Advances and Challenges in Cell-Free Incorporation of Unnatural Amino Acids Into Proteins.

Incorporation of unnatural amino acids (UNAAs) into proteins currently is an active biological research area for various fundamental and applied science. In this context, cell-free synthetic biology (CFSB) has been developed and recognized as a robust testing and biomanufacturing platform for highly efficient UNAA incorporation. It enables the orchestration of unnatural biological machinery toward an exclusive user-defined objective of unnatural protein synthesis. This review aims to overview the principles of cell-free unnatural protein synthesis (CFUPS) systems, their advantages, different UNAA incorporation approaches, and recent achievements. These have catalyzed cutting-edge research and diverse emerging applications. Especially, present challenges and future trends are focused and discussed. With the development of CFSB and the fusion with other advanced next-generation technologies, CFUPS systems would explicitly deliver their values for biopharmaceutical applications.