Understanding glycolysis in Escherichia coli : a systems approach using nuclear magnetic resonance spectroscopy

[摘要] ENGLISH ABSTRACT: This dissertation explores the behaviour and regulation of central carbon metabolism in Escherichiacoli K12 W3110 under fermentative microaerobic conditions. To achieve this, an integrative systemsmodelling approach was adopted, which is introduced in Chapter 1 along with a review ofmetabolism in E. coli. An open-source software suite NMRPy, developed using the Python programminglanguage, is presented in Chapter 2. NMRPy provides a host functions for basic processing,analysis and visualisation of Nuclear Magnetic Resonance (NMR) spectroscopy data. In additionto this, NMRPy offers specialised functions for the deconvolution of arrayed reaction time series,which proved indispensable to the research presented in this dissertation. NMRPy presents an easyto use, extensible tool for both routine and advanced use. In Chapter 3, a novel methodology ispresented which was developed for the effective and comprehensive determination of enzyme kineticparameters for systems biology using NMR. In contrast to traditional enzyme kinetic assaymethods, this new methodology is less labour-intensive and yields significantly more informationper experiment. By fitting kinetic equations to real time NMR data, dynamic changes in substrates,products and allosteric modifiers are quantified and allowed to inform the parameter fittingprocedure. These data contain information on cooperative substrate binding, reversibility, productinhibition and allosteric effects. The proposed methodology is applied to the study of the first twoenzymes of the glycolytic pathway. In Chapter 4, the construction, parameterisation and validationof a number of kinetic models of glycolysis in E. coli under microaerobic conditions is detailed.To model the lower half of glycolysis, a similar technique was adopted as in Chapter 3, in whichmodels representing the reactions from triosephosphate isomerase to pyruvate kinase were parameterisedby fitting them to a collection of 31P NMR reaction time series. This approach extendsthe methodology to enzyme sub-networks, yielding data that encompass the full complexity of thenetwork regulatory interactions. The verified kinetic models were subjected to scrutiny, the resultsof which are presented in Chapter 5. The value of the modelling approach is demonstrated by theease with which cumbersome in vivo experiments can be performed in silico. A structural analysisof the model topology was conducted, elucidating the elementary flux modes of fermentative glycolysisin E. coli, and identifying a futile cycle around PEP carboxylase and PEP carboxykinase.Model steady-state behaviour and control properties were explored in silico under various degreesof ATP demand and oxygen availability and a number of hypotheses are presented, explainingthe regulation of free energy in E. coli, and the metabolic responses of E. coli to changing redoxdemands. Amongst other things, the results demonstrated that the glucose importing phosphoenolpyruvate:phosphotransferase pathway controlled glycolytic flux, and that under microaerobicconditions E. coli is able to regulate redox balance not only by balancing flux between acetate andethanol, but also by altering the balance of flux between acetate and lactate at the pyruvate formatelyase/lactate dehydrogenase branch point. This study demonstrates the value of an integratedcomputational and experimental systems approach to exploring biological phenomena.

[发布机构] Stellenbosch University