Thesis (MSc)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: Glycerol metabolism is paramount to the physiological adaptation by Saccharomyces
cerevisiae to hyper-osmotic stress conditions. Glycerol metabolism also -plays a
fundamental role in maintaining a redox state favourable for growth under fermentative
conditions. All aspects of the relationship between redox balancing and glycerol
metabolism are not yet fully defined and attempts to manipulate this relationship, i.e., to
increase or decrease glycerol yields from fermentation, result in a redox disturbance that
is often detrimental to other aspects of metabolism. Another aspect of glycerol
metabolism that is not thoroughly understood, is how the various parameters of the
glycerol synthesis pathway, each independently and in conjunction with each other,
control the rate at which glycerol is synthesized. Addressing these questions has been the
topic of this thesis. In this regard, the theory of metabolic control analysis (MeA) was
adopted and calculations were performed with the aid of an experimentally validated
kinetic model.
To ascertain the in vivo substrate, product, coenzyme and known modifier
concentrations of the glycerol synthesis pathway, reliable techniques to halt
metabolism, extract and measure these metabolites had to be established. The
metabolite concentrations constitute a portion of the parameters of the pathway
and are necessary to construct a detailed kinetic model. Measuring the
concentration of an intracellular metabolite enzymatically requires the cell extract
to have an adequate quantity of the metabolite in question. This may be achieved
by concentrating the cells, before extracting the metabolite, by means of rapid
filtration. Then by freezing the cells with liquid nitrogen, metabolism can be halted
instantly. It was found that when metabolites were measured, yields were largely
dependent on the method of extraction, since different metabolites are sensitive
to different pH and temperature conditions. Methods of extraction found to be
reliable for the metabolites of interest in this study are presented in Chapter 3.
Metabolic control coefficients calculated by the model helped identify the
parameters that control flux through the glycerol synthesis pathway most rigidly.
The first reaction of the pathway, catalyzed by NAO+-dependent glycerol 3- phosphate dehydrogenase, had a flux control coefficient ( c; )of 0.83 to 0.87 and
exercises the majority of control of flux through the pathway, while the
subsequent reaction, catalyzed by glycerol 3-phosphatase, had far less control
(C:2 = 0.13 to 0.17).
The response coefficients (RJ
) of various parameter metabolites indicate [x]
that flux through the pathway is most responsive to the concentration of the
substrate, DHAP (RJ = 0.48 to 0.69), followed by the concentration of the [DHAP]
inhibitor, ATP (RJ =-0.21 to -0.5). Interestingly, the model also predicts that
[ATP]
the pathway responds far more severely to the ATP/ADP ratio than to the
NADH/NAD ratio, because of the weak response coefficient attributed to NADH
(RJ = 0.03 to 0.08). Thus, the model suggests that the targets most strategic [NADH]
for altering glycerol synthesis would be the Vmax of the glycerol 3-phosphate
dehydrogenase reaction and the concentrations of DHAP and ATP. Ideally, the
approach would entail manipulating each of these parameters to their optimal
levels in conjunction with each other, with the least detrimental physiological
effect possible. / AFRIKAANSE OPSOMMING: Gliserolmetabolisme is noodsaaklik tydens die fisiologiese aanpassing van gis
onder hiperosmotiese strestoestande. Dit speelook 'n fundamentele rol in die
handhawing van 'n voordelige redokstoestand, tydens groei onder fermentatiewe
kondisies. Alle aspekte rondom die verwantskap tussen redoksbalansering en
gliserolmetabolisme is nog nie ten volle gedefinieer nie en pogings om hierdie
verwantskap te manipuleer, m.a.w. om gliserolproduksie tydens fermentasie te
verhoog of verlaag, het In redoksversteuring tot gevolg, wat dikwels nadelig
teenoor ander aspekte van metabolisme is. Verder is die meganisme van hoe
verskeie parameters in die gliserolsintese pad, beide afsonderlik en gesamentlik,
die tempo van gliserolsintese beheer, nie ten volle duidelik nie. Die doel van
hierdie studie was dus om die bogenoemde onduidelikhede te probeer verklaar.
In hierdie verband is die teorie van metaboliese kontrole analise (MeA) gebruik,
en berekeninge is uitgevoer met behulp van 'n eksperimenteel gevalideerde
kinetiese model.
Ten einde, die in vivo substraat-, produk-, koensiem-, asook bekende
aktiveerder en inhibeerder-konsentrasies van die gliserolsintese pad te bepaal,
moes betroubare tegnieke ontwikkel word om die metabolisme vinnig te stop en
sodoende metaboliete te ekstraheer en te meet. Dié metabolietkonsentrasies
vorm 'n deel van die parameters in die pad, en word dus benodig om 'n
gedetailleerde kinetiese model saam te stel. Die ensiematiese bepaling van
intrasellulêre metabolietkonsentrasies vereis dat die selekstrak genoegsame
hoeveelhede van die metaboliete bevat. Dit kan verkry word deur die selle te
konsentreer deur middel van 'n vinnige filtrasiestap voordat ekstraksie van die
metaboliete plaasvind. Metabolisme word onmiddellik gestop deur die selle te
vries met vloeibare stikstof. Die metaboliet-hoeveelhede het grootliks afgehang
van die ekstraksie-metode gebruik, aangesien verskillende metaboliete gevoelig
is vir verskeie pH en temperatuurkondisies. Betroubare ekstraksiemetodes vir die
metaboliete van belang vir hierdie studie word aangedui in Hoofstuk 3 van die
tesis. Metaboliese kontrole koëeffisiënte wat met die model bereken is, het
daardie parameters geïdentifiseer wat die fluksie deur die gliserolsintese pad die
meeste beïnvloed. pie eerste reaksie in die pad, wat deur NAD+-afhanklike
gliserol 3-fosfaat dehidrogenase gekataliseer word, besit 'n fluksie kontrolekoëeffisiënt
( C~) van 0.83 tot 0.87, en oefen die grootste beheer uit oor fluksie
deur die pad. Die daaropvolgende reaksie, gekataliseer deur gliserol 3-fosfatase,
handhaaf minder kontrole oor fluksie deur die pad (C;2 = 0.13 tot 0.17).
Responskoëeffisiënte (RJ
) van verskeie parametermetaboliete dui [x)
daarop dat die fluksie deur die pad die sterkste deur substraat (DHAP)
konsentrasie beïnvloed word (RJ = 0.48 tot 0.69), gevolg deur inhibeerder
[DHA?)
(ATP) konsentrasie (RJ =-0.21 tot -0.5). Daarbenewens dui die model oak aan
[AT?)
dat die pad meer sensitief is teenoor die ATP/ADP verhouding, relatief tot die
NADH/NAD+ verhouding, wat die gevolg is van die klein responskoëeffisiënt
teenoor NADH (RJ =0.03 tot 0.08). Die model suggereer dus dat die Vmaks
[NADH)
van die gliserol 3-fosfaat dehidrogenase reaksie, asook die DHAP en ATP
konsentrasies, die mees strategiese teikens vir manipulering van gliserolsintese
behoort te wees. Die ideale benadering sal dus wees om al hierdie parameters in
samehang met mekaar te kan manipuleer tot huloptimale vlakke, met die minste
ontwrigting van die sel se fisiologie.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/52657 |
| Date | 12 1900 |
| Creators | Cronwright, Garth Rupert, 1974- |
| Contributors | Prior, B. A., Rohwer, J. M., Stellenbosch University. Faculty of Science. Dept. of Microbiology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | xii, 75 p. : ill. |
| Rights | Stellenbosch University |
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