Repositioning the Leader Peptide in Graspetide Biosynthesis

Area of Science:

• Synthetic biology
• Biochemistry
• Natural product synthesis

Background:

• Ribosomally synthesized and post-translationally modified peptides (RiPPs) exhibit diverse structures, including mechanically interlocked architectures.
• Tailoring enzymes modify precursor peptides guided by N-terminal leader sequences to produce mature RiPPs.

Purpose of the Study:

• To investigate the effect of repositioning the leader sequence of fuscimiditide precursor peptide from N-terminal to C-terminal.
• To explore the generation of novel RiPP structures using leader peptide engineering.

Main Methods:

• Enzymatic modification and cyclization of precursor peptides with native and C-terminal leader sequences.
• In cellulo and in vitro assays using graspetide synthetase ThfB.
• Generation of chimeric RiPPs using engineered precursor peptides.

Main Results:

• Substrate-selective post-translational modification by ThfB was retained with the C-terminal leader sequence.
• A modest 2-fold reduction in modification rate was observed upon leader sequence repositioning.
• Chimeric RiPPs were successfully generated, incorporating modifications from both lasso peptide and graspetide families.

Conclusions:

• Repositioning RiPP leader sequences is a viable strategy for generating novel peptide products.
• This approach expands the structural diversity of RiPPs, enabling access to new mechanically interlocked molecules.
• Leader peptide engineering offers a powerful tool for creating new-to-nature peptide architectures.