Determination of flux through different metabolite pathways in Saccharomyces cerevisiae by ¹H‐NMR and ¹³C‐NMR spectroscopy

Abstract

We propose an experimental approach combining 'H-NMR and ' 3C-NMR spectroscopy to investigate metabolite flux in cells under physiological conditions and present a mathematical model giving the relationships between the following different parameters: 13C fractional enrichment, fluxes in competing pathways, metabolite concentration and experimental time. This model has been used for determining the absolute and/or relative values of five fluxes involving pyruvate, ethanol, acetyl-CoA and glutamate via the Krebs cycle in glucose-grown repressed Saccharomyces cerevisiae cells fed with [1-' 3C]glucose and/or unlabeled ethanol. The glucose consumption and the production of various compounds such as ethanol, glycerol, trehalose etc. were studied qualitatively and/or quantitatively as a function of time. The 13C fractional enrichment of [2-'3C]ethanol was determined by observing the proton resonance of the methyl group. Addition of 25 mM unlabeled ethanol shows no significant effect on the glucose consumption or the production of any metabolites. However unlabeled ethanol exerts a strong influence on the enrichment of glutamate C4, but only induces an insignificant change on glutamate C2 and C3. Apart from the fact that ethanol is a potential precursor of acetyl-CoA as expected, these results indicate that (a) the probability for citrate and 2-oxoglutarate to make one turn or more in the Krebs cycle is negligible and (b) the scrambling between C4 and C3 via the glyoxylate shunt is virtually absent.The flux of ethanol formation from pyruvate is about three-times and nine-times greater than that of ethanol consumption and acetyl-CoA formation, respectively, from pyruvate via pyruvate dehydrogenase. Without addition of unlabeled ethanol, the ratio of the integrated resonance of glutamate (C2 + C3)/C4 reflecting the activity of pyruvate carboxylase relative to that of citrate synthase, is about 1 .I. By comparing the absolute values of the different fluxes, it was found that 88% of the glucose was used to synthetize ethanol but the observed concentration of ethanol in the supernatant represents only 58% of the glucose consumption. The validity of the present model was supported by the data obtained from similar experiments using unlabeled ethanol and non-NMR techniques. Now that the investigation of cellular metabolism is in rapid progress, interest in understanding regulatory processes through measurement of the metabolite flux along the various pathways in cells is growing considerably. In recent years, thanks to the use of specifically labeled substrates, 13C-NMR was found to be a powerful tool for flux evaluation in isolated cells 11 -101, tissues [ll-151 and perfused organs [16-241 even though the I3C nucleus is much less sensitive than 'H and 31P. Many studies [I, 2, 9-11, 14, 16, 18-23] have been devoted to the flow of pyruvate and acetyl-CoA into the Krebs cycle frequently using the 13C fractional enrichment of some labeled compounds as experimental parameters. Chance et al.[I81 have developed a mathematical mode...

The Fate of Acetic Acid during Glucose Co-Metabolism by the Spoilage Yeast Zygosaccharomyces bailii

“…In the first spectrum, glutamate was labelled primarily on C 4 and to a lower extent on C 2 and C 3 , as expected for the metabolism of [2- 13 C]acetyl-CoA through the Krebs cycle. Furthermore, the last spectrum in the sequence clearly showed the typical multiplet pattern in the glutamate resonances originated by several turns of the cycle, as previously described [22]–[24], indicating that in the presence of glucose, acetic acid was metabolised through the Krebs cycle.…”

“…Several works have been published concerning the determination in S. cerevisiae of the metabolic fluxes through the Krebs and glyoxylate cycles by 13 C-NMR [22]–[24], [29]. In these studies it was possible to assume that all acetyl-CoA entering these cycles was labelled.…”

“…The acetyl-CoA besides being produced through acetic acid (labelled) metabolism could also be produced from glucose (unlabelled) by the pyruvate dehydrogenase (PDH) complex or by the so called PDH bypass [30]. Furthermore, [2− 13 C]acetyl-CoA once entering either the Krebs or glyoxylate cycles gives rise to 13 C-glutamate [22]. The contribution of the glyoxylate cycle to the metabolism of acetyl-CoA can be ruled out since it has been shown that isocitrate lyase and malate syntethase activities under the experimental conditions used are absent [8].…”

Xylose fermentation by Saccharomyces cerevisiae

“…It is reasonable to assume that this low ATP yield together with the relatively slow xylose metabolism causes the growth arrest on xylose observed in the absence of respiration. Xylitol production from xylose in S. cerevisiae has recently been shown in X Y L 1 transformants (Tran-Dinh et al 1991). In this case however the accumulation of xylitol is due to the absence of the XDH reaction.…”

Physiological Engineering

“…With the develop ment of powerful NMR methods it is, however, possible to obtain good estimates of the in-vivo fluxes, e.g. using 13 C-labelled substrates [Tran-Dinh et al, 1991;Marz et al, 1996].…”