Role of the LolP cytochrome P450 monooxygenase in loline alkaloid biosynthesis

Martin J. Spiering; Jerome R. Faulkner; Dong Xiu Zhang; Caroline Machado; Robert B. Grossman; Christopher L. Schardl

doi:10.1016/j.fgb.2008.07.001

Role of the LolP cytochrome P450 monooxygenase in loline alkaloid biosynthesis

Martin J. Spiering, Jerome R. Faulkner, Dong Xiu Zhang, Caroline Machado, Robert B. Grossman, Christopher L. Schardl

Research output: Contribution to journal › Article › peer-review

28 Scopus citations

Abstract

The insecticidal loline alkaloids, produced by Neotyphodium uncinatum and related endophytes, are exo-1-aminopyrrolizidines with an ether bridge between C-2 and C-7. Loline alkaloids vary in methyl, acetyl, and formyl substituents on the 1-amine, which affect their biological activity. Enzymes for key loline biosynthesis steps are probably encoded by genes in the LOL cluster, which is duplicated in N. uncinatum, except for a large deletion in lolP2. The role of lolP1 was investigated by its replacement with a hygromycin B phosphotransferase gene. Compared to wild type N. uncinatum and an ectopic transformant, ΔlolP1 cultures had greatly elevated levels of N-methylloline (NML) and lacked N-formylloline (NFL). Complementation of ΔlolP1 with lolP1 under control of the Emericella nidulans trpC promoter restored NFL production. These results and the inferred sequence of LolP1 indicate that it is a cytochrome P450, catalyzing oxygenation of an N-methyl group in NML to the N-formyl group in NFL.

Original language	English
Pages (from-to)	1307-1314
Number of pages	8
Journal	Fungal Genetics and Biology
Volume	45
Issue number	9
DOIs	https://doi.org/10.1016/j.fgb.2008.07.001
State	Published - Sep 2008

Bibliographical note

Funding Information:
This work was supported by US National Science Foundation Grant MCB-0213217 and U.S. Department of Agriculture Grant 200506271031. We thank Walter Hollin, Alfred D. Byrd, and LaTasha S. Williams for valuable technical support. Sequence analyses were conducted in the University of Kentucky Advanced Genetic Technologies Center. This is Kentucky Agricultural Experiment Station publication number 08-12-090, published with the approval of the director.

Keywords

Epichloë
Grass endophyte
Loline alkaloids
N-Formylation
Neotyphodium uncinatum
P450 monooxygenase
Pyrrolizidine

ASJC Scopus subject areas

Microbiology
Genetics

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10.1016/j.fgb.2008.07.001

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title = "Role of the LolP cytochrome P450 monooxygenase in loline alkaloid biosynthesis",

abstract = "The insecticidal loline alkaloids, produced by Neotyphodium uncinatum and related endophytes, are exo-1-aminopyrrolizidines with an ether bridge between C-2 and C-7. Loline alkaloids vary in methyl, acetyl, and formyl substituents on the 1-amine, which affect their biological activity. Enzymes for key loline biosynthesis steps are probably encoded by genes in the LOL cluster, which is duplicated in N. uncinatum, except for a large deletion in lolP2. The role of lolP1 was investigated by its replacement with a hygromycin B phosphotransferase gene. Compared to wild type N. uncinatum and an ectopic transformant, ΔlolP1 cultures had greatly elevated levels of N-methylloline (NML) and lacked N-formylloline (NFL). Complementation of ΔlolP1 with lolP1 under control of the Emericella nidulans trpC promoter restored NFL production. These results and the inferred sequence of LolP1 indicate that it is a cytochrome P450, catalyzing oxygenation of an N-methyl group in NML to the N-formyl group in NFL.",

keywords = "Epichlo{\"e}, Grass endophyte, Loline alkaloids, N-Formylation, Neotyphodium uncinatum, P450 monooxygenase, Pyrrolizidine",

