Sunday, May 4, 2008
2-38

In vitro 13C labeling for simultaneous identification and quantification of central carbon intermediates in genetically modified Saccharomyces yeast capable of  glucose/xylose co-fermentation using reverse phase liquid chroma

Miroslav Sedlak1, Wenchu Yang2, Nathan S. Mosier1, Nancy W. Y. Ho3, and Jiri Adamec2. (1) Lorre/abe, Purdue University, Potter Engineering Center, 500 Central Drive, West Lafayette, IN 47907, (2) Bindley Bioscience Center, Purdue University, 1203 West State Street, West Lafayette, IN 47907, (3) LORRE/Chemical Engineering, Purdue University, 500 Central Dr, West Lafayette, IN 47907

Comprehensive analysis of intracellular metabolites involved in central carbon metabolism is the key to understanding cellular metabolism. However, using reversed phase liquid chromatography-mass spectrometry (RPLC-MS) to perform this task is difficult because metabolite diversity in biological samples prevent accurate RPLC separation. Furthermore, precise metabolite quantization is very difficult due to the lack of commercially available stable isotope-coded standards.  A new MS quantization method, named Global Isotope-labeled Internal Standard (GILISA) was developed for quantitative analysis. Through the use of aniline-13C6-labeled internal standards, accurate MS quantification could be readily achieved. Thirty-two common metabolites from yeast lysate involved in glycolysis, the pentose phosphate pathway, and tricarboxylic acid cycle were unambiguously identified and quantified with relative standard deviations smaller than 10%. This method provides a convenient, robust, and high throughput tool for intracellular metabolite analysis.  This paper reports the use of this newly developed GILSA method to indentify metabolic bottlenecks in genetically engineered S. cerevisiae 424A(LNH-ST) yeast capable of  effectively co-fermenting glucose and xylose to ethanol.

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