This thesis extends the work of Crimmins (1995) on the control of glycolytic flux in yeast by the enzymes 6-phosphofructo-1-kinase and pyruvate kinase (Pyk1p). This study also examines the influence of Pf1kp and Pyk1p upon yeast resistance to the weak acid preservative, benzoic acid. In <I>Saccharomyces cerevisiae</I>, Pyk1p is encoded by <I>PYK1</I>, and the α and β subunits of Pf1kp are encoded by <I>PFK1</I> and <I>PFK2</I>, respectively. To test the influence of these genes upon glycolytic control, an isogenic set of <I>S. cerevisiae</I> mutants were utilised in which <I>PYK1, PFK1</I> and <I>PFK2</I> expression is dependent on the <I>PGK1</I> promoter. Increased Pf1k levels had little effect upon rates of glucose utilisation or ethanol production during fermentative growth. However, overexpressing Pyk1p resulted in an increased growth rate and an increase in glycolytic flux. This suggests that Pyk1p, but not Pf1kp, exerts some degree of control over the glycolytic flux under these conditions. The effects of reducing Pf1kp and Pyk1p levels were also studied by placing <I>PYK1, PFK1</I> and <I>PFK2</I> under the control of the weak <I>PGK1Δuas</I> promoter. The double Pf1kp mutant showed no significant changes in doubling time, ethanol production or glucose consumption. However, a mutant with a 3-fold reduction ion Pyk1p levels displayed slower growth rates and reduced glycolytic flux. In addition, there was an imbalance in the carbon flow in this mutant, with reductions in ethanol and glycerol production evident, along with increased TCA cycle activity. Hence, while Pf1kp levels did not affect cell physiology significantly under the conditions studied, reduced Pyk1p levels seemed to disturb glycolytic flux and carbon flow. Decreased Pf1kp levels caused an increase in the sensitivity of yeast cells to benzoate, whereas the Pyk1p mutant was not affected. This confirmed that benzoic acid specifically inhibits Pf1kp rather than glycolysis in general.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:301297 |
| Date | January 1999 |
| Creators | Pearce, Amanda K. |
| Publisher | University of Aberdeen |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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