Determination of the in vivo NAD:NADH ratio in Saccharomyces cerevisiae under anaerobic conditions, using alcohol dehydrogenase as sensor reaction

With the current quantitative metabolomics techniques, only whole‐cell concentrations of NAD and NADH can be quantified. These measurements cannot provide information on the in vivo redox state of the cells, which is determined by the ratio of the free forms only. In this work we quantified free NAD:NADH ratios in yeast under anaerobic conditions, using alcohol dehydrogenase (ADH) and the lumped reaction of glyceraldehyde‐3‐phosphate dehydrogenase and 3‐phosphoglycerate kinase as sensor reactions. We showed that, with an alternative accurate acetaldehyde determination method, based on rapid sampling, instantaneous derivatization with 2,4 diaminophenol hydrazine (DNPH) and quantification with HPLC, the ADH‐catalysed oxidation of ethanol to acetaldehyde can be applied as a relatively fast and simple sensor reaction to quantify the free NAD:NADH ratio under anaerobic conditions. We evaluated the applicability of ADH as a sensor reaction in the yeast Saccharomyces cerevisiae, grown in anaerobic glucose‐limited chemostats under steady‐state and dynamic conditions. The results found in this study showed that the cytosolic redox status (NAD:NADH ratio) of yeast is at least one order of magnitude lower, and is thus much more reduced, under anaerobic conditions compared to aerobic glucose‐limited steady‐state conditions. The more reduced state of the cytosol under anaerobic conditions has major implications for (central) metabolism. Accurate determination of the free NAD:NADH ratio is therefore of importance for the unravelling of in vivo enzyme kinetics and to judge accurately the thermodynamic reversibility of each redox reaction. Copyright © 2015 John Wiley & Sons, Ltd.

[1]  Christopher J. R. Loewen,et al.  Faculty Opinions recommendation of Genome-wide analysis of intracellular pH reveals quantitative control of cell division rate by pH(c) in Saccharomyces cerevisiae. , 2016 .

[2]  J. Winther,et al.  Non-invasive In-cell Determination of Free Cytosolic [NAD+]/[NADH] Ratios Using Hyperpolarized Glucose Show Large Variations in Metabolic Phenotypes* , 2013, The Journal of Biological Chemistry.

[3]  Franco J. Vizeacoumar,et al.  Genome-wide analysis of intracellular pH reveals quantitative control of cell division rate by pHc in Saccharomyces cerevisiae , 2012, Genome Biology.

[4]  Enrico Gratton,et al.  NADH distribution in live progenitor stem cells by phasor-fluorescence lifetime image microscopy. , 2012, Biophysical journal.

[5]  Jens Nielsen,et al.  De novo sequencing, assembly and analysis of the genome of the laboratory strain Saccharomyces cerevisiae CEN.PK113-7D, a model for modern industrial biotechnology , 2012, Microbial Cell Factories.

[6]  X. Guan,et al.  An optimized method for the measurement of acetaldehyde by high-performance liquid chromatography. , 2012, Alcoholism, clinical and experimental research.

[7]  J. Heijnen,et al.  An in vivo data-driven framework for classification and quantification of enzyme kinetics and determination of apparent thermodynamic data. , 2011, Metabolic engineering.

[8]  J. Heijnen,et al.  Intracellular metabolite determination in the presence of extracellular abundance: Application to the penicillin biosynthesis pathway in Penicillium chrysogenum , 2010, Biotechnology and bioengineering.

[9]  D. Peričin,et al.  Binding of coenzymes to yeast alcohol dehydrogenase , 2010 .

[10]  J. Heijnen,et al.  A comprehensive method for the quantification of the non-oxidative pentose phosphate pathway intermediates in Saccharomyces cerevisiae by GC-IDMS. , 2009, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences.

[11]  J. Pronk,et al.  A three‐way proteomics strategy allows differential analysis of yeast mitochondrial membrane protein complexes under anaerobic and aerobic conditions , 2009, Proteomics.

[12]  J. Heijnen,et al.  Quantitative evaluation of intracellular metabolite extraction techniques for yeast metabolomics. , 2009, Analytical chemistry.

[13]  Peter J.T. Verheijen,et al.  Data Reconciliation and Error Detection , 2009 .

[14]  J. Heijnen,et al.  Simultaneous quantification of free nucleotides in complex biological samples using ion pair reversed phase liquid chromatography isotope dilution tandem mass spectrometry. , 2009, Analytical biochemistry.

[15]  Jim Berg,et al.  A genetically encoded fluorescent reporter of ATP/ADP ratio , 2008, Nature Methods.

[16]  J. Albertyn,et al.  The alcohol dehydrogenases of Saccharomyces cerevisiae: a comprehensive review. , 2008, FEMS yeast research.

[17]  J. Heijnen,et al.  Determination of the cytosolic free NAD/NADH ratio in Saccharomyces cerevisiae under steady‐state and highly dynamic conditions , 2008, Biotechnology and bioengineering.

[18]  Jean-Marc Daran,et al.  Control of the Glycolytic Flux in Saccharomyces cerevisiae Grown at Low Temperature , 2007, Journal of Biological Chemistry.

