The important roles that protein glycosylation plays in modulating the activities and efficacies of protein therapeutics have motivated the development of synthetic glycosylation systems in living bacteria and in vitro. A key challenge is the lack of glycosyltransferases that can efficiently and site-specifically glycosylate desired target proteins without the need to alter primary amino acid sequences at the acceptor site. Here, we report an efficient and systematic method to screen a library of glycosyltransferases capable of modifying comprehensive sets of acceptor peptide sequences in parallel. This approach is enabled by cell-free protein synthesis and mass spectrometry of self-assembled monolayers and is used to engineer a recently discovered prokaryotic N-glycosyltransferase (NGT). We screened 26 pools of site-saturated NGT libraries to identify relevant residues that determine polypeptide specificity and then characterized 122 NGT mutants, using 1052 unique peptides and 52,894 unique reaction conditions. We define a panel of 14 NGTs that can modify 93% of all sequences within the canonical X-1-N-X+1-S/T eukaryotic glycosylation sequences as well as another panel for many noncanonical sequences (with 10 of 17 non-S/T amino acids at the X+2 position). We then successfully applied our panel of NGTs to increase the efficiency of glycosylation for three protein therapeutics. Our work promises to significantly expand the substrates amenable to in vitro and bacterial glycoengineering.
Keywords: cell-free protein synthesis; glycosylation; glycosyltransferase; high-throughput; synthetic biology; therapeutics.