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

Hongxia Zhao, Wenlong Ding, Jia Zang, Yang Yang, Chao Liu, Linzhen Hu, Yulin Chen, Guanglong Liu, Yu Fang, Ying Yuan and Shixian Lin ()
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Hongxia Zhao: Zhejiang University
Wenlong Ding: Zhejiang University
Jia Zang: Zhejiang University
Yang Yang: Nanjing University
Chao Liu: Zhejiang University
Linzhen Hu: Zhejiang University
Yulin Chen: Zhejiang University
Guanglong Liu: Zhejiang University
Yu Fang: Zhejiang University
Ying Yuan: Zhejiang University School of Medicine
Shixian Lin: Zhejiang University

Nature Communications, 2021, vol. 12, issue 1, 1-12

Abstract: Abstract 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.

Date: 2021
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DOI: 10.1038/s41467-021-27399-x

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