Molecular networking assisted discovery and biosynthesis elucidation of the antimicrobial spiroketals epicospirocins

Two pairs of dibenzospiroketal racemates, (±)-epicospirocin A ( 1a/1b ) and (±)-1- epi -epicospirocin A ( 2a/2b ), and two (+)-enantiomers of aspermicrones, ent -aspermicrone B ( 3b ) and ent -aspermicrone C ( 4b ), together with two hemiacetal epimeric mixtures, epicospirocin B/1- epi -epicospirocin B ( 5/6 ) and epicospirocin C/1- epi -epicospirocin C ( 7/8 ), were investigated from the phytopathogenic fungus Epicoccum nigrum 09116 via MS/MS molecular networking guided isolation and chiral separation for the first time. A plausible epicospirocin biosynthetic pathway was elucidated through in silico gene function annotation together with knock-out experiments. This is the first report that has applied MS/MS molecular networking to identify intermediates correlated with a biosynthetic pathway. A series of new antimicrobial dibenzospiroketals epicospirocins, and their biosynthetic pathway, were investigated via molecular networking from phytopathogenic fungi EN09116 for the first time.