Affinity of single S-cerevisiae cells to 2-NBDglucose under changing substrate concentrations

Background: Saccharomyces cerevisiae is a widely employed microorganism in biotechnological processes. Since proliferation and product formation depend on the capacity of the cell to access and metabolize a carbon source, a technique was developed to enable for analyzing the S. cerevisiae H155 cells' affinity to extracellular glucose concentrations. Methods: The fluorescent glucose analogue 2-NBDglucose was employed as a functional parameter to analyze the cells' affinity to glucose. Structural parameters (proliferation, neutral lipid content, granularity, and cell size) were also investigated. Cells were grown both in batches and in chemostat regimes. Results: The 2-NBDglucose uptake in individual cells proceeds in a time- and concentration-dependent manner and is affected by respiratory and respirofermentative modes of growth. The process is inhibited by D-glucose, D-fructose, D-mannose, and sucrose, but not L-glucose, D-galactose or lactose; maltose is a weak inhibitor. The affinity of the individual cells to 2-NBDglucose was found to be high at low extracellular glucose concentrations, and weak at high concentrations. An additional, underlying pattern in the cells' affinity to glucose was detected, illustrated by the recurrent appearance of two subpopulations showing distinctly differing quantities of this substrate. Conclusions: A multiparameter flow cytometry approach is presented that enables, for the first time, for analysis of the affinity of individual S. cerevisiae cells to glucose. Besides the adjustment of the yeast cell metabolism to extracellular glucose concentrations by altering their affinity to glucose, at least one further mechanism is clearly involved. Two subpopulations of cells were resolved, with different affinities not correlated with other cellular parameters measured. (C) 2004 Wiley-Liss, Inc.