Abstract
Applying enzymes from unique biosynthetic pathways in a combinatorial fashion to garner new-to-nature products has been a long-standing goal of synthetic biology. Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a family of natural products that comprise a broad array of chemical and structural diversity, including mechanically interlocked molecular architectures. Guided by supramolecular recognition of an N-terminal leader sequence, the core region of a precursor peptide is decorated by tailoring enzymes to generate a mature RiPP. Through repositioning the leader sequence of the fuscimiditide precursor peptide C-terminal to the core, we have shown that substrate-selective post-translational modification by the graspetide synthetase, ThfB, is retained in cellulo and in vitro. Reconstitution of the ThfB-mediated cyclization of precursors with the native N-terminal or repositioned C-terminal leader sequence in vitro revealed a modest 2-fold reduction in the rate of enzymatic modification upon repositioning the leader. Rearrangement of the precursor peptide enabled the generation of chimeric RiPP products that were decorated by both lasso peptide and graspetide family enzymes guided by two leader sequences (one N-terminal and one C-terminal). This leader peptide engineering strategy unlocks access to mechanically interlocked peptide products with post-translational modifications not seen in nature, expanding the structural diversity possible in RiPPs.