Abstract #5

Metabolomic analysis of respiring yeast by comprehensive GCXGC-TOFMS: the missing link between a dynamic transcriptome and growth on non-fermentable carbon sources. Kenneth M. Dombek¹, Rachel E. Mohler², Robert E. Synovec², Elton T. Young¹. 1) Department of Biochemistry, University of Washington, Seattle, WA; 2) Department of Chemistry, University of Washington, Seattle, WA.
   The transcription factors Adr1 and Cat8 orchestrate a transcriptional program that allows the yeast Saccharomyces cerevisiae to grow on non-fermentable carbon sources. Transcriptome and ChIP on CHIP analyses indicate that Adr1 and Cat8 directly regulate genes involved in feeding non-fermentable carbon into central carbon metabolism. Adr1 also regulates the genes of peroxisomal biogenesis and fatty acid b-oxidation, while Cat8 regulates the genes of gluconeogenesis and the glyoxylate cycle. This suggests that the impaired ability of adr1 and cat8 mutants to grow on non-fermentable carbon sources is due to an inadequate flow of carbon through these pathways, assuming that the observed changes in gene expression reflect predictable metabolomic changes. To test this assumption, we are utilizing comprehensive two-dimensional gas chromatography coupled to time of flight mass spectrometry (GCXGC-TOFMS) to analyze the yeast metabolome. As a proof of principle, metabolite extracts of yeast cells fermenting glucose were compared to extracts of respiring cells oxidizing ethanol. Principal component analysis followed by parallel factor analysis in conjunction with the LECO ChromaTOF software identified and quantified twenty-six differentiating metabolites. Sugar phosphates of glycolysis were significantly more abundant in fermenting cells, whereas, TCA cycle intermediates were more abundant in respiring cells. These results are consistent with the redirection of carbon flow from glycolysis to the TCA cycle and gluconeogenesis upon shifting from the fermention of glucose to the oxidation of ethanol. These results are also consistent with transcriptome data showing that TCA cycle genes are more highly expressed in respiring cells than in fermenting cells. This methodology will now be used to analyze the ADR1- and CAT8-dependent metabolomes.

Return to YGM 2006 Home at SGD