Biosynthesis of Xyloketals: Furanochromene Skeleton Formed via Heterodimerization of Two Orsellinic Acid-Branched Metabolites

The furanochromene scaffold is a privileged structural motif widely distributed in bioactive natural products, with fungi-derived xyloketals standing out for their unique 2,3,3a,9a-tetrahydro-4<i>H</i>-furo[2,3-<i>b</i>]chromene skeleton and potent cardiovascular protective activity. Previously, the furanochromene skeleton of xyloketals has long been accepted to be derived from a hybrid polyketide-terpenoid pathway, yet this hypothesis has not been experimentally validated. Here, we addressed this knowledge gap by elucidating the biosynthetic pathway of simpliketals A-D, four xyloketal congeners isolated from <i>Simplicillium</i> sp. AHK071-01. Our key findings establish that the furanochromene skeleton of xyloketals is formed via heterodimerization of two metabolites branched from orsellinic acid, revealing their complete polyketide origin, which contradicts the previous proposal. Notably, we identified a critical <i>O</i>-methyltransferase SipD that plays an essential role in both metabolic branches. Specifically, its selective methylation of orsellinaldehyde blocks the formation of reactive <i>ortho</i>-quinone methide and ensures that the biosynthetic cascade proceeds toward flavoprotein monooxygenase SipE-mediated Baeyer-Villiger oxidation to generate an unusual oxepinone intermediate, which is essential for producing the reactive enone intermediate for final dimerization to form the xyloketal furanochromene skeleton.