Modeling and simulation of the main metabolism in Escherichia coli and its several single-gene knockout mutants with experimental verification

Tuty Asmawaty Abdul Kadir 0 Ahmad A Mannan 2 Andrzej M Kierzek 2 Johnjoe McFadden 2 Kazuyuki Shimizu 0 1 0 Dept. of Bioscience and Bioinformatics, Kyushu Institute of Technology , Iizuka, Fukuoka 820-8502 , Japan 1 Institute for Advanced Biosciences, Keio University , Tsuruoka, Yamagata 997-0017 , Japan 2 Fac. Of Health and Medical Sciences, AW Building, University of Surrey , Guilford Surrey GU2 7TE , UK Background: It is quite important to simulate the metabolic changes of a cell in response to the change in culture environment and/or specific gene knockouts particularly for the purpose of application in industry. If this could be done, the cell design can be made without conducting exhaustive experiments, and one can screen out the promising candidates, proceeded by experimental verification of a select few of particular interest. Although several models have so far been proposed, most of them focus on the specific metabolic pathways. It is preferred to model the whole of the main metabolic pathways in Escherichia coli, allowing for the estimation of energy generation and cell synthesis, based on intracellular fluxes and that may be used to characterize phenotypic growth. Results: In the present study, we considered the simulation of the main metabolic pathways such as glycolysis, TCA cycle, pentose phosphate (PP) pathway, and the anapleorotic pathways using enzymatic reaction models of E. coli. Once intracellular fluxes were computed by this model, the specific ATP production rate, the specific CO2 production rate, and the specific NADPH production rate could be estimated. The specific ATP production rate thus computed was used for the estimation of the specific growth rate. The CO2 production rate could be used to estimate cell yield, and the specific NADPH production rate could be used to determine the flux of the oxidative PP pathway. The batch and continuous cultivations were simulated where the changing patterns of extracellular and intra-cellular metabolite concentrations were compared with experimental data. Moreover, the effects of the knockout of such pathways as Ppc, Pck and Pyk on the metabolism were simulated. It was shown to be difficult for the cell to grow in Ppc mutant due to low concentration of OAA, while Pck mutant does not necessarily show this phenomenon. The slower growth rate of the Ppc mutant was properly estimated by taking into account the lower specific ATP production rate. In the case of Pyk mutant, the enzyme level regulation was made clear such that Pyk knockout caused PEP concentration to be up-regulated and activated Ppc, which caused the increase in MAL concentration and backed up reduced PYR through Mez, resulting in the phenotypic growth characteristics similar to the wild type. Conclusions: It was shown to be useful to simulate the main metabolism of E. coli for understanding metabolic changes inside the cell in response to specific pathway gene knockouts, considering the whole main metabolic pathways. The comparison of the simulation result with the experimental data indicates that the present model could simulate the effect of the specific gene knockouts to the changes in the metabolisms to some extent. - Background One of the most challenging goals of metabolic engineering is to design the cell metabolism based on the analysis of metabolic regulation. For this, it is strongly desired to develop a mathematical model which can describe the dynamic behaviour of the cell in response to the changes in the culture environment and/or specific genetic modifications. Although an attempt has been made to develop a platform for the whole cell model [1], the total cell model has not yet been developed. If such a model could be developed, it becomes possible to check the metabolism of a specific gene knockout on the metabolism and fermentation characteristics without conducting many exhaustive experiments, and allow for the screening out of the preferred candidates for performance improvement, followed by experimental verification only for the selected candidates. Some of the mathematical models which can describe the dynamic behaviour of the intracellular metabolite concentrations of the central metabolic pathways have been developed for Saccharomyces cerevisiae [2-4]. The measurement of the intracellular metabolite concentrations for the pulse addition of glucose during continuous culture has been made, and the time profile was compared to the predicted dynamic simulation together with model parameter identification [3,5-7]. The kinetic equations for the glycolysis and the pentose phosphate (PP) pathway have also been developed for E. coli to simulate the transient data obtained by the fast sampling system [8]. These models do not contain TCA cycle and the fermentative pathways, and thus cannot simulate the typical aerobic batch culture. In the present research, therefore, we considered several kinetic models for the TCA cycle, anapleorotic pathways as well as the glycolysis and the PP pathway to simulate the time profiles of the batch and continuous cultures. Moreover, most of the kinetic models developed so far can express only enzyme level regulation due to the change in the concentrations of substrate and product as well as various effectors. Thus, the application of the conventional model is limited in practice to some extent. Recently, several mathematical models which describe the effects of global regulators on the metabolic pathway reactions for catabolite repression for substrate uptake [9] and for suc mutant for glutamate production [10] have been proposed, which pay attention to particular pathways. Recently, we estimated the flux changes during batch culture of E. coli based on 13C-labeling experiment using CE-TOF/MS [11]. It is quite important to estimate the flux changes of the main metabolic pathways, allowing opportunity for the proper analysis of the energy metabolism and cell synthesis. Although 13C-metabolic flux analysis has proven to be quite useful [12,13], it is a method of the analysis of a static physiological state of the organism and does not have predictability characteristics. It is highly important and indeed useful to be able to predict cell growth characteristics. In the present study, therefore, we attempted to develop a new model for the cell growth rate with the advantages of considering the metabolic fluxes and an enzymatic model. Furthermore, incorporating the relationship between ATP production rate obtained by the intracellular fluxes of the main metabolic pathways and the cell growth rate together with some rule-based approach for gene-level regulation. Once we could simulate the whole main metabolic pathways, we may be able to compute CO2 production rate and NAD(P)H production rate as well as ATP production rate. In particular, we attempted to simulate several single-gene knockout mutants to show the utility of the model and its limitations. Some of the experiments were also conducted to ve (...truncated)