Biosynthesis of Phenylnannolone A, a MDR Reversal Agent from <em>Nannocystis pusilla</em>

Abstract

Myxobacteria are gliding bacteria known to produce an immense diverse spectrum of natural products with unique modes of action. <em>Nannocystis pusilla</em> B150 is a myxobacterial representative, which is able to synthesize polyketides, named phenylnannolones. Phenylnannolone A showed inhibitory activity towards the ABCB1 gene product p-glycoprotein and reverses daunorubicin resistance in cultured cancer cells. Feeding studies with labeled precursors revealed, besides three acetates, a butyrate and a phenylalanine-derived starter unit, also the unprecedented incorporation of a C-2 carbon atom derived from acetate. This labeling pattern suggested novel biochemical reactions. To decipher the biochemical reactions leading to the formation of phenylnannolone A, the biosynthetic gene cluster for phenylnannolone A was identified and sequenced; this revealed a gene encoding for a Polyketide Synthase (PKS), whose domain order is consistent with the putative biosynthesis that we postulated for phenylnannolone A. However, the putative loading module comprises an NRPS-like loading module that activates the aromatic starter unit. Investigations on the functionality and substrate selectivity of the loading module were tested in a γ-¹⁸O₄-ATP pyrophosphate exchange assay and showed the significant and specific activation of cinnamic acid by the AMP-ligase.