title: Ether bridge formation in loline alkaloid biosynthesis.
abstract: 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.
authors.affiliation: Department of Plant Pathology, 201F Plant Sciences Building, 1405 Veterans Drive, University of Kentucky, Lexington, KY 40546-0312, USA.
authors.forename: Juan
authors.initials: J
authors.lastname: Pan
authors.affiliation: Department of Chemistry, 339 Chemistry-Physics Building, 505 Rose Street, University of Kentucky, Lexington, KY 40506-0055, USA.
authors.forename: Minakshi
authors.initials: M
authors.lastname: Bhardwaj
authors.affiliation: Department of Plant Pathology, 201F Plant Sciences Building, 1405 Veterans Drive, University of Kentucky, Lexington, KY 40546-0312, USA.
authors.forename: Jerome R
authors.initials: JR
authors.lastname: Faulkner
authors.affiliation: Department of Plant Pathology, 201F Plant Sciences Building, 1405 Veterans Drive, University of Kentucky, Lexington, KY 40546-0312, USA.
authors.forename: Padmaja
authors.initials: P
authors.lastname: Nagabhyru
authors.affiliation: The Samuel Roberts Noble Foundation, Forage Improvement Division, 2510 Sam Noble Parkway, Ardmore, OK 73401-2124, USA.
authors.forename: Nikki D
authors.initials: ND
authors.lastname: Charlton
authors.affiliation: Graduate Center for Toxicology, 521 Biopharmacy Building, 1000 South Limestone, University of Kentucky, Lexington, KY 40536-0293, USA.
authors.forename: Richard M
authors.initials: RM
authors.lastname: Higashi
authors.affiliation: Department of Chemistry, 339 Chemistry-Physics Building, 505 Rose Street, University of Kentucky, Lexington, KY 40506-0055, USA.
authors.forename: Anne-Frances
authors.initials: AF
authors.lastname: Miller
authors.affiliation: The Samuel Roberts Noble Foundation, Forage Improvement Division, 2510 Sam Noble Parkway, Ardmore, OK 73401-2124, USA.
authors.forename: Carolyn A
authors.initials: CA
authors.lastname: Young
authors.affiliation: Department of Chemistry, 339 Chemistry-Physics Building, 505 Rose Street, University of Kentucky, Lexington, KY 40506-0055, USA.
authors.forename: Robert B
authors.initials: RB
authors.lastname: Grossman
authors.affiliation: Department of Plant Pathology, 201F Plant Sciences Building, 1405 Veterans Drive, University of Kentucky, Lexington, KY 40546-0312, USA. Electronic address: schardl@uky.edu.
authors.forename: Christopher L
authors.initials: CL
authors.lastname: Schardl
mesh_terms: D000470:Alkaloids / Q000096:biosynthesis* / Q000737:chemistry; D004987:Ethers / Q000737:chemistry / Q000378:metabolism*; D015394:Molecular Structure; D010105:Oxygenases / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000470:Alkaloids; D004987:Ethers; D010105:Oxygenases; C433124:iron(II) oxygenase; C059478:loline
keywords: Clavicipitaceae; Epichloë spp.; Genome sequencing; Grass symbionts; Loline alkaloids; Oxoglutarate/iron-dependent dioxygenase; Pyrrolizidines; exo-1-Acetamidopyrrolizidine; lolO