author = "Spiering, {Martin J.} and Faulkner, {Jerome R.} and Zhang, {Dong Xiu} and Caroline Machado and Grossman, {Robert B.} and Schardl, {Christopher L.}",

note = "Funding Information: This work was supported by US National Science Foundation Grant MCB-0213217 and U.S. Department of Agriculture Grant 200506271031. We thank Walter Hollin, Alfred D. Byrd, and LaTasha S. Williams for valuable technical support. Sequence analyses were conducted in the University of Kentucky Advanced Genetic Technologies Center. This is Kentucky Agricultural Experiment Station publication number 08-12-090, published with the approval of the director.",

year = "2008",

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language = "English",

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pages = "1307--1314",

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AU - Spiering, Martin J.

AU - Faulkner, Jerome R.

AU - Zhang, Dong Xiu

AU - Machado, Caroline

AU - Grossman, Robert B.

AU - Schardl, Christopher L.

N1 - Funding Information: This work was supported by US National Science Foundation Grant MCB-0213217 and U.S. Department of Agriculture Grant 200506271031. We thank Walter Hollin, Alfred D. Byrd, and LaTasha S. Williams for valuable technical support. Sequence analyses were conducted in the University of Kentucky Advanced Genetic Technologies Center. This is Kentucky Agricultural Experiment Station publication number 08-12-090, published with the approval of the director.

PY - 2008/9

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N2 - The insecticidal loline alkaloids, produced by Neotyphodium uncinatum and related endophytes, are exo-1-aminopyrrolizidines with an ether bridge between C-2 and C-7. Loline alkaloids vary in methyl, acetyl, and formyl substituents on the 1-amine, which affect their biological activity. Enzymes for key loline biosynthesis steps are probably encoded by genes in the LOL cluster, which is duplicated in N. uncinatum, except for a large deletion in lolP2. The role of lolP1 was investigated by its replacement with a hygromycin B phosphotransferase gene. Compared to wild type N. uncinatum and an ectopic transformant, ΔlolP1 cultures had greatly elevated levels of N-methylloline (NML) and lacked N-formylloline (NFL). Complementation of ΔlolP1 with lolP1 under control of the Emericella nidulans trpC promoter restored NFL production. These results and the inferred sequence of LolP1 indicate that it is a cytochrome P450, catalyzing oxygenation of an N-methyl group in NML to the N-formyl group in NFL.

AB - The insecticidal loline alkaloids, produced by Neotyphodium uncinatum and related endophytes, are exo-1-aminopyrrolizidines with an ether bridge between C-2 and C-7. Loline alkaloids vary in methyl, acetyl, and formyl substituents on the 1-amine, which affect their biological activity. Enzymes for key loline biosynthesis steps are probably encoded by genes in the LOL cluster, which is duplicated in N. uncinatum, except for a large deletion in lolP2. The role of lolP1 was investigated by its replacement with a hygromycin B phosphotransferase gene. Compared to wild type N. uncinatum and an ectopic transformant, ΔlolP1 cultures had greatly elevated levels of N-methylloline (NML) and lacked N-formylloline (NFL). Complementation of ΔlolP1 with lolP1 under control of the Emericella nidulans trpC promoter restored NFL production. These results and the inferred sequence of LolP1 indicate that it is a cytochrome P450, catalyzing oxygenation of an N-methyl group in NML to the N-formyl group in NFL.

KW - Epichloë

KW - Grass endophyte

KW - Loline alkaloids

KW - N-Formylation

KW - Neotyphodium uncinatum

KW - P450 monooxygenase

KW - Pyrrolizidine

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JO - Fungal Genetics and Biology

JF - Fungal Genetics and Biology

IS - 9

ER -

Role of the LolP cytochrome P450 monooxygenase in loline alkaloid biosynthesis

Abstract

Bibliographical note

Keywords

ASJC Scopus subject areas

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