Preparation of a ribosomally synthesized fungal peptide toxin precursor and its in-vitro cyclization

Duleepa Pathiraja; Juan Byun; Saeyoung Lee; In Geol Choi

doi:10.1016/j.jbiotec.2019.12.004

Preparation of a ribosomally synthesized fungal peptide toxin precursor and its in-vitro cyclization

Duleepa Pathiraja, Juan Byun, Saeyoung Lee, In Geol Choi

Department of Biotechnology

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

1 Citation (Scopus)

Abstract

Amatoxins are ribosomally synthesized and post-translationally modified peptides (RiPPs) found in poisonous mushrooms. These cyclic peptides are potent inhibitors of RNA polymerase II transcriptional activity. Though the macrocyclization of amatoxin is extensively studied, little is known about its subsequent post-translational modifications. However, studies and the potential use of amatoxins has been deterred by the scarcity of the mushroom biomass. To overcome this issue, we sought to produce the α-amanitin in Escherichia coli. Genes encoding the amanitin precursor peptide (AMA1) and prolyl oligopeptidase (POPB) were separately cloned and expressed in E. coli. Fusion tags were attached to candidate proteins to improve expression and solubility. Purified AMA1 was processed in vitro by POPB, and the formation of cyclic α-amanitin was confirmed by HPLC and MALDI/TOF mass spectroscopy. Our strategy can be applied to the mass production of the α-amanitin, allowing α-amanitin to be investigated as a promising lead compound in drug development.

Original language	English
Pages (from-to)	124-129
Number of pages	6
Journal	Journal of Biotechnology
Volume	308
DOIs	https://doi.org/10.1016/j.jbiotec.2019.12.004
Publication status	Published - 2020 Jan 20

Bibliographical note

Funding Information:
We would like to thank Dr. Byeongnam Min, Mr. Hongjae Park and Ms. Youngeun Chun for their invaluable support throughout this study. This study was supported by School of Life Sciences and Biotechnology for BK21 PLUS, Korea University. Appendix A

Funding Information:
This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. NRF-2019R1A2C1089704 ) and New and Renewable Energy Core Technology Program of the Korea Institute of Energy Technology Evaluation and Planning (KETEP) grants from the Ministry of Trade, Industry and Energy (No. 20173010092460 ).

Publisher Copyright:
© 2019 Elsevier B.V.

Keywords

Cyclic peptide
Prolyl oligopeptidase
RiPP
α-Amanitin

ASJC Scopus subject areas

Biotechnology
Bioengineering
Applied Microbiology and Biotechnology

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10.1016/j.jbiotec.2019.12.004

Cite this

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title = "Preparation of a ribosomally synthesized fungal peptide toxin precursor and its in-vitro cyclization",

abstract = "Amatoxins are ribosomally synthesized and post-translationally modified peptides (RiPPs) found in poisonous mushrooms. These cyclic peptides are potent inhibitors of RNA polymerase II transcriptional activity. Though the macrocyclization of amatoxin is extensively studied, little is known about its subsequent post-translational modifications. However, studies and the potential use of amatoxins has been deterred by the scarcity of the mushroom biomass. To overcome this issue, we sought to produce the α-amanitin in Escherichia coli. Genes encoding the amanitin precursor peptide (AMA1) and prolyl oligopeptidase (POPB) were separately cloned and expressed in E. coli. Fusion tags were attached to candidate proteins to improve expression and solubility. Purified AMA1 was processed in vitro by POPB, and the formation of cyclic α-amanitin was confirmed by HPLC and MALDI/TOF mass spectroscopy. Our strategy can be applied to the mass production of the α-amanitin, allowing α-amanitin to be investigated as a promising lead compound in drug development.",

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N1 - Funding Information: We would like to thank Dr. Byeongnam Min, Mr. Hongjae Park and Ms. Youngeun Chun for their invaluable support throughout this study. This study was supported by School of Life Sciences and Biotechnology for BK21 PLUS, Korea University. Appendix A Funding Information: This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. NRF-2019R1A2C1089704 ) and New and Renewable Energy Core Technology Program of the Korea Institute of Energy Technology Evaluation and Planning (KETEP) grants from the Ministry of Trade, Industry and Energy (No. 20173010092460 ). Publisher Copyright: © 2019 Elsevier B.V.

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N2 - Amatoxins are ribosomally synthesized and post-translationally modified peptides (RiPPs) found in poisonous mushrooms. These cyclic peptides are potent inhibitors of RNA polymerase II transcriptional activity. Though the macrocyclization of amatoxin is extensively studied, little is known about its subsequent post-translational modifications. However, studies and the potential use of amatoxins has been deterred by the scarcity of the mushroom biomass. To overcome this issue, we sought to produce the α-amanitin in Escherichia coli. Genes encoding the amanitin precursor peptide (AMA1) and prolyl oligopeptidase (POPB) were separately cloned and expressed in E. coli. Fusion tags were attached to candidate proteins to improve expression and solubility. Purified AMA1 was processed in vitro by POPB, and the formation of cyclic α-amanitin was confirmed by HPLC and MALDI/TOF mass spectroscopy. Our strategy can be applied to the mass production of the α-amanitin, allowing α-amanitin to be investigated as a promising lead compound in drug development.

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Preparation of a ribosomally synthesized fungal peptide toxin precursor and its in-vitro cyclization

Abstract

Bibliographical note

Keywords

ASJC Scopus subject areas

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