Two distinct pathways for trehalose assimilation in the yeast Saccharomyces cerevisiae

Abstract

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.

FIG. 1.

Growth curves of CEN.PK113-7D and mutant strains on trehalose medium at pH 4.8. Cultures were carried out in 2-liter bioreactors as described in Materials and Methods. The culture was started with 0.08 mg (equivalent dry mass) of cells from an overnight preculture in YN glucose medium. (A) Wild-type and ath1 mutant strains; (B) nth1, agt1, ath1 nth1, and ath1 agt1 mutant strains. Dry mass: wild type (♦), ath1 (•), nth1 (*), agt1(▴), ath1 nth1 (▪), and ath1 agt1(□). Trehalose: wild type (⋄), ath1 (○), and agt1 (▵). The μ_max values calculated from fitting curves (solid lines) were 0.07 h⁻¹ (r² = 0.990) for the wild type, 0.035 h⁻¹ (r² = 0.994) for the ath1 mutant, 0.037 h⁻¹ (r² = 0.987) for the nth1 mutant, and 0.040 h⁻¹ (r² = 0.991) for the agt1 mutant. The dashed line is the theoretical projection of the exponential fitting curve from the ath1 mutant strain.

FIG. 2.

Growth curves of CEN.PK113-7D and mutant strains that overexpress ATH1, AGT1, and NTH1 on trehalose medium at pH 4.8. Procedures were as described in the legend to Fig. 1. (A) Wild type (dry mass, ♦) and CENPK113-5D transformed with pGRSd-ATH1 (dry mass, •; trehalose, ○); (B) ath1 (dry mass, -), ath1 ura mutant strain transformed with pGRSd-AGT1 (dry mass, ▴; transport activity, ▵) and ath1 ura his mutant strain transformed with both pGRSd-AGT1 and pGRSd-NTH1 (dry mass, ▪; transport activity, □). The μ_max values calculated from exponential fitting curves were 0.07 h⁻¹ (r² = 0.990) for the wild type, 0.14 h⁻¹ (r² = 0.994) for the ura/pGRSd-ATH1 strain, 0.13 h⁻¹ (r² = 0.994) for the ath1 ura/pGRSd-AGT1 strain, and 0.13 h⁻¹ (r² = 0.998) for the ath1 ura his/pGRSd-AGT1/pGRSd-NTH1 strain. Dashed lines are theoretical projections of exponential fitting curves from ath1 mutant strains transformed with pGRSd-AGT1 and pGRSd-AGT1 plus pGRSd-NTH1 (B).