Repositioning the Leader Peptide in Graspetide Biosynthesis

TL;DR

Scientists repositioned a leader peptide in a biosynthetic pathway to create new peptide structures with expanded chemical diversity.

Contribution

A novel leader peptide engineering strategy enables the synthesis of chimeric RiPPs with interlocked structures not found in nature.

Findings

Repositioning the leader sequence retained enzymatic modification by ThfB in cellulo and in vitro.

Chimeric RiPP products were generated using two leader sequences and multiple tailoring enzymes.

The strategy enables access to new mechanically interlocked peptides with expanded structural diversity.

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.

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