# Dual-probe genome mining identifies citrulassin N, a novel citrulline modified lasso peptide from Streptomyces sp. NAX00255

## TL;DR

Scientists discovered a new lasso peptide, citrulassin N, using a dual-probe method to find rare modified peptides in bacteria.

## Contribution

A dual-probe genome-mining strategy was developed to identify rare citrulline-modified lasso peptides.

## Key findings

- Citrulassin N is a novel citrulline-modified lasso peptide from Streptomyces sp. NAX00255.
- The dual-probe approach successfully identified a rare RiPP with a PAD-catalyzed modification.
- Structural analysis confirmed the unique citrulline modification and lasso topology.

## Abstract

Lasso peptides are a structurally distinctive class of ribosomally synthesized and post-translationally modified peptides (RiPPs) featuring a threaded rotaxane topology that confers remarkable thermal stability and protease resistance. Citrulassins represent a rare subgroup of lasso peptides distinguished by a citrulline residue generated through peptidylarginine deiminase (PAD)-catalyzed deimination of arginine. Prior to the identification of citrulassin A, such a modification had not been observed in RiPPs, and notably, the PAD-encoding gene is located outside the canonical lasso peptide biosynthetic gene cluster (BGC).

Here, we developed a dual-probe genome-mining strategy that integrates homology searches for both the lasso peptide cyclase (CitC) and a PAD homolog to selectively prioritize candidate producers from the IFB bacterial genome database. Guided by this strategy, fermentation and targeted isolation led to the discovery of citrulassin N (1) from Streptomyces sp. NAX00255.

Comprehensive structural elucidation using NMR spectroscopy and tandem mass spectrometry confirmed citrulassin N as a novel citrulline-modified lasso peptid.

This study expands the structural diversity of citrulline-containing lasso peptides, demonstrates the utility of a dual-probe genome-mining approach for identifying RiPPs with rare post-translational modifications, and provides a practical framework for the targeted discovery of functionally decorated RiPP natural products.