Orthogonal synthetases for polyketide precursors

Abstract

The absence of orthogonal aminoacyl-tRNA synthetases that accept non-L--amino acids is the primary bottleneck hindering the in vivo translation of sequence-defined hetero-oligomers. Here we report PylRS enzymes that accept -hydroxy acids, -thio acids, N-formyl-L--amino acids, and -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 -carboxyl acid substituent. This work emphasizes the potential of PylRS-derived enzymes for acylating tRNA with monomers whose -substituent diverges significantly from the -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.