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Treffer: Hijacking a bacterial ABC transporter for genetic code expansion.

Title:
Hijacking a bacterial ABC transporter for genetic code expansion.
Authors:
Iype T; Department of Chemistry and Applied Biosciences (D-CHAB), ETH Zurich, Zurich, Switzerland., Fottner M; Department of Chemistry and Applied Biosciences (D-CHAB), ETH Zurich, Zurich, Switzerland., Böhm P; Department of Chemistry and Applied Biosciences (D-CHAB), ETH Zurich, Zurich, Switzerland., Piedrafita C; Center for Protein Assemblies, TUM School of Natural Sciences, Department of Bioscience, Technical University of Munich, Garching, Germany., Möller Y; Department of Chemistry and Applied Biosciences (D-CHAB), ETH Zurich, Zurich, Switzerland., Groll M; Center for Protein Assemblies, TUM School of Natural Sciences, Department of Bioscience, Technical University of Munich, Garching, Germany., Lang K; Department of Chemistry and Applied Biosciences (D-CHAB), ETH Zurich, Zurich, Switzerland. kathrin.lang@org.chem.ethz.ch.; Center for Protein Assemblies, TUM School of Natural Sciences, Department of Bioscience, Technical University of Munich, Garching, Germany. kathrin.lang@org.chem.ethz.ch.
Source:
Nature [Nature] 2025 Nov; Vol. 647 (8091), pp. 1045-1053. Date of Electronic Publication: 2025 Oct 15.
Publication Type:
Journal Article
Language:
English
Journal Info:
Publisher: Nature Publishing Group Country of Publication: England NLM ID: 0410462 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1476-4687 (Electronic) Linking ISSN: 00280836 NLM ISO Abbreviation: Nature Subsets: MEDLINE
Imprint Name(s):
Publication: Basingstoke : Nature Publishing Group
Original Publication: London, Macmillan Journals ltd.
MeSH Terms:
ATP-Binding Cassette Transporters*/metabolism , ATP-Binding Cassette Transporters*/genetics , ATP-Binding Cassette Transporters*/chemistry , Escherichia coli*/genetics , Escherichia coli*/metabolism , Genetic Code*/genetics , Amino Acids*/metabolism , Amino Acids*/genetics , Amino Acids*/chemistry , Escherichia coli Proteins*/metabolism , Escherichia coli Proteins*/genetics , Bacterial Proteins*/metabolism , Bacterial Proteins*/genetics, Directed Molecular Evolution ; Biological Transport
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Substance Nomenclature:
0 (ATP-Binding Cassette Transporters)
0 (Amino Acids)
0 (Escherichia coli Proteins)
0 (Bacterial Proteins)
Entry Date(s):
Date Created: 20251015 Date Completed: 20251127 Latest Revision: 20251201
Update Code:
20251202
PubMed Central ID:
PMC12657241
DOI:
10.1038/s41586-025-09576-w
PMID:
41094137
Database:
MEDLINE

Weitere Informationen

The site-specific encoding of non-canonical amino acids (ncAAs) provides a powerful tool for expanding the functional repertoire of proteins <sup>1-4</sup> . Its widespread use for basic research and biotechnological applications is, however, hampered by the low efficiencies of current ncAA incorporation strategies. Here we reveal poor cellular ncAA uptake as a main obstacle to efficient genetic code expansion and overcome this bottleneck by hijacking a bacterial ATP-binding cassette (ABC) transporter <sup>5</sup> to actively import easily synthesizable isopeptide-linked tripeptides that are processed into ncAAs within the cell. Using this approach, we enable efficient encoding of a variety of previously inaccessible ncAAs, decorating proteins with bioorthogonal <sup>6</sup> and crosslinker <sup>7</sup> moieties, post-translational modifications <sup>8,9</sup> and functionalities for chemoenzymatic conjugation. We then devise a high-throughput directed evolution platform to engineer tailored transporter systems for the import of ncAAs that were historically refractory to efficient uptake. Customized Escherichia coli strains expressing these evolved transporters facilitate single and multi-site ncAA incorporation with wild-type efficiencies. Additionally, we adapt the tripeptide scaffolds for the co-transport of two different ncAAs, enabling their efficient dual incorporation. Collectively, our study demonstrates that engineering of uptake systems is a powerful strategy for programmable import of chemically diverse building blocks.
(© 2025. The Author(s).)

Competing interests: K.L, M.F. and T.I are named as inventors on a pending patent application EP24205590.3. The other authors declare no competing interests.