Orthogonal synthetases for polyketide precursors

Schepartz, A.; Fricke, R.; Swenson, C.; Roe, L. T.; Hamlish, N.; Ad, O.; Smaga, S.; Gee, C. L.

doi:10.1101/2022.02.28.482149

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bioRxiv · 10.1101/2022.02.28.482149

Orthogonal synthetases for polyketide precursors

Schepartz, A.·Fricke, R.·Swenson, C.·Roe, L. T.·Hamlish, N.·Ad, O.·Smaga, S.·Gee, C. L.

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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.

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Schepartz, A., Fricke, R., Swenson, C., Roe, L. T., Hamlish, N., Ad, O., Smaga, S., Gee, C. L.. 2022-03-01. Orthogonal synthetases for polyketide precursors. https://doi.org/10.1101/2022.02.28.482149

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