AUTHOR=Fischer Jonathan T. , Söll Dieter , Tharp Jeffery M. 

TITLE=Directed Evolution of Methanomethylophilus alvus Pyrrolysyl-tRNA Synthetase Generates a Hyperactive and Highly Selective Variant

JOURNAL=Frontiers in Molecular Biosciences

VOLUME=9

YEAR=2022

URL=https://www.frontiersin.org/journals/molecular-biosciences/articles/10.3389/fmolb.2022.850613

DOI=10.3389/fmolb.2022.850613

ISSN=2296-889X

ABSTRACT=<p>Pyrrolysyl-tRNA synthetase (PylRS) is frequently used for site-specific incorporation of noncanonical amino acids (ncAAs) into proteins. Recently, the active site of <italic>Methanomethylophilus alvus</italic> PylRS (<italic>Ma</italic>PylRS) has been rationally engineered to expand its substrate compatibility, enabling the incorporation of difficult ncAAs. However, mutations beyond the active site that enhance the enzymatic properties of <italic>Ma</italic>PylRS have not been reported. We utilized phage-assisted non-continuous evolution (PANCE) to evolve <italic>Ma</italic>PylRS to efficiently incorporate <italic>N</italic><sup>ε</sup>-Boc-<sc>l</sc>-lysine (BocK). Directed evolution yielded several mutations outside of the active site that greatly improve the activity of the enzyme. We combined the most effective mutations to generate a new PylRS variant (PylRS<sub>opt</sub>) that is highly active and selective towards several lysine and phenylalanine derivatives. The mutations in PylRS<sub>opt</sub> can be used to enhance previously engineered PylRS constructs such as <italic>Ma</italic>PylRS<sub>N166S</sub>, and PylRS<sub>opt</sub> is compatible in applications requiring dual ncAA incorporation and substantially improves the yield of these target proteins.</p>