Functional Survey for Heterologous Sugar Transport Proteins, Using Saccharomyces cerevisiae as a Host

ABSTRACTMolecular transport is a key process in cellular metabolism. This step is often limiting when using a nonnative carbon source, as exemplified by xylose catabolism inSaccharomyces cerevisiae. As a step toward addressing this limitation, this study seeks to characterize monosaccharide transport preference and efficiency. A group of 26 known and putative monosaccharide transport proteins was expressed in a recombinantSaccharomyces cerevisiaehost unable to transport several monosaccharides. A growth-based assay was used to detect transport capacity across six different carbon sources (glucose, xylose, galactose, fructose, mannose, and ribose). A mixed glucose-and-xylose cofermentation was performed to determine substrate preference. These experiments identified 10 transporter proteins that function as transporters of one or more of these sugars. Most of these proteins exhibited broad substrate ranges, and glucose was preferred in all cases. The broadest transporters confer the highest growth rates and strongly prefer glucose. This study reports the first molecular characterization of the annotated XUT genes ofScheffersomyces stipitisand open reading frames from the yeastsYarrowia lipolyticaandDebaryomyces hansenii.Finally, a phylogenetic analysis demonstrates that transporter function clusters into three distinct groups. One particular group comprised ofD. hanseniiXylHPandS. stipitisXUT1andXUT3demonstrated moderate transport efficiency and higher xylose preferences.

Silica Nanochannel Surface Effect on Monosaccharide Transport

The interface of silica nanochannel of 10 nm was studied by molecular modeling and experimental methods. Molecular Dynamics study on glucose solution revealed that 2–3 nm of interface solution to silica walls has reduced glucose diffusivity. That reduction affects the effective diffusivity of glucose in silica nanochannel. Experimental results show Fickian-like release of glucose through 13 nm nanochannel. Molecular modeling and experimental results suggest that glucose is not sufficiently confined to possess non-Fickian behavior.

α- and β-Monosaccharide transport in human erythrocytes

Equilibrative sugar uptake in human erythrocytes is characterized by a rapid phase, which equilibrates 66% of the cell water, and by a slow phase, which equilibrates 33% of the cell water. This behavior has been attributed to the preferential transport of β-sugars by erythrocytes (Leitch JM, Carruthers A. Am J Physiol Cell Physiol 292: C974–C986, 2007). The present study tests this hypothesis. The anomer theory requires that the relative compartment sizes of rapid and slow transport phases are determined by the proportions of β- and α-sugar in aqueous solution. This is observed with d-glucose and 3- O-methylglucose but not with 2-deoxy-d-glucose and d-mannose. The anomer hypothesis predicts that the slow transport phase, which represents α-sugar transport, is eliminated when anomerization is accelerated to generate the more rapidly transported β-sugar. Exogenous, intracellular mutarotase accelerates anomerization but has no effect on transport. The anomer hypothesis requires that transport inhibitors inhibit rapid and slow transport phases equally. This is observed with the endofacial site inhibitor cytochalasin B but not with the exofacial site inhibitors maltose or phloretin, which inhibit only the rapid phase. Direct measurement of α- and β-sugar uptake demonstrates that erythrocytes transport α- and β-sugars with equal avidity. These findings refute the hypothesis that erythrocytes preferentially transport β-sugars. We demonstrate that biphasic 3- O-methylglucose equilibrium exchange kinetics refute the simple carrier hypothesis for protein-mediated sugar transport but are compatible with a fixed-site transport mechanism regulated by intracellular ATP and cell shape.