Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Metabolic networks of cellular systems are complex, in that there are numerous components with
multiple non-linear interactions. To understand how these networks work they are often split into
manageable pieces and studied individually. However, an individual part is unable to account for
the complex properties of systems. In order to study these interactions the eld of systems biology
has developed. Systems biology makes use of computers to construct models as a method to
describe aspects of living systems. Using cellular pathways, kinetic models of metabolic pathways
can be constructed and used as a tool to study the biological systems and provide a quantitative
description. This thesis describes the quantitative analysis of a bacterium using a systems biology
approach.
Zymomonas mobilis is a rod shaped, Gram-negative, non-mobile facultative anaerobe and has
one of the fastest observed fermentations, yet least energy e cient extractions found in nature.
Furthermore it is the only known micro-organism to use the Entner-Doudoro (2-keto-3-deoxy-6-
phosphogluconate) pathway anaerobically. The low energy yield of fermentation in Z. mobilis is
a result of the usage of the Entner-Doudoro glycolytic pathway, which has half the energy yield
per mol substrate compared to the well known Embden-Meyerhof-Parnas glycolytic pathway.
The work presented in this thesis forms part of a larger project to compare glycolytic regulation
in di erent micro-organisms Z. mobilis, Escherichia coli, Saccharomyces cerevisiae and Lactococcus
lactis. These organisms were chosen based on their usage of di erent glycolytic mechanisms.
Kinetic models are suitable tools to draw a comparison between these organisms. The emphasis
here is on the construction of a kinetic model of the Entner-Doudoroff glycolytic pathway as it
occurs in Z. mobilis. The aim of this thesis was to characterise as many of the Entner-Doudoro pathway enzymes
as possible, under standard conditions. This was done using enzyme assays, to obtain the kinetic
parameters of each of the enzymes. Microtitre plate assays were used to characterise most of the
enzymes of the Entner-Doudoro pathway. However, not all characterisations could be done using
plate assay methods, as some intermediates were not commercially available to perform coupled
assays. Nuclear magnetic resonance (NMR) spectroscopy was used to characterise these enzymes.
These experimentally obtained parameters were then incorporated in a mathematical framework.
Time simulations on the initial model were unable to reach a steady-state, with a build up of
metabolic intermediates. A secondary model was constructed (using calculated maximal activities)
which allowed us to identify discrepancies in the initial model. This showed that the experimentally
determined maximal activities of three enzymes in lower glycolysis were unrealistically low,
which might be due to protein denaturation by sonication.
A nal model was constructed which incorporated a correction factor for these three enzymes.
The models' predicted output (steady-state concentrations and
ux) was compared to that of either literature or experimentally determined values, as a method to validate the model. The
model output compared well to literature values. The constructed and partially validated kinetic
model was then used as an analytical tool to identify points of control and regulation of glycolysis
in Z. mobilis.
The model presented in this work was also compared to published models. Our model relies
much less on literature obtained values, and uses kinetic parameters experimentally determined
under the same conditions. The parameters of the published models were obtained from the
literature and in many instances, the assay conditions for these parameters were set-up to yield
the maximum activity under non-physiological conditions. Furthermore, the number of excluded or
assumed parameters is much less in our model. However, introduction of a milder, more predictable
extraction technique for preparing cell lysates, should be considered for future work, to obtain the
parameters that was not determined during this study. The published models do include reactions
not included in our model (e.g ATP metabolism), which should be considered for inclusion, as we
strive to construct a detailed kinetic model of glycolysis in Z. mobilis in the future. / AFRIKAANSE OPSOMMING: Sellul^ere metaboliese netwerke is komplekse stelsels, omdat hulle bestaan uit talle komponente met
verskeie nie-lineêre interaksies. Om die funksionering van hierdie netwerke te verstaan, word hulle
dikwels in hanteerbare stukke verdeel en individueel bestudeer. 'n Enkele komponent is egter nie in
staat om die komplekse eienskappe van sulke stelsels te verklaar nie. Die veld van sisteembiologie
het ontwikkel met die doel om sulke stelsels te bestudeer. Sisteembiologie maak gebruik van
rekenaarmodelle as 'n metode om aspekte van lewende sisteme te beskryf. Kinetiese modelle van
metaboliese paaie word gebou en gebruik as gereedskap om die biologiese stelsels te bestudeer en
'n kwantitatiewe beskrywing te bekom. Hierdie tesis beskryf die kwantitatiewe ontleding van 'n
bakterie deur middel van 'n sisteembiologiese benadering.
