Respiration-Dependent Utilization of Sugars in Yeasts: a Determinant Role for Sugar Transporters

105

116

The yeast Saccharomyces cerevisiae predominantly ferments glucose to ethanol at high external glucose concentrations, irrespective of the presence of oxygen. In contrast, at low external glucose concentrations and in the presence of oxygen, as in a glucose-limited chemostat, no ethanol is produced. The importance of the external glucose concentration suggests a central role for the affinity and maximal transport rates of yeast's glucose transporters in the control of ethanol production. Here we present a series of strains producing functional chimeras between the hexose transporters Hxt1 and Hxt7, each of which has distinct glucose transport characteristics. The strains display a range of decreasing glycolytic rates resulting in a proportional decrease in ethanol production. Using these strains, we show for the first time that at high glucose levels, the glucose uptake capacity of wild-type S. cerevisiae does not control glycolytic flux during exponential batch growth. In contrast, our chimeric Hxt transporters control the rate of glycolysis to a high degree. Strains whose glucose uptake is mediated by these chimeric transporters will undoubtedly provide a powerful tool with which to examine in detail the mechanism underlying the switch between fermentation and respiration in S. cerevisiae and will provide new tools for the control of industrial fermentations.

Section: Discussionsupporting

confidence: 82%

Role of Hexose Transport in Control of Glycolytic Flux in Saccharomyces cerevisiae

Elbing

Larsson

Bill

et al. 2004

105

116

“…In practice, the Kluyver effect in K. lactis is linked to limiting sugar uptake, which results in a low glycolytic flux, since overexpression of S. cerevisiae sugar transporters enables K. lactis to ferment maltose (17). In the case of S. cerevisiae on trehalose, this effect is dependent not only on trehalose uptake by the Agt1p transporter (16, this study) but also on the extracellular Ath1p (this study).…”

Section: Discussionmentioning

confidence: 80%

Two Distinct Pathways for Trehalose Assimilation in the Yeast Saccharomyces cerevisiae

Jules

Guillou

François

et al. 2004

141

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 ...

“…By introducing multiple permease genes, Goffrini et al enabled Kluyveromyces lactis to grow on galactose and raffinose without respiration (17). Ostergaard et al were able to increase galactose consumption and respirofermentative activity in S. cerevisiae by altering the regulatory network of the cell (42).…”

Section: Discussionmentioning

confidence: 99%

Saccharomyces cerevisiae Engineered for Xylose Metabolism Exhibits a Respiratory Response

Liu¹,

Laplaza

Jeffries

2004

153

143

Native strains of Saccharomyces cerevisiae do not assimilate xylose. S. cerevisiae engineered for D-xylose utilization through the heterologous expression of genes for aldose reductase (XYL1), xylitol dehydrogenase (XYL2), and D-xylulokinase (XYL3 or XKS1) produce only limited amounts of ethanol in xylose medium. In recombinant S. cerevisiae expressing XYL1, XYL2, and XYL3, mRNA transcript levels for glycolytic, fermentative, and pentose phosphate enzymes did not change significantly on glucose or xylose under aeration or oxygen limitation. However, expression of genes encoding the tricarboxylic acid cycle, respiration enzymes (HXK1, ADH2, COX13, NDI1, and NDE1), and regulatory proteins (HAP4 and MTH1) increased significantly when cells were cultivated on xylose, and the genes for respiration were even more elevated under oxygen limitation. These results suggest that recombinant S. cerevisiae does not recognize xylose as a fermentable carbon source and that respiratory proteins are induced in response to cytosolic redox imbalance; however, lower sugar uptake and growth rates on xylose might also induce transcripts for respiration. A petite respiration-deficient mutant (°) of the engineered strain produced more ethanol and accumulated less xylitol from xylose. It formed characteristic colonies on glucose, but it did not grow on xylose. These results are consistent with the higher respiratory activity of recombinant S. cerevisiae when growing on xylose and with its inability to grow on xylose under anaerobic conditions.