Heterologous biosynthesis of elsinochrome A sheds light on the formation of the photosensitive perylenequinone system.

Perylenequinones are a class of aromatic polyketides characterised by a highly conjugated pentacyclic core, which confers them with potent light-induced bioactivities and unique photophysical properties. Despite the biosynthetic gene clusters for the perylenequinones elsinochrome A (), cercosporin () and hypocrellin A () being recently identified, key biosynthetic aspects remain elusive. Here, we first expressed the intact gene cluster encoding from the wheat pathogen heterologously in on a yeast-fungal artificial chromosome (YFAC). This led to the identification of a novel flavin-dependent monooxygenase, ElcH, responsible for oxidative enolate coupling of a perylenequinone intermediate to the hexacyclic dihydrobenzo()perylenequinone in . In the absence of ElcH, the perylenequione intermediate formed a hexacyclic cyclohepta()perylenequinone system an intramolecular aldol reaction resulting in and a novel hypocrellin with opposite helicity to . Theoretical calculations supported that and resulted from atropisomerisation upon formation of the 7-membered ring. Using a bottom-up pathway reconstruction approach on a tripartite YFAC system developed in this study, we uncovered that both a berberine bridge enzyme-like oxidase ElcE and a laccase-like multicopper oxidase ElcG are involved in the double coupling of two naphthol intermediates to form the perylenequinone core. Gene swapping with the homologs from the biosynthetic pathway of showed that cognate pairing of the two classes of oxidases is required for the formation of the perylenequinone core, suggesting the involvement of protein-protein interactions.