Zymomonas Mobilis is 'n staafvormige, Gram-negatiewe, nie-mobiele fakultatiewe ana erobe,
en het een van die vinnigste waargenome fermentasies, maar met die minste energie-doeltre ende
ekstraksie wat in die natuur aangetref word. Verder is dit die enigste bekende mikro-organisme wat
die Entner-Doudoro (2-keto-3-dioksi-6-fosfoglukonaat) pad ana erobies gebruik. Die lae-energieopbrengs
van fermentasie in Z. mobilis is 'n gevolg van die gebruik van die Entner-Doudoro
metaboliese pad, wat die helfte van die energie-opbrengs per mol substraat lewer, in vergelyking
met die bekende Embden-Meyerhof-Parnas pad.
Die werk wat in hierdie tesis aangebied word, vorm deel van 'n groter projek om glikolitiese
regulering in verskillende mikro-organismes te vergelyk, naamlik Z. mobilis, Escherichia coli, Sac-
charomyces en Lactococcus lactis. Hierdie organismes is gekies op grond van hul gebruik van
verskillende glikolitiese meganismes. Kinetiese modellering is 'n handige metode om 'n vergelyking
tussen hierdie organismes te trek. Hierdie werk fokus op die bou van 'n kinetiese model van
die Entner-Doudoro glikolitiese metaboliese pad soos dit in Z. mobilis voorkom. Die doel van hierdie tesis was om so veel moontlik van die Entner-Doudoro ensieme onder
standaard-toestande te karakteriseer. Die kinetiese parameters van elk van die ensieme is met
behulp van ensimatiese essai's bepaal. Vir die meeste essai's is 96-put mikrotiterplate gebruik,
maar nie al die karakteriserings kon met behulp van hierdie metode gedoen word nie, omdat
sommige intermediate nie kommersieel beskikbaar was om gekoppelde essai's mee uit te voer nie.
Kernmagnetiese resonansie (KMR) spektroskopie is gebruik om hierdie ensieme te karakteriseer.
Die eksperimenteel bepaalde parameters is opgeneem in 'n wiskundige raamwerk. Tydsimulasies
op die aanvanklike model was nie in staat om 'n bestendige toestand te bereik nie, omdat
metaboliete opgebou het. 'n Sekond^ere model is gebou (met behulp van berekende maksimale
aktiwiteite) wat ons toegelaat om teenstrydighede in die aanvanklike model te identi seer. Dit het
getoon dat die eksperimenteel bepaalde maksimale aktiwiteite van drie ensieme in die laer gedeelte
van glikolise te laag was, waarskynlik as gevolg van prote en denaturering tydens die ultrasoniese
disintegrasieproses. 'n Finale model is gebou waarin 'n korreksiefaktor vir hierdie drie ensieme opgeneem is. Die
modelle se voorspelde uitset (bestendige toestand konsentrasies en
uksie) is vergelyk met waardes
uit die literatuur of wat ons self bepaal het, as 'n metode om die model te valideer. Die model uitset
was in goeie ooreenstemming met hierdie waardes. Die gedeeltelik gevalideerde kinetiese model is
voorts gebruik as 'n analitiese instrument om beheer en regulering van glikolise in Z. mobilis te
ondersoek.
Die model wat in hierdie werk ontwikkel is, is ook vergelyk met die vorige gepubliseerde modelle.
Ons model berus baie minder op waardes uit die wetenskaplike literatuur, en maak gebruik
van parameters wat eksperimenteel bepaal is, onder identiese toestande. Die parameters van die
gepubliseerde modelle is meesal verkry uit die literatuur, en in baie gevalle was die eksperimentele
kondisies vir hierdie analises opgestel om die maksimale aktiwiteit te lewer onder nie- siologiese
toestande. Verder bevat ons model minder parameters wat of uitgesluit is of wie se waardes aangeneem
moes word. In toekomstige werk sal daar egter klem gel^e moet word op 'n minder wisselvallige
ekstraksietegniek vir die verkryging van selekstrakte, om sodoende parameters te identi seer wat
nie in hierdie werk bepaal kon word nie.
Die gepubliseerde modelle sluit ook reaksies in wat nie ingesluit is in ons model nie (bv. ATP
metabolisme). Hierdie sou in ag geneem moet word vir insluiting in 'n toekomstige uitgebreide
model, om daarna te streef om 'n gedetailleerde kinetiese model van glikolise in Z. mobilis te bou.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/96033 |
| Date | 12 1900 |
| Creators | Van Staden, Charles Theo |
| 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 | Unknown |
| Type | Thesis |
| Format | xiv, 69 p. |
| Rights | Stellenbosch University |
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