Evolution-Guided Engineering of Elongation Factor-Tu for Incorporation of Noncanonical Amino Acids

Site-specific incorporation of noncanonical amino acids (ncAA) has emerged as a promising tool for synthesizing proteins with novel biological properties. However, the breadth of ncAA incorporation systems is limited by incompatibility between ncAA and endogenous translational machinery. Specifically, Elongation Factor Tu (EF-Tu), a critical translational component that proofreads and delivers the aminoacyl-tRNAs to the ribosome, has been a major barrier for ncAA incorporation. Here we employ reconstructing evolutionary adaptive paths (REAP) analysis and create a site-saturation library based on 48 positions of EF-Tu that potentially modulate its recognition of ncAA-tRNA complexes. Using in vivo antibiotic resistance-based selection assays and fluorescence-based screening assays, we isolate 18 EF-Tu variants that could incorporate either O-phospho-L-serine or 4-azido-L-phenylalanine via an amber codon. To further evaluate EF-Tu variants that have shown substrate-promiscuity in vivo, we construct an in vitro assessment system based on the PURExpress system (New England Biolabs). We then examine EF-Tu variants’ abilities to incorporate exotic ncAAs that are not compatible with aminoacyl-tRNA synthetases with the help of flexizymes. These in vitro assays allow us to interrogate EF-Tu variants for their individual abilities to incorporate ncAAs. Collectively, these findings support an evolutionary approach to identify poly-specific or ncAA-specific EF-Tus and broaden the scope of ncAA incorporation.