Dynamic modeling of the central carbon metabolism of Escherichia coli

Abstract

Application of metabolic engineering principles to the rational design of microbial production processes crucially depends on the ability to describe quantitatively the systemic behavior of the central carbon metabolism to redirect carbon fluxes to the product-forming pathways. Despite the importance for several production processes, development of an essential dynamic model for central carbon metabolism of Escherichia coli has been severely hampered by the current lack of kinetic information on the dynamics of the metabolic reactions. Here we present the design and experimental validation of such a dynamic model, which, for the first time, links the sugar transport system (i.e., phosphotransferase system [PTS]) with the reactions of glycolysis and the pentose-phosphate pathway. Experimental observations of intracellular concentrations of metabolites and cometabolites at transient conditions are used to validate the structure of the model and to estimate the kinetic parameters. Further analysis of the detailed characteristics of the system offers the possibility of studying important questions regarding the stability and control of metabolic fluxes.

Development and application of a differential method for reliable metabolome analysis in Escherichia coli

“…From the above it is clear that total broth extraction, as used in several published studies (Chassagnole et al, 2002, Schaub et al, 2006 method is that metabolism is instantaneously stopped and that all compounds which are present outside the cells can be effectively removed. However, according to several studies quenching with cold aqueous methanol prior to separation might lead to additional leakage of intracellular metabolites from the cells, especially in case of bacteria.…”

“…In vivo kinetic studies rely partly on rapid dynamic perturbation experiments during which a steady-state chemostat culture is perturbed and the subsequent response in both extracellular and intracellular metabolites is monitored (Theobald et al, 1997;Chassagnole et al, 2002;Visser et al, 2002;Mashego et al, 2004;Wu et al, 2005). Taking into account the high turnover rate of most intracellular metabolites, proper measurement of their concentrations requires rapid broth sampling and instantaneous quenching of enzyme activity.…”

“…Centrifugation of the broth/quenching liquid mixture is done at temperatures below -20 o C (Schaefer et al, 1999). Liquid nitrogen was also used for quenching of Escherichia coli (Chassagnole et al, 2002), as well as the above mentioned rapid heating of the broth by means of a heat exchanger (Schaub et al, 2006). The disadvantage of these methods is that removal of extracellular metabolites is not possible.…”

“…However, as they also mentioned in their work, the application of glycerol leads to additional problems because it is difficult to remove from the samples Subsequently the quenched cells have to be extracted to release the intracellular metabolites. For Escherichia coli perchloric acid extraction has been the most widely used procedure (Schaefer et al, 1999;Buchholz 2001Buchholz , 2002Chassagnole et al, 2002). In some cases different methods have been used for the extraction of different classes of metabolites, for example alkaline extraction for NADH and NADPH (Chassagnole et al, 2002).…”

“…For Escherichia coli perchloric acid extraction has been the most widely used procedure (Schaefer et al, 1999;Buchholz 2001Buchholz , 2002Chassagnole et al, 2002). In some cases different methods have been used for the extraction of different classes of metabolites, for example alkaline extraction for NADH and NADPH (Chassagnole et al, 2002). Maharjan and Ferenci (2003) investigated the influence of different extraction methods i.e.…”