Thesis (MSc)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: Scientists and biochemical engineers alike are very interested in the control and regulation
of free-energy metabolism in micro-organisms, whether the findings purely
satisfy scientific curiosity or translate into the meeting of biotechnology company
deadlines. We used a rather fundamental approach to investigate experimentally
the control and regulation of yeast free-energy metabolism in anaerobic chemostat
cultures using supply-demand analysis. This conceptually simple, quantitative
framework, however, may lead to insight into the control properties of various
metabolic pathways to be used in biotechnological applications.
Supply-demand analysis is based on the theoretical framework of metabolic control
analysis (MCA).Sections (of arbitrary size) of a metabolic pathway are grouped
together around a linking metabolite. Those steps that produce the intermediate
are combined into the supply block while the reactions that remove/consume the
intermediate are grouped together as the demand. The elasticity coefficients of
the supply and demand blocks (with regard to the linking metabolite concentration)
can be used to determine the flux and concentration control coefficients by
using the traditional MCAsummation and connectivity theorems. Supply and demand
rate characteristics are a powerful visual approach to determine and display
the control structure of the pathway under consideration and sets supply-demand
analysis apart from traditional top-down analysis.
Our first tool of analysis was a structured kinetic model of yeast growing in a chemos tat, constructed by using methods developed in our research group for
modelling systems with variable volumes. Independent perturbations of the linking
metabolite concentration resulted in a control profile where the control resided
mainly in the demand (flux control coefficient of 0.92), as a result of a large negative
supply elasticity. This elasticity, however, varied greatly under different conditions,
leading to increased flux control by the supply in some cases.
We extended our research to an experimental setup of Saccharomyces cerevisiae
growing in a glucose-limited chemos tat supplemented with yeast extract as
a source of carbon intermediates. This allowed glucose to act solely as the freeenergy
source, as confirmed by balancing the glucose flux with the fluxes towards
the fermentation products, ethanol and carbon dioxide. We obtained the supply
rate characteristic by perturbing the ATPdemand through the addition of benzoate,
which uncouples the proton gradient across the cell membrane. The demand rate
characteristic was obtained by perturbing the ATP supply through changes in the
dilution rate and thus the residual glucose concentration in the fermentor. The
concentrations of ATPand ADPwere measured using a luciferase bioluminescence
assay, while the fermentation products were measured with HPLCand C02 with
an acoustic off-gas analyser. For our experimental conditions the flux-control of
energy metabolism resided predominantly in the supply with respect to the linking
metabolite [ATP]/[ADP](chosen as an indication of the free-energy state of the cell),
i.e. a flux control coefficient of 0.90. Further, the [ATP]/[ADP]was under strong
homeostatic control, as evidenced by the low [ATP]/[ADP]control coefficients of ±
0.12.
We adjusted the structured kinetic model by varying strategic parameters, so
that the results resembled the experimental observations more closely. However,
the kinetics of our core model seem to be too simplistic to capture fully the extent
of regulation displayed by the experimental system. The model did, however, reveal
the regulatory importance of glucose transport into the cell. We conclude that the
control and regulation of free energy metabolism in yeast strongly depend on the culturing conditions and on the steady state being analysed. / AFRIKAANSE OPSOMMING: Wetenskaplikes sowel as biochemiese ingenieurs is dikwels geïnteresseerd in die
beheer en regulering van vry-energie metabolisme in mikro-organismes, hetsy die
bevindinge suiwer wetenskaplike nuuskierigheid bevredig of die haalbaarheid van
biotegnologie-maatskappy-mikpunte beteken. Ons het 'n redelik fundamentele benadering
gevolg om die beheer en regulering van vry-energie metabolisme in gis
eksperimenteel te bepaal in anaerobiese chemostaatkulture met behulp van aanbod-
aanvraag analise. Dit is 'n konseptueel eenvoudige, kwantitatiewe raamwerk
met die potensiaal om insig te gee in die beheereienskappe van verskeie metaboliese
paaie wat nuttig kan wees in biotegnologiese toepassings.
