Directed Evolution of <i>Candidatus Methanomethylophilus alvus</i> Pyrrolysyl-tRNA Synthetase for the Genetic Incorporation of Two Different Noncanonical Amino Acids in One Protein

The genetic code expansion technique is a powerful chemical biology tool to install noncanonical amino acids (ncAAs) in proteins. As a key enzyme for this technique, pyrrolysyl-tRNA synthetase (PylRS), coupled with its cognate amber suppressor tRNA^Pyl, has been engineered for the genetic incorporation of more than 200 ncAAs. Using PylRS clones from different archaeal origins, two ncAAs have also been genetically encoded in one protein. In this work, we show that the C41AU mutant of tRNA^Pyl from <i>Candidatus Methanomethylophilus alvus</i> (CmatRNA^Pyl) is catalytically inert toward PylRS from <i>Methanosarcina mazei</i> (MmPylRS) but has weak activity toward PylRS from <i>Ca. M. alvus</i> (CmaPylRS). To improve the catalytic efficiency of CmaPylRS toward CmatRNA^Pyl-C41AU, we conducted a directed evolution of CMaPylRS by randomizing its coding sequence, followed by the screening of active mutant clones. After three rounds of randomization and screening, we identified 4 mutations, Y16F/N57D/E161G/N182I, that improve the catalytic efficiency of CMaPylRS toward CMatRNA^Pyl-C41AU. This new clone, named R3-14, coupling with CmatRNA^Pyl-C41AU to recognize an amber codon, has been successfully used together with an evolved MmPylRS clone, coupling with a mutant <i>M. mazei</i> tRNA^Pyl to recognize an ochre codon, to genetically incorporate two different ncAAs, <i>N</i> ^ε-(<i>t</i>-butoxycarbonyl)-lysine and <i>N</i> ^ε-acetyl-lysine, into one model protein.