In Vivo Production of Diverse β‐Amino Acid‐Containing Proteins

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 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.

Adding α,α-disubstituted and β-linked monomers to the genetic code of an organism

“…In future work we will leverage the cellular acylation of tRNAs with ncMs to enable translation-based selections for orthogonal ribosomes 34 , 41 – 43 that can polymerize ncMs that are poor substrates for natural translation, and facilitate the encoded cellular synthesis of polymers composed of more diverse ncMs. The repertoire of non-canonical polymers may be further enhanced by leveraging post-translational modifications and ligations 44 – 49 .…”