An analysis of glycerol synthesis by Saccharomyces cerevisiae

Cronwright, Garth Rupert, 1974- (2002-12)

Thesis (MSc)--University of Stellenbosch, 2002.

Thesis

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.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/52657
This item appears in the following collections: