Physiological and molecular analysis of the stress response of Saccharomyces cerevisiae imposed by strong inorganic acid with implication to industrial fermentations.

AIMS: This work aimed to identify the molecular mechanism that allows yeast cells to survive at low pH environments such as those of bioethanol fermentation. METHODS AND RESULTS: The industrial strain JP1 cells grown at pH 2 was evaluated by microarray analysis showing that most of the genes induced at low pH were part of the general stress response (GSR). Further, an acid-tolerant yeast mutant was isolated by adaptive selection that was prone to grow at low pH in inorganic but weak organic acid. It showed higher viability under acid-temperature synergistic treatment. However, it was deficient in some physiological aspects that are associated with defects in protein kinase A (PKA) pathway. Microarray analysis showed the induction of genes involved in inhibition of RNA and protein synthesis. CONCLUSIONS: The results point out that low pH activates GSR, mainly heat shock response, that is important for long-term cell survival and suggest that a fine regulatory PKA-dependent mechanism that might affect cell cycle in order to acquire tolerance to acid environment. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings might guide the construction of a high-fermentative stress-tolerant industrial yeast strain that can be used in complex industrial fermentation processes.

Effects of temperature shifts on the metabolism of trehalose in Neurospora crassa wild type and a trehalase-deficient (tre) mutant. Evidence against the participation of periplasmic trehalase in the catabolism of intracellular trehalose.

The effects of temperature shifts on the metabolism of trehalose in Neurospora crassa were studied in conidiospore germlings of a wild type strain, and of a mutant (tre), deficient in the activity of periplasmic trehalase. When the temperature of the medium was raised from 30 degrees C to 45 degrees C both strains accumulated trehalose, either in media supplemented with glucose or with glycerol as carbon sources. The profiles of glycolysis metabolites suggested that at 45 degrees C glycolysis was inhibited at the level of the phosphofructokinase-1 reaction, while that of fructose-1,6-bisphosphatase was active, thus explaining how the flux of carbon from glucose or glycerol was channeled to trehalose synthesis at that temperature. This assumption was also supported by the changes in levels of fructose-2,6-bisphosphate, which dropped during the incubation at 45 degrees C. The opposite phenomena were observed when the cultures were reincubated at 30 degrees C and glycolysis was strongly activated. Surprisingly, the intracellular pool of trehalose of the mutant decreased after reincubation at 30 degrees C at the same rate observed for the wild type (about 25.0 nmol/min per mg protein) despite its low trehalase activity (about 5.0 nmol/min per mg protein). Labeling experiments using [U-14C]-glucose demonstrated that both the wild type and the mutant metabolized internally the trehalose pool, without detectable leakage of glucose or trehalose into the external medium. Cells submitted to heat shock in glycerol-supplemented medium and resuspended at 30 degrees in the absence of an exogenous carbon source and in the presence of the glycolysis inhibitor 2-deoxyglucose accumulated high levels of free intracellular glucose, indicating that trehalose was hydrolysed internally. This suggested the existence of a cytosolic regulatory trehalase in Neurospora crassa, but all efforts to detect such activity in cell extracts have been unsuccessful so far. Altogether, these results argued against the participation of the periplasmic trehalase of N. crassa in the catabolism of intracellular trehalose. They are also conflictant with the enzyme/substrate decompartmentation hypothesis, earlier suggested as a way of explaining the mobilization of endogenous trehalose reserves accumulated in fungal spores (reviewed in Thevelein 1984, Microbiol. Rev. 48, 42-59).

Effects of temperature shifts on the activities of Neurospora crassa glycogen synthase, glycogen phosphorylase and trehalose-6-phosphate synthase.

