Measurement of fast dynamic intracellular pH in <i>Saccharomyces cerevisiae</i> using benzoic acid pulse

Kresnowati, Made Tri Ari Penia; Suarez-Mendez, Camilo A.; Groothuizen, M.K.; Winden, Wouter A. van; Heijnen, Joseph J.

doi:10.1002/bit.21179

Cited by 22 publications

(28 citation statements)

References 41 publications

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“…Because the intracellular pH is close to neutral (19,20), virtually all fumaric acid inside the cells is present as the double-charged form, while the concentration of undissociated acid is negligible. Hence, at a low extracellular pH, the driving force is always from outside the cells to inside and not reversible.…”

Section: Resultsmentioning

confidence: 99%

pH-Dependent Uptake of Fumaric Acid in Saccharomyces cerevisiae under Anaerobic Conditions

Jamalzadeh

Verheijen

Heijnen

et al. 2012

Appl Environ Microbiol

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Microbial production of C 4 dicarboxylic acids from renewable resources has gained renewed interest. The yeast Saccharomyces cerevisiae is known as a robust microorganism and is able to grow at low pH, which makes it a suitable candidate for biological production of organic acids. However, a successful metabolic engineering approach for overproduction of organic acids requires an incorporation of a proper exporter to increase the productivity. Moreover, low-pH fermentations, which are desirable for facilitating the downstream processing, may cause back diffusion of the undissociated acid into the cells with simultaneous active export, thereby creating an ATP-dissipating futile cycle. In this work, we have studied the uptake of fumaric acid in S. cerevisiae in carbon-limited chemostat cultures under anaerobic conditions. The effect of the presence of fumaric acid at different pH values (3 to 5) has been investigated in order to obtain more knowledge about possible uptake mechanisms. The experimental results showed that at a cultivation pH of 5.0 and an external fumaric acid concentration of approximately 0.8 mmol · liter ؊1 , the fumaric acid uptake rate was unexpectedly high and could not be explained by diffusion of the undissociated form across the plasma membrane alone. This could indicate the presence of protein-mediated import. At decreasing pH levels, the fumaric acid uptake rate was found to increase asymptotically to a maximum level. Although this observation is in accordance with proteinmediated import, the presence of a metabolic bottleneck for fumaric acid conversion under anaerobic conditions could not be excluded.

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Section: Resultsmentioning

confidence: 99%

pH-Dependent Uptake of Fumaric Acid in Saccharomyces cerevisiae under Anaerobic Conditions

Jamalzadeh

Verheijen

Heijnen

et al. 2012

Appl Environ Microbiol

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“…The well-documented ability of S. cerevisiae to rapidly increase its glycolytic flux (47, 50) without a need for enzyme synthesis (48) allows fast adaptation to environmental conditions that affect central carbon metabolism, such as weak acid stress (20) and temperature changes (39). In the latter case, it has recently been proposed that the apparent "overcapacity" of glycolysis that is often observed when S. cerevisiae is grown at standard laboratory growth temperatures may reflect an evolutionary adaptation to diurnal temperature cycling in the natural environments of this yeast (39).…”

Section: Discussionmentioning

confidence: 99%

“…Therefore, translation of the highly abundant glycolytic proteins may be strongly affected by the cellular energy status. The experimental system used in this study is well suited for studying the relationship between ATP status and induction of glycolytic proteins, e.g., by additional studies under aerobic conditions or by increasing the ATP demand by adding a weak acid, such as benzoate (8,20,53).…”

Section: Discussionmentioning

confidence: 99%

Dynamics of Glycolytic Regulation during Adaptation of Saccharomyces cerevisiae to Fermentative Metabolism

