Yeast Homologous Recombination Cloning Leading to the Novel Peptides Ambactin and Xenolindicin

Authors

  • Olivia Schimming,

    1. Merck Stiftungsprofessur für Molekulare Biotechnologie, Fachbereich Biowissenschaften, Goethe Universität Frankfurt, Max-von-Laue Strasse 9, 60438 Frankfurt am Main (Germany)
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  • Florian Fleischhacker,

    1. Merck Stiftungsprofessur für Molekulare Biotechnologie, Fachbereich Biowissenschaften, Goethe Universität Frankfurt, Max-von-Laue Strasse 9, 60438 Frankfurt am Main (Germany)
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  • Friederike I. Nollmann,

    1. Merck Stiftungsprofessur für Molekulare Biotechnologie, Fachbereich Biowissenschaften, Goethe Universität Frankfurt, Max-von-Laue Strasse 9, 60438 Frankfurt am Main (Germany)
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  • Prof. Dr. Helge B. Bode

    Corresponding author
    1. Merck Stiftungsprofessur für Molekulare Biotechnologie, Fachbereich Biowissenschaften, Goethe Universität Frankfurt, Max-von-Laue Strasse 9, 60438 Frankfurt am Main (Germany)
    • Merck Stiftungsprofessur für Molekulare Biotechnologie, Fachbereich Biowissenschaften, Goethe Universität Frankfurt, Max-von-Laue Strasse 9, 60438 Frankfurt am Main (Germany)===

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Abstract

Heterologous production of GameXPeptide A (1), as well as of the novel peptide natural products ambactin (2) and xenolindicins A–C (3 ac), was achieved by using the “overlap extension PCR-yeast homologous recombination” (ExRec) method. ExRec cloning is based on the ability of yeast to assemble overlapping DNA fragments into functional plasmids. Here we used this technique to clone a total of 15 biosynthesis gene clusters from Photorhabdus and Xenorhabdus with sizes of up to 45 kb. The structures of the novel compounds 2 and 3 a, which were produced in Escherichia coli, were elucidated by detailed MS and bioinformatics analysis, and additionally confirmed by their chemical synthesis.

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