In vivo and in vitro investigations provide insights into maleidride biosynthesis in fungi†

Maleidrides are a family of polyketide-derived natural products isolated from filamentous fungi, that can exhibit significant bioactivities. These compounds are classified according to the size of their central carbocyclic ring, to which one or more maleic anhydride moieties are attached. The studies described herein provide important insights into maleidride biosynthesis, in particular the pathways to the nonadrides scytalidin and castaneiolide, and the octadride zopfiellin. We propose a supportive role for isochorismatase-like enzymes, which are commonly encoded within maleidride biosynthetic gene clusters, in facilitating α-ketoglutarate dependent dioxygenase-mediated catalysis. This is evidenced by gene deletions as well as enzyme assays, for two maleidride biosynthetic pathways: that of zopfiellin, from Diffractella curvata; and of scytalidin, from Scytalidium album. These experiments collectively underscore the significance of the isochorismatase-like enzymes in the catalytic process of α-ketoglutarate dependent dioxygenases. Feeding studies with either scytalidin or an unsaturated analogue to D. curvata ΔzopPKS both gave the 5,6-diol, castaneiolide and the structure was confirmed by NMR and X-ray crystallography. Furthermore, a putative biosynthetic gene cluster for castaneiolide biosynthesis was identified from a de novo genome assembly of the native producer, Macrophoma castaneicola.