Ether bridge formation in loline alkaloid biosynthesis

Juan Pan, Minakshi Bhardwaj, Jerome R. Faulkner, Padmaja Nagabhyru, Nikki D. Charlton, Richard M. Higashi, Anne Frances Miller, Carolyn A. Young, Robert B. Grossman, Christopher L. Schardl

Research output: Contribution to journalArticlepeer-review

37 Scopus citations

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.

Original languageEnglish
Pages (from-to)60-68
Number of pages9
JournalPhytochemistry
Volume98
DOIs
StatePublished - Feb 2014

Bibliographical note

Funding Information:
We thank Timothy D. Phillips for providing Danthonia spicata with Atkinsonella hypoxylon , Sladana Bec for preparation of A. hypoxylon DNA for genome sequencing, Jennifer S. Webb, Emily Gay and Cagney Coomer at the University of Kentucky AGTC facility for genome and PCR product sequencing, Jolanta Jaromczyk for genome assembly, John May at the University of Kentucky ERTL facility for GC–MS analysis, and Johanna E. Takach for sequencing E. amarillans lolO segments. Capable assistance was also provided by Walter Hollin, Julien Nolleau, and Trevor Kellen. This research was supported by USDA-CSREES Grants 2009-11131030 and 2012-6701319384, National Science Foundation Grants EF-0523661 and EPS-0814194, and NIH-NIGMS Grant R01GM086888, and by The Samuel Roberts Noble Foundation, Ardmore, Oklahoma. NMR instruments used in this research were obtained with funds from NSF's CRIF program (CHE-997841) and the University of Kentucky's Research Challenge Trust Fund. This is publication number 13-12-147 of the Kentucky Agricultural Experiment Station, published with approval of the Director.

Keywords

  • Clavicipitaceae
  • Epichloë spp.
  • Genome sequencing
  • Grass symbionts
  • Loline alkaloids
  • Oxoglutarate/iron-dependent dioxygenase
  • Pyrrolizidines
  • exo-1-Acetamidopyrrolizidine
  • lolO

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Plant Science
  • Horticulture

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