Metabolic oligosaccharide engineering (MOE) using biocatalysts

Metabolic oligosaccharide engineering (MOE) involves the exploitation of carbohydrate biosynthetic pathways in bacterial and mammalian cells with the aim of reengineering cell surface glycoproteins. Classically, cells are fed sugar precursors bearing latently reactive functional groups, these are almost always azides which can be modified with fluorescent/or purification tags using staudinger ligations, Cu-catalysed click reactions, or strain promoted click reactions. This technique has facilitated fluorescent imaging inside live cells, and even dynamically inside zebrafish embryos. MOE has also been used to map, detect and enrich glycoproteins, specifically GlcNAc modified and sialic acid capped glycoproteins. ManAc derivatives bearing azide groups are metabolised by the mammalian biosynthetic machinery and incorporated into glycoproteins as modified sialic acids. Ac4Glc/GalNAz can be similarly metabolised and incorporated as O-GlcNAc sugars via salvage pathways, however the levels of incorporation are low (due to "biological crosstalk") and the Glc/GalNAz moieties are also incorporated into N-glycans and O-mucins preventing the specific detection of O-GlcNAcylated proteins, which are increasingly important biological targets, and play prominent roles in disease etiology. In this project we will reinvigorate glycoprotein detection by using biocatalysts for MOE and use a combination of directed evolution and mining of gram-positive bacterial metogenomes to develop a universal suite of enzyme variants capable of specific detection of distinct sugars in live cells.