Construction of an engineered yeast with glucose-inducible emission of green fluorescence from the cell surface

An engineered yeast with emission of fluorescence from the cell surface was constructed. Cell surface engineering was applied to display a visible rep orter molecule, green fluorescent protein (GFP). A glucose inducible promot er GAPDH as a model promoter was selected to control the expression of the reporter gene in response to environmental changes. The GFP gene was fused with the gene encoding the C-terminal half of x-agglutinin of Saccharomyces cerevisiae having a glycosylphosphatidylinositol anchor attachment signal sequence. A secretion signal sequence of the fungal glucoamylase precursor protein was connected to the N-terminal of GFP. This designed gene was inte grated into the TRP1 locus of the chromosome of S. cerevisiae with homologo us recombination. Fluorescence microscopy demonstrated that the transforman t cells emitted green fluorescence derived from functionally expressed GFP involved in the fusion molecule. The surface display of GFP was further ver ified by immunofluorescence labeling with a polyclonal antibody (raised in rabbits) against GFP as the first antibody and Rhodamine Red-X-conjugated g oat anti-rabbit IgG as the second antibody which cannot penetrate into the cell membrane. The display of GFP on the cell surface was confirmed using a confocal laser scanning microscope and by measuring fluorescence in each c ell fraction obtained after the subcellular fractionation. As GFP was prove d to be displayed as an active form on the cell surface, selection of promo ters will endow yeast cells with abilities to respond to changes in environ mental conditions, including nutrient concentrations in the media, through the emission of fluorescence.