Metabolic pathway engineering of the toluene degradation pathway
This thesis addresses the problem of how to examine a metabolic pathway and identify what are the key elements, specifically with respect to rate-limitation. The aim is to be able to analyze a pathway, identify the bottlenecks and implement genetic modifications to remove these bottlenecks. This is done by defining the system of interest and developing a predictive model using kinetic data. The model predictions can then be verified using fermentation data and genetic techniques to make the appropriate changes for improved performance. The test system chosen for this study was the TOL meta-cleavage pathway for the degradation of benzoate. This system was chosen on the basis of the application of pathway engineering principles to other systems. The modelling strategy and software was developed using principles from metabolic control theory and biochemical systems theory. By applying this to the TOL pathway using kinetic data, the control coefficients for the pathway were obtained as well as the system parameters required for the optimization of the pathway. The simulated results obtained from this model must be validated by experiment. Errors can arise both from incorrect assumptions in the model and from the fact that the kinetic data taken from individual in vitro experiments may not be applicable to the in vivo system. The effect of the presence of the TOL pathway on the behaviour of E.coli JM107 during fermentation was investigated and the transient concentration data necessary to identify the bottlenecks in the pathway measured. This data is then used to calculate the flux control coefficients for the TOL pathway. The predictive results were verified by the fermentation data which identified the first two enzymes in the pathway as having significant flux control coefficients. This final chapter also addresses the issue of flux analysis, that is, the calculation of the fluxes in the system to determine where fluxes to unwanted by-products occur and to indicate points of control. A graphical user interface is used to provide a user-friendly and intuitive means of building and customising metabolic pathways which can then be interfaced with instrumentation to provide on-line flux analysis.