Dual-Probe Genome Mining Identifies Citrulassin N, a Novel Citrulline Modified Lasso Peptide from Streptomyces sp. NAX00255

Zi-Ru Wang^1,2Chao Zeng¹Zhang-Yuan Yan²Dan Feng^1*Zi-Fei Xu^3*

• ¹Affiliated Hospital of Jiangsu University, Zhenjiang, China
• ²State Key Laboratory of Pharmaceutical Biotechnology, Institute of Functional Biomolecules, Nanjing University School of Life Sciences, Nanjing, China
• ³State Key Laboratory of Phytochemistry and Natural Medicines, Chinese Academy of Sciences Kunming Institute of Botany, Kunming, China

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 peptide. 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.