@article{83ba98b2aabd4190a0d37dfb7ebb5a6a,
title = "Dissection of goadsporin biosynthesis by in vitro reconstitution leading to designer analogues expressed in vivo",
abstract = "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.",
author = "Taro Ozaki and Kona Yamashita and Yuki Goto and Morito Shimomura and Shohei Hayashi and Shumpei Asamizu and Yoshinori Sugai and Haruo Ikeda and Hiroaki Suga and Hiroyasu Onaka",
note = "Funding Information: We appreciate the assistance provided by Mizuho Nakaho and Yukari Kurokawa at Toyama Prefectural University, Hisashi Okamoto at The University of Tokyo for experiments, Yasuharu Kato at The University of Tokyo for technical supports in the in vitro experiments and Dr Joseph Rogers at The University of Tokyo for proof-reading. We also appreciate the LC-MS analysis support by Dr Mamoru Komatsu and Dr Kiyoko T. Miyamoto at Kitasato University, and Dr Naoya Oku at Toyama Prefectural University. This research was supported in part by a grant-in-aid from IFO, Institute for Fermentation, Osaka (to H.O., S.A. and T.O.), JSPS KAKENHI grant-in-aid for Young Scientists B (no. 26850043 to T.O. and no. 26850044 to S.A.), JSPS KAKENHI grant-in-aid for Challenging Exploratory Research (15K12739 to Y.G.), JST CREST of Molecular Technologies to H.S, JSPS KAKENHI (no. 25108707 to H.O.) and JSPS KAKENHI for Scientific Research on Innovative Areas (no. JP16H06444 to H.S, Y.G. and H.O.) Publisher Copyright: {\textcopyright} The Author(s) 2017.",
year = "2017",
month = feb,
day = "6",
doi = "10.1038/ncomms14207",
language = "English",
volume = "8",
journal = "Nature Communications",
issn = "2041-1723",
publisher = "Nature Publishing Group",
}