| An experimental approach of untargeted high performance liquid chromatography-mass spectrometry (LC-MS), metabolomics, isotopic tracers, and biochemical assays has identified the DET1 (decreased ergosterol transport) gene in Saccharomyces cerevisiae as one that has impaired use of NADPH. The deletion of the DET1 gene selectively accumulates NADPH across media conditions, co-fitness mutants, and physical interaction binding partners. A created overexpression strain has depleted NADPH and lower levels of pentose phosphate pathway intermediates. An increased pool of NADPH can contribute to increased reducing power needed for bioengineering microorganisms to produce biofuels and electron rich products via the enzymatic conversion of a fermentable carbon substrate. NADPH is involved in many reactions and is a cofactor throughout metabolism. Det1Delta cells exhibit drug sensitivities to compounds in sterol biosynthesis, consistent with previous literature linking the DET1 gene to sterol transport and uptake. Det1Delta cells are confirmed to have phosphatase activity against known substrates when purified protein is bound to IgG. There is a defect in Det1Delta cells in recovery from oxidative stress exposure. Under oxidative stress, NADPH still accumulates in Det1Delta cells, with the remainder of metabolism displaying a general oxidative stress response.;The phenomena of nutrient integration and metabolic effects of nutrient perturbation are examined in wild type Saccharomyces cerevisiae. By analyzing the dynamics of intracellular metabolite concentrations in response to changes in nutrient availability, an understanding of cellular metabolic regulation can be obtained. Upon nutrient upshift from limited media, a return to whole composition homeostasis is observed. After nitrogen upshift, the primary result is increased metabolite abundances of amino acids, with glutamine as a key intermediate in nitrogen assimilation, nucleotide triphosphates, and S-adenosyl-methionine. alpha-ketoglutarate is depleted, and it may function as a nitrogen transporter in connection with central carbon metabolism. SAM pathway mutants exhibit similar metabolomic profiles to wild type. Upon phosphate upshift, there are increased metabolite abundances of nucleotides, glycolytic intermediates, and pentose phosphate pathway intermediates, with depleted abundances of purine nucleosides. Carbon flow for nitrogen and phosphate upshift remains similar for central carbon metabolism. |
