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Yeast Homologous Recombination Cloning Leading to the Novel Peptides Ambactin and Xenolindicin
Author(s) -
Schimming Olivia,
Fleischhacker Florian,
Nollmann Friederike I.,
Bode Helge B.
Publication year - 2014
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201402065
Subject(s) - homologous recombination , cloning (programming) , plasmid , escherichia coli , heterologous , yeast , biology , genetics , photorhabdus , gene , saccharomyces cerevisiae , dna , library , homologous chromosome , molecular cloning , recombinant dna , biochemistry , peptide sequence , 16s ribosomal rna , computer science , programming language
Heterologous production of GameXPeptide A ( 1 ), as well as of the novel peptide natural products ambactin ( 2 ) and xenolindicins A–C ( 3 a – c ), 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.