2-Hydroxyglutarate (2HG) is an atypical chiral metabolite that accumulates, under its D- and/or L-enantiomeric form(s), in several neurometabolic diseases as well as in certain types of cancer. Given its role in cancer, D-2HG is designated as an oncometabolite and both D-2HG and L-2HG are believed to have neurotoxic properties. The mechanisms through which 2HG leads to cell transformation or neurodegeneration remain, however, poorly understood. In addition, the fact that patients with 2HG-accumulating gliomas have a better prognosis than patients with non 2HG-accumulating gliomas as well as recent observations showing that 2HG increases the life span of C. elegans worms and inhibits mTOR signalling in human cells suggest that the role of 2HG in pathophysiological processes is more complex than what is currently believed.In the described research project, we aim to further elucidate the genetic network involved in D-2HG formation and degradation in yeast. Our recently published discovery of several enzymes participating in D-2HG metabolism in yeast suggests the existence of a coupling between D-2HG metabolism and the mitochondrial respiratory chain in these cells. Gene regulatory properties of the enzyme that we showed to play a major role in D-2HG degradation also suggest that toxic effects of D-2HG may become more prominent in mitochondrially deficient cells. In this project, we aim at providing experimental support for these hypotheses and address further questions that have been opened by our prior work on 2HG metabolism. Our primary objectives are: (1) The elucidation of the role of D-2HG in mitochondrial dysfunction and aging, (2) the identification of additional genes involved in D-2HG metabolism or the regulation thereof, and (3) the integration of D-2HG into a central carbon network model of yeast that will be validated experimentally. The methodologies used include high-throughput growth and aging phenotyping screens with selected yeast mutants, LC-MS-based metabolomics analyses in cell extracts and cultivation media, including targeted 2HG assays, genetic linkage mapping in the progeny of natural yeast variants producing abnormally high or low levels of D-2HG, and LC-MS-based isotopic-tracer analyses to validate new metabolic network model predictions. We expect that a number of the molecular mechanisms connecting D-2HG to mitochondrial function and of the new D-2HG genes identified in this project will be conserved in humans. The mid- to long-term goals of the project are to provide a solid basis for the engineering of yeast models and for the rational design of therapeutic strategies for D-2HG associated diseases.