A central challenge in expanding the genetic code of cells to incorporate non-canonical amino acids into proteins is the scalable discovery of aminoacyl-tRNA synthetase (aaRS)-tRNA pairs that are orthogonal in their aminoacylation specificity. Here we computationally identify candidate orthogonal tRNAs from millions of sequences and develop a rapid, scalable approach -named tRNA Extension (tREX) -to determine the in vivo aminoacylation status of tRNAs. Using tREX, we test 243 candidate tRNAs in Escherichia coli and identify 71 orthogonal tRNAs, covering 16 isoacceptor classes, and 23 functional orthogonal tRNA-cognate aaRS pairs. We discover five orthogonal pairs, including three highly active amber suppressors, and evolve new amino acid substrate specificities for two pairs. Finally, we use tREX to characterize a matrix of 64 orthogonal synthetase-orthogonal tRNA specificities. This work expands the number of orthogonal pairs available for genetic code expansion and provides a pipeline for the discovery of additional orthogonal pairs and a foundation for encoding the cellular synthesis of non-canonical biopolymers.Genetic code expansion enables the cellular synthesis of modified proteins via the cotranslational incorporation of non-canonical amino acids 1, 2 . Orthogonal aaRS-tRNA pairs are crucial to genetic code expansion. These pairs consist of (1) a synthetase that efficiently aminoacylates its cognate tRNA, but minimally aminoacylates endogenous tRNAs in the host organism, and (2) a tRNA that is a substrate for its cognate synthetase but is a poor substrate for endogenous synthetases 3,4 . Derivatives of orthogonal pairs that recognize blank codons (most commonly, the amber stop codon) and selectively use non-canonical amino acids (ncAAs) have been used to site-specifically incorporate numerous ncAAs into proteins 3,4 . Despite many years of effort, a limited number of orthogonal aaRS-tRNA pairs *

It is widely hypothesized that removing cellular transfer RNAs (tRNAs)—making their cognate codons unreadable—might create a genetic firewall to viral infection and enable sense codon reassignment. However, it has been impossible to test these hypotheses. In this work, following synonymous codon compression and laboratory evolution in Escherichia coli, we deleted the tRNAs and release factor 1, which normally decode two sense codons and a stop codon; the resulting cells could not read the canonical genetic code and were completely resistant to a cocktail of viruses. We reassigned these codons to enable the efficient synthesis of proteins containing three distinct noncanonical amino acids. Notably, we demonstrate the facile reprogramming of our cells for the encoded translation of diverse noncanonical heteropolymers and macrocycles.

The direct genetically encoded cell-based synthesis of non-natural peptide and depsipeptide macrocycles is an outstanding challenge. Here we programme the encoded synthesis of 25 diverse non-natural macrocyclic peptides, each containing two non-canonical amino acids, in Syn61Δ3-derived cells; these cells contain a synthetic Escherichia coli genome in which the annotated occurrences of two sense codons and a stop codon, and the cognate transfer RNAs (tRNAs) and release factor that normally decode these codons, have been removed. We further demonstrate that pyrrolysyl-tRNA synthetase/tRNA pairs from distinct classes can be engineered to direct the co-translational incorporation of diverse alpha hydroxy acids, with both aliphatic and aromatic side chains. We define 49 engineered mutually orthogonal pairs that recognize distinct non-canonical amino acids or alpha hydroxy acids and decode distinct codons. Finally, we combine our advances to programme Syn61Δ3-derived cells for the encoded synthesis of 12 diverse non-natural depsipeptide macrocycles, which contain two non-canonical side chains and either one or two ester bonds.