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

Author

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  • Taro Ozaki

    (Graduate School of Agricultural and Life Sciences, The University of Tokyo
    Present address: Department of Chemistry, Graduate School of Science, Hokkaido University, Sapporo, Hokkaido 060-0810, Japan)

  • Kona Yamashita

    (Graduate School of Agricultural and Life Sciences, The University of Tokyo)

  • Yuki Goto

    (Graduate School of Science, The University of Tokyo)

  • Morito Shimomura

    (Graduate School of Agricultural and Life Sciences, The University of Tokyo)

  • Shohei Hayashi

    (Graduate School of Agricultural and Life Sciences, The University of Tokyo
    Present address: Department of Agricultural and Forest Sciences, Faculty of Life and Environmental Science, Shimane University, 1060 Nishikawatsu, Matsue, Shimane 690-8504, Japan)

  • Shumpei Asamizu

    (Graduate School of Agricultural and Life Sciences, The University of Tokyo)

  • Yoshinori Sugai

    (Graduate School of Agricultural and Life Sciences, The University of Tokyo)

  • Haruo Ikeda

    (Kitasato Institute for Life Sciences, Kitasato University)

  • Hiroaki Suga

    (Graduate School of Science, The University of Tokyo)

  • Hiroyasu Onaka

    (Graduate School of Agricultural and Life Sciences, The University of Tokyo
    Biotechnology Research Center, Toyama Prefectural University)

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.

Suggested Citation

  • Taro Ozaki & Kona Yamashita & Yuki Goto & Morito Shimomura & Shohei Hayashi & Shumpei Asamizu & Yoshinori Sugai & Haruo Ikeda & Hiroaki Suga & Hiroyasu Onaka, 2017. "Dissection of goadsporin biosynthesis by in vitro reconstitution leading to designer analogues expressed in vivo," Nature Communications, Nature, vol. 8(1), pages 1-13, April.
    • Handle: RePEc:nat:natcom:v:8:y:2017:i:1:d:10.1038_ncomms14207
    DOI: 10.1038/ncomms14207
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    Cited by:

    1. Zeng-Fei Pei & Lingyang Zhu & Satish K. Nair, 2023. "Core-dependent post-translational modifications guide the biosynthesis of a new class of hypermodified peptides," Nature Communications, Nature, vol. 14(1), pages 1-13, December.
    2. Wan-Qiu Liu & Xiangyang Ji & Fang Ba & Yufei Zhang & Huiling Xu & Shuhui Huang & Xiao Zheng & Yifan Liu & Shengjie Ling & Michael C. Jewett & Jian Li, 2024. "Cell-free biosynthesis and engineering of ribosomally synthesized lanthipeptides," Nature Communications, Nature, vol. 15(1), pages 1-13, December.

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