Natural Products Discovery from Cultured Cyanobacteria Guided by Phenotypic and Chemoinformatic Analyses

Cyanobacteria produce structurally diverse natural products with a wide range of applications. In natural products discovery, biological activity guided isolation has been an original paradigm and is still a widely utilized strategy today. The process to discover new natural products is undergoing major changes as multi-omics strategies are integrated with classical approaches. In this study, we applied mass spectrometry (MS) based metabolomics and genomics to a bioactivity-guided research pipeline to improve the discovery and production of new and bioactive metabolites from cultured cyanobacteria. In one example, bioassay-guided isolation led to the identification of aulosirazole, from terrestrial Nostoc sp. UIC 10771, a known natural product which was found to act through a promising mechanism against ovarian cancer cells. Using a media study guided by comparative metabolomics, we observed that phosphate limitation and sulfate supplementation improved the yield of new analogs, aulosirazole B and C, allowing for their isolation and structural characterization using 1D and 2D NMR and DFT calculations. In a second approach, a molecular phylogenetic analysis was used to prioritize the investigation of Nostoc sp. UIC 10607, a bioactive sponge-associated strain that is taxonomically related to cyanobacteria of pharmaceutical relevance. From this strain, the new cytotoxic depsipeptides, menominin A and B, were obtained using bioassay-, NMR-, and MS-guided isolation and were characterized using 1D and 2D NMR, J-based configurational analysis, chiral HPLC, and Mosher’s ester analysis. The menominin biosynthetic gene cluster was identified and annotated. Finally, MS-based comparative metabolomics using statistical and chemoinformatic analyses was used to investigate changes in the chemical profile of five bioactive Nostocales strains. Environmentally relevant culture conditions were applied and include nitrogen limitation, phosphate limitation, iron supplementation, and heat shock. Using this approach, both new and known metabolites were detected, including metabolites which were not previously identified using a bioassay-guided approach. Enhanced metabolite production was observed for each strain under unique culture requirements and multiple culture conditions were needed to stimulate production of all observed chemical classes. This study integrated phenotypic screening and multiple informatics-guided strategies to discover unique and bioactive natural products from cultured cyanobacteria.