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

Hongxia Zhao,Chao Liu,Shixian Lin,Jia Zang,Ying Yuan,Linzhen Hu,Guanglong Liu,Wenlong Ding,Yang Yang,Yulin Chen,Yu Fang

doi:10.1038/s41467-021-27399-x

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.

Highlights

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
It’s critical to have an in vivo site-specific UAA incorporation system with comparable efficiency and similar protein yield when UAAs are used in place of natural amino acids (NAAs)
Engineered aaRS/tRNA pairs with such low activity has difficulty competing with the endogenous protein termination machinery at the amber codon

Summary

Introduction

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. Site-specific incorporation of biophysical probes, bioorthogonal handles, cross-linkers, cage groups, and natural post-translational modifications into proteins has been successfully used for a wide range of fundamental studies and advanced applications[8,9,10,11,12,13,14,15,16] In this context, it’s critical to have an in vivo site-specific UAA incorporation system with comparable efficiency and similar protein yield (or in this study referred to as wild-type like efficiency) when UAAs are used in place of natural amino acids (NAAs). Our work provides a general strategy for in vitro and in vivo application of UAA-dependent synthetic auxotrophs

Methods

Results

Conclusion