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

Taro Ozaki,Yuki Goto,Hiroyasu Onaka,Yoshinori Sugai,Kona Yamashita,Morito Shimomura,Haruo Ikeda,Shumpei Asamizu,Hiroaki Suga,Shohei Hayashi

doi:10.1038/ncomms14207

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.

Highlights

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
GS belongs to a family of ribosomally synthesized and post-translationally modified peptides (RiPPs)[4], which are generally produced via ribosomal synthesis of a precursor peptide and subsequent post-translational modifications (PTMs)
Since the PTM enzymes involved in RiPPs biosynthesis pathways often tolerate minor alterations of the substrate sequences, some analogues of RiPPs have been successfully expressed by engineering of the wild-type precursor peptide genes in appropriate host cells[17,18,19,20]

Summary

Introduction

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. 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 the precursor peptide from its synthetic DNA template and 52 mutants, enabling us to dissect the modification determinants of GodA for each enzyme. GS belongs to a family of ribosomally synthesized and post-translationally modified peptides (RiPPs)[4], which are generally produced via ribosomal synthesis of a precursor peptide and subsequent post-translational modifications (PTMs). Since the PTM enzymes involved in RiPPs biosynthesis pathways often tolerate minor alterations of the substrate sequences, some analogues of RiPPs have been successfully expressed by engineering of the wild-type precursor peptide genes in appropriate host cells[17,18,19,20]. With regard to the biosynthesis of GS, we have not yet witnessed comprehensive investigations of the substrate tolerance of the PTM enzymes

Methods

Results

Conclusion