Fig. 3: Directed evolution of PylRS variants with specificity for hydroxy acids with aromatic side chains.
From: Genetically programmed cell-based synthesis of non-natural peptide and depsipeptide macrocycles
a, Structures of hydroxy acids with aromatic side chains attached to the beta carbon, 14–16, used in this study. b, Strategy to generate PylRS variants with specificity towards hydroxy acids with aromatic side chains. Directed evolution of MmPylRS (light grey) yielded variants MmPylRS(PheOH_1) and MmPylRS(PheOH_6), which selectively incorporate 14 (phenyllactic acid). Further directed evolution of MmPylRS(PheOH_6), using libraries that mutate amino acids in the enzyme involved in recognizing the side chain of the substrate led to the discovery of ArOH-RS. This variant enables the incorporation of hydroxy acids 15 and 16 that bear substituents on the phenyl ring. c, Activity of MmPylRS(PheOH_1), MmPylRS(PheOH_6) and MmPylRS(ArOH) with hydroxy acids that bear aromatic side chains (14–16). The production of sfGFP was measured in Syn61∆3(ev5) cells that contained the indicated aaRS and cognate tRNA, an sfGFP-3-TCG gene and the indicated hydroxy acid. Error bars represent standard deviation from the mean of three biological replicates. d, sfGFP-His6 was produced in the presence of 14, 15 and 16 in DH10B cells transformed with sfGFP-3-TAG, the indicated aaRS and cognate tRNA. The identity of the monomer incorporated was verified by ESI–MS. In all three cases, only the mass that corresponded to hydroxy acid incorporation followed by hydrolysis of the ester bond at position 3 was detected, which confirmed the selectivity of each aaRS for the indicated hydroxy acid.