Brink

Canelas

Gulik

et al. 2008

Appl Environ Microbiol

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The ability of baker's yeast (Saccharomyces cerevisiae) to rapidly increase its glycolytic flux upon a switch from respiratory to fermentative sugar metabolism is an important characteristic for many of its multiple industrial applications. An increased glycolytic flux can be achieved by an increase in the glycolytic enzyme capacities (V max ) and/or by changes in the concentrations of low-molecular-weight substrates, products, and effectors. The goal of the present study was to understand the time-dependent, multilevel regulation of glycolytic enzymes during a switch from fully respiratory conditions to fully fermentative conditions. The switch from glucose-limited aerobic chemostat growth to full anaerobiosis and glucose excess resulted in rapid acceleration of fermentative metabolism. Although the capacities (V max ) of the glycolytic enzymes did not change until 45 min after the switch, the intracellular levels of several substrates, products, and effectors involved in the regulation of glycolysis did change substantially during the initial 45 min (e.g., there was a buildup of the phosphofructokinase activator fructose-2,6-bisphosphate). This study revealed two distinct phases in the upregulation of glycolysis upon a switch to fermentative conditions: (i) an initial phase, in which regulation occurs completely through changes in metabolite levels; and (ii) a second phase, in which regulation is achieved through a combination of changes in V max and metabolite concentrations. This multilevel regulation study qualitatively explains the increase in flux through the glycolytic enzymes upon a switch of S. cerevisiae to fermentative conditions and provides a better understanding of the roles of different regulatory mechanisms that influence the dynamics of yeast glycolysis.Baker's yeast (Saccharomyces cerevisiae) can rapidly switch between respiratory and fermentative sugar metabolism in response to changes in the availability of oxygen and fermentable sugars. This metabolic flexibility is likely to have arisen during evolution in environments where there are strong temporal and/or spatial fluctuations in sugar and oxygen levels. Upon transfer from respiratory to fermentative conditions, S. cerevisiae can, within a short time, tremendously increase its catabolic rates and start accumulating ethanol, as well as smaller amounts of acetate, succinate, and glycerol (50). In the natural, evolutionary context, this may have helped this organism rapidly monopolize sugars and create a hostile environment for competing microorganisms. This metabolic flexibility of S. cerevisiae is also an important characteristic for its multiple industrial applications. For instance, yeast biomass starved for glucose during storage has to rapidly adapt to a very high sugar concentration when it is added to bread dough or wort. Ideally, S. cerevisiae cells should be able to increase their CO 2 and ethanol production rates within minutes following their introduction into a fresh production medium. Upon such a transfer, the pathways responsi...

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“…As yeast cells are rather small, and thus inappropriate for the direct measurement of intracellular pH (pH in ) by microelectrodes, several indirect methods have been developed: the redistribution of radioactively labelled weak acids or bases (Rottenberg, 1979), 31 P nuclear magnetic resonance (NMR) (Gillies et al, 1981), calculation from the changes in extracellular pH after the addition of a weak acid (Kresnowati et al, 2007), loading cells with pyranine by electroporation (Pena et al, 1995) and several other fluorescence techniques that offer continuous and fast pH in measurements (Breeuwer and Abee, 2000;Lanz et al, 1999;Sureshkumar and Mutharasan, 1994).…”

Section: Introductionmentioning

confidence: 99%

New applications of pHluorin—measuring intracellular pH of prototrophic yeasts and determining changes in the buffering capacity of strains with affected potassium homeostasis

Marešová

Hošková

Urbánková

et al. 2010

Yeast

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AbstractpHluorin is a pH-sensitive variant of green fluorescent protein for measuring intracellular pH (pH in ) in living cells. We constructed a new pHluorin plasmid with the dominant selection marker KanMX. This plasmid allows pH measurements in cells without auxotrophic mutations and/or grown in chemically indefinite media. We observed differing values of pH in for three prototrophic wild-types. The new construct was also used to determine the pH in in strains differing in the activity of the plasma membrane Pma1 H + -ATPase and the influence of glucose on pH in . We describe in detail pHluorin measurements performed in a microplate reader, which require much less hands-on time and much lower cell culture volumes compared to standard cuvettes measurements. We also utilized pHluorin in a new method of measuring the buffering capacity of yeast cell cytosol in vivo, shown to be ca. 52 mM/pH for wild-type yeast and moderately decreased in mutants with affected potassium transport.

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Measurement of fast dynamic intracellular pH in Saccharomyces cerevisiae using benzoic acid pulse

Cited by 22 publications

References 41 publications

pH-Dependent Uptake of Fumaric Acid in Saccharomyces cerevisiae under Anaerobic Conditions

pH-Dependent Uptake of Fumaric Acid in Saccharomyces cerevisiae under Anaerobic Conditions

Dynamics of Glycolytic Regulation during Adaptation of Saccharomyces cerevisiae to Fermentative Metabolism

New applications of pHluorin—measuring intracellular pH of prototrophic yeasts and determining changes in the buffering capacity of strains with affected potassium homeostasis

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