Measurement of intracellular metabolites of primary metabolism and adenine nucleotides in chemostat cultivated Penicillium chrysogenum

• Published in: Biotechnology and bioengineering 2006-05, Vol.94 (1), p.159-166
• Main Authors: Nasution, U., van Gulik, W.M., Kleijn, R.J., van Winden, W.A., Proell, A., Heijnen, J.J.
• Format: Article
• Language: English
• Subjects: Adenine Nucleotides - metabolism; Biological and medical sciences; Biomass; Bioreactors - microbiology; Biotechnology; Carbon Isotopes; chemostat; Chromatography, Ion Exchange; Citric Acid Cycle; Culture Media - chemistry; Fundamental and applied biological sciences. Psychology; Glycolysis; in vivo; Isotope Labeling; Kinetics; Metabolism; metabolome; Penicillin; Penicillium chrysogenum; Penicillium chrysogenum - genetics; Penicillium chrysogenum - growth & development; Penicillium chrysogenum - metabolism; quenching; Radioisotope Dilution Technique; rapid sampling; Reference Standards; Saccharomyces cerevisiae; Saccharomyces cerevisiae - metabolism; Spectrometry, Mass, Electrospray Ionization; Yeast
• ISSN: 0006-3592; 1097-0290
• DOI: 10.1002/bit.20842

An experimental platform has been developed for rapid sampling and quenching of chemostat cultivated Penicillium chrysogenum broth for metabolome analysis in highly dynamic experiments, aimed at the elucidation of the in vivo kinetic properties of metabolism. The sampling and quenching protocol available from Saccharomyces cerevisiae had to be modified for Penicillium chrysogenum mainly because of its filamentous character. Intracellular metabolites of glycolysis, TCA cycle, and adenine nucleotides were measured with isotope dilution mass spectrometry (IDMS) using a U‐13C‐labeled metabolite mix produced from yeast cells as internal standard. By addition of the U‐13C internal standard mix prior to the metabolite extraction procedure, partial degradation of metabolites as well as non‐linearity and drift of the LC‐MS/MS could be successfully compensated for. It was found that there is a serious matrix effect on metabolite extraction between different organisms, which is however completely corrected for by the IDMS approach. Intracellular metabolites could be analyzed with standard deviations of around 5%. A comparison of the metabolite levels between Saccharomyces cerevisiae and Penicillium chrysogenum showed both significant similarities and large differences, which seem to be related to the presence of the penicillin pathway. © 2006 Wiley Periodicals, Inc.