Discovery and Characterization of Actinosynnelassin: An Anti-<i>Pseudomonas fluorescens</i> Lasso Peptide Derived from a Large Precursor Open Reading Frame

Lasso peptides, a unique class of ribosomally synthesized and post-translationally modified peptide (RiPP), are challenging to synthesize chemically, making the discovery of new peptides and their biosynthetic pathways essential. This study reports the discovery and characterization of a novel lasso peptide, actinosynnelassin, from <i>Actinosynnema pretiosum</i> subsp. <i>auranticum</i> DSM 44131. By overexpressing an endogenous TetR/AcrR family regulator and employing OSMAC (One Strain Many Compounds)-guided fermentation screening, several endogenous secondary metabolite biosynthetic gene clusters (BGCs) were activated, resulting in the isolation of actinosynnelassin. The 3D structure of actinosynnelassin, confirmed by nuclear magnetic resonance (NMR) NOE-derived distance constraints, features a 9-aa macrolactam ring, a 6-aa loop, and a 2-aa tail, with the ring encircling the tail between three aromatic bulkier residues. The minimal inhibitory concentration (MIC) tests indicate that actinosynnelassin inhibits several Gram-positive bacteria and <i>Pseudomonas fluorescens</i>, making it the first reported lasso peptide to inhibit <i>P. fluorescens</i>. The predicted open reading frame (ORF) of the precursor peptide may be translated into a 331-aa fusion protein featuring an N-terminal AraC/XylS family transcriptional regulator, making it longer than typical lasso precursors. Thus, discovering this large precursor ORF enhances our understanding of lasso peptide BGCs with unusual architectures and enables the finding of other unique lasso peptides.