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 . ![Figure][1]</img> 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. ### Competing Interest Statement C.C.L. is a co-founder of Eira Bio, which uses OrthoRep for protein engineering. P.J.A. is an inventor on a patent that covers enzymatic synthesis of tyrosine analogs from analogs of phenol and serine (US12421534). The remaining authors declare no competing interests. NIH, R35GM136297, F32GM156066 JSPS, 202260318, 25K00105 [1]: pending:yes

Kinetic proofreading is a class of error-correcting mechanisms in biology that expend energy to avoid mistakes during replication, transcription, and translation. Proofreading is typically assumed to ...

Mutational rates of bacterial evolution are increased using an error-prone orthogonal system.

Honoring Dr. Andrew D. Hanson, Eminent Scholar and UF Hort Sci Professor, whose visionary research and mentorship shaped plant biology.

Discovering and optimizing reactions is central to synthetic chemistry. However, chemical reactions are traditionally screened using relatively low-throughput methods, prohibiting exploration of diver...

LaccID, an engineered laccase, enables hydrogen-peroxide-free proximity labeling and electron microscopy (EM) in mammalian cells. Notably, LaccID is selectively active at the cell surface, enabling th...

Systems that perform continuous hypermutation of designated genes without compromising the integrity of the host genome can substantially accelerate the evolution of new or enhanced protein functions....

University of California, Irvine is hiring. Apply now!

In an experimental multicopy plasmid evolution system that allows manipulation of mutation rate and copy number, low copy number promoted enrichment of beneficial alleles whereas high copy number main...

Genetic code expansion (GCE) enables the incorporation of new amino acids into proteins. Here, authors demonstrate the rapid OrthoRep-driven evolution of aminoacyl-tRNA synthetases for GCE, increasing...

The emergence and evolution of new gene functions is central to biology, yet experimental tools that allow us to prospectively probe and understand this process are lacking. While systems for continuo...

Biomedical research impacts all of us. I became curious about how drugs work after my mom was diagnosed with rheumatoid arthritis when I was a small boy. She received “gold shots” as treatment, which ...

The move halts a large slice of money for most universities and research institutions virtually overnight, imperiling vital research in everything from cancer to heart disease.

Efficient methods for diversifying genes of interest (GOIs) are essential in protein engineering. For example, OrthoRep, a yeast-based orthogonal DNA replication system that achieves the rapid in vivo...

University of California, Irvine is hiring. Apply now!

Loveless, Carlson and colleagues describe peCHYRON, a DNA recorder that propagates an unending chain of insertions. Each link contains a randomizable 3-bp ‘signature’ sequence that serves to record a ...

When nature evolves a gene over eons at scale, it produces a diversity of homologous sequences with patterns of conservation and change that contain rich structural, functional, and historical informa...