Biosynthesis of Halogenated Tryptophans for Protein Engineering Using Genetic Code Expansion

Abstract

AbstractGenetic Code Expansion technology offers significant potential in incorporating noncanonical amino acids into proteins at precise locations, allowing for the modulation of protein structures and functions. However, this technology is often limited by the need for costly and challenging‐to‐synthesize external noncanonical amino acid sources. In this study, we address this limitation by developing autonomous cells capable of biosynthesizing halogenated tryptophan derivatives and introducing them into proteins using Genetic Code Expansion technology. By utilizing inexpensive halide salts and different halogenases, we successfully achieve the selective biosynthesis of 6‐chloro‐tryptophan, 7‐chloro‐tryptophan, 6‐bromo‐tryptophan, and 7‐bromo‐tryptophan. These derivatives are introduced at specific positions with corresponding bioorthogonal aminoacyl‐tRNA synthetase/tRNA pairs in response to the amber codon. Following optimization, we demonstrate the robust expression of proteins containing halogenated tryptophan residues in cells with the ability to biosynthesize these tryptophan derivatives. This study establishes a versatile platform for engineering proteins with various halogenated tryptophans.