Abstract 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 α‐keto‐β‐amino acids. Backbone modifications influence diverse protein properties, yet the toolkit to install β‐amino acids is limited. Here we report their leader‐independent incorporation into proteins in E. coli. Integrating an 11‐residue splice tag into six different proteins permitted the site‐selective introduction of β‐residues 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.
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