[19]  K. Entian,et al.  25 Yeast Genetic Strain and Plasmid Collections , 2007 .

[20]  R. Veech,et al.  The determination of the redox states and phosphorylation potential in living tissues and their relationship to metabolic control of disease phenotypes , 2006, Biochemistry and molecular biology education : a bimonthly publication of the International Union of Biochemistry and Molecular Biology.

[21]  J. Heijnen,et al.  Quantitative analysis of the microbial metabolome by isotope dilution mass spectrometry using uniformly 13C-labeled cell extracts as internal standards. , 2005, Analytical biochemistry.

[22]  Robert N. Goldberg,et al.  Thermodynamics of enzyme-catalyzed reactions - a database for quantitative biochemistry , 2004, Bioinform..

[23]  U. Theobald,et al.  Dynamics of orthophosphate in yeast cytoplasm , 1996, Biotechnology Letters.

[24]  G. Stanley,et al.  Inhibition and stimulation of yeast growth by acetaldehyde , 1993, Biotechnology Letters.

[25]  J. Heijnen,et al.  Critical evaluation of sampling techniques for residual glucose determination in carbon‐limited chemostat culture of Saccharomyces cerevisiae , 2003, Biotechnology and bioengineering.

[26]  X. Parés,et al.  Properties and functional significance of Saccharomyces cerevisiae ADHVI. , 2003, Chemico-biological interactions.

[27]  X. Parés,et al.  Characterization of a Saccharomyces cerevisiae NADP(H)-dependent alcohol dehydrogenase (ADHVII), a member of the cinnamyl alcohol dehydrogenase family. , 2002, European journal of biochemistry.

[28]  X. Parés,et al.  Characterization of the Saccharomyces cerevisiae YMR318C (ADH6) gene product as a broad specificity NADPH-dependent alcohol dehydrogenase: relevance in aldehyde reduction. , 2002, The Biochemical journal.

[29]  J J Heijnen,et al.  Improved rapid sampling for in vivo kinetics of intracellular metabolites in Saccharomyces cerevisiae. , 2001, Biotechnology and bioengineering.

[30]  Barbara M. Bakker,et al.  The Mitochondrial Alcohol Dehydrogenase Adh3p Is Involved in a Redox Shuttle in Saccharomyces cerevisiae , 2000, Journal of bacteriology.

[31]  E. Conte,et al.  Sample preparation for the analysis of flavors and off-flavors in foods. , 2000, Journal of chromatography. A.

[32]  Pronk,et al.  Regulation of fermentative capacity and levels of glycolytic enzymes in chemostat cultures of Saccharomyces cerevisiae. , 2000, Enzyme and microbial technology.

[33]  A. D. de Graaf,et al.  Dynamic in vivo 31P nuclear magnetic resonance study of Saccharomyces cerevisiae in glucose‐limited chemostat culture during the aerobic–anaerobic shift , 2000, Yeast.

[34]  U. Karst,et al.  Hydrazine reagents as derivatizing agents in environmental analysis – a critical review , 2000, Fresenius' journal of analytical chemistry.

[35]  R. Tsien,et al.  green fluorescent protein , 2020, Catalysis from A to Z.

[36]  M. Reuss,et al.  In vivo analysis of metabolic dynamics in Saccharomyces cerevisiae : I. Experimental observations. , 1997, Biotechnology and bioengineering.

[37]  N. Pamment,et al.  Liquid injection gas chromatography and the ALDH assay overestimate free acetaldehyde in complex fermentation media , 1997 .

[38]  M. Chalfie.  GREEN FLUORESCENT PROTEIN , 1995, Photochemistry and photobiology.

[39]  V. Novelli,et al.  Liquid chromatographic determination of 2-furaldehyde and 5-hydroxymethyl-2-furaldehyde in beer☆ , 1995 .

[40]  W. A. Scheffers,et al.  Physiology of Saccharomyces cerevisiae in anaerobic glucose-limited chemostat cultures. , 1990, Journal of general microbiology.

[41]  D. Pilgrim,et al.  Isolation and DNA sequence of ADH3, a nuclear gene encoding the mitochondrial isozyme of alcohol dehydrogenase in Saccharomyces cerevisiae , 1985, Molecular and cellular biology.

[42]  J. Delcour,et al.  AN ENZYMATIC ASSAY FOR THE DETERMINATION OF ACETALDEHYDE IN BEERS , 1982 .

[43]  C. Bernofsky,et al.  Mitochondrial alcohol dehydrogenase from Saccharomyces cerevisiae. , 1971, Biochimica et biophysica acta.

[44]  H. Krebs,et al.  The redox state of free nicotinamide-adenine dinucleotide phosphate in the cytoplasm of rat liver. , 1969, The Biochemical journal.

[45]  H. Krebs,et al.  The redox state of free nicotinamide-adenine dinucleotide in the cytoplasm and mitochondria of rat liver. , 1967, The Biochemical journal.

[46]  EDWIN C. Webb.  The Enzymes , 1961, Nature.

[47]  Britton Chance,et al.  Studies on Liver Alcohol Dehydrogenase. II. The Kinetics of the Compound of Horse Liver Alcohol Dehydrogenase and Reduced Diphosphopyridine Nucleotide. , 1951 .