Saccharomyces cerevisiae tolerance to hyper-osmolarity in aerobic glucose-limited chemostat culture

Different mechanisms contribute to regulate cellular functions in order to cope with threats from physiological stress conditions. As a fundamental response to balance excess water loss and restore turgor, Saccharomyces cerevisiae subjected to increased osmolarity accelerate intracellular glycerol biosynthesis and accumulation as a compatible solute. This study assessed cellular response to sorbitol-induced osmolarity in aerobic glucose-limited chemostat culture at various levels of the glycerol flux. Cell number declined slightly without any substantial increase in dry weight and total protein contents following exposure to 1M sorbitol that lasted for 90 min. On the other hand, total glycerol levels increased over time in different yeast cultivations corresponding with enhanced glycerol-3-phosphate dehydrogenase 1 (GPD1) transcript levels, which led to a steady and intensified protein product. These results support literature evidence of accumulated intracellular glycerol regulation at different levels and further increase curiosity to understand yeast tolerance in various applications including bakery, brewery and wine making or leavening of bread underdifferent osmotic conditions.

Down-regulation of TORC2-Ypk1 signaling promotes MAPK-independent survival under hyperosmotic stress

In eukaryotes, exposure to hypertonic conditions activates a MAPK (Hog1 in Saccharomyces cerevisiae and ortholog p38 in human cells). In yeast, intracellular glycerol accumulates to counterbalance the high external osmolarity. To prevent glycerol efflux, Hog1 action impedes the function of the aquaglyceroporin Fps1, in part, by displacing channel co-activators (Rgc1/2). However, Fps1 closes upon hyperosmotic shock even in hog1∆ cells, indicating another mechanism to prevent Fps1-mediated glycerol efflux. In our prior proteome-wide screen, Fps1 was identified as a target of TORC2-dependent protein kinase Ypk1 (<xref ref-type="bibr" rid="bib30">Muir et al., 2014</xref>). We show here that Fps1 is an authentic Ypk1 substrate and that the open channel state of Fps1 requires phosphorylation by Ypk1. Moreover, hyperosmotic conditions block TORC2-dependent Ypk1-mediated Fps1 phosphorylation, causing channel closure, glycerol accumulation, and enhanced survival under hyperosmotic stress. These events are all Hog1-independent. Our findings define the underlying molecular basis of a new mechanism for responding to hypertonic conditions.

Effects of centrifugal stress on cell disruption and glycerol leakage from Dunaliella salina

Dunaliella salina accumulates large amounts of intracellular glycerol in response to the increases in salt concentration, thus is a potential source for producing fuel grade glycerol as an alternative to biodiesel-derived crude glycerol. D. salina lacks a cell wall; therefore the mode of harvesting Dunaliella cells is critical to avoid cell disruption caused by extreme engineering conditions. This study explored cell disruption and glycerol leakage of D. salina under various centrifugal stresses during cell harvesting. Results show a centrifugal g-force lower than 5000 g caused little cell disruption, while a g-force higher than 9000 g led to ~40% loss of the intact cells and glycerol yields from the recovered algal pellets. Theoretical calculations of the centrifugal stresses that could rupture Dunaliella cells were in agreement with the experimental results, indicating optimisation of centrifugation conditions is important for recovering intact cells of D. salina enriched in glycerol.

Response to high osmotic conditions and elevated temperature in Saccharomyces cerevisiae is controlled by intracellular glycerol and involves coordinate activity of MAP kinase pathways

In the yeast Saccharomyces cerevisiae, response to an increase in external osmolarity is mediated by the HOG (high osmolarity glycerol) MAP kinase pathway. HOG pathway mutant strains display osmosensitive phenotypes. Recently evidence has been obtained that the osmosensitivity of HOG pathway mutants is reduced during growth at elevated temperature (37 °C). A notable exception is the ste11ssk2ssk22 mutant, which displays hypersensitivity to osmotic stress at 37 °C. This paper reports that overexpression of FPS1 or GPD1 (encoding the glycerol transport facilitator and glycerol-3-phosphate dehydrogenase, respectively, and both affecting intracellular glycerol levels) reduces the hypersensitivity to osmotic stress of ste11ssk2ssk22 at 37 °C. Although in this particular HOG pathway mutant a correlation between suppression of the phenotype and glycerol content could be demonstrated, the absolute level of intracellular glycerol per se does not determine whether a strain is osmosensitive or not. Rather, evidence was obtained that the glycerol level may have an indirect effect, viz. by influencing signalling through the PKC (protein kinase C) MAP kinase pathway, which plays an important role in maintenance of cellular integrity. In order to validate the data obtained with a HOG pathway mutant strain for wild-type yeast cells, MAP kinase signalling under different growth conditions was examined in wild-type strains. PKC pathway signalling, which is manifest at elevated growth temperature by phosphorylation of MAP kinase Mpk1p, is rapidly lost when cells are shifted to high external osmolarity conditions. Expression of bck1-20 or overexpression of WSC3 in wild-type cells resulted in restoration of PKC signalling. Both PKC and HOG signalling, cell wall phenotypes and high osmotic stress responses in wild-type cells were found to be influenced by the growth temperature. The data taken together indicate the intricate interdependence of growth temperature, intracellular glycerol, cell wall structure and MAP kinase signalling in the hyperosmotic stress response of yeast.