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, 13 (12), 1253-1260

Continuous Directed Evolution of aminoacyl-tRNA Synthetases


Continuous Directed Evolution of aminoacyl-tRNA Synthetases

David I Bryson et al. Nat Chem Biol.

Erratum in


Directed evolution of orthogonal aminoacyl-tRNA synthetases (AARSs) enables site-specific installation of noncanonical 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 noncanonical 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.


Figure 1
Figure 1. Overview of PACE positive selections for the continuous evolution of AARS activity and the noncanonical amino acids used in this study
(a) Strategies for linking AARS activity to the expression of gene III, which encodes the pIII protein required for phage to be infectious. In strategy 1, AARS-catalyzed aminoacylation of an amber suppressor tRNA enables translation of full-length T7 RNAP from a transcript containing a premature amber stop codon. T7 RNAP subsequently drives expression of gene III from the T7 promoter (PT7). In strategy 2, orthogonal aminoacylation permits full-length translation of pIII from gene III mRNA containing a premature amber stop codon. (b) Diagram of PACE with selection strategy 1 plasmids shown. The accessory plasmid (AP) encodes gene III and the amber suppressor tRNA. The complementary plasmid (CP) encodes T7 RNAP. The mutagenesis plasmid (MP) increases the rate of evolution during PACE through arabinose-induced production of mutagenic proteins. The selection phage encodes all phage genes except gene III, which is replaced by the evolving AARS gene. SPs capable of catalyzing aminoacylation of the amber suppressor tRNA in the host E. coli result in production of pIII protein. Under continuous dilution in the fixed-volume vessel (the “lagoon”), phage that trigger the production of pIII propagate faster than the rate of dilution, resulting in the continuous enrichment of SPs encoding active AARS variants. (c) Non-canonical amino acids used in this study.
Figure 2
Figure 2. Evolution of AARS activity during mock PACE
(a) p-NFRS was challenged to aminoacylate the amber suppressor tRNA in the absence of its cognate ncAA substrate, p-NF, over 48 h of positive selection PACE conducted in two separate lagoons (L1 and L2). Enhanced mutagenesis from the MP was supplied in L2 only. Phage titers of L2 (green) rapidly increased after 16 h, while titers in L1 (magenta) were relatively stable throughout the evolution. (b) Mutations in PACE-evolved clones and the relative amino acid substrate specificities of clones from L2. Relative aminoacylation activity was compared in the PACE host strain by measuring the luminescence signal resulting from amber suppression of a premature stop codon at position 361 of a luciferase gene (luxAB). More coding mutations were obtained in phage isolates from L2, in which the MP provided enhanced mutagenesis, and every characterized L2 mutant emerged from PACE with increased activity on endogenous amino acids (no ncAA) compared to the progenitor enzyme, p-NFRS. Values and error bars in b reflect the mean and s.d. of at least three independent biological replicates.
Figure 3
Figure 3. Continuous evolution and characterization of chimeric pyrrolysyl-tRNA synthetase (chPylRS) variants with enhanced aminoacylation activity
(a) PACE was performed in three segments designed to gradually increase selection stringency. The first two segments (Pyl-1 and Pyl-2) used the selection requiring amber suppression of two stop codons in T7 RNAP, and the final segment (Pyl-3) used the selection requiring direct amber suppression of stop codons in gene III. The number of stop codons in the gene required for each selection and the concentration of BocK substrate are shown above the phage titer graph. Dotted lines (black) indicate transfer of evolved phage from the end of each PACE segment into the subsequent segment. Colored triangles indicate convergence toward the specified mutations. (b,c) The relative expression of luciferase containing BocK at position 361 resulting from aminoacylation by progenitor enzyme, chPylRS, compared to evolved variants from the end of PACE segment Pyl-1 (b) or compared to variants containing only the consensus mutations from the end of each PACE segment (c). Label colors correspond to PACE segments in a. (d) The relative efficiency of multisite BocK incorporation into sfGFP resulting from aminoacylation by chPylRS variants with or without beneficial mutations discovered in PACE (V31I, T56P, H62Y, and A100E; IPYE). (e) The relative efficiency of AcK incorporation at position 2 of sfGFP resulting from aminoacylation by AcK3RS variants with or without transplanted mutations from PACE. Values and error bars in be reflect the mean and s.d. of at least three independent biological replicates.
Figure 4
Figure 4. Evolution of AARS variants from dual positive- and negative-selection PACE with greatly improved amino acid specificity
(a) Strategy for linking undesired aminoacylation to gene III-neg expression, which encodes the pIII-neg protein. When undesired aminoacylation occurs in the negative selection, pIII-neg is produced, impeding progeny phage infectivity. In the absence of undesired aminoacylation, only pIII is produced, resulting in infectious phage progeny. Negative-selection stringency is modulated by ATc concentration. (b) Diagram of dual-selection PACE using simultaneous positive and negative selections. Evolving phage are continuously cross-seeded between positive and negative selection lagoons at a 50-fold dilution. (c) The relative site-specific incorporation efficiency of either endogenous amino acids (no ncAA), p-NF, or p-IF at position 39 of sfGFP resulting from aminoacylation by p-NFRS, p-IFRS, or evolved variants from PACE (Iodo.1, Iodo.5, Iodo.7, and Iodo.8). (d) Predicted position of mutations evolved during dual-selection PACE. The shown crystal structure is the p-NFRS protein sequence aligned to PDB 2AG6 (ref. 37), which is the crystal structure of an AARS that has the identical protein sequence of p-IFRS and is bound to the ncAA substrate, p-bromo-L-phenylalanine (dark blue). The colored spheres in the crystal structure correspond to the colored mutations in the table to the left. Active-site residues within a 5 Å radius around the ncAA substrate are colored gray. Values and error bar in c reflect the mean and s.d. of at least three independent biological replicates.

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