In Vitro Genetic Code Reprogramming for the Expansion of Usable Noncanonical Amino Acids.

Genetic code reprogramming has enabled us to ribosomally incorporate various nonproteinogenic amino acids (npAAs) into peptides in vitro. The repertoire of usable npAAs has been expanded to include not only l-α-amino acids with noncanonical sidechains but also those with noncanonical backbones. Despite successful single incorporation of npAAs, multiple and consecutive incorporations often suffer from low efficiency or are even unsuccessful. To overcome this stumbling block, engineering approaches have been used to modify ribosomes, EF-Tu, and tRNAs. Here, we provide an overview of these in vitro methods that are aimed at optimal expansion of the npAA repertoire and their applications for the development of de novo bioactive peptides containing various npAAs.