was markedly repressed (93% reduction) in both GLPK and DKI strains.…”

“…In addition, glpK mutation effects (as shown in Fig. 3d), including changes in transporter proteins, gluconeogenesis, TCA cycle, glyoxylate cycle, acetate consumption and carbon-wasting mechanism have previously been similarly associated with decreased levels of cAMP, catabolite repression and increased growth rate in E. coli 21,26 . Moreover, most changes observed in the RPOC strain (containing no additional non-adaptive mutations) are overall dominant and well reflected in the DKI strain ( Fig.…”

Aerobic Glycolysis: Meeting the Metabolic Requirements of Cell Proliferation

“…Blood cells infected with malaria also exhibit much higher glucose uptake than normal blood cells (Bryant et al 1964). While glucose fermentation is a common feature of many proliferating cells, it is not required for proliferation; aerobic yeasts such as Y. lipolytica rely solely on respiration (Christen & Sauer 2010), and E. coli do not undergo fermentation under aerobic conditions, only excreting the fermentative product acetate when glucose uptake exceeds a maximum respiration rate (Valgepea et al 2010, Xu et al 1999. Thus, evolution has devised more than one metabolic solution to support cell proliferation.…”

Global dynamics of Escherichia coli phosphoproteome in central carbon metabolism under changing culture conditions

“…This mechanism was the first example of phosphorylation control reported in a prokaryote [67]. Here we show that the glyoxylate shunt is in addition to other mechanisms of control [68], controlled by a cascade mechanism of posttranslational modification [69] where, in addition to Icd, the ratio of phosphorylation for enzymes Mdh, Pck and AcnB change when cells are grown on acetate. Our data suggests that unphosphorylation of Mdh, Pck and AcnB plays a key role in activating and controlling the glyoxylate shunt.…”