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Cueto-Rojas HF, Maleki Seifar R, Ten Pierick A, et al. Accurate Measurement of the in vivo Ammonium Concentration in Saccharomyces cerevisiae. Metabolites. 2016;6(2)doi: 10.3390/metabo6020012.
Cueto-Rojas, H. F., Maleki Seifar, R., Ten Pierick, A., Heijnen, S. J., & Wahl, A. (2016). Accurate Measurement of the in vivo Ammonium Concentration in Saccharomyces cerevisiae. Metabolites, 6(2), . https://doi.org/10.3390/metabo6020012
Cueto-Rojas, Hugo F, et al. "Accurate Measurement of the in vivo Ammonium Concentration in Saccharomyces cerevisiae." Metabolites vol. 6,2 (2016). doi: https://doi.org/10.3390/metabo6020012
Cueto-Rojas HF, Maleki Seifar R, Ten Pierick A, Heijnen SJ, Wahl A. Accurate Measurement of the in vivo Ammonium Concentration in Saccharomyces cerevisiae. Metabolites. 2016 Apr 23;6(2). doi: 10.3390/metabo6020012. PMID: 27120628; PMCID: PMC4931543.
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Metabolites. 2016 Apr 23;6(2). doi: 10.3390/metabo6020012.

Accurate Measurement of the in vivo Ammonium Concentration in Saccharomyces cerevisiae.

Metabolites

Hugo F Cueto-Rojas, Reza Maleki Seifar, Angela Ten Pierick, Sef J Heijnen, Aljoscha Wahl

Affiliations

  1. Cell Systems Engineering Group, Department of Biotechnology, Delft University of Technology, 2628 BC Delft, The Netherlands. [email protected].
  2. Cell Systems Engineering Group, Department of Biotechnology, Delft University of Technology, 2628 BC Delft, The Netherlands. [email protected].
  3. Cell Systems Engineering Group, Department of Biotechnology, Delft University of Technology, 2628 BC Delft, The Netherlands. [email protected].
  4. Cell Systems Engineering Group, Department of Biotechnology, Delft University of Technology, 2628 BC Delft, The Netherlands. [email protected].
  5. Cell Systems Engineering Group, Department of Biotechnology, Delft University of Technology, 2628 BC Delft, The Netherlands. [email protected].

PMID: 27120628 PMCID: PMC4931543 DOI: 10.3390/metabo6020012

Abstract

Ammonium (NH₄⁺) is the most common N-source for yeast fermentations, and N-limitation is frequently applied to reduce growth and increase product yields. While there is significant molecular knowledge on NH₄⁺ transport and assimilation, there have been few attempts to measure the in vivo concentration of this metabolite. In this article, we present a sensitive and accurate analytical method to quantify the in vivo intracellular ammonium concentration in Saccharomyces cerevisiae based on standard rapid sampling and metabolomics techniques. The method validation experiments required the development of a proper sample processing protocol to minimize ammonium production/consumption during biomass extraction by assessing the impact of amino acid degradation-an element that is often overlooked. The resulting cold chloroform metabolite extraction method, together with quantification using ultra high performance liquid chromatography-isotope dilution mass spectrometry (UHPLC-IDMS), was not only more sensitive than most of the existing methods but also more accurate than methods that use electrodes, enzymatic reactions, or boiling water or boiling ethanol biomass extraction because it minimized ammonium consumption/production during sampling processing and interference from other metabolites in the quantification of intracellular ammonium. Finally, our validation experiments showed that other metabolites such as pyruvate or 2-oxoglutarate (αKG) need to be extracted with cold chloroform to avoid measurements being biased by the degradation of other metabolites (e.g., amino acids).

Keywords: UHPLC-IDMS; in vivo quantification; intracellular ammonium; metabolomics; rapid sampling

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