Enzymatic Engineering of Live Bacterial Cell Surfaces Using Butelase 1

Abstract Butelase‐mediated ligation (BML) can be used to modify live bacterial cell surfaces with diverse cargo molecules. Surface‐displayed butelase recognition motif NHV was first introduced at the C‐terminal end of the anchoring protein OmpA on E. coli cells. This then served as a handle of BML for the functionalization of E. coli cell surfaces with fluorescein and biotin tags, a tumor‐associated monoglycosylated peptide, and mCherry protein. The cell‐surface ligation reaction was achieved at low concentrations of butelase and the labeling substrates. Furthermore, the fluorescein‐labeled bacterial cells were used to show the interactions with cultured HeLa cells and with macrophages in live transgenic zebrafish, capturing the latter's powerful phagocytic effect in action. Together these results highlight the usefulness of butelase 1 in live bacterial cell surface engineering for novel applications.