Current textbook knowledge holds that the structural scope of ribosomal biosynthesis is based exclusively on α-amino acid backbone topology. Here we report the genome-guided discovery of bacterial pathways that posttranslationally create β-amino acid-containing products. The transformation is widespread in bacteria and is catalyzed by an enzyme belonging to a previously uncharacterized radical -adenosylmethionine family. We show that the β-amino acids result from an unusual protein splicing process involving backbone carbon-carbon bond cleavage and net excision of tyramine. The reaction can be used to incorporate diverse and multiple β-amino acids into genetically encoded precursors in In addition to enlarging the set of basic amino acid components, the excision generates keto functions that are useful as orthogonal reaction sites for chemical diversification.

The wide range of moieties installed in ribosomally synthesized and post-translationally modified peptides (RiPPs) suggests largely untapped potential for protein engineering. However, many RiPP maturases recognize target peptide precursors through an N-terminal leader sequence that is challenging to adapt to proteins. We have recently reported a family of enzymes that splice XYG sites in RiPPs to install αketo-β-amino acids. Backbone modifications influence diverse protein properties, yet the toolkit to install βamino acids is limited. Here we report their leaderindependent incorporation into proteins in E. coli. Integrating an 11-residue splice tag into six different proteins permitted the site-selective introduction of βresidues in vivo. The motif fusion at C-, N-terminal, and internal positions yielded various β-residues. Our approach complements the few existing methods to introduce β-amino acids or ketone-bearing moieties, suggesting diverse applications in chemical biology.

The tetrazine ligation is an inverse electron-demand Diels–Alder reaction widely used for bioorthogonal modifications due to its versatility, site specificity and fast reaction kinetics. A major limitation has been the incorporation of dienophiles in biomolecules and organisms, which relies on externally added reagents. Available methods require the incorporation of tetrazine-reactive groups by enzyme-mediated ligations or unnatural amino acid incorporation. Here we report a tetrazine ligation strategy, termed TyrEx (tyramine excision) cycloaddition, permitting autonomous dienophile generation in bacteria. It utilizes a unique aminopyruvate unit introduced by post-translational protein splicing at a short tag. Tetrazine conjugation occurs rapidly with a rate constant of 0.625 (15) M−1 s−1 and was applied to produce a radiolabel chelator-modified Her2-binding Affibody and intracellular, fluorescently labelled cell division protein FtsZ. We anticipate the labelling strategy to be useful for intracellular studies of proteins, as a stable conjugation method for protein therapeutics, as well as other applications.

The wide range of moieties installed in ribosomally synthesized and post-translationally modified peptides (RiPPs) suggests largely untapped potential for protein engineering. However, many RiPP maturases recognize target peptide precursors through an N-terminal leader sequence that is challenging to adapt to proteins. We have recently reported a family of enzymes that splice XYG sites in RiPPs to install αketo-β-amino acids. Backbone modifications influence diverse protein properties, yet the toolkit to install βamino acids is limited. Here we report their leaderindependent incorporation into proteins in E. coli. Integrating an 11-residue splice tag into six different proteins permitted the site-selective introduction of βresidues in vivo. The motif fusion at C-, N-terminal, and internal positions yielded various β-residues. Our approach complements the few existing methods to introduce β-amino acids or ketone-bearing moieties, suggesting diverse applications in chemical biology.