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

Source abstract

Abstract By evolving the N‐terminal domain of Methanosarcina mazei pyrrolysyl‐tRNA synthetase (PylRS) that directly interacts with tRNA Pyl , a mutant clone displaying improved amber‐suppression efficiency for the genetic incorporation of N ϵ ‐( tert ‐butoxycarbonyl)‐ l ‐lysine threefold more than the wild type was identified. The identified mutations were R19H/H29R/T122S. Direct transfer of these mutations to two other PylRS mutants that were previously evolved for the genetic incorporation of N ϵ ‐acetyl‐ l ‐lysine and N ϵ ‐(4‐azidobenzoxycarbonyl)‐ l ‐δ, ϵ ‐dehydrolysine also improved the incorporation efficiency of these two noncanonical amino acids. As the three identified mutations were found in the N‐terminal domain of PylRS that was separated from its catalytic domain for charging tRNA Pyl with a noncanonical amino acid, they could potentially be introduced to all other PylRS mutants to improve the 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.