Progress Towards the Directed Evolution of an Efficient Aminoacyl-tRNA Synthetase for the Incorporation of Benzyl-Acyl-Hydrazyl-Lysine

The amino acids that make up a protein are responsible for their structure and function, which can include uses as therapeutics, catalysts, and materials. Expanding the genetic code to incorporate non-canonical amino acids (ncAAs) could enable novel protein functions not found in nature. One such ncAA is benzyl-acyl-hydrazyl-lysine (BAHK), which contains a hydrazine group capable of forming a reversible covalent bond with other ketone-containing ncAAs such as p-acetyl-phenylalanine. This could allow for modulation of protein activity via reversible intramolecular bonding. However, incorporating BAHK and similar ncAAs into proteins is inefficient because protein translation processes that incorporate ncAAs into proteins must be designed such that its components operate independently of native translation machinery. We are addressing this challenge by using directed evolution to engineer an aminoacyl-tRNA synthetase (aaRS) that more efficiently aminoacylates BAHK onto its tRNA, resulting in improved BAHK incorporation into proteins. To do this, we used the Methanomethylophilus alvus pyrrolysyl-tRNA synthetase (M. alvus pylRS) as a template to create a library of mutants by identifying 8 target residues in the aaRS binding pocket and randomly mutated them via overlap extension PCR, with the aim of modifying it to better accommodate BAHK. The effectiveness of this effort was evaluated by transforming the library into Escherichia coli bacteria and sequencing the aaRS plasmids in several dozen of the resulting colonies. We found each contained synthetase genes with unique combinations of mutations in the target residues, demonstrating the quality of the library. We are now developing a streamlined system of selections using a strain of E. coli containing a plasmid coding for green fluorescent protein (GFP) and chloramphenicol acetyltransferase (CAT) that have amber (TAG) stop codons in the middle. We will utilize Cell-Free Protein Synthesis (CFPS) to test the activity of the resulting mutants in vitro with GFP fluorescence. Using CFPS, we discovered the wild type M. alvus pylRS and an engineered version differed in activity toward the ncAAs azido lysine (AZK) and Z-lysine (ZK). We plan to use this difference to perform mock selections to fine tune this system before using BAHK. If successful, this work will expand the library of available synthetases for to incorporate BAHK into proteins and give us insights into the functionality of the M. Alvus pylRS that could be applicable to other synthetases.