Heterologous Expression in Anabaena of the Columbamide Pathway from the Cyanobacterium Moorena bouillonii and Production of New Analogs.

Columbamides are chlorinated acyl amide natural products, several of which exhibit cannabinomimetic activity. These compounds were originally discovered from a culture of the filamentous marine cyanobacterium Moorena bouillonii PNG5-198 collected from the coastal waters of Papua New Guinea. The columbamide biosynthetic gene cluster (BGC) had been identified using bioinformatics, but not confirmed by experimental evidence. Here, we report the heterologous expression in Anabaena ( Nostoc ) PCC 7120 of the 28.5 kb BGC that encodes for columbamide biosynthesis. The production of columbamides in Anabaena is investigated under several different culture conditions, and several new columbamide analogs are identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and nuclear magnetic resonance (NMR). In addition to previously characterized columbamides A, B, and C, new columbamides I-M are produced in these experiments, and the structure of the most abundant monochlorinated analog, columbamide K ( 11 ), is fully characterized. The other new columbamide analogs are produced in only small quantities, and structures are proposed based on high-resolution-MS, MS/MS, and 1 H NMR data. Overexpression of the pathway's predicted halogenases resulted in increased productions of di- and trichlorinated compounds. The most significant change in production of columbamides in Anabaena is correlated with the concentration of NaCl in the medium.