Orthogonal synthetases for polyketide precursors

The absence of orthogonal aminoacyl-tRNA synthetases that accept non-L-α-amino acids is the primary bottleneck hindering the in vivo translation of sequence-defined hetero-oligomers. Here we report PylRS enzymes that accept α-hydroxy acids, α-thio acids, N -formyl-L-α-amino acids, and α-carboxyl acid monomers (malonic acids) that are formally precursors to polyketide natural products. These monomers are all accommodated and accepted by the translation apparatus in vitro . High-resolution structural analysis of the complex between one such PylRS enzyme and a meta -substituted 2-benzylmalonate derivative reveals an active site that discriminates pro -chiral carboxylates and accommodates the large size and distinct electrostatics of an α-carboxyl acid substituent. This work emphasizes the potential of PylRS-derived enzymes for acylating tRNA with monomers whose α-substituent diverges significantly from the α-amine embodied in proteinogenic amino acids. These enzymes could act in synergy with natural or evolved ribosomes to generate diverse sequence-defined non-protein hetero-oligomers.