Ether bridge formation in loline alkaloid biosynthesis

The LolO non-heme iron oxygenase is required to form the ether bridge of loline alkaloids. •Genomes were sequenced for four fungi with three different loline alkaloid profiles.•Different loline alkaloid profiles were associated with differences in loline alkaloid biosynthesis gene clusters.•The LolO non-heme iron oxygenase is required for loline ether bridge formation.•An exo-1-acetamidopyrrolizidine is a likely pathway intermediate in loline alkaloid biosynthesis. Lolines are potent insecticidal agents produced by endophytic fungi of cool-season grasses. These alkaloids are composed of a pyrrolizidine ring system and an uncommon ether bridge linking carbons 2 and 7. Previous results indicated that 1-aminopyrrolizidine was a pathway intermediate. We used RNA interference to knock down expression of lolO, resulting in the accumulation of an alkaloid identified as exo-1-acetamidopyrrolizidine based on high-resolution MS and NMR. Genomes of endophytes differing in alkaloid profiles were sequenced, revealing that those with mutated lolO accumulated exo-1-acetamidopyrrolizidine but no lolines. Heterologous expression of wild-type lolO complemented a lolO mutant, resulting in the production of N-acetylnorloline. These results indicated that the non-heme iron oxygenase, LolO, is required for ether bridge formation, probably through oxidation of exo-1-acetamidopyrrolizidine.