Efficient genetic code expansion without host genome modifications.

Autor: Costello A; Department of Chemistry, The Scripps Research Institute, La Jolla, CA, USA.; Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA, USA., Peterson AA; Department of Chemistry, The Scripps Research Institute, La Jolla, CA, USA.; Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA, USA., Lanster DL; Department of Chemistry, The Scripps Research Institute, La Jolla, CA, USA.; Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA, USA.; Doctoral Program in Chemical and Biological Sciences, The Scripps Research Institute, La Jolla, CA, USA., Li Z; Department of Chemistry, The Scripps Research Institute, La Jolla, CA, USA.; Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA, USA.; Doctoral Program in Chemical and Biological Sciences, The Scripps Research Institute, La Jolla, CA, USA., Carver GD; Department of Molecular Biology, Princeton University, Princeton, NJ, USA., Badran AH; Department of Chemistry, The Scripps Research Institute, La Jolla, CA, USA. ahbadran@scripps.edu.; Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA, USA. ahbadran@scripps.edu.
Jazyk: angličtina
Zdroj: Nature biotechnology [Nat Biotechnol] 2024 Sep 11. Date of Electronic Publication: 2024 Sep 11.
DOI: 10.1038/s41587-024-02385-y
Abstrakt: Supplementing translation with noncanonical amino acids (ncAAs) can yield protein sequences with new-to-nature functions but existing ncAA incorporation strategies suffer from low efficiency and context dependence. We uncover codon usage as a previously unrecognized contributor to efficient genetic code expansion using non-native codons. Relying only on conventional Escherichia coli strains with native ribosomes, we develop a plasmid-based codon compression strategy that minimizes context dependence and improves ncAA incorporation at quadruplet codons. We confirm that this strategy is compatible with all known genetic code expansion resources, which allowed us to identify 12 mutually orthogonal transfer RNA (tRNA)-synthetase pairs. Enabled by these findings, we evolved and optimized five tRNA-synthetase pairs to incorporate a broad repertoire of ncAAs at orthogonal quadruplet codons. Lastly, we extend these resources to an in vivo biosynthesis platform that can readily create >100 new-to-nature peptide macrocycles bearing up to three unique ncAAs. Our approach will accelerate innovations in multiplexed genetic code expansion and the discovery of chemically diverse biomolecules.
(© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)
Databáze: MEDLINE
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