Directed-evolution of translation system for efficient unnatural amino acids incorporation and generalizable synthetic auxotroph construction

作者：Hongxia Zhao, Wenlong Ding, Jia Zang, Yang Yang, Chao Liu, Linzhen Hu, Yulin Chen, Guanglong Liu, Fang Yu, Ying Yuan, Shixian Lin · 发表于：Nature Communications · 年份：2021 · DOI：10.1038/s41467-021-27399-x · 被引用次数：66 · 研究领域：RNA and protein synthesis mechanisms、RNA modifications and cancer、Genomics and Phylogenetic Studies

Site-specific incorporation of unnatural amino acids (UAAs) with similar incorporation efficiency to that of natural amino acids (NAAs) and low background activity is extremely valuable for efficient synthesis of proteins with diverse new chemical functions and design of various synthetic auxotrophs. However, such efficient translation systems remain largely unknown in the literature. Here, we describe engineered chimeric phenylalanine systems that dramatically increase the yield of proteins bearing UAAs, through systematic engineering of the aminoacyl-tRNA synthetase and its respective cognate tRNA. These engineered synthetase/tRNA pairs allow single-site and multi-site incorporation of UAAs with efficiencies similar to those of NAAs and high fidelity. In addition, using the evolved chimeric phenylalanine system, we construct a series of E. coli strains whose growth is strictly dependent on exogenously supplied of UAAs. We further show that synthetic auxotrophic cells can grow robustly in living mice when UAAs are supplemented.