Initiation of Protein Synthesis with Non‐Canonical Amino Acids In Vivo

Abstract

Abstract By transplanting identity elements into E. coli tRNA fMet , we have engineered an orthogonal initiator tRNA (itRNA Ty2 ) that is a substrate for Methanocaldococcus jannaschii TyrRS. We demonstrate that itRNA Ty2 can initiate translation in vivo with aromatic non‐canonical amino acids (ncAAs) bearing diverse sidechains. Although the initial system suffered from low yields, deleting redundant copies of tRNA fMet from the genome afforded an E. coli strain in which the efficiency of non‐canonical initiation equals elongation. With this improved system we produced a protein containing two distinct ncAAs at the first and second positions, an initial step towards producing completely unnatural polypeptides in vivo. This work provides a valuable tool to synthetic biology and demonstrates remarkable versatility of the E. coli translational machinery for initiation with ncAAs in vivo.