Mammalian Cell Surface Display for High‐Throughput Protein Engineering of Glycosyltransferases

High-throughput methods are needed to study structure-function relationships of mammalian glycosyltransferases due to their essential role in assembling the glycocalyx. Mutations in these enzymes result in Congenital Disorders of Glycosylation, and improvements of enzyme properties can yield new biocatalysts. In this manuscript, a cell-based glycosyltransferase activity assay is developed using mammalian surface display. It is demonstrated that coupling this approach with click chemistry enables rapid quantification of glycosyltransferase activity. Screening of 1680 different pig ST3Gal1 mutants yielded α(2,3)sialyltransferases with improved enzymatic properties. Using endogenous cell-surface substrates, the method is extended to other human sialyltransferases, including ST6Gal1, ST3Gal4, and ST6GalNAc2. Additionally, the approach is used to screen putative sialyltransferases from diverse organisms not characterized in the CAZy (Carbohydrate-Active EnZymes) database. Overall, a facile, robust, high-throughput, low-cost method is presented to study glycosyltransferase structure-function relationships.