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Dynamic in vivo 31 P nuclear magnetic resonance study of Saccharomyces cerevisiae in glucose‐limited chemostat culture during the aerobic–anaerobic shift
Author(s) -
Gonzalez Benjamin,
de Graaf Albert,
Renaud Michel,
Sahm Hermann
Publication year - 2000
Publication title -
yeast
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.923
H-Index - 102
eISSN - 1097-0061
pISSN - 0749-503X
DOI - 10.1002/(sici)1097-0061(200004)16:6<483::aid-yea542>3.0.co;2-e
Subject(s) - chemostat , biology , biochemistry , extracellular , intracellular ph , pyruvate kinase , glycolysis , intracellular , anaerobic exercise , chemiosmosis , phosphoglycerate kinase , anaerobic glycolysis , oxidative phosphorylation , atp synthase , biophysics , metabolism , enzyme , physiology , genetics , bacteria
The purpose of this work was to analyse in vivo the influence of sudden oxygen depletion on Saccharomyces cerevisiae , grown in glucose‐limited chemostat culture, using a recently developed cyclone reactor coupled with 31 P NMR spectroscopy. Before, during and after the transition, intracellular and extracellular phosphorylated metabolites as well as the pHs in the different cellular compartments were monitored with a time resolution of 2.5 min. The employed integrated NMR bioreactor system allowed the defined glucose‐limited continuous cultivation of yeast at a density of 75 g DW/l and a p O 2of 30% air saturation. A purely oxidative metabolism was maintained at all times. In vivo 31 P NMR spectra obtained were of excellent quality and even allowed the detection of phosphoenolpyruvate (PEP). During the switch from aerobic to anaerobic conditions, a rapid, significant decrease of intracellular ATP and PEP levels was observed and the cytoplasmic pH decreased from 7.5 to 6.8. This change, which was accompanied by a transient influx of extracellular inorganic phosphate (P i ), appeared to correlate linearly with the decrease of the ATP concentration, suggesting that the cause of the partial collapse of the plasma membrane pH gradient was a reduced availability of ATP. The complete phosphorous balance established from our measurement data showed that polyphosphate was not the source of the increased intracellular P i . The derived intracellular P i , ATP and ADP concentration data confirmed that the glycolytic flux at the level of glyceraldehyde‐3‐phosphate dehydrogenase, 3‐phosphoglycerate kinase and enolase enzymes is mainly controlled by thermodynamic constraints. Copyright © 2000 John Wiley & Sons, Ltd.