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

Taro Ozaki, Kona Yamashita, Yuki Goto, Morito Shimomura, Shohei Hayashi, Shumpei Asamizu, Yoshinori Sugai, Haruo Ikeda, Hiroaki Suga () and Hiroyasu Onaka ()
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Taro Ozaki: Graduate School of Agricultural and Life Sciences, The University of Tokyo
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
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

Nature Communications, 2017, vol. 8, issue 1, 1-13

Abstract: 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.

Date: 2017
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DOI: 10.1038/ncomms14207

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