Furuhata, Y. | Search bioRxiv

bioRxiv2026

Continuous hypermutation and evolution of noncanonical amino acid synthases

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 (TmTyrS) 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 TmTyrS 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 O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=50 SRC="FIGDIR/small/711232v1_ufig1.gif" ALT="Figure 1"> View larger version (10K): org.highwire.dtl.DTLVardef@a7fe66org.highwire.dtl.DTLVardef@12eed57org.highwire.dtl.DTLVardef@1dff391org.highwire.dtl.DTLVardef@1efc4f0_HPS_FORMAT_FIGEXP M_FIG C_FIG 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.

synthetic biology↗

bioRxiv2024

Directed evolution of aminoacyl-tRNA synthetases through in vivo hypermutation

Genetic code expansion (GCE) has become a critical tool in biology by enabling the site-specific incorporation of non-canonical amino acids (ncAAs) into proteins. Central to GCE is the development of orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pairs wherein engineered aaRSs recognize chosen ncAAs and charge them onto tRNAs that decode blank codons (e.g., the amber stop codon). Many orthogonal aaRS/tRNA pairs covering a wide range of ncAAs have been generated by directed evolution, yet the evolution of new aaRS/tRNA pairs by standard strategies remains a labor-intensive process that often produces aaRS/tRNA pairs with suboptimal ncAA incorporation efficiencies. In this study, we present a strategy for evolving aaRSs that leverages OrthoRep to drive their continuous hypermutation in yeast. We demonstrate our strategy in 8 independent aaRS evolution campaigns starting from 4 different aaRS/tRNA parents targeting 7 distinct ncAAs. We observed the rapid evolution of multiple novel aaRSs capable of incorporating an overall range of 13 ncAAs tested into proteins in response to the amber codon. Some evolved systems reached efficiencies for amber codon-specified ncAA-dependent translation comparable to translation with natural amino acids specified by sense codons in yeast. Additionally, we discovered a surprising aaRS that evolved to self-regulate its own expression for greater dependency on ncAAs for translation. These findings demonstrate the potential of OrthoRep-driven aaRS evolution platforms in supporting the continued growth of GCE technologies.

synthetic biology↗