An analysis of glycerol synthesis by Saccharomyces cerevisiae

[摘要] ENGLISH ABSTRACT:Glycerol metabolism is paramount to the physiological adaptation by Saccharomycescerevisiae to hyper-osmotic stress conditions. Glycerol metabolism also -plays afundamental role in maintaining a redox state favourable for growth under fermentativeconditions. All aspects of the relationship between redox balancing and glycerolmetabolism are not yet fully defined and attempts to manipulate this relationship, i.e., toincrease or decrease glycerol yields from fermentation, result in a redox disturbance thatis often detrimental to other aspects of metabolism. Another aspect of glycerolmetabolism that is not thoroughly understood, is how the various parameters of theglycerol synthesis pathway, each independently and in conjunction with each other,control the rate at which glycerol is synthesized. Addressing these questions has been thetopic of this thesis. In this regard, the theory of metabolic control analysis (MeA) wasadopted and calculations were performed with the aid of an experimentally validatedkinetic model.To ascertain the in vivo substrate, product, coenzyme and known modifierconcentrations of the glycerol synthesis pathway, reliable techniques to haltmetabolism, extract and measure these metabolites had to be established. Themetabolite concentrations constitute a portion of the parameters of the pathwayand are necessary to construct a detailed kinetic model. Measuring theconcentration of an intracellular metabolite enzymatically requires the cell extractto have an adequate quantity of the metabolite in question. This may be achievedby concentrating the cells, before extracting the metabolite, by means of rapidfiltration. Then by freezing the cells with liquid nitrogen, metabolism can be haltedinstantly. It was found that when metabolites were measured, yields were largelydependent on the method of extraction, since different metabolites are sensitiveto different pH and temperature conditions. Methods of extraction found to bereliable for the metabolites of interest in this study are presented in Chapter 3.Metabolic control coefficients calculated by the model helped identify theparameters 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 andexercises the majority of control of flux through the pathway, while thesubsequent 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 thesubstrate, 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 theNADH/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-phosphatedehydrogenase reaction and the concentrations of DHAP and ATP. Ideally, theapproach would entail manipulating each of these parameters to their optimallevels in conjunction with each other, with the least detrimental physiologicaleffect possible.

[发布机构] Stellenbosch University