Fig. 1

Schematic view of cell-based high-throughput screening strategies based on fluorescently-labeled substrates. In the original strategy, a single fluorescent substrate was used to screen a library of mutant sialyltransferases. First, fluorescently-labeled monosaccharides are transported into the cell by a transmembrane sugar transport protein (step 1). After an incubation period during which the fluorescently-labeled substrates may be modified enzymatically (step 2), unreacted substrates are removed using a washing step (step 3). Cells containing catalytically-active glycosyltransferases retain the fluorescent product inside the cell, now as part of a polysaccharide. Finally, desired cells (with high fluorescence, hence high-level polysaccharide production) are screened by rapid sorting techniques such as fluorescence activated cell sorting (FACS) (step 4), followed by sequencing (step 5)