Rational deployment of molecular markers for the benzenediol lactones and related compounds biosynthesis in filamentous fungi

Fungal benzenediol lactones (BDLs) are hydrophobic secondary metabolites that include the mycotoxin zearalenone, the phytotoxin curvularin, and related compounds such as cytosporones and cladosporin. The extensive diversity of macrolactones likely reflects long-standing microbial competition for resource-limited niches. We screened 97 fungal isolates for highly reducing polyketide synthases (HR-PKSs) implicated in BDL biosynthesis, designed degenerate markers for conserved HR-PKS domains, and validated them in silico against 1,039 reference genomes. To automate candidate selection, we developed a customizable pipeline for classifying nucleotide or protein sequences by phylogenetic placement on a curated reference tree (https://github.com/gkoczyk/micro_phyloplace). Phylogenetic placement of amplicons against a large set of reference PKSs identified putative BDL/BDL-like compound producers among known and previously uninvestigated genera in <i>Diaporthales</i> (<i>Coniella</i>, <i>Diaporthe</i>, <i>Diaporthella</i>, <i>Valsa</i>), <i>Hypocreales</i> (<i>Fusarium</i>, <i>Ilyonectria</i>, <i>Pochonia</i>), as well as <i>Curvularia</i>, <i>Penicillium</i>, <i>Phoma</i> and <i>Talaromyces</i> spp., demonstrating utility for environmental screening. Our analysis underscores both the efficacy and challenges of amplification with degenerate primers; nevertheless, phylogenetic placement offers a viable, low-cost screen for targeted subsets of highly diverse gene families.