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Natl. Acad. Sci. U.S.A."],"published-print":{"date-parts":[[2026,9,15]]},"abstract":"<jats:p>\n                    Genetic code expansion introduces new-to-nature chemical moieties into ribosomally synthesized proteins. In practice, the scope of functional groups that can be accessed using this method is often limited by noncanonical amino acid (ncAA) availability. Producing ncAAs directly in cells can circumvent poor ncAA uptake or commercial unavailability, but limited enzymes suitable for this application exist. In vitro evolution campaigns have yielded synthetically useful \u201cncAA synthases,\u201d but these enzymes are optimized for preparative-scale synthesis and their activities often do not translate well to cellular biosynthesis. Thus, expanding strategies to engineer enzymes specifically for ncAA production within cells will benefit further implementation of genetic code expansion. Here, we use phage-assisted noncontinuous and continuous evolution to evolve enzymes for improved synthesis of noncanonical tyrosine derivatives in\n                    <jats:italic toggle=\"yes\">Escherichia coli<\/jats:italic>\n                    . Using simple serial passaging, we uncovered mutations that doubled the production of an expensive ncAA, 3-methoxytyrosine, by tyrosine phenol lyase, and furthermore evolved variants that enable 3-iodotyrosine biosynthesis, a transformation the parent enzyme is unable to perform in cells. Additionally, we evolved a recently reported tyrosine synthase for improved production of 3-halogenated tyrosines, identifying variants that exhibit high activity even at low substrate concentrations owing to a ~eightfold reduction in K\n                    <jats:sub>M<\/jats:sub>\n                    . Our results demonstrate that phage assisted evolution can be used to rapidly improve the activity of enzymes for ncAA production in cells.\n                  <\/jats:p>","DOI":"10.1073\/pnas.2614167123","type":"journal-article","created":{"date-parts":[[2026,9,10]],"date-time":"2026-09-10T17:02:15Z","timestamp":1789059735000},"update-policy":"https:\/\/doi.org\/10.1073\/pnas.cm10313","source":"Crossref","is-referenced-by-count":1,"title":["Phage-assisted continuous evolution of enzymes for noncanonical tyrosine biosynthesis"],"prefix":"10.1073","volume":"123","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8471-2341","authenticated-orcid":false,"given":"James S.","family":"Andon","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/01y2jtd41","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Biochemistry, University of 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Andon A. Behera Maps of plasmids used in this work. Zenodo. https:\/\/doi.org\/10.5281\/zenodo.21534616. 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