Department of Chemical and Biological Engineering, Korea University, Seoul, Korea.
J Appl Microbiol. 2012 Dec;113(6):1468-78. doi: 10.1111/jam.12013. Epub 2012 Oct 9.
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.
随着色谱 - 质谱联用技术和快速采样技术的进步，细胞内（IC）代谢物的分析研究得到了发展。我们应用 IC 代谢物分析来表征 GCY1 在酿酒酵母甘油（GLY）分解代谢途径中的作用。
构建了破坏或过表达 GLY 分解代谢基因（如 GCY1、DAK1 和 DAK2）的菌株。在不同通气条件下培养这些菌株，并使用新型快速采样端口快速淬火。通过气相色谱/质谱法分析菌株中 GLY、二羟丙酮（DHA）、甘油 3-磷酸和二羟丙酮磷酸的 IC 浓度。在微需氧条件下，在 DHA 激酶基因缺陷型菌株中，gcy1 基因缺失菌株中未检测到 DHA，但积累了 225.91 μmol g DCW(-1)。此外，在 DHA 激酶缺陷型菌株中过表达 GCY1 可使 DHA 增加 16.1%。
代谢组学研究表明，GCY1 基因产物在酿酒酵母中作为 GLY 脱氢酶发挥作用，特别是在微需氧条件下。
成功地在酿酒酵母中展示了 GLY 异化途径的代谢组学研究，并通过结果解释了 GCY1 的功能。
