Phage-assisted continuous evolution of enzymes for noncanonical tyrosine biosynthesis

Abstract

Significance Genetic code expansion enables the incorporation of diverse chemical functionalities into proteins, yet its utility is often limited by the availability of noncanonical amino acids (ncAAs). While in-cell ncAA biosynthesis can address this challenge, existing enzymes often lack the activity required for efficient cellular ncAA production and can be limited in substrate scope. We show that phage-assisted evolution can be used to rapidly optimize enzymes for the synthesis of noncanonical tyrosine derivatives in Escherichia coli. Our evolved variants exhibited significantly improved activities on known substrates and expanded activity to new substrates, enabling the synthesis of ncAAs previously inaccessible by the parent enzymes. This platform provides a powerful approach for the rapid engineering of ncAA synthases amenable to use in cells.