New approaches to enzymatic glycoside synthesis through directed evolution

The expanding field of glycobiology requires tools for the synthesis of structurally defined oligosaccharides and glycoconjugates, while any potential therapeutic applications of sugar-based derivates would require access to substantial quantities of such compounds. Classical chemical approaches are not well suited for such large-scale syntheses, thus enzymatic approaches are sought. Traditional routes to the enzymatic assembly of oligosaccharides have involved the use of either Nature's own biosynthetic enzymes, the glycosyl transferases, or glycosidases run in transglycosylation mode. However, each approach has drawbacks that have limited its application. Glycosynthases are mutant glycosidases in which the catalytic nucleophile has been replaced by mutation, inactivating them as hydrolases. When used in conjunction with glycosyl fluorides of the opposite anomeric configuration to that of the substrate, these enzymes function as highly efficient transferases, frequently giving stoichiometric yields of products. Further improvements can be obtained through directed evolution of the gene encoding the enzyme in question, but this requires the ability to screen very large libraries of catalysts. In this review we survey new screening methods for the formation of glycosidic linkages using high-throughput techniques, such as FACS, chemical complementation, and robot-assisted ELISA assays. Enzymes were evolved to have higher catalytic activity with their natural substrates, to show altered substrate specificities or to be promiscuous for efficient application in oligosaccharide, glycolipid, and glycoprotein synthesis. (C) 2010 Elsevier Ltd. All rights reserved.