Continuous directed evolution of aminoacyl-tRNA synthetases to alter amino acid specificity and enhance activity

Functions of various biotherapeutics can often be enhanced by modifying individual amino acid residues. However, the current approaches rely on existing natural amino acid chemistry. Approaches that provide new chemical “handles” will add versatility to modifications with an ultimate aim to enhance efficacy. An exciting way to provide these new sites in vivo, is through the incorporation of non-canonical amino acids (ncAA’s) into biotherapeutics. ncAA’s can be designed while keeping in mind the chemistry needed for subsequent modification.

There are four components that are crucial in ncAA incorporation and are (i) the ncAA itself; (ii) the codon that codes for the ncAA; (ii) the tRNA that recognizes the targeted codon and (iv) the aminoacyl tRNA synthetases (AARSs) that ligate the ncAA to the tRNA. Of these four components, generating novel AARS is the most challenging and generation of novel variants has been hampered by the long selection time needed in laboratory-based evolution of existing AARS.

In order to catalyze the development of AARSs, the Liu lab has adopted Phage-Assisted Continuous Evolution (PACE) for AARS evolution. The ability to rapidly move through a single evolution cycle, coupled with the ability to perform hundreds of such cycles, makes PACE a great tool for AARS evolution. Application of this technology to pyrolosyl-tRNA synthetase (PyIRS) led to the discovery of novel variants with 45 fold greater enzymatic efficiency. In addition, when this system was used to translate a model protein (sGFP), 10 fold increases in yields were observed. Lastly, the PACE system was used to increase selectivity such that a PACE-derived variant of tyrosyl –tRNA synthetase demonstrated a 23 fold higher specificity for the desired substrate p-iodo-L-phenylalanine. These results (Nat Chem Biol. 2017 Dec;13(12):1253-1260) establish PACE as a robust method for evolving AARSs.


Expansion of the genetic code through ncAA’s can facilitate the incorporation of features into proteins that are useful in research as well as in therapeutic product development. Genetically encoding photo cross-linkers, spectroscopic probes, can be used to enhance our understanding of the natural environment in which proteins exist. On the therapeutic front, amino-acid modifications that mimic post-translational modifications can lead to improved thermostability and chemostability. Various applications of genetic code expansion are only beginning to be appreciated and tools such as PACE derived AARS will undoubtedly play an enabling and important role in unlocking its full potential.