Pyrrolysine aminoacyl-tRNA synthetase sequences after directed evolution using the PANCE method

In this study, we used phage-associated directed rapid evolution (PANCE) to generate novel pyrrolysine aminoacyl-tRNA synthetase (aaRS) variants capable of aminoacylating pyrrolysine tRNA with noncanonical amino acids (ncAAs). These amino acids included tyrosine and phenylalanine derivatives such as O-methyl-L-tyrosine, 4-azido-L-phenylalanine, O-allyl-L-tyrosine, and 2-chloro-L-phenylalanine. The aaRS sequences were introduced into the genome of bacteriophage M13. Mutagenesis of the sequences was dependent on the activity of a mutagenesis plasmid transformed into E. coli strain S2060 cells. The synthetases underwent four selection rounds, each including positive and negative selection steps. Finally, MiSeq (Illumina) sequencing was performed for the synthetases, the activity of which had been previously confirmed by fluorescence analysis upon incorporation of the corresponding ncAA into the sfGFP protein, and the selectivity by mass spectrometry analysis of sfGFP.