Flux analysis and control of the central metabolic pathways in Escherichia coli

Abstract

The growth of the bacterial cell involves the co-ordination of the fluxes of carbon into a considerable diversity of products that are the components of the cell. Fortunately the monomers from which the cell's polymers are made are themselves synthesised from a relatively small group of precursors that are the products of the central metabolic pathways. This simplification renders cell metabolism accessible to flux analysis, a method for handling experimental data to derive metabolic fluxes. Through such analysis of the growth of Escherichia coli ML308 on 11 single carbon sources in batch, turbidostat or chemostat culture general patterns are discernible. Most significant among these are that growth on different carbon sources is achieved without any obvious enzyme acting as a regulator of metabolic flux, except when acetate is the sole source of carbon. In this case a junction is created at which iso citrate dehydrogenase (ICDH) and isocitrate lyase (ICL) compete for their common substrate and this competition is resolved by partial inactivation of ICDH to match flux through ICL and this balance limits growth rate. In this sense, flux through ICDH and ICL is 'rate-limiting'. Uptake of six of the remaining carbon inputs exceeds the capacity of the central metabolic pathways (CMPs) to sustain flux to the precursors required for growth and the CMPs are balanced by excretion of acetate. Restriction of carbon uptake by chemostat progressively diminishes growth rate and acetate excretion until acetate excretion is prevented. For the four remaining carbon sources, uptake is apparently restricted and the products are biomass, carbon dioxide and water. Carbon sources feeding the phosphorylated parts of the CMPs flux relatively more carbon to precursors (Pre-C) than CO2 when compared with carbon sources which feed into the non-phosphorylated pathways. Pre-C/CO2 ratios for the former are 1.73-3.91 and for the latter are 0.46-0.78. Flux analysis of all 11 carbon sources shows that there is an overabundant supply of 'energy' (ATP + [2H]), generated by the CMPs, in all phenotypes and conditions down to a glucose chemostat at mu of 0.72. This excess energy is a thermodynamic inefficiency which must be dissipated as heat. E. coli ML308 probably evolved in circumstances of 'feast' and 'famine'. The two strategies selected (excretion of surplus carbon and restriction of mu) would appear to be defences against 'feast'. Presumably there are defences against 'famine'. These are not made obvious by flux analysis but allosteric control of irreversible enzymes would protect pools of essential nutrients from rapid depletion on the sudden onset of 'famine'.