Aanbod-aanvraag analise is gebaseer op die teoretiese onderbou van metaboliese
kontrole-analise (MKA).Dele (van arbitrêre grootte) van 'n metaboliese pad word
gegroepeer rondom 'n verbindingsmetaboliet. Die stappe wat die intermediaat produseer
word gekombineer as die aanbod terwyl die reaksies wat die intermediaat
verbruik, saamgegroepeer word as die aanvraag. Die elastisiteitskoëffisiënte
van die aanbod en aanvraag blokke (met betrekking tot die verbindingsmetabolietkonsentrasie)
kan gebruik word om die fluksie en konsentrasie kontrolekoëffisiënte
te bereken met behulp van die sommasie en konnektwiteit teoremas van MKA.Aanbod
en aanvraag snelheidskenmerkgrafieke is 'n treffende visuele benadering om
die kontroleprofiel van die betrokke metaboliese pad te bepaal en te vertoon. Hierdie
kenmerk onderskei aanbod-aanvraag analise van bo-na-onder analise. Die eerste deel van ons ondersoek het behels 'n gestruktureerde kinetiese model
(van gis wat groei in 'n chemostaat) met behulp van metodes wat in ons groep ontwikkel
is om sisteme met variërende volumes te modelleer. Onafhanklike perturbasies
van die verbindingsmetaboliet konsentrasie het gelei tot 'n kontroleprofiel
waar die kontrole hoofsaaklik in die aanvraag gesetel was (fluksie kontrolekoëffisiënt
van 0.92), as gevolg van 'n groot negatiewe aanbod-elastisiteit. Hierdie elastisiteit
kan egter grootliks varieer tydens verskillende kondisies, wat lei tot 'n toenemende
fluksle-beheer deur die aanbod in sommige gevalle.
Ons het ons navorsing uitgebrei na 'n eksperimentele opstelling van Saccharomyces
cerevisiae wat groei in 'n glukose-gelimiteerde chemostaat, aangevul met
gisekstrak as 'n bron van koolstof-Intermediate. Dit bring mee dat glukose slegs
as energiebron dien; dit is wel bevestig deur balanse op te stel van die koolstoffluksie
vanaf glukose na koolstofdioksied en etanol as die fermentasieprodukte.
Die aanbod snelheidskenmerkgrafiek is gegenereer deur die aanvraag van ATP
te manipuleer deur middel van toevoeging van bensoaat, wat die protongradiënt
oor die selmembraan ontkoppel. Die snelheidskenmerkgrafiek Vir die aanvraag is
gegenereer deur die aanbod van ATP te manipuleer deur middel van 'n variasie in
die verdunningstempo en sodoende die residuele glukose konsentrasie in die fermentor.
Die konsentrasies van ATPen ADPis bepaal deur middel van 'n lusiferase
bioluminessensie-essai, terwyl die fermentasieprodukte met 'n HPLCen CO2 met 'n
akoestiese aflaatgasanaliseerder gemeet is. Vir die betrokke eksperimentele toestande
was die flukste-kontrole van energiemetabolisme oorwegend in die aanbod
met betrekking tot die verbindingsmetaboliet, [ATP]/[ADP](gekies as aanduiding
van die vrye-energiestatus van die sel), naamlik 'n fluksie kontrolekoëffisiënt van
0.90. Verder was die [ATP]/[ADP]onder sterk homeostatiese beheer soos duidelik
blyk uit die lae [ATP]j[ADP] kontrolekoëffisiënte van ± 0.12.
Ons het die gestruktureerde kinetiese model aangepas deur strategiese parameters
te verander om sodoende die eksperimentele gedrag te probeer naboots. Die
kinetika van ons kernmodel blyk egter te simplisties te wees om die volle omvang van die regulering van die eksperimentele sisteem te vertoon. Die model het egter
die belang van glukose transport oor die selmembraan aan die lig gebring. Ons kom
tot die gevolgtrekking dat die beheer en regulering van vrye-energie metabolisme in
gis sterk afhang van die groeitoestande sowel as die spesifieke bestendige toestand
wat ondersoek word.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/53607 |
| Date | 12 1900 |
| Creators | Kroukamp, Marthinus |
| Contributors | Rohwer, J. M., Snoep, J. 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 | 73 p. : ill. |
| Rights | Stellenbosch University |
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