Development of quantitative metabolomics for <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Accurate, reliable and reproducible measurement of intracellular metabolite levels has become important for metabolic studies of microbial cell factories. A first critical step for metabolomic studies is the establishment of an adequate quenching and washing protocol, which ensures effective arrest of all metabolic activity and removal of extracellular metabolites, without causing leakage of metabolites from the cells. Five different procedures based on cold methanol quenching and cell separation by filtration were tested for metabolomics of Pichia pastoris regarding methanol content and temperature of the quenching solution as key parameters. Quantitative evaluation of these protocols was carried out through mass balance analysis, based on metabolite measurements in all sample fractions, those are whole broth, quenched and washed cells, culture filtrate and quenching and washing solution. Finally, the optimal method was used to study the time profiles of free amino acid and central carbon metabolism intermediates in glucose-limited chemostat cultures. Acceptable recoveries (>90%) were obtained for all quenching procedures tested. However, quenching at −27°C in 60% v/v methanol performed slightly better in terms of leakage minimization. We could demonstrate that five residence times under glucose limitation are enough to reach stable intracellular metabolite pools. Moreover, when comparing P. pastoris and S. cerevisiae metabolomes, under the same cultivation conditions, similar metabolite fingerprints were found in both yeasts, except for the lower glycolysis, where the levels of these metabolites in P. pastoris suggested an enzymatic capacity limitation in that part of the metabolism.