Continuous directed evolution of aminoacyl-tRNA synthetases

By D. Bryson, Chenguang Fan, Li-Tao Guo, Corwin A. Miller, Dieter Söll, David R. Liu. 2017

226 citations recorded by the source corpus.

Directed evolution of orthogonal aminoacyl-tRNA synthetases (AARSs) enables site-specific installation of non-canonical amino acids (ncAAs) into proteins. Traditional evolution techniques typically produce AARSs with greatly reduced activity and selectivity compared to their wild-type counterparts. We designed phage-assisted continuous evolution (PACE) selections to rapidly produce highly active and selective orthogonal AARSs through hundreds of generations of evolution. PACE of a chimeric Methanosarcina spp. pyrrolysyl-tRNA synthetase (PylRS) improved its enzymatic efficiency (kcat/KMtRNA) 45-fold compared to the parent enzyme. Transplantation of the evolved mutations into other PylRS-derived synthetases improved yields of proteins containing non-canonical residues up to 9.7-fold. Simultaneous positive and negative selection PACE over 48 h greatly improved the selectivity of a promiscuous Methanocaldococcus jannaschii tyrosyl-tRNA synthetase variant for site-specific incorporation of p-iodo-L-phenylalanine. These findings offer new AARSs that increase the utility of orthogonal translation systems and establish the capability of PACE to efficiently evolve orthogonal AARSs with high activity and amino acid specificity.

Subfields: Molecular Biology

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Evolved Cas9 variants with broad PAM compatibility and high DNA specificity (2018, positive): “This phage-assisted non-continuous evolution (PANCE) system 19,21 preferentially replicates Cas9 variants that bind a greater variety of PAM sequences, similar to PACE, but with lower stringency since there is no outflow of phage.”