Conidiospore germlings of Neurospora crassa submitted to a heat shock at 45 degrees C accumulate trehalose and degrade glycogen. The opposite occurs upon reincubation at a physiologic temperature (30 degrees C). These observations suggest a temperature-dependent mechanism for the preferential synthesis of one or the other sugar reserve. Here we show that concomitant with these shifts of temperature, occurred reversible changes in the activities of glycogen synthase and phosphorylase. Glycogen synthase was inactivated at 45 degrees C while phosphorylase was activated. The reverse was true when the cells were shifted back to 30 degrees C. Addition of cycloheximide did not prevent the reversible enzymatic changes, which remained stable after gel filtration. Apparently, the effects of temperature shifts occurred at the level of reversible covalent enzymatic modifications. Trehalose-6-phosphate synthase properties were also affected by temperature. For instance, the enzyme was less sensitive to in vitro inhibition by inorganic phosphate at 50 degrees C than at 30 degrees C. Fructose-6-phosphate partially relieved the inhibitory effect of phosphate at 30 degrees C but not at 50 degrees C. These effects of the assay temperature, inorganic phosphate, and fructose-6-phosphate, on trehalose-6-phosphate synthase activity, were more evident for crude extracts obtained from heat-shocked cells. Altogether, these results may contribute to explain the preferential accumulation of trehalose 45 degrees C, or that of glycogen at 30 degrees C.

Neutral trehalases catalyse intracellular trehalose breakdown in the filamentous fungi Aspergillus nidulans and Neurospora crassa.

A cAMP-activatable Ca2+-dependent neutral trehalase was identified in germinating conidia of Aspergillus nidulans and Neurospora crassa. Using a PCR approach, A. nidulans and N. crassa genes encoding homologues of the neutral trehalases found in several yeasts were cloned and sequenced. Disruption of the AntreB gene encoding A. nidulans neutral trehalase revealed that it is responsible for intracellular trehalose mobilization at the onset of conidial germination, and that this phenomenon is partially involved in the transient accumulation of glycerol in the germinating conidia. Although trehalose mobilization is not essential for the completion of spore germination and filamentous growth in A. nidulans, it is required to achieve wild-type germination rates under carbon limitation, suggesting that intracellular trehalose can partially contribute the energy requirements of spore germination. Furthermore, it was shown that trehalose accumulation in A. nidulans can protect germinating conidia against an otherwise lethal heat shock. Because transcription of the treB genes is not increased after a heat shock but induced upon heat shock recovery, it is proposed that, in filamentous fungi, mobilization of trehalose during the return to appropriate growth is promoted by transcriptional and post-translational regulatory mechanisms, in particular cAMP-dependent protein kinase-mediated phosphorylation.

Expression of escherichia coli otsA in a Saccharomyces cerevisiae tps1 mutant restores trehalose 6-phosphate levels and partly restores growth and fermentation with glucose and control of glucose influx into glycolysis.

The TPS1 gene, encoding trehalose-6-phosphate synthase (TPS), exerts an essential control on the influx of glucose into glycolysis in the yeast Saccharomyces cerevisiae. The deletion of TPS1 causes an inability to grow on glucose because of a hyperaccumulation of sugar phosphates and depletion of ATP and phosphate. We show that expression of the Escherichia coli homologue, otsA, in a yeast tps1 mutant results in high TPS activity. Although the trehalose 6-phosphate (Tre6P) level during exponential growth on glucose was at least as high as in a wild-type yeast strain, growth on glucose was only partly restored and the lag phase was much longer. Measurement of the glycolytic metabolites immediately after the addition of glucose showed that in spite of a normal Tre6P accumulation there was still a partial hyperaccumulation of sugar phosphates. Strong elevation of the Tre6P level by the additional deletion of the TPS2 gene, which encodes Tre6P phosphatase, was not able to cause a strong decrease in the sugar phosphate levels in comparison with the wild-type strain. In addition, in chemostat experiments the short-term response to a glucose pulse was delayed, but normal metabolism was regained over a longer period. These results show that Tre6P synthesis from a heterologous TPS enzyme can to some extent restore the control of glucose influx into glycolysis and growth on glucose in yeast. However, they also indicate that the yeast TPS enzyme, as opposed to the E. coli otsA gene product, is able to increase the efficiency of the Tre6P control on glucose influx into yeast glycolysis.