The yeast Saccharomyces cerevisiae can synthesize trehalose and also use this disaccharide as a carbon source for growth. However, the molecular mechanism by which extracellular trehalose can be transported to the vacuole and degraded by the acid trehalase Ath1p is not clear. By using an adaptation of the assay of invertase on whole cells with NaF, we showed that more than 90% of the activity of Ath1p is extracellular, splitting of the disaccharide into glucose. We also found that Agt1p-mediated trehalose transport and the hydrolysis of the disaccharide by the cytosolic neutral trehalase Nth1p are coupled and represent a second, independent pathway, although there are several constraints on this alternative route. First, the AGT1/MAL11 gene is controlled by the MAL system, and Agt1p was active in neither non-maltose-fermenting nor maltose-inducible strains. Second, Agt1p rapidly lost activity during growth on trehalose, by a mechanism similar to the sugar-induced inactivation of the maltose permease. Finally, both pathways are highly pH sensitive and effective growth on trehalose occurred only when the medium was buffered at around pH 5.0. The catabolism of trehalose was purely oxidative, and since levels of Ath1p limit the glucose flux in the cells, batch cultures on trehalose may provide a useful alternative to glucose-limited chemostat cultures for investigation of metabolic responses in yeast. Trehalose [␣-D-glucopyranosyl-(1-1)-␣-D-glucopyranoside] is a nonreducing disaccharide of glucose discovered in 1832 byWiggers in a mushroom crop and later in many other fungi, plants, and insects (13). In the yeast Saccharomyces cerevisiae, trehalose can accumulate up to 15% of the cell dry mass, depending on the growth conditions, the stage of the life cycle, or environmental stress (2, 27), and for this reason it has been considered a storage carbohydrate (15,27). Moreover, the ability of this disaccharide to protect proteins and biological membranes against adverse conditions suggests that it also plays a stress-protectant role in this yeast (49,50) and probably in other organisms that produce it (14).Intracellular levels of trehalose result from a well-controlled balance between enzymatic synthesis and degradation. In the yeast S. cerevisiae, the synthesis of trehalose is catalyzed by a UDP-glucose-dependent trehalose synthase (TPS) protein complex encoded by four genes. TPS1 and TPS2 encode trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase, respectively, while TPS3 and TSL1 code for two regulatory subunits of the TPS complex (for a review, see reference 15). Hydrolysis of trehalose into glucose can be carried out by at least two enzymes: a cytosolic or neutral trehalase encoded by NTH1 (26) and a vacuolar or acid trehalase (25, 28) encoded by ATH1 (12). The yeast genome also harbors the NTH2 gene, whose product is 77% identical to Nth1p, but until now, no trehalase activity has been associated with this protein (37). The neutral trehalase Nth1p is responsible for the intracellular mobilization ...

The purpose of this study was to explore the role of glycogen and trehalose in the ability of Saccharomyces cerevisiae to respond to a sudden rise of the carbon flux. To this end, aerobic glucose-limited continuous cultures were challenged with a sudden increase of the dilution rate from 0.05 to 0.15 h(-1). Under this condition, a rapid mobilization of glycogen and trehalose was observed which coincided with a transient burst of budding and a decrease of cell biomass. Experiments carried out with mutants defective in storage carbohydrates indicated a predominant role of glycogen in the adaptation to this perturbation. However, the real importance of trehalose in this response was veiled by the unexpected phenotypes harboured by the tps1 mutant, chosen for its inability to synthesize trehalose. First, the biomass yield of this mutant was 25% lower than that of the isogenic wild-type strain at dilution rate of 0.05 h(-1), and this difference was annulled when cultures were run at a higher dilution rate of 0.15 h(-1). Second, the tps1 mutant was more effective to sustain the dilution rate shift-up, apparently because it had a faster glycolytic rate and an apparent higher capacity to consume glucose with oxidative phosphorylation than the wild type. Consequently, a tps1gsy1gsy2 mutant was able to adapt to the dilution rate shift-up after a long delay, likely because the detrimental effects from the absence of glycogen was compensated for by the tps1 mutation. Third, a glg1Deltaglg2Delta strain, defective in glycogen synthesis because of the lack of the glycogen initiation protein, recovered glycogen accumulation upon further deletion of TPS1. This recovery, however, required glycogen synthase. Finally, we demonstrated that the rapid breakdown of reserve carbohydrates triggered by the shift-up is merely due to changes in the concentrations of hexose-6-phosphate and UDPglucose, which are the main metabolic effectors of the rate-limiting enzymes of glycogen and trehalose pathways.

No abstract

An industrial spectrophotometer was used as a very accurate on-line biomass sensor to investigate f-t dynamic changes in yeast culture in the range of 0.5-5 g/l. High sensitive variation in biomass concentration of 0.015 g/l was detected. A fast dynamic response is induced in a steady state continuous culture of Saccharomyces cerevisiae by an acetate pulse and biomass concentration profire clearly determined by this sensor.