Engineered orthogonal translation systems from metagenomic libraries expand the genetic code

Abstract Genetic code expansion with non-canonical amino acids (ncAAs) opens new opportunities for the function and design of proteins by broadening their chemical repertoire. Unfortunately, ncAA incorporation is limited both by a small collection of orthogonal aminoacyl-tRNA synthetases (aaRSs) and tRNAs and by low-throughput methods to discover them. Here, we report the discovery, characterization, and engineering of a UGA suppressing orthogonal translation system mined from metagenomic data. We developed an integrated computational and experimental pipeline to profile the orthogonality of >200 tRNAs, test >1,250 combinations of aaRS:tRNA pairs, and identify the AP1 TrpRS:tRNATrpUCA as an orthogonal pair that natively encodes tryptophan at the UGA codon. We demonstrate that the AP1 TrpRS:tRNATrpUCA is highly active in cell-free and cellular contexts. We then use Ochre, a genomically recoded Escherichia coli strain that lacks UAG and UGA codons, to engineer an AP1 TrpRS variant capable of 5-hydroxytryptophan incorporation at an open UGA codon. We anticipate that our strategy of integrating metagenomic bioprospecting with cell-free screening and cell-based engineering will accelerate the discovery and optimization of orthogonal translation systems for genetic code expansion. Competing Interest Statement K.S., M.T.A.N., P.I.P., J.F.B., F.J.I., and M.C.J. have filed invention disclosures based on the work presented. M.C.J. and F.J.I. have a financial interest in Pearl Bio, Inc. The interests of M.C.J. are reviewed and managed by Stanford University and Northwestern University in accordance with their competing interest policies. The interests of F.J.I. are reviewed and managed by Yale University in accordance with their competing interest policies. Data and materials availability All data is available upon request.