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

Doctoral thesis, 2003

This thesis focuses on the physiology and control of the glycolysis and glycerol metabolism in the yeast Sacchromyces cerevisiae. 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 TDH-genes), while the reduction of dihydroxyacetone phosphate (DHAP) to glycerol 3-phosphate (G3P) is catalysed by the glycerol 3-phosphate dehydrogenases (encoded by the GPD-genes). Both the Gpd- and Tdh-enzymes catalyse redox reactions using either part of the redox couple, NADH/NAD+, as cofactor. The products of the GPD and TDH 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 GPD1 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 GPD2 gene causes a decrease of the growth rate and an increase of the TDH1 gene expression under anaerobic conditions. Deletion of the TDH1 gene (but not of TDH2 or TDH3) in a gpd2Δ strain can partially suppress the anaaerobic growth defect of the gpd2Δ strain. To understand the link between the Gpd- and the Tdh-reactions, we have made all combinatorial mutants between the GPD and TDH genes. In the present work, we have shown that the earlier reported lethal mutant of glycolysis, tdh2Δ tdh3Δ, is suppressed by disruption of the GPD genes. Our work has revealed that the glycolytic flow of the lethal tdh2Δ tdh3Δ 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 S. cerevisiae are examined and discussed.