Influence of OSMAC-Based Cultivation in Metabolome and Anticancer Activity of Fungi Associated with the Brown Alga Fucus vesiculosus.

The fungi associated with marine algae are prolific sources of metabolites with high chemical diversity and bioactivity. In this study, we investigated culture-dependent fungal communities associated with the Baltic seaweed Fucus vesiculosus. Altogether, 55 epiphytic and endophytic fungi were isolated and identified. Twenty-six strains were selected for a small-scale One-Strain-Many-Compounds (OSMAC)-based fermentation in four media under solid and liquid culture regimes. In total, 208 fungal EtOAc extracts were tested for anticancer activity and general cytotoxicity. Ten most active strains (i.e., 80 extracts) were analyzed for their metabolome by molecular networking (MN), in-silico MS/MS fragmentation analysis (ISDB⁻UNPD), and manual dereplication. Thirty-six metabolites belonging to 25 chemical families were putatively annotated. The MN clearly distinguished the impact of culture conditions in chemical inventory and anticancer activity of the fungal extracts that was often associated with general toxicity. The bioactivity data were further mapped into MN to seek metabolites, exclusively expressed in the active extracts. This is the first report of cultivable fungi associated with the Baltic F. vesiculosus that combined an OSMAC and an integrated MN-based untargeted metabolomics approaches for efficient assessment and visualization of the impact of the culture conditions on chemical space and anticancer potential of the fungi.

Imaging the Unimaginable: Desorption Electrospray Ionization - Imaging Mass Spectrometry (DESI-IMS) in Natural Product Research.

Imaging mass spectrometry (IMS) has recently established itself in the field of "spatial metabolomics." Merging the sensitivity and fast screening of high-throughput mass spectrometry with spatial and temporal chemical information, IMS visualizes the production, location, and distribution of metabolites in intact biological models. Since metabolite profiling and morphological features are combined in single images, IMS offers an unmatched chemical detail on complex biological and microbiological systems. Thus, IMS-type "spatial metabolomics" emerges as a powerful and complementary approach to genomics, transcriptomics, and classical metabolomics studies. In this review, we summarize the current state-of-the-art IMS methods with a strong focus on desorption electrospray ionization (DESI)-IMS. DESI-IMS utilizes the original principle of electrospray ionization, but in this case solvent droplets are rastered and desorbed directly on the sample surface. The rapid and minimally destructive DESI-IMS chemical screening is achieved at ambient conditions and enables the accurate view of molecules in tissues at the µm-scale resolution. DESI-IMS analysis does not require complex sample preparation and allows repeated measurements on samples from different biological sources, including microorganisms, plants, and animals. Thanks to its easy workflow and versatility, DESI-IMS has successfully been applied to many different research fields, such as clinical analysis, cancer research, environmental sciences, microbiology, chemical ecology, and drug discovery. Herein we discuss the present applications of DESI-IMS in natural product research.

Chemical analysis of the Alphaproteobacterium strain MOLA1416 associated with the marine lichen Lichina pygmaea.

Alphaproteobacterium strain MOLA1416, related to Mycoplana ramosa DSM 7292 and Chelativorans intermedius CC-MHSW-5 (93.6% 16S rRNA sequence identity) was isolated from the marine lichen, Lichina pygmaea and its chemical composition was characterized by a metabolomic network analysis using LC-MS/MS data. Twenty-five putative different compounds were revealed using a dereplication workflow based on MS/MS signatures available through GNPS (https://gnps.ucsd.edu/). In total, ten chemical families were highlighted including isocoumarins, macrolactones, erythrinan alkaloids, prodiginines, isoflavones, cyclohexane-diones, sterols, diketopiperazines, amino-acids and most likely glucocorticoids. Among those compounds, two known metabolites (13 and 26) were isolated and structurally identified and metabolite 26 showed a high cytotoxic activity against B16 melanoma cell lines with an IC50 0.6 ± 0.07 µg/mL.

Multiple Streptomyces species with distinct secondary metabolomes have identical 16S rRNA gene sequences.

Microbial diversity studies using small subunit (SSU) rRNA gene sequences continue to advance our understanding of biological and ecological systems. Although a good predictor of overall diversity, using this gene to infer the presence of a species in a sample is more controversial. Here, we present a detailed polyphasic analysis of 10 bacterial strains isolated from three coastal lichens Lichina confinis, Lichina pygmaea and Roccella fuciformis with SSU rRNA gene sequences identical to the type strain of Streptomyces cyaneofuscatus. This analysis included phenotypic, microscopic, genetic and genomic comparisons and showed that despite their identical SSU rRNA sequences the strains had markedly different properties, and could be distinguished as 5 different species. Significantly, secondary metabolites profiles from these strains were also found to be different. It is thus clear that SSU rRNA based operational taxonomy units, even at the most stringent cut-off can represent multiple bacterial species, and that at least for the case of Streptomyces, strain de-replication based on SSU gene sequences prior to screening for bioactive molecules can miss potentially interesting novel molecules produced by this group that is notorious for the production of drug-leads.