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.Wh...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 1.91μmol/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.展开更多
基金supported by National Key R&D Program of China(2024YFA0919900)National Natural Science Foundation of China(No.81803403)CAMS Innovation Fund for Medical Sciences(CIFMS,No.2021-I2M-1-029,China).
文摘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 1.91μmol/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.
