Induction of Antibacterial Metabolites by Co-Cultivation of Two Red-Sea-Sponge-Associated Actinomycetes sp. UR56 and sp. EG49.

Liquid chromatography coupled with high resolution mass spectrometry (LC-HRESMS)-assisted metabolomic profiling of two sponge-associated actinomycetes, sp. UR56 and 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 (), phenazine-1,6-dicarboxylic acid mono methyl ester (phencomycin; ), phenazine-1-carboxylic acid (tubermycin; ), N-(2-hydroxyphenyl)-acetamide (), and -anisamide (). Subsequently, the antibacterial, antibiofilm, and cytotoxic properties of these metabolites (-, , and ) 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 DNA gyrase-B and pyruvate kinase as possible molecular targets together with binding mode studies showed that compounds - 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.