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

doi:10.1016/j.phytochem.2013.11.015

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 journal › Article › peer-review

43 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 language	English
Pages (from-to)	60-68
Number of pages	9
Journal	Phytochemistry
Volume	98
DOIs	https://doi.org/10.1016/j.phytochem.2013.11.015
State	Published - 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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10.1016/j.phytochem.2013.11.015

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@article{bcf4f07601374fc785c397b74b028368,

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.",

keywords = "Clavicipitaceae, Epichlo{\"e} spp., Genome sequencing, Grass symbionts, Loline alkaloids, Oxoglutarate/iron-dependent dioxygenase, Pyrrolizidines, exo-1-Acetamidopyrrolizidine, lolO",

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

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. ",

year = "2014",

month = feb,

doi = "10.1016/j.phytochem.2013.11.015",

language = "English",

volume = "98",

pages = "60--68",

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T1 - Ether bridge formation in loline alkaloid biosynthesis

AU - Pan, Juan

AU - Bhardwaj, Minakshi

AU - Faulkner, Jerome R.

AU - Nagabhyru, Padmaja

AU - Charlton, Nikki D.

AU - Higashi, Richard M.

AU - Miller, Anne Frances

AU - Young, Carolyn A.

AU - Grossman, Robert B.

AU - Schardl, Christopher L.

N1 - 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.

PY - 2014/2

Y1 - 2014/2

N2 - 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.

AB - 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.

KW - Clavicipitaceae

KW - Epichloë spp.

KW - Genome sequencing

KW - Grass symbionts

KW - Loline alkaloids

KW - Oxoglutarate/iron-dependent dioxygenase

KW - Pyrrolizidines

KW - exo-1-Acetamidopyrrolizidine

KW - lolO

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U2 - 10.1016/j.phytochem.2013.11.015

DO - 10.1016/j.phytochem.2013.11.015

M3 - Article

C2 - 24374065

AN - SCOPUS:84892928323

SN - 0031-9422

VL - 98

SP - 60

EP - 68

JO - Phytochemistry

JF - Phytochemistry

ER -

Ether bridge formation in loline alkaloid biosynthesis

Abstract

Bibliographical note

Keywords

ASJC Scopus subject areas

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