Production of epicocconone and acetosellin in <i>Epicoccum nigrum</i> KACC 40642 and characterization of their biosynthetic gene cluster

Epicocconone (ECN), reported from a strain of Epicoccum nigrum, is a dihydro-azaphilone (-AZ) with a triene hexaketidederived -lactone ring and possesses notable fluorescence property.AZ biosynthesis involves nonreducing-type fungal polyketide synthase with a reductive release domain (NR-fPKS-R).ECN adducts of primary amine compounds display enhanced and wavelength-shifted fluorescence, making ECN a useful staining reagent for proteins.Acetosellin (ACN) was separately reported from another strain of E. nigrum and was proposed to be produced through the reductive cyclization/aromatization of ECN.This study showed that E. nigrum KACC 40642 produces both ECN and ACN.Specifically, a non-sporulating white derivative (whi) of the epipyrone non-producer (epnA) displayed enhanced production of these compounds and was used for their isolation.ACN was produced in routine potato dextrose agar culture, whereas ECN production was achieved when an extractive culture with a hydrophobic resin was applied.ECN biosynthetic gene cluster (BGC) was identified in the E. nigrum genome sequence.A knockout study of its component ecnD, acyltransferase gene, confirmed its role in ECN/ACN biosynthesis by revealing a loss of ECN/ACN production and the production of intermediates-derived compounds (termed ECND and ED-NP-Y) in the ecnD knockout strain.ECN BGC information was used to localize the hypothetical bulgarialactone (BLN) BGC.The divergence between ECN and BLN biosynthesis is discussed based on differences in dehydrogenase/reductase genes between the two BGCs.The findings of this study support a previous proposal that the dihydro-AZ nucleus controls the regiospecificity of -lactone ring formation.