Genomic and Metabolite Analysis-Driven Revision of Basidiomycete Natural Product Biosynthetic Pathway

The biosynthetic study of secondary metabolites in ectomycorrhizal mushrooms has been hindered by limited mycelial growth and transcriptional silencing of biosynthetic genes under artificial conditions. Ustalic acid, a Na⁺/K⁺-ATPase inhibitor and a common cause of mushroom poisoning in Japan, was originally identified in the fruiting body of Tricholoma kakishimeji and was thought to be absent in its mycelium. Here, we demonstrate for the first time the production of ustalic acid in the cultured mycelia of T. stans and T. albobrunneum, two species that were recently segregated from T. ustale by phylogenetic analyses. LC-MS profiling also detected polyporic acid and additional ions consistent with oxidized derivatives of polyporic acid but failed to identify phlebiarubrone, which was previously considered as the biosynthetic precursor of ustalic acid. Genome sequencing and bioinformatic analysis of the sequence information identified a candidate biosynthetic gene cluster for ustalic acid. This cluster was named the uta cluster, and heterologous expression of the uta genes in Aspergillus nidulans afforded polyporic acid, supporting its role in the scaffold assembly. While the downstream biosynthetic steps remain to be investigated, this work establishes the basis for future biochemical and genetic studies of ustalic acid biosynthesis.