Ribosomally synthesized and post-translationally modified peptides, RiPPs, are a class of natural products made by bacteria, plants, and fungi that have drawn intense research interest for their structural diversity and potent bioactivity. Among fungal RiPPs, the MSDIN family was thought to follow a single architectural blueprint: a precursor protein bearing an N-terminal leader sequence that guides recognition and cyclization by prolyl oligopeptidase B, POPB, an enzyme specific to Amanita species. The MSDIN family, named for a conserved five-residue leader motif, encompasses the amatoxins and phallotoxins responsible for the lethal toxicity of death cap mushrooms, and more than 40 predicted MSDIN genes have been identified in Amanita phalloides, the world's deadliest mushroom. Yet the chemical products of most of these genes remain unidentified, hinting at hidden biosynthetic mechanisms.
Researchers in the Pringle and Keller Groups at the University of Wisconsin-Madison and the Drott Group at the USDA-ARS Cereal Disease Laboratory, published in PNAS, combined chemistry-guided metabolite isolation with an updated bioinformatic pipeline to test whether A. phalloides harbors non-canonical MSDIN genes. The work was led by Dr. Sung Chul Park, a postdoctoral researcher in the Keller Group. They extracted and purified cyclic peptides from field-collected mushrooms and characterized nine compounds by nuclear magnetic resonance, NMR, spectroscopy, Marfey's analysis for absolute configuration, and tandem mass spectrometry. They then updated existing genome-mining scripts to relax constraints on leader sequence presence and the conserved C-terminal proline residue, screening 88 A. phalloides genomes for non-canonical MSDIN genes. RNA sequencing of wild mushrooms collected from both native European and invasive Californian populations provided quantitative data on differential MSDIN expression across ranges.
The team confirmed structures for nine monocyclic peptides and linked five to MSDIN genes with non-canonical architectures. Three categories of non-canonical genes emerged: leaderless, lacking any N-terminal leader sequence; prolineless, lacking the conserved C-terminal proline in the core; and leaderless-prolineless, lacking both features. Cycloamanides C1/C2 and D1/D2 were traced to a single genomic locus encoding the leaderless core sequences MLGFLVLP and MLGFLPLP, two alleles differing by a single valine-to-proline substitution. mRNA sequencing confirmed the absence of any upstream start codon in any reading frame, establishing that this locus is genuinely leaderless. Cycloamanide A was matched to a prolineless gene, demonstrating that the conserved C-terminal proline, previously considered essential for POPB-catalyzed cyclization, may not be an absolute requirement. Transcriptomic profiling revealed that the leaderless cycloamanide C1/C2 transcript and the prolineless cycloamanide A transcript ranked among the 15 most highly expressed genes in Amanita mushrooms, expressed 1,000 to 10,000 times more than most canonical MSDIN transcripts. Both were significantly more highly expressed in invasive Californian populations than in native European populations, and cycloamanide C1/C2 expression correlated positively with mushroom maturity specifically in the invasive range. A phylogenetic survey of 644 Agaricales genomes confirmed that leaderless MSDINs are restricted to the genus Amanita and represent ancient, conserved lineages that have diversified independently across the lethal Amanita clade.
These findings overturn a foundational assumption in fungal natural product biosynthesis: leader peptides are not absolute prerequisites for RiPP synthesis in fungi. Current genome-mining algorithms built on these assumptions require revision to capture the full scope of fungal RiPP diversity. The markedly elevated expression of leaderless and prolineless MSDINs in invasive A. phalloides populations raises the provocative question of whether these peptides contribute to the ecological success of this mushroom in new habitats. Confirming the structures of these non-canonical compounds also opens pathways for probing their molecular targets, an avenue already proven productive with amatoxin-based antibody-drug conjugates now advancing through clinical trials for cancer treatment.
