Biology subjects

Svenningsen, T.

Publications and source records attributed to Svenningsen, T..

3 recordsLinked to original sources

bioRxiv2026

An Unusual Follower Peptide is Required for Biosynthesis of the Antibiotic Lasso Peptide Triculamin

Triculamin is a potent antibiotic lasso peptide first isolated in 1967. Previous studies have demonstrated that its biosynthesis follows a non-canonical logic unlike any other lasso peptide. In this study, we investigate the role of the unusual follower peptide and demonstrate that it is essential for efficient biosynthesis. Using structural prediction and targeted mutations of key conserved residues, we hypothesize that the interactions between the follower peptide and the macrocyclase create an enzyme-substrate complex that ensures delivery of the core peptide to the enzyme active site. Moreover, we demonstrate that analogs of the lasso peptide can be produced by modifying the core peptide, highlighting the substrate promiscuity of the lasso macrocyclase and identifying lysine-3 in the lasso peptide ring as the site of acetylation. Lastly, we achieve successful heterologous expression in Burkholderia sp. FERM 3421, which proves to be a superior heterologous host.

synthetic biology↗

bioRxiv2025

Biosynthesis, Structure, and Antibiotic Properties of Gelatinamin A, a Triculamin-like Lasso Peptide

Lasso peptides are structurally unique natural products endowed with high thermal and proteolytic stability, making them attractive as scaffolds for drug discovery. Recently, a new class of lasso peptides containing a second macrocycle, formed between a lysine sidechain and the C-terminus, was discovered, resulting in an even more compact architecture. Here, we report the first NMR structure of the class V lasso peptide, gelatinamin A. Using heterologous expression of the gelatinamin biosynthetic gene cluster (BGC) in Bacillus subtilis, we delineated the biosynthetic pathway through targeted gene deletions. We expressed and characterized the predicted transpeptidase, GelP, that catalyzes the formation of an isopeptide bond between Lys2 and the C-terminus and mediates the reversible conversion of gelatinamin B to gelatinamin A. In addition, we characterize GelT, an N-acetyltransferase that inactivates lasso peptide antimicrobial activity by acetylating a key lysine residue. Furthermore, we demonstrate that gelatinamin is highly potent against several important pathogens and that the activity is strongly bicarbonate-dependent. Finally, we propose a complete biosynthetic pathway for gelatinamin. The structural insight of gelatinamin A and the functional characterization of GelP provide the foundation for future discovery of class V lasso peptides and for engineering transpeptidases to modify other lasso peptide scaffolds.

biochemistry↗

bioRxiv2024

Convergent Evolution of the Antimycobacterial Lasso Peptide Triculamin

Triculamin is a ribosomally synthesized and post-translationally modified peptide (RiPP) lasso peptide with potent antimycobacterial activity, produced by an unusual, non-canonical biosynthetic gene cluster (BGC). In this study, we elucidate the biosynthetic pathway of triculamin through heterologous expression and show that the biosynthesis proceeds in the presence of a precursor (triA), macrocyclase (triC), and acetyltransferase (triT). Through in vitro triT acetylation and bioactivity assays, we show that acetylation functions as a resistance mechanism. Genomic searches of triculamin BGC genes across bacteria show that triculamin is more widely distributed than previously anticipated, as triculamin-like core peptides are found in at least three phyla in contrast to previously described lasso peptides that are typically restricted to one phylum. Triculamin BGCs with both canonical and non-canonical RiPP biosynthetic genes were identified. Two strains containing canonical triculamin-like BGCs were chemically characterized and shown to produce the novel triculamin-like lasso peptides palmamin and gelatinamin, the latter of which appears to have an unprecedented additional ring formation. Detailed phylogenetic investigation of the macrocyclases from triculamin-like BGCs suggests that these molecules are products of convergent evolution. These findings broaden the evolutionary and functional landscape of lasso peptides, revealing their unexpected diversification and cross-phylum distribution.

biochemistry↗