Evolving the N-Terminal Domain of Pyrrolysyl-tRNA Synthetase for Improved Incorporation of Noncanonical Amino Acids

Abstract

<p class="first" id="P1">By evolving the <i>N</i>-terminal domain of <i>Methanosarcina mazei</i> pyrrolysyl-tRNA synthetase (PylRS) that directly interacts with tRNA ^Pyl, a mutant clone that displays improved amber suppression efficiency for the genetic incorporation of <i>N</i> ^ε-( <i>tert</i>-butoxycarbonyl)-L-lysine three-fold more than the wild type was identified. The identified mutations are R19H/H29R/T122S. Direct transfer of these mutations to two other PylRS mutants that were previously evolved for the genetic incorporation of <i>N</i> ^ε-acetyl- L-lysine and <i>N ^ε- </i>(4-azidobenzoxycarbonyl)- L-δ,ε-dehydrolysine respectively also improved the incorporation efficiency of these two noncanonical amino acids. Since the three identified mutations are in the <i>N-</i>terminal domain of PylRS that is separated from its catalytic domain for charging tRNA ^Pyl with a noncanonical amino acid, they can be potentially introduced to all other PylRS mutants for improving incorporation efficiency of their corresponding noncanonical amino acids. Therefore, it represents a general strategy to optimize the pyrrolysine incorporation system-based noncanonical amino acid mutagenesis. </p><p id="P2">By evolving the <i>N</i>-terminal domain of pyrrolysyl-tRNA synthetase, mutations that improve the genetic incorporation of <i>N ^ε </i>-( <i>tert</i>-butoxycarbonyl)-L-lysine at amber codon were identified. These mutations can be directly transferred to PylRS mutants for improve incorporation efficiency of their corresponding noncanonical amino acids. </p><p id="P3"> <div class="figure-container so-text-align-c"> <img alt="" class="figure" src="/document_file/e86a9c9c-c97f-44b2-b839-c20f11e9252c/PubMedCentral/image/nihms966132u1.jpg"/> </div> </p>