The transport and regulation of maltose utilization by Torulaspora delbrueckii, one of the most abundant non-Saccharomyces species present in home-made corn and rye bread dough, has been investigated. A DNA fragment containing the MAL11 gene from T. delbrueckii (TdMAL11) was isolated by complementation cloning in Saccharomyces cerevisiae. DNA sequence analysis revealed the presence of an open reading frame (ORF) of 1884 bp, encoding a 627-amino acid membrane protein, which displays high homology to other yeast maltose transporters. Upstream of TdMAL11, the DNA insert contained a partial ORF (TdMAL12) on the opposite strand, which showed high similarity to the S. cerevisiae MAL12 gene. Sequence analysis, Northern blot and transport measurements indicated that TdMAL11 expression is regulated by the carbon source. Attempts to disrupt TdMAL11 revealed the presence of two functional MAL loci. Disruption of a single copy decreased the V(max) of maltose transport, but not the K(m), whereas the double disruption abolished the uptake of this sugar in T. delbrueckii.

Recently, the academic interest in the yeast Torulaspora delbrueckii has increased notably due to its high resistance to several types of stress, including salt and osmotic imbalance. However, the molecular mechanisms underlying these unusual properties are poorly understood. In Saccharomyces cerevisiae, the high-salt response is mediated by calcineurin, a conserved Ca 2؉ /calmodulin-modulated protein phosphatase that regulates the transcriptional factor Crz1p. Here, we cloned the T. delbrueckii TdCRZ1 gene, which encodes a putative zinc finger transcription factor homologue to Crz1p. Consistent with this, overexpression of TdCRZ1 enhanced the salt tolerance of S. cerevisiae wild-type cells and suppressed the sensitivity phenotype of cnb1⌬ and crz1⌬ mutants to monovalent and divalent cations. However, T. delbrueckii cells lacking TdCrz1p showed phenotypes distinct from those previously observed in S. cerevisiae crz1⌬ mutants. Quite remarkably, Tdcrz1-null cells were insensitive to high Na ؉ and were more Li ؉ tolerant than wild-type cells. Clearly, TdCrz1p was not required for the salt-induced transcriptional activation of the TdENA1 gene, encoding a putative P-type ATPase homologue to the main S. cerevisiae Na ؉ pump ENA1. Furthermore, T. delbrueckii cells were insensitive to the immunosuppressive agents FK506 and cyclosporine A, both in the presence and in the absence of NaCl. Signaling through the calcineurin/Crz1 pathway appeared to be essential only on high-Ca 2؉ /Mn 2؉ media. Hence, T. delbrueckii and S. cerevisiae differ in the regulatory circuits and mechanisms that drive the adaptive response to salt stress.

The response of Saccharomyces cerevisiae and freeze-tolerant Torulaspora delbrueckii strains to osmotic stress and their CO2 production capacity in sweet and frozen-sweet dough has been examined. T. delbrueckii strains, IGC5321 and IGC5323 showed higher leavening ability than Saccharomyces, specially after exposure to hyperosmotic stress of bread dough containing 20% sucrose and 2% salt added. In addition, Torulaspora and especially T. delbrueckii IGC5321 exhibited no loss of CO2 production capacity during freeze-thaw stress. Overall, these results appeared to indicate that Torulaspora cells are more tolerant than Saccharomyces to osmotic stress of bread dough. This trait correlated with a low invertase activity, a slow rate of trehalose mobilisation and the ability to respond rapidly to osmotic stress. Growth behaviour on high osmotic synthetic media was also examined. Cells of the IGC5321 strain showed intrinsic osmotolerance and ion toxicity resistance. However, T. delbrueckii IGC5323 exhibited a clear phenotype of osmosensitivity. Hence, this characteristic may not be essential or the only determinant for leavening ability in salted high-sugar dough.