| Summary: | Liquid chromatography coupled with high resolution mass spectrometry (LC-HRESMS)-assisted metabolomic profiling of two sponge-associated actinomycetes, <i>Micromonospora</i> sp. UR56 and <i>Actinokineospora</i> sp. EG49, revealed that the co-culture of these two actinomycetes induced the accumulation of metabolites that were not traced in their axenic cultures. Dereplication suggested that phenazine-derived compounds were the main induced metabolites. Hence, following large-scale co-fermentation, the major induced metabolites were isolated and structurally characterized as the already known dimethyl phenazine-1,6-dicarboxylate (<b>1</b>), phenazine-1,6-dicarboxylic acid mono methyl ester (phencomycin; <b>2</b>), phenazine-1-carboxylic acid (tubermycin; <b>3</b>), N-(2-hydroxyphenyl)-acetamide (<b>9</b>), and <i>p</i>-anisamide (<b>10</b>). Subsequently, the antibacterial, antibiofilm, and cytotoxic properties of these metabolites (<b>1</b>–<b>3</b>, <b>9</b>, and <b>10</b>) were determined in vitro. All the tested compounds except 9 showed high to moderate antibacterial and antibiofilm activities, whereas their cytotoxic effects were modest. Testing against <i>Staphylococcus</i> DNA gyrase-B and pyruvate kinase as possible molecular targets together with binding mode studies showed that compounds <b>1</b>–<b>3</b> could exert their bacterial inhibitory activities through the inhibition of both enzymes. Moreover, their structural differences, particularly the substitution at C-1 and C-6, played a crucial role in the determination of their inhibitory spectra and potency. In conclusion, the present study highlighted that microbial co-cultivation is an efficient tool for the discovery of new antimicrobial candidates and indicated phenazines as potential lead compounds for further development as antibiotic scaffold.
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