Abstract
The analytical study of intracellular (IC) metabolites has developed with advances in chromatography-linked mass spectrometry and fast sampling procedures. We applied the IC metabolite analysis to characterize the role of GCY1 in the glycerol (GLY) catabolic pathway in Saccharomyces cerevisiae. Strains with disrupted or overexpressing GLY catabolic genes such as GCY1, DAK1 and DAK2 were constructed. The strains were cultivated under different aeration conditions and quickly quenched using a novel rapid sampling port. IC concentrations of GLY, dihydroxyacetone (DHA), glycerol 3-phosphate and dihydroxyacetone phosphate were analysed in the strains by gas chromatography/mass spectrometry. DHA was not detected in the gcy1 gene-disrupted strain but accumulated 225.91 μmol g DCW(-1) in a DHA kinase gene-deficient strain under micro-aerobic conditions. Additionally, a 16.1% increase in DHA occurred by overexpressing GCY1 in the DHA kinase-deficient strain. Metabolic profiling showed that the GCY1 gene product functions as a GLY dehydrogenase in S. cerevisiae, particularly under micro-aerobic conditions. Metabolic profiling of the GLY dissimilation pathway was successfully demonstrated in S. cerevisiae, and the function of GCY1 was explained by the results.
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