From Phenols to Proteins: One‐Pot Biosynthesis and Genetic Encoding of Chalcogen‐Containing Tyrosine Analogues

ABSTRACT Expanding the genetic code with unnatural amino acids (UAAs) offers powerful opportunities to engineer proteins with novel redox and catalytic functions, but is often limited by the need for multistep UAA synthesis and inefficient cellular uptake. Here, we report an integrated biosynthetic–genetic incorporation strategy for chalcogen‐containing proteins from the respective phenols. Structure‐guided engineering of tyrosine phenol lyase (TPL) enabled the enzymatic production of 3‐methoxy‐, 3‐methylthio‐, and 3‐methylseleno‐L‐tyrosine (MeSeY) directly in living cells. Using evolved orthogonal aminoacyl‐tRNA synthetases, these analogues were site‐specifically incorporated into green fluorescent protein (GFP), as confirmed by fluorescence assays, spectroscopy, and mass spectrometry. We further established a one‐pot in vivo system that unifies analogue biosynthesis with translation, reducing precursor requirements and cellular toxicity. This work introduces selenium as a genetically encoded handle for protein engineering and establishes a scalable strategy that couples biocatalysis with genetic code expansion to access redox‐active designer proteins. Importantly, installation of MeSeY at the GFP chromophore residue Tyr66 provides redox‐responsive fluorescence. In a circularly permuted GFP (cpGFP) scaffold, improved chromophore accessibility enables reversible redox switching under H 2 O 2 /thiol cycling.