Abstract
Genetic code expansion (GCE) enables the site-specific incorporation of noncanonical amino acids (ncAAs) into proteins but is constrained by reliance on exogenously supplied chiral ncAAs. Achieving intracellular ncAA biosynthesis would enable more scalable and cost-effective GCE. Here, we report the continuous hypermutation and evolution of amino acid synthases that produce high levels of ncAAs inside yeast, thus supporting GCE from simple ncAA precursors. We encoded an engineered 'tyrosine synthase' ( Tm TyrS) on an error-prone orthogonal DNA replication system (OrthoRep) and selected variants based on ncAA biosynthesis from readily available phenol analogs and intracellular L-serine. Our selection employed orthogonal ncAA-specific aminoacyl-tRNA synthetases (aaRSs) as biosensors whereby target ncAA production leads to aminoacylation of an amber suppressor tRNA and the translation of a selectable reporter containing an amber stop codon. Our evolution successfully yielded Tm TyrS variants that efficiently produced 3-iodo-, 3-bromo-, 3-chloro-, and 3-methyl-L-tyrosine, enabling amber codon-specified ncAA-dependent translation, in some cases at levels comparable to sense codon-specified natural amino acid translation. This work reduces barriers for expressing proteins containing substituted tyrosines. Moreover, because aaRSs can themselves be evolved (including with OrthoRep) for a flexible range of ncAA specificities, these results establish an end-to-end framework for evolving ncAA biosynthetic enzymes in vivo . GRAPHICAL ABSTRACT: We describe an OrthoRep-driven platform for evolving noncanonical amino acid (ncAA) synthases. Hypermutation of ncAA synthase genes enables evolution of ncAA biosynthesis from simple precursors, while intracellular ncAA production is linked to fluorescence via an orthogonal aaRS/tRNA system, allowing FACS enrichment of improved variants through iterative cycles.