Activity of the α-glucoside transporter Agt1 in Saccharomyces cerevisiae cells during dehydration-rehydration events.

Microbial cells can enter a state of anhydrobiosis under desiccating conditions. One of the main determinants of viability during dehydration-rehydration cycles is structural integrity of the plasma membrane. Whereas much is known about phase transitions of the lipid bilayer, there is a paucity of information on changes in activity of plasma membrane proteins during dehydration-rehydration events. We selected the α-glucoside transporter Agt1 to gain insights into stress mechanisms/responses and ecophysiology during anhydrobiosis. As intracellular water content of S. cerevisiae strain 14 (a strain with moderate tolerance to dehydration-rehydration) was reduced to 1.5 g water/g dry weight, the activity of the Agt1 transporter decreased by 10-15 %. This indicates that functionality of this trans-membrane and relatively hydrophobic protein depends on water. Notably, however, levels of cell viability were retained. Prior incubation in the stress protectant xylitol increased stability of the plasma membrane but not Agt1. Studies were carried out using a comparator yeast which was highly resistant to dehydration-rehydration (S. cerevisiae strain 77). By contrast to S. cerevisiae strain 14, there was no significant reduction of Agt1 activity in S. cerevisiae strain 77 cells. These findings have implications for the ecophysiology of S. cerevisiae strains in natural and industrial systems.

Anhydrobiosis in yeast: cell wall mannoproteins are important for yeast Saccharomyces cerevisiae resistance to dehydration.

The state of anhydrobiosis is linked with the reversible delay of metabolism as a result of strong dehydration of cells, and is widely distributed in nature. A number of factors responsible for the maintenance of organisms' viability in these conditions have been revealed. This study was directed to understanding how changes in cell wall structure may influence the resistance of yeasts to dehydration-rehydration. Mutants lacking various cell wall mannoproteins were tested to address this issue. It was revealed that mutants lacking proteins belonging to two structurally and functionally unrelated groups (proteins non-covalently attached to the cell wall, and Pir proteins) possessed significantly lower cell resistance to dehydration-rehydration than the mother wild-type strain. At the same time, the absence of the GPI-anchored cell wall protein Ccw12 unexpectedly resulted in an increase of cell resistance to this treatment; this phenomenon is explained by the compensatory synthesis of chitin. The results clearly indicate that the cell wall structure/composition relates to parameters strongly influencing yeast viability during the processes of dehydration-rehydration, and that damage to cell wall proteins during yeast desiccation can be an important factor leading to cell death. Copyright © 2016 John Wiley & Sons, Ltd.

The role of glycerol transporters in yeast cells in various physiological and stress conditions.

Small and uncharged glycerol is an important molecule for yeast metabolism and osmoadaptation. Using a series of S. cerevisiae BY4741-derived mutants lacking genes encoding a glycerol exporter (Fps1p) and/or importer (Stl1p) and/or the last kinase of the HOG pathway (Hog1p), we studied their phenotypes and various physiological characteristics with the aim of finding new roles for glycerol transporters. Though the triple mutant hog1Δ stl1Δ fps1Δ was viable, it was highly sensitive to various stresses. Our results showed that the function of both Stl1p and Fps1p transporters contributes to the cell ability to survive during the transfer into the state of anhydrobiosis, and that the deletion of FPS1 decreases the cell's tolerance of hyperosmotic stress. The deletion of STL1 results in a slight increase in cell size and in a substantial increase in intracellular pH. Taken together, our results suggest that the fluxes of glycerol in both directions across the plasma membrane exist in yeast cells simultaneously, and the export or import predominates according to the actual specific conditions.

Immobilisation increases yeast cells' resistance to dehydration-rehydration treatment.

This study was performed with the goal of revealing if the dehydration procedure used in our new immobilisation method noticeably decreases the viability of yeast cells in immobilised preparations. Various yeasts were used in this research: Saccharomyces cerevisiae cells that were rather sensitive to dehydration and had been aerobically grown in an ethanol-containing medium, a recombinant strain of S. cerevisiae grown in aerobic conditions which were completely non-resistant to dehydration and an anaerobically grown bakers' yeast strain S. cerevisiae, as well as a fairly resistant Pichia pastoris strain. Experiments performed showed that immobilisation of all these strains essentially increased their resistance to a dehydration-rehydration treatment. The increase of cells' viability (compared with control cells dehydrated in similar conditions) was from 30 to 60%. It is concluded that a new immobilisation method, which includes a dehydration stage, does not lead to an essential loss of yeast cell viability. Correspondingly, there is no risk of losing the biotechnological activities of immobilised preparations. The possibility of producing dry, active yeast preparations is shown, for those strains that are very sensitive to dehydration and which can be used in biotechnology in an immobilised form. Finally, the immobilisation approach can be used for the development of efficient methods for the storage of recombinant yeast strains.

Effects of yeast immobilization on bioethanol production.

The current study evaluated a newer method, which includes a dehydration step, of immobilizing Saccharomyces cerevisiae L-77 and S. cerevisiae L-73 onto hydroxylapatite and chamotte ceramic supports. The efficiency of cell immobilization on chamotte was significantly higher than hydroxylapatite. Immobilized yeast preparations were investigated for their ethanol-producing capabilities. The glucose concentration in a fermentation medium was 100 mg/mL. Immobilized preparations produced the same amount of ethanol (48 ± 0.5 mg/mL) as free cells after 36 H of fermentation. During the early stages of fermentation, immobilized yeast cells produced ethanol at a higher rate than free cells. Yeast preparations immobilized on both supports (hydroxylapatite and chamotte) were successfully used in six sequential batch fermentations without any loss of activity. The chamotte support was more stable in the fermentation medium during these six cycles of ethanol production. In addition to the high level of ethanol produced by cells immobilized on chamotte, the stability of this support and its low cost make it a promising material for biotechnologies associated with ethanol production.

Potassium uptake system Trk2 is crucial for yeast cell viability during anhydrobiosis.

Yeasts grow at very different potassium concentrations, adapting their intracellular cation levels to changes in the external environment. Potassium homeostasis is maintained with the help of several transporters mediating the uptake and efflux of potassium with various affinities and mechanisms. In the model yeast Saccharomyces cerevisiae, two uptake systems, Trk1 and Trk2, are responsible for the accumulation of a relatively high intracellular potassium content (200-300 mM) and the efflux of surplus potassium is mediated by the Tok1 channel and active exporters Ena ATPase and Nha1 cation/proton antiporter. Using a series of deletion mutants, we studied the role of individual potassium transporters in yeast cell resistance to dehydration. The Trk2 transporter (whose role in S. cerevisiae physiology was not clear) is important for cell viability in the stationary phase of growth and, moreover, it plays a crucial role in the yeast survival of dehydration/rehydration treatments. Mutants lacking the TRK2 gene accumulated significantly lower amounts of potassium ions in the stationary culture growth phase, and these lower amounts correlated with decreased resistance to dehydration/rehydration stress. Our results showed Trk2 to be the major potassium uptake system in stationary cells, and potassium content to be a crucial parameter for desiccation survival.