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

ABSTRACT: Background: The 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. Results: In 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 1/h) 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 1/h 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 1/h leading to total Acs inhibition and fast accumulation of acetate. Conclusion: This 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.

ORGANISM(S): Escherichia coli str. K-12 substr. MG1655

PROVIDER: GSE23920 | GEO | 2010/09/02

SECONDARY ACCESSION(S): PRJNA130523

REPOSITORIES: GEO

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Project description:Background: The 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. Results: In 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 1/h) 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 1/h 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 1/h leading to total Acs inhibition and fast accumulation of acetate. Accumulation of acetate was also coupled to excretion of products such as orotate and N-carbomoyl-L-aspartate making it a novel carbon spilling mechanism in E. coli. Conclusion: This 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. Reference samples at specific growth rate (μ) 0.11 1/h were compared to the ones acquired at μ 0.21, 0.26, 0.31, 0.36, 0.40 and 0.48 1/h

Project description:A Synthetic Gene-Metabolic Oscillator Reference: Fung et al; Nature (2005) 435:118-122 Name of kinetic law Reaction Glycolytic flux, V_gly: nil -> AcCoA; Flux to TCA cycle/ETOH, V_TCA: AcCoA -> TCA/EtOH; HOAc ex/import,reversible, V_out: HOAc -> HOAc_E V_Pta: AcCoA + Pi -> AcP + CoA reversible, V_Ack: AcP + ADP -> OAc + ATP V_Acs: OAC + ATP -> AcCoA +PPi Acetic acid-base equillibrium, reversible, V_Ace: OAC + H -> HOAc Expression of LacI, R_LacI: nil -> LacI Expression of Acs, R_Acs: nil -> Acs Expression of Pta, R_Pta: nil -> Pta LacI degradation, R_dLacI: LacI -> nil Acs degradation, R_dAcs: Acs -> nil Pta degradation, R_dPta: Pta -> nil For this model the differential equation for V_Ace was changed from: C*(AcP*H-K_eq*OAC) with C = 100 in the supplemental material to C*(OAc*H-K_eq*HOAc) with C = 100, as in Bulter et. al; PNAS(2004),101,2299-2304 , and a value for K_eq of 5*10^-4 after communication with the authors. translated to SBML by: Lukas Endler(luen at tbi.univie.ac.at), Christoph Flamm (xtof at tbi.univie.ac.at) Biomodels Curation The model reproduces 3a of the paper for glycolytic flux Vgly = 0.5. The authors have agreed that the values on Y-axis are marked wrong and hence there is a discrepancy between model simulation results and the figure. Also, note that the values of concentration and time are in dimensionless units. The model was successfully tested on MathSBML and Jarnac.

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

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