Phenylspirodrimanes are a class of structurally diverse meroterpenoids, including the bioactive dimer stachybocin A (1) and the high-reactivity monomer stachybotrydial (2), which are isolated from the genus Stachybotrys. Whereas the biosynthetic pathway of these phenylspirodrimane meroterpenoids has remained elusive. Herein, we deciphered the complete biosynthetic pathway of 2 with unprecedented two gene clusters and five discrete genes by genome mining, gene inactivation, heterologous expression, biochemical experiments, and especially combining with transcriptome-based hierarchical clustering and expression correlation analyses. Totally, 11 genes for the phenylspirodrimane core skeleton formation, 8′-methyl oxidation, and 3-OH epimerization were efficiently discovered and functionally characterized. Notably, these biosynthetic genes are distributed across seven distinct regions, with a rare combination of multiple gene clusters and genes outside the clusters. Bioactivity assays revealed that four intermediates 6−8, and 9a exhibited significant inhibitory effect on the inactivated state hNaV1.2 channels with IC50 values of 0.15, 0.04, 0.28, and 1.91 μmol/L, respectively. These findings expand our understanding of phenylspirodrimane-type meroterpenoid biosynthesis and underscore the utility of transcriptome-based hierarchical clustering and expression correlation analyses for identifying unclustered biosynthetic genes in fungi.
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