Abstract
Genetic code expansion (GCE) has significantly enhanced the diversity of proteins in the biological world, leading to a wide range of applications. Despite the advances in GCE, the cost of noncanonical amino acids (ncAAs) remains one of the major obstacles for large-scale production. In situ biosynthesis of ncAAs from commercial precursors offers a promising solution to this challenge, yet only a few biosynthetic pathways have been reported. Here, we present a platform that couples the biosynthesis of aromatic ncAAs with genetic code expansion in E. coli, enabling the production of proteins and peptides containing ncAAs. Forty ncAAs are synthesized from aryl aldehydes by the biosynthetic pathway, while nineteen ncAAs are incorporated into superfolder GFP using three orthogonal translation systems. The platform's versatility is demonstrated by the production of macrocyclic peptides and antibody fragments. We envision that the platform will facilitate the production of peptides, enzymes, and antibody fragments containing ncAAs.