(724h) Analysis of Central Metabolic Reaction Rates Via Non-Stationary 13C Isotope Study Combined With Kinetic Model in Yeast

Calculation of metabolic reaction rates in the central carbon metabolic pathways (glycolysis, pentose phosphate pathway and TCA cycle) can improve our understanding about microbial physiology of metabolically engineered strains. This has become possible by recently introduced non-staionary ¹³C metabolite profiling, which directly measures isotope tracer conversion rates of metabolites. For this purpose, a few critical experimental steps should be established and examined, such as proper sampling protocol including quenching and extraction, the verification of metabolite leakage, and the assessment of appropriate mass peaks corresponding to the derivatized metabolites. Also, enzyme kinetic models should be hired for the calculation of metabolic conversion rates from the dynamic ¹³C isotope data.

In this study, we performed comprehensive analysis of intermediate metabolites with quadruple gas chromatography mass spectrometry (GC-MS) and calculation of metabolic reaction rates in glycolysis, pentose phosphate pathway and TCA cycle in yeast. Firstly, non-stationary metabolite profiling methods were set up for central carbon metabolites analysis from Saccharomyces cerevisiae and Kluyveromyces marxianus. For example, a bioreactor with fast sampling device was designed, metabolite leakage was tested by mass balance analysis among different types of sample, and the proper mass peaks corresponding target metabolites were assessed via statistical approach. Secondly, isotope-based dynamic studies were performed with uniformly labeled ¹³C glucose. From time series isotopic studies, conversion of glucose into the central metabolites were well demonstrated. Lastly, kinetic model of each pathway enzymes were used to calculate metabolic reaction rates. The results well demonstrated the difference of glucose utilizing metabolism between S. cerevisiae and K. marxianus.