Recombinant thiopeptides containing noncanonical amino acids
Xiaozhou Luo1, Claudio Zambaldo1, Tao Liu2
1Department of Chemistry, The Scripps Research Institute, La Jolla, CA 92037; Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037;
Summary
Researchers engineered thiopeptides, a class of antimicrobial peptides, to include noncanonical amino acids (ncAAs). This innovation allows for enhanced structural diversity and functional probing of these important biomolecules.
Area of Science:
- Biochemistry and Molecular Biology
- Synthetic Biology
- Antimicrobial Peptides
Background:
- Thiopeptides are a significant subclass of ribosomally synthesized and post-translationally modified peptides (RiPPs).
- These peptides possess complex structures and exhibit potent biological activities, notably antimicrobial properties.
Purpose of the Study:
- To generate novel thiopeptide variants incorporating noncanonical amino acids (ncAAs).
- To enable site-specific functionalization of thiopeptides for structural and activity studies.
- To explore the application of this methodology to other RiPP classes.
Main Methods:
- Introduction of orthogonal amber suppressor aminoacyl-tRNA synthetase/tRNA pairs into Bacillus cereus.
- Engineering a thiocillin producer strain for ncAA incorporation.
- Postbiosynthetic modification of ncAA-containing thiopeptides with biophysical probes (fluorophores, photo-cross-linkers).
Main Results:
- Successful generation of thiopeptides containing site-specifically incorporated ncAAs.
- Demonstration of bioorthogonal chemical reactivity in ncAA-containing thiopeptide variants.
- Facilitation of postbiosynthetic modification with probes, enabling detailed biological activity analysis.
Conclusions:
- The developed method allows for the creation of structurally diverse thiopeptides via ncAA incorporation.
- This approach provides a powerful tool for probing the biological activity of thiopeptides.
- The strategy is adaptable for expanding the chemical diversity of other RiPP families.
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