Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan.
Department of Chemistry, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan.
Nat Commun. 2017 Feb 6;8:14207. doi: 10.1038/ncomms14207.
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.
Goadsporin (GS) 是核糖体合成和翻译后修饰肽 (RiPPs) 的成员，其肽主链含有 N 端乙酰基、六个唑和两个脱水丙氨酸。尽管已经定义了参与 GS 生物合成的酶，但各自的酶如何控制特定修饰的原理仍不清楚。在这里，我们使用在“灵活”体外翻译系统中重组的酶报告了 GS 的一锅合成，称为 FIT-GS 系统。该系统使我们不仅能够从其合成 DNA 模板制备前体肽，还能够制备 52 种突变体，从而使我们能够剖析每个酶的 GodA 修饰决定因素。体外知识还使我们能够成功地在体内生产设计的 GS 类似物。本工作中展示的方法也适用于其他 RiPP 生物合成，使我们能够轻松快速地研究修饰事件的原理。
