A structurally conserved helix enables leader-independent tyramine splicing of proteins.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are natural products that feature diverse modifications. They show a wide range of biological activities and are therefore of great interest for drug discovery and protein engineering. An unusual modification found in spliceotide RiPPs is the installation of β-amino acid residues with diverse side chains, generated by backbone excision of a tyramine moiety derived from tyrosine. We have previously shown that the modification can be adapted to protein engineering to greatly expand the set of amino acid residues and to introduce unique reaction centers for site-directed modification. To understand requirements for splicease-substrate interactions, we investigated the role of a RiPP recognition element (RRE) in spliceotide biosynthesis and provide evidence that it acts as an activator and enables leader-independent protein splicing. We leveraged this knowledge to engineer a simplified splicease system derived from B26 that processes splice tags introduced into proteins with high efficiency. This work expands the toolbox for peptide and protein engineering and contributes to an understanding of substrate recognition in RiPP biosynthesis.