Abstract
Methanol quenching and fast filtration, the two most common sampling protocols in microbial metabolome analysis, were validated for intracellular amino acid analysis in phylogenetically different yeast strains comprising Saccharomyces cerevisiae, Kluyveromyces marxianus, Pichia pastoris, Schizosaccharomyces pombe and Zygosaccharomyces bailii. With only few exceptions for selected amino acids, all yeasts exhibited negligible metabolite leakage during quenching with 60% cold buffered methanol. Slightly higher leakage was observed with increasing methanol content in the quenching solution. Fast filtration resulted in identical levels for intracellular amino acids in all strains tested. The results clearly demonstrate the validity of both approaches for leakage-free sampling of amino acids in yeast.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Bolten CJ, Kiefer P, Letisse F, Portais JC, Wittmann C (2007) Sampling for metabolome analysis of microorganisms. Anal Chem 79:3843–3849
de Koning W, van Dam K (1992) A method for the determination of changes of glycolytic metabolites in yeast on a subsecond time scale using extraction at neutral pH. Anal Biochem 204:118–123
Gonzalez B, Francois J, Renaud M (1997) A rapid and reliable method for metabolite extraction in yeast using boiling buffered ethanol. Yeast 13:1347–1355
Hans MA, Heinzle E, Wittmann C (2001) Quantification of intracellular amino acids in batch cultures of Saccharomyces cerevisiae. Appl Microbiol Biotechnol 56:776–779
Hollemeyer K, Velagapudi VR, Wittmann C, Heinzle E (2007) Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for metabolic flux analyses using isotope-labeled ethanol. Rapid Commun Mass Spectrom 21:336–342
Jensen NB, Jokumsen KV, Villadsen J (1999) Determination of the phosphorylated sugars of the Embden–Meyerhoff–Parnas pathway in Lactococcus lactis using a fast sampling technique and solid phase extraction. Biotechnol Bioeng 63:356–362
Kiefer P, Heinzle E, Zelder O, Wittmann C (2004) Comparative metabolic flux analysis of lysine-producing Corynebacterium glutamicum cultured on glucose or fructose. Appl Environ Microbiol 70:229–239
Lange HC, Heijnen JJ (2001) Statistical reconciliation of the elemental and molecular biomass composition of Saccharomyces cerevisiae. Biotechnol Bioeng 75:334–344
Lange HC, Eman M, van Zuijlen G, Visser D, van Dam JC, Frank J, de Mattos MJ, Heijnen JJ (2001) Improved rapid sampling for in vivo kinetics of intracellular metabolites in Saccharomyces cerevisiae. Biotechnol Bioeng 75:406–415
Loret MO, Pedersen L, Francois J (2007) Revised procedures for yeast metabolites extraction: application to a glucose pulse to carbon-limited yeast cultures, which reveals a transient activation of the purine salvage pathway. Yeast 24:47–60
Nasution U, van Gulik WM, Kleijn RJ, van Winden WA, Proell A, Heijnen JJ (2006) Measurement of intracellular metabolites of primary metabolism and adenine nucleotides in chemostat cultivated Penicillium chrysogenum. Biotechnol Bioeng 94:159–166
Ruijter GJG, Visser J (1996) Determination of intermediary metabolites in Aspergillus niger. J Microbiol Methods 25:295–302
Saez MJ, Lagunas R (1976) Determination of intermediary metabolites in yeast. Critical examination of the effect of sampling conditions and recommendations for obtaining true levels. Mol Cell Biochem 13:73–78
Villas-Boas SG, Bruheim P (2007) Cold glycerol-saline: the promising quenching solution for accurate intracellular metabolite analysis of microbial cells. Anal Biochem 370:87–97
Villas-Boas SG, Hojer-Pedersen J, Akesson M, Smedsgaard J, Nielsen J (2005) Global metabolite analysis of yeast: evaluation of sample preparation methods. Yeast 22:1155–1169
Wallace PG, Pedler SM, Wallace JC, Berry MN (1994) A method for the determination of the cellular phosphorylation potential and glycolytic intermediates in yeast. Anal Biochem 222:404–408
Weibel KE, Mor JR, Fiechter A (1974) Rapid sampling of yeast cells and automated assays of adenylate, citrate, pyruvate and glucose-6-phosphate pools. Anal Biochem 58:208–216
Wittmann C, Hans M, Bluemke W (2002) Metabolic physiology of aroma-producing Kluyveromyces marxianus. Yeast 19:1351–1363
Wittmann C, Krömer JO, Kiefer P, Binz T, Heinzle E (2004) Impact of the cold shock phenomenon on quantification of intracellular metabolites in bacteria. Anal Biochem 327:135–139
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Bolten, C.J., Wittmann, C. Appropriate sampling for intracellular amino acid analysis in five phylogenetically different yeasts. Biotechnol Lett 30, 1993–2000 (2008). https://doi.org/10.1007/s10529-008-9789-z
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10529-008-9789-z