earticle

영어

As development of analytical method we have chance to look into interest cell directly. NMR and mass device combined with chromatography made it possible mine structural and quantitative information of intracellular metabolite what we interest. Glycerol is an interesting metabolite which get involved in stress response mechanism such as HOG pathway in S. cerevisiae. So studies of anabolic and catabolic pathway of glycerol in S. cerevisiae were focused in early days. But there still remain unknown mechanism and function of genes such as GCY1 putative NADP (+) glycerol dehydrogenase. In this study, we identified role of GCY1 gene among glycerol dissimilation pathway by metabolic engineering and analyzed glycerol and its derived metabolites by GC/MS. Homologous recombination PCR method and pRS423 expression vector were used for disruption or overexpression of interested genes such as GCY1, DAK1 and DAK2. Developed strains were fermented under different oxygen contained conditions by 1L volume bioreactor with sampling device for quick sampling and pretreatment before analaysis. Intracellular glycerol, dihydroxyacetone, glycerol 3-phosphate and dihydroxyacetone phosphate were analyzed by GC/MS and ethanol was analyzed by HPLC. Dihydroxyacetone was not detected in gcy1 gene disrupted strain and it was accumulated in dihydroxyacetone kinase deficient strain. Lastly, dihydroxyacetone were GCY1 gene overexpressed based on dihydroxyacetone kinase deficient strain increased 510% than wild type strain under microaerobic cultivation. Via these gene manipulation and analysis we identify role of GCY1 gene as a glycerol dehydrogenase under oxygen limited condition in S. cerevisiae.