Structure revision of cryptosporioptides and determination of the genetic basis for dimeric xanthone biosynthesis in fungi

Three novel dimeric xanthones, cryptosporioptides A-C were isolated from <i>Cryptosporiopsis</i> sp. 8999 and their structures elucidated. Methylation of cryptosporioptide A gave a methyl ester with identical NMR data to cryptosporioptide, a compound previously reported to have been isolated from the same fungus. However, HRMS analysis revealed that cryptosporioptide is a symmetrical dimer, not a monomer as previously proposed, and the revised structure was elucidated by extensive NMR analysis. The genome of <i>Cryptosporiopsis</i> sp. 8999 was sequenced and the dimeric xanthone (<i>dmx</i>) biosynthetic gene cluster responsible for the production of the cryptosporioptides was identified. Gene disruption experiments identified a gene (<i>dmxR5</i>) encoding a cytochrome P450 oxygenase as being responsible for the dimerisation step late in the biosynthetic pathway. Disruption of <i>dmxR5</i> led to the isolation of novel monomeric xanthones. Cryptosporioptide B and C feature an unusual ethylmalonate subunit: a hrPKS and acyl CoA carboxylase are responsible for its formation. Bioinformatic analysis of the genomes of several fungi producing related xanthones, <i>e.g.</i> the widely occurring ergochromes, and related metabolites allows detailed annotation of the biosynthetic genes, and a rational overall biosynthetic scheme for the production of fungal dimeric xanthones to be proposed.