Mutasynthesis of halogenated aurachins in escherichia coli Sebastian Kruth 1 , Cindy Zimmermann 2 , Lina Schibajew 1 , Jörg Pietruszka 2,3 , MarkusNett 1 1 TU Dortmund University, Germany, 2 Heinrich-Heine-University Düsseldorf at Forschungszentrum Jülich, Germany, 3 Forschungszentrum Jülich GmbH, Germany The aurachins are a family of prenylated quinolone antibiotics, which were first discovered in the myxobacterium Stigmatella aurantiaca . [1] Bioactivity testing revealed that these natural products possess potent antiplasmodial activities, but also high cytotoxicity. [2,3] Therefore, there is a great interest in the generation of unnatural aurachin derivatives with a larger therapeutic window. In this study, we aimed to produce such compounds by fermentation. The key reaction in aurachin biosynthesis is the prenylation of a quinoline precursor by the membrane-bound farnesyltransferase AuaA. [4] Preliminary evidence indicates that this enzyme exhibits broad substrate tolerance, which enables the modification of the aurachin scaffold by precursor-directed biosynthesis. [5] To avoid a competition with the natural precursor, we decided to express AuaA in the heterologous host Escherichia coli and to supplement cultures of the recombinant strain with synthetically prepared quinolines (Figure 1).
Figure 1: Biosynthesis of unnatural aurachins in AuaA-expressing E. coli cells after feeding of substituted 4-hydroxy-2-methyl- quinolines. By screening a library of bicistronic design elements, we could find the optimal expression level for auaA in E. coli and reached an aurachin titer of 1 mg·L -1 . Through the reconstitution of the mevalonate pathway in E. coli , the titer was further raised to over 16 mg·L -1 . With this optimized production strain at hand, we fed synthetically prepared halogenated quinolines and successfully isolated chlorinated and bromated aurachin derivatives. References 1. B. Kunze, G. Höfle, H. Reichenbach, J. Antibiot. 1987 , 40 , 258–265. 2. G. Höfle, B. Kunze, J. Nat. Prod. 2008 , 71 , 1843–1849. 3. G. Höfle, B. Böhlendorf, T. Fecker, F. Sasse, B. Kunze, J. Nat. Prod. 2008 , 71 , 1967–1969. 4. E. Stec, D. Pistorius, R. Müller, S. M. Li, ChemBioChem 2011 , 12 , 1724–1730. 5. A. Sester, K. Stüer‐Patowsky, W. Hiller, F. Kloss, S. Lütz, M. Nett, ChemBioChem 2020 , 21 , 2268–2273.
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