Sugar transporter research focuses on the sugar uptake into cells. Under certain physiological conditions, however, the intracellular accumulation and secretion of carbohydrates (efflux) are relevant processes in many cell types. Currently, no cell-based system is available for specifically investigating glucose efflux. Therefore, we designed a system based on a hexose transporter-deficient Saccharomyces cerevisiae strain, in which the disaccharide maltose is provided as a donor of intracellular glucose. By deleting the hexokinase genes, we prevented the metabolization of glucose, and thereby achieved the accumulation of growth-inhibitory glucose levels inside the cells. When a permease mediating glucose efflux is expressed in this system, the inhibitory effect is relieved proportionally to the capacity of the introduced transporter. The assay is thereby suitable for screening of transporters and quantitative analyses of their glucose efflux capacities. Moreover, by simultaneous provision of intracellular glucose and extracellular xylose, we investigated how each sugar influences the transport of the other one from the opposite side of the membrane. Thereby, we could show that the xylose transporter variant Gal2N376F is insensitive not only to extracellular but also to intracellular glucose. Considering the importance of sugar transporters in biotechnology, the assay could facilitate new developments in a variety of applications.
Keywords: hxt0 strain; competitive inhibition of xylose transport by glucose; glucose secretion; screening system; sugar efflux; xylose transport.
© The Author(s) 2022. Published by Oxford University Press on behalf of FEMS.