Functional studies of the <i>TDH</i> and <i>GPD</i> gene-products in the glycolytic flux and glycerol metabolism of <i>Saccharomyces cerevisiae</i>

This thesis focuses on the physiology and control of the glycolysis and glycerol metabolism in the yeast <i>Sacchromyces cerevisiae</i>. The study is based on the relation ship between the first two reactions around the branch-point of the upper and lower part of glycolysis and the glycerol pathway. In these reactions, the oxidation of glyceraldehyde 3-phosphate (GAP) to 1,3-bisphospho glycerate (1,3-BFG) is catalysed by the glyceraldehyde 3-phosphate dehydrogenases (encoded by the <i>TDH</i>-genes), while the reduction of dihydroxyacetone phosphate (DHAP) to glycerol 3-phosphate (G3P) is catalysed by the glycerol 3-phosphate dehydrogenases (encoded by the <i>GPD</i>-genes). Both the Gpd- and Tdh-enzymes catalyse redox reactions using either part of the redox couple, NADH/NAD<font size="-1">⁺</font>, as cofactor. The products of the <i>GPD</i> and <i>TDH</i> genes are involved in many different aspects of yeast physiology such as; maintenance of redoxbalance, osmoregulation, lipid biosynthesis, and protection against reactive oxygen species (ROS). The <i>GPD1</i> gene is mainly important when the cells are subjected to osmotic stress and deletion of this gene does not affect the growth rate aerobically or anaerobically under “normal” growth conditions. In contrast, deletion of the <i>GPD2</i> gene causes a decrease of the growth rate and an increase of the <i>TDH1</i> gene expression under anaerobic conditions. Deletion of the <i>TDH1</i> gene (but not of <i>TDH2</i> or <i>TDH3</i>) in a <i>gpd2&#916;</i> strain can partially suppress the anaaerobic growth defect of the <i>gpd2&#916;</i> strain. To understand the link between the Gpd- and the Tdh-reactions, we have made all combinatorial mutants between the <i>GPD</i> and<i> TDH</i> genes. In the present work, we have shown that the earlier reported lethal mutant of glycolysis, <i>tdh2&#916; tdh3&#916;</i>, is suppressed by disruption of the <i>GPD</i> genes. Our work has revealed that the glycolytic flow of the lethal <i>tdh2&#916; tdh3&#916;</i> mutant has a limiting step at the Tdh-reaction and that the Tdh1p is fully capable to perform this reaction if it expressed at sufficient levels. Further, the kinetic properties of Tdh-isoenzymes are elucidated and the pivotal role of the Tdh- and Gpd-enzymes in metabolism of <i>S. cerevisiae</i> are examined and discussed