Coupling Enzyme Activity to Host Survival via Genetic Code Expansion: An In Vivo Evolution System for Biocatalysts

Abstract Survival-based selection facilitates the exploration of enzyme mutational space, but coupling the activity of noncanonical amino acid (ncAA)-producing biocatalysts to host survival remains challenging. Here, we report an in vivo selection platform that links biocatalyst activity to host cell survival via genetic code expansion. In this system, the intracellularly generated ncAA serves as a substrate for an orthogonal aminoacyl-tRNA synthetase (aaRS). Its incorporation triggers the readthrough of an amber (TAG) stop codon to yield a functional chloramphenicol resistance protein, allowing cells harboring active biocatalyst variants to survive under antibiotic selection. The applicability of this approach was validated using three biocatalysts: tyrosine phenol-lyase (TPL), tryptophan synthase β-subunit (TrpB), and a hydrolytic carbamoylase (SmLcar). Using this directed evolution system, we evolved TPL and TrpB to process previously inactive substrates, enabling the gram-scale preparative biosynthesis of target ncAAs with final titers reaching 36.8 g/L. Furthermore, because cell survival requires both in vivo ncAA synthesis and its subsequent orthogonal translation, this dual dependency enables the simultaneous selection of upstream biocatalysts and downstream aaRS variants. Leveraging this mechanism, we performed the selection of TPL and chPylRS libraries simultaneously, yielding enzyme pairs for the generation and recognition of target ncAAs. This platform provides a powerful tool for the directed evolution of ncAA-producing biocatalysts.