Biosynthesis of (+)-palitantin requires homologs of alkyl salicylaldehyde-forming enzymes without involvement of an aromatic intermediate.

(+)-Palitantin, a bioactive fungal polyketide from Penicillium palitans, features a densely functionalized cyclohexanone core with multiple stereocenters. Here, we elucidate its biosynthetic pathway by gene deletion experiments as well as in vivo and in vitro reconstitution of the key steps. The formation of the cyclohexanone scaffold in palitantin is catalyzed by homologs of alkyl salicylaldehyde-forming enzymes without involving an aromatic intermediate. In contrast, the products of known alkyl salicylaldehyde-forming enzymes are aromatic derivatives, including that in the non-aromatic trichoxide pathway. Furthermore, the cupin protein PaltE enhances the formation of an unstable cyclohexenone intermediate for stereospecific ene reduction by PaltF and ensures efficient flux toward (+)-palitantin. Subsequent aldehyde reduction by the SDR PaltG and stereospecific hydroxylation by the P450 enzyme PaltD complete the pathway and establish the stereochemically complex decoration on the cyclohexanone scaffold. Our work expands the functional scope of alkyl salicylaldehyde-forming enzymes and provides a highly programmed strategy for natural product biosynthesis.