Hybridorubrins A-D, novel azaphilone heterodimers from stromata of Hypoxylon fragiforme and insights into the biosynthetic machinery for azaphilone diversification.

Abstract

The diversity of azaphilones in stromatal extracts of the fungus Hypoxylon fragiforme was investigated and linked to their biosynthetic machineries using bioinformatics. Nineteen azaphilone-type compounds were isolated and characterized by NMR spectroscopy and mass spectrometry, with their absolute stereoconfigurations assigned using Mosher ester analysis and ECD spectroscopy. Four unprecedented bisazaphilones, named hybridorubrins A-D ( 1 - 4 ), were elucidated, in addition to new fragirubrins F-G ( 5 - 6 ) and various known mitorubrin derivatives. Only the hybridorubrins, which are composed of mitorubrin and fragirubrin moieties, exhibited strong inhibition of Staphylococcus aureus biofilm formation. Analysis of the genome of H. fragiforme revealed the presence of two separate biosynthetic gene clusters (BGC) hfaza1 and hfaza2 responsible for azaphilone formation. While the hfaza1 BGC likely encodes the assembly of the backbone and addition of fatty acid moieties to yield the ( R )-configured series of fragirubrins, the hfaza2 BGC contains the necessary genes to synthesise the widely distributed ( S )-mitorubrins. This study is the first example of two distant cross-acting fungal BGC collaborating to produce two families of azaphilones and bisazaphilones derived thereof.