Global analysis of fungal biosynthetic gene clusters reveals the diversification of diketopiperazine biosynthesis.

Fungi are a vast reservoir of structurally diverse natural products, yet their biosynthetic potential remains underexplored. Here, we present the most comprehensive fungal biosynthetic gene cluster (BGC) atlas, comprising 303,983 BGCs predicted from 13,125 fungal genomes, revealing numerous underexplored taxa harboring extensive biosynthetic diversity. These BGCs were classified into 43,984 gene cluster families (GCFs), of which 99.6 % remain uncharacterized. Gene-centric analysis revealed the presence of 359 cyclodipeptide synthases (CDPSs) of three distinct subcategories and 9,482 nonribosomal peptide synthetases (NRPSs) responsible for diketopiperazine biosynthesis in the fungal BGC atlas. Notably, 304 type I CDPSs were exclusively found in Fusarium, with one confirmed to be responsible for the synthesis of cyclo(Leu-Ile) and cyclo(Pro-Leu). Bioinformatics analysis suggests that three newly identified indole diketopiperazine alkaloids, isolated from a marine-derived Aspergillus strain, are synthesized by an NRPS. This study presents the most comprehensive fungal BGC atlas and highlights the diversification of diketopiperazine biosynthesis in fungi.