The highly osmo-and cryotolerant yeast species Torulaspora delbrueckii is an important case study among the non-Saccharomyces yeast species. The strain T. delbrueckii PYCC 5321, isolated from traditional corn and rye bread dough in northern Portugal, is considered particularly interesting for the baking industry. This paper reports the sugar utilization patterns of this strain, using media with glucose, maltose and sucrose, alone or in mixtures. Kinetics of growth, biomass and ethanol yields, fermentation and respiration rates, hydrolase activities and sugar uptake rates were used to infer the potential applied relevance of this yeast in comparison to a conventional baker's strain of Saccharomyces cerevisiae. The results showed that both maltase and maltose transport in T. delbrueckii were subject to glucose repression and maltose induction, whereas invertase was subject to glucose control but not dependent on sucrose induction. A comparative analysis of specific sugar consumption rates and transport capacities suggests that the transport step limits both glucose and maltose metabolism. Specific rates of CO 2 production and O 2 consumption showed a significantly higher contribution of respiration to the overall metabolism in T. delbrueckii than in S. cerevisiae. This was reflected in the biomass yields from batch cultures and could represent an asset for the large-scale production of the former species. This work contributes to a better understanding of the physiology of a non-conventional yeast species, with a view to the full exploitation of T. delbrueckii by the baking industry.

The HSP12 gene encodes one of the two major small heat-shock proteins of Saccharomyces cerevisiae and is induced under different conditions, such as low and high temperatures, osmotic or oxidative stress and high sugar or ethanol concentrations. However, few studies could demonstrate any correlation between HSP12 deletion or overexpression and a phenotype of sensitivity/resistance, making it difficult to attribute a role for Hsp12p under several of these stress conditions. We investigated the possible role of Hsp12p in yeast freezing tolerance. Contrary to what would be expected, the hsp12 null mutant when subjected to prolonged storage at "20 6C showed an increased resistance to freezing when compared with the isogenic wild-type strain. Because the mutant strain displayed a higher intracellular trehalose concentration than the wildtype, which could mask the effect of manipulating HSP12, we overexpressed the HSP12 gene in a trehalose-6-phosphate synthase (TPS1) null mutant. The tps1D strain overexpressing HSP12 showed an increase in resistance to freezing storage, indicating that Hsp12p plays a role in freezing tolerance in a way that seems to be interchangeable with trehalose. In addition, we show that overexpression of HSP12 in this tps1D strain also increased resistance to heat shock and that absence of HSP12 compromises the ability of yeast cells to accumulate high levels of trehalose in response to a mild heat stress. INTRODUCTIONBecause freezing is one of the major abiotic stresses, the adaptation mechanisms that preserve cells at subzero temperatures are extremely important in the development of technology for the cryopreservation of life. Preservation of cell activity is also a very important issue in frozendough technology. This process is well established in the modern baking industry, as it can more easily supply ovenfresh bakery products to consumers while improving labour conditions. Nevertheless, storage of frozen bread dough may lead to the loss of cell viability of baker's yeast as well as of its baking capacity, and consequently to economic losses (Alves-Araú jo et al., 2004;Randez-Gil et al., 1999). In spite of their importance, limited information is available about the mechanisms and determinants of freezing resistance and cold responses in yeast (Kandror & Goldberg, 1997;Kandror et al., 2004;Odani et al., 2003;Zarka et al., 2003). When yeast cells are cultured at 4 u C for a long period, several heat-shock proteins (HSPs) are induced (Homma et al., 2003), suggesting that the induction of these genes might be necessary for adjustment to cold resistance. Considerable evidence indicates that the intracellular level of trehalose may determine the survival response of yeasts under extreme environmental conditions (Diniz-Mendes et al., 1999;Hottiger et al., 1987;Singer & Lindquist, 1998;Van Dijck et al., 1995;Wiemken, 1990). In general, there is wide consensus that trehalose can serve as a stress protectant when yeast cells are confronted with high or low temperatures (Attfield, 1987;Hottiger et ...

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SFT and RA modulate the trafficking of SGLT1 to the BBM and may contribute to the control of plasma glucose.

The freeze stress responses to prolonged storage at -20 degrees C in Torulaspora delbrueckii PYCC5323 were investigated. In this yeast, no loss of cell viability was observed for at least 120 days during freezing at -20 degrees C, whereas a loss of 80% was observed in a commercial baker's yeast after 15 days. In the former strain, freeze resistance was dependent on an adaptation process. The primary cell target of freeze stress was the plasma membrane, preservation of its integrity being related with a lower increase of lipid peroxidation and with a higher resistance to H(2)O(2), but not with the intracellular trehalose concentration.