Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: This dissertation explores the behaviour and regulation of central carbon metabolism in Escherichia
coli K12 W3110 under fermentative microaerobic conditions. To achieve this, an integrative systems
modelling approach was adopted, which is introduced in Chapter 1 along with a review of
metabolism in E. coli. An open-source software suite NMRPy, developed using the Python programming
language, is presented in Chapter 2. NMRPy provides a host functions for basic processing,
analysis and visualisation of Nuclear Magnetic Resonance (NMR) spectroscopy data. In addition
to 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 easy
to use, extensible tool for both routine and advanced use. In Chapter 3, a novel methodology is
presented which was developed for the effective and comprehensive determination of enzyme kinetic
parameters for systems biology using NMR. In contrast to traditional enzyme kinetic assay
methods, this new methodology is less labour-intensive and yields significantly more information
per 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 fitting
procedure. These data contain information on cooperative substrate binding, reversibility, product
inhibition and allosteric effects. The proposed methodology is applied to the study of the first two
enzymes of the glycolytic pathway. In Chapter 4, the construction, parameterisation and validation
of 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 which
models representing the reactions from triosephosphate isomerase to pyruvate kinase were parameterised
by fitting them to a collection of 31P NMR reaction time series. This approach extends
the methodology to enzyme sub-networks, yielding data that encompass the full complexity of the
network regulatory interactions. The verified kinetic models were subjected to scrutiny, the results
of which are presented in Chapter 5. The value of the modelling approach is demonstrated by the
ease with which cumbersome in vivo experiments can be performed in silico. A structural analysis
of the model topology was conducted, elucidating the elementary flux modes of fermentative glycolysis
in 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 degrees
of ATP demand and oxygen availability and a number of hypotheses are presented, explaining
the regulation of free energy in E. coli, and the metabolic responses of E. coli to changing redox
demands. Amongst other things, the results demonstrated that the glucose importing phosphoenolpyruvate:
phosphotransferase pathway controlled glycolytic flux, and that under microaerobic
conditions E. coli is able to regulate redox balance not only by balancing flux between acetate and
ethanol, but also by altering the balance of flux between acetate and lactate at the pyruvate formate
lyase/lactate dehydrogenase branch point. This study demonstrates the value of an integrated
computational and experimental systems approach to exploring biological phenomena. / AFRIKAANSE OPSOMMING: In hierdie proefskrif word die gedrag en regulering van die sentrale koolstofmetabolisme in Escherichia
coli K12 W3110 onder fermenterende mikro-a¨erobiese toestande ondersoek. Dit is moontlik
gemaak deur ’n ge¨ıntegreerde stelsel-modelleringsbenadering, wat in Hoofstuk 1 bekendgestel
word. D´ıe hoofstuk verskaf ook ’n oorsig van die metabolisme in E. coli. ’n Oopbron-kodepakket
NMRPy, wat in die programmeringstaal Python ontwikkel is, word in Hoofstuk 2 beskryf. NMRPy
verskaf ’n aantal funksies vir die basiese verwerking, analise en visualisering van Kern-Magnetiese
Resonansie (KMR) spektroskopiese data, sowel as gespesialiseerde funksies vir die dekonvolusie van
opeenvolgende reaksie-tydreekse. Hierdie funksionaliteit was onontbeerlik vir die verdere navorsing
in hierdie proefskrif. Hoofstuk 3 beskryf die ontwikkeling van ’n nuwe metodiek vir die omvangryke
bepaling van ensiem-kinetiese parameters vir sisteembiologie, deur van KMR gebruik te maak.
In teenstelling tot tradisionele ensiem-kinetiese essai-metodes, is hierdie nuwe metodologie minder
arbeidsintensief en lewer dit beduidend meer inligting per eksperiment. Deur die kinetiese vergelykings
op tydsafhanklike KMR data te pas, word dinamiese veranderinge in substrate, produkte en
allosteriese effektors gekwantifiseer en hierdie inligting gebruik in die passingsprosedure. Die data
bevat inligting oor ko¨operatiewe substraatbinding, omkeerbaarheid, produkinhibisie en allosteriese
effekte. Die voorgestelde metodologie word toegepas op die karakterisering van die eerste twee
glikolitiese ensieme. In Hoofstuk 4 word die konstruksie, parameterisering en validering van ’n
aantal kinetiese modelle van glikolise in E. coli onder mikro-a¨erobiese toestande uiteengesit. Die
waarde van die modelleringsbenadering lˆe in die gemak waarmee omslagtige in vivo eksperimente
in silico uitgevoer kan word. Om die onderste helfte van die glikolitiese pad te modelleer word ’n
soortgelyke tegniek as in Hoofstuk 3 gebruik. Modelle van die reaksies vanaf triosefosfaat-isomerase
tot by pirovaat-kinase is geparameteriseer deur dit op ’n versameling 31P KMR-tydreekse te pas.
Hierdie benadering brei bostaande metodologie uit tot ensiem-subnetwerke en genereer data wat
die volle kompleksiteit van regulerende interaksies in die netwerk insluit. Die geverifieerde modelle
word in Hoofstuk 5 noukeurig ondersoek. ’n Strukturele analise van die modeltopologie word onderneem
om die elementˆere fluksie-modes van fermentatiewe glikolise in E. coli te verklaar, sowel
as om ’n futiele siklus rondom fosfo¨enolpirovaat karboksilase en fosfo¨enolpirovaat karboksikinase
te identifiseer. Die bestendige-toestandsgedrag en kontrole-eienskappe word in silico ondersoek
onder toestande van verskeie ATP beladings en suurstofbeskikbaarheid. ’n Aantal hipoteses word voorgelˆe, wat die regulering van vry energie in E. coli, sowel as die metaboliese reaksies van E. coli
onder veranderende redoks-vereistes kan verklaar. Onder andere dui die resultate daarop dat die
fosfo¨enolpirovaat:fosfotransferase sisteem (wat verantwoordelik is vir glukose-opname in die sel) die
glikolitiese fluksie beheer en dat E. coli onder mikro-a¨erobiese toestande die redoksbalans nie net
tussen asetaat en etanol kan reguleer nie, maar ook die deur wysiging van die fluksie-balans tussen
asetaat en laktaat rondom die pirovaat-formiaat-liase/laktaatdehidrogenase vertakkingspunt. Hierdie
studie toon die waarde van ’n ge¨ıntegreerde rekenaarmatige en eksperimentele sisteembenadering
om biologiese verskynsels te ondersoek.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/85730 |
| Date | 12 1900 |
| Creators | Eicher, Johann Josef |
| Contributors | Rohwer, Johann M., Snoep, Jacky L., Stellenbosch University. Faculty of Science. Dept. of Biochemistry. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | ix, 188 p. : ill. |
| Rights | Stellenbosch University |
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