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Dissection of goadsporin biosynthesis by in vitro reconstitution leading to designer analogues expressed in vivo

Goadsporin (GS) is a member of ribosomally synthesized and post-translationally modified peptides (RiPPs), containing an N-terminal acetyl moiety, six azoles and two dehydroalanines in the peptidic main chain. Although the enzymes involved in GS biosynthesis have been defined, the principle of how t...

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Autores principales: Ozaki, Taro, Yamashita, Kona, Goto, Yuki, Shimomura, Morito, Hayashi, Shohei, Asamizu, Shumpei, Sugai, Yoshinori, Ikeda, Haruo, Suga, Hiroaki, Onaka, Hiroyasu
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5303826/
https://www.ncbi.nlm.nih.gov/pubmed/28165449
http://dx.doi.org/10.1038/ncomms14207
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author Ozaki, Taro
Yamashita, Kona
Goto, Yuki
Shimomura, Morito
Hayashi, Shohei
Asamizu, Shumpei
Sugai, Yoshinori
Ikeda, Haruo
Suga, Hiroaki
Onaka, Hiroyasu
author_facet Ozaki, Taro
Yamashita, Kona
Goto, Yuki
Shimomura, Morito
Hayashi, Shohei
Asamizu, Shumpei
Sugai, Yoshinori
Ikeda, Haruo
Suga, Hiroaki
Onaka, Hiroyasu
author_sort Ozaki, Taro
collection PubMed
description Goadsporin (GS) is a member of ribosomally synthesized and post-translationally modified peptides (RiPPs), containing an N-terminal acetyl moiety, six azoles and two dehydroalanines in the peptidic main chain. Although the enzymes involved in GS biosynthesis have been defined, the principle of how the respective enzymes control the specific modifications remains elusive. Here we report a one-pot synthesis of GS using the enzymes reconstituted in the ‘flexible' in vitro translation system, referred to as the FIT–GS system. This system allows us to readily prepare not only the precursor peptide from its synthetic DNA template but also 52 mutants, enabling us to dissect the modification determinants of GodA for each enzyme. The in vitro knowledge has also led us to successfully produce designer GS analogues in vivo. The methodology demonstrated in this work is also applicable to other RiPP biosynthesis, allowing us to rapidly investigate the principle of modification events with great ease.
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spelling pubmed-53038262017-02-27 Dissection of goadsporin biosynthesis by in vitro reconstitution leading to designer analogues expressed in vivo Ozaki, Taro Yamashita, Kona Goto, Yuki Shimomura, Morito Hayashi, Shohei Asamizu, Shumpei Sugai, Yoshinori Ikeda, Haruo Suga, Hiroaki Onaka, Hiroyasu Nat Commun Article Goadsporin (GS) is a member of ribosomally synthesized and post-translationally modified peptides (RiPPs), containing an N-terminal acetyl moiety, six azoles and two dehydroalanines in the peptidic main chain. Although the enzymes involved in GS biosynthesis have been defined, the principle of how the respective enzymes control the specific modifications remains elusive. Here we report a one-pot synthesis of GS using the enzymes reconstituted in the ‘flexible' in vitro translation system, referred to as the FIT–GS system. This system allows us to readily prepare not only the precursor peptide from its synthetic DNA template but also 52 mutants, enabling us to dissect the modification determinants of GodA for each enzyme. The in vitro knowledge has also led us to successfully produce designer GS analogues in vivo. The methodology demonstrated in this work is also applicable to other RiPP biosynthesis, allowing us to rapidly investigate the principle of modification events with great ease. Nature Publishing Group 2017-02-06 /pmc/articles/PMC5303826/ /pubmed/28165449 http://dx.doi.org/10.1038/ncomms14207 Text en Copyright © 2017, The Author(s) http://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/
spellingShingle Article
Ozaki, Taro
Yamashita, Kona
Goto, Yuki
Shimomura, Morito
Hayashi, Shohei
Asamizu, Shumpei
Sugai, Yoshinori
Ikeda, Haruo
Suga, Hiroaki
Onaka, Hiroyasu
Dissection of goadsporin biosynthesis by in vitro reconstitution leading to designer analogues expressed in vivo
title Dissection of goadsporin biosynthesis by in vitro reconstitution leading to designer analogues expressed in vivo
title_full Dissection of goadsporin biosynthesis by in vitro reconstitution leading to designer analogues expressed in vivo
title_fullStr Dissection of goadsporin biosynthesis by in vitro reconstitution leading to designer analogues expressed in vivo
title_full_unstemmed Dissection of goadsporin biosynthesis by in vitro reconstitution leading to designer analogues expressed in vivo
title_short Dissection of goadsporin biosynthesis by in vitro reconstitution leading to designer analogues expressed in vivo
title_sort dissection of goadsporin biosynthesis by in vitro reconstitution leading to designer analogues expressed in vivo
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5303826/
https://www.ncbi.nlm.nih.gov/pubmed/28165449
http://dx.doi.org/10.1038/ncomms14207
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