Dissection of goadsporin biosynthesis by in vitro reconstitution leading to designer analogues expressed in vivo.
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.
Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan.
Taro
T
Ozaki
Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan.
Kona
K
Yamashita
Department of Chemistry, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan.
Yuki
Y
Goto
Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan.
Morito
M
Shimomura
Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan.
Shohei
S
Hayashi
Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan.
Shumpei
S
Asamizu
Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan.
Yoshinori
Y
Sugai
Kitasato Institute for Life Sciences, Kitasato University, Sagamihara, Kanagawa 252-0373, Japan.
Haruo
H
Ikeda
Department of Chemistry, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan.
Hiroaki
H
Suga
Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan.|Biotechnology Research Center, Toyama Prefectural University, Imizu, Toyama 939-0398, Japan.
Hiroyasu
H
Onaka
D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D015195:Drug Design; D036341:Intercellular Signaling Peptides and Proteins; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D012270:Ribosomes / Q000201:enzymology*; D013302:Streptomyces / Q000235:genetics / Q000378:metabolism*
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; C449027:goadsporin