Isolation and characterization of theLGT1gene encoding a low-affinity glucose transporter fromTorulaspora delbrueckii

Abstract

Torulaspora delbrueckii PYCC 5321 displayed a mediated glucose transport activity best fitted assuming a biphasic Michaelis-Menten kinetics with a low-and a highaffinity component. A genomic library of this yeast strain was used to transform a mutant of Saccharomyces cerevisiae deficient in glucose transport. Sequence analysis of a DNA fragment cloned, revealed the presence of a 1704 bp length ORF. This ORF, named LGT1, displayed a high homology to yeast glucose transporter genes. Functional characterization of the LGT1 gene product in S. cerevisiae revealed that it encodes a low-affinity transporter, able to mediate the uptake of glucose and fructose. In consonance with this, expression of LGT1 in S. cerevisiae was high in media containing 4% of glucose and almost undetectable in galactose as sole carbon source. In the absence of glucose, repression of LGT1 expression required the transcription factor Rgt1p. However, a functional Rgt1p does not appear to be required for a full induction of LGT1 at high glucose levels. Deletion of the gene coding for the general repressor Mig1p had no effect on LGT1 expression, but additional disruption of MIG2 in a mig1 background indicated that Mig2p or both Mig1p and Mig2p in a redundant way, act as repressors of LGT1 expression at high glucose concentrations. The GeneBank Accession No. for LGT1 is AY598344.

The Emerging Role of the Yeast Torulaspora delbrueckii in Bread and Wine Production: Using Genetic Manipulation to Study Molecular Basis of Physiological Responses

“…So far, only one glucose transporter has been identified in T. delbrueckii, the low-affinity glucose transporter LGT1 (50). Southern blot analysis of the T. delbrueckii genome revealed the existence of several genes with high similarity to LGT1, suggesting there are several hexose transporters in this yeast, which hampered disruption of the LGT1 gene.…”

“…Based on this assumption, multiple hexose transporters with different affinities for glucose probably exist in T. delbrueckii. This yeast displays a mediated glucose transport activity best fitted assuming a biphasic Michaelis-Menten kinetics with a low-(apparent Km = 8.32 ± 0.55 mM) and a high-affinity component (apparent Km = 1.30 ± 0.34 mM) (50). A kinetic compatible with the presence of these two components was observed in either glucose-, frutose-or maltose-grown cells.…”

“…For Saccharomyces cerevisiae, these values were similar to those obtained in mixed-sugar media, but for T. delbrueckii the values obtained in mixed-sugar media with maltose (Glucose-Maltose (G-M) and Sucrose-Maltose (S-M) mixtures) were again slightly lower. Since analysis of the specific sugar consumption rates and transport capacities suggested that sugar transfer rates into the cell limit fermentation efficiency (50), the lower growth rates obtained in the presence of maltose were probably a result of inhibition of glucose uptake by maltose, which is known to occur in both S. cerevisiae (51) and T. delbrueckii (7). In G-M mixtures, maltose was consumed only after glucose was exhausted from the medium, and the increase in maltose transport and maltase activities clearly correlated with the beginning of maltose consumption.…”

“…A kinetic compatible with the presence of these two components was observed in either glucose-, frutose-or maltose-grown cells. Aiming to identify glucose transporters in T. delbrueckii, a complementation screen of a S. cerevisiae hexose transport-null mutant strain (71) yielded a genomic DNA fragment containing a gene encoding Lgt1p, a low-affinity glucose transporter (50). When expressed in the hxt null strain, Lgt1p exhibited an apparent Km value for glucose of 36.5 ± 3.1 mM, in the range of the low-affinity component, and a Vmax of 1.1 ± 0.04 nmol/s/mg dry weight.…”

“…Evidence of the low efficiency of T. delbrueckii homologous recombination emerged when disruption of the TdMAL11 gene was attempted (95); however, in that case it was possible to screen the transformants through phenotypic analysis, since TdMAL11 null mutants exhibit deficient growth on medium containing maltose as the sole carbon source. However, it was not possible to use the same strategy to disrupt LGT1, the first gene identified as coding for a hexose transporter in T. delbrueckii, since there was evidence that other hexose transporters exist in this yeast (50). Therefore, we could not screen for potential T. delbrueckii LGT1 disruptants by searching for a clear-cut phenotype, because loss of LGT1 might be compensated by the activities of other genes and was thus not expected to impair glucose growth capacity.…”