Continuous directed evolution of aminoacyl-tRNA synthetases

Abstract

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.

Crystal structures reveal an elusive functional domain of pyrrolysyl-tRNA synthetase

“…Since the active site is located in the CTD 14,26 one must assume that the communication of the presence of both domains occurs through the tRNA. Supplementary Figure 6c also shows the importance of mutations (S158N, G343D) endowing the 24B CTD with substantial in vivo activity, which the wild-type chPylRS CTD lacks 19 . This may be explained by increased tRNA Pyl binding caused by the S158N mutation 15 (Supplementary Fig.…”

“…However, the NTD is not active by itself (Supplementary Fig. 6c) 19 . These data are in line with the fact that the NTD is required for in vivo PylRS activity 12 and that mutations in the NTD enhance pylS -dependent UAG 15 (ref.…”

“…For this purpose, we modified the effective phage-assisted continuous evolution (PACE) method 16 , which does not require prior structural knowledge for protein evolution and has been successfully applied to evolution of such proteins and enzymes as polymerases 16 , receptor binding proteins 17 , proteases 18 , and aaRSs 19 . One cornerstone of this technique is its customized continuous flow machine, uniquely utilizing a two-chambered system which houses Escherichia coli cells infected with M13 bacteriophage inside a secondary ‘lagoon’ vessel.…”

Evolved Cas9 variants with broad PAM compatibility and high DNA specificity

“…The resulting host cells were incubated overnight with SP containing ω(I12N)–dCas9. 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.…”

Playing with the Molecules of Life

“…29, 30 One recent exciting example involves the use of rapid phage-assisted continuous evolution (PACE) methodology to evolve highly efficient and selective aaRSs. 31 The x-ray crystal structures of a number of these evolved aaRSs reveal remarkable plasticity in the amino acid binding site such that a limited number of mutations can substantially alter the active site structure and specificity. 32, 33 More recently, it has been discovered that as a consequence of other ncAAs being absent from the negative selection, some aaRSs exhibit a degree of polyspecificity towards structurally related ncAAs.…”