ALBERT

Description: Microbial co‐cultivation is employed for awakening silent biosynthetic gene clusters (BGCs) to enhance chemical diversity. However, the selection of appropriate partners for co‐cultivation remains a challenge. Furthermore, competitive interactions involving the suppression of BGCs or upregulation of known, functional metabolite(s) during co‐cultivation efforts is also common. Herein, we performed an alternative approach for targeted selection of the best co‐cultivation pair. Eight marine sediment‐derived fungi were classified as strong or weak, based on their anti‐phytopathogenic potency. The fungi were co‐cultured systematically and analyzed for their chemical profiles and anti-phytopathogenic activity. Based on enhanced bioactivity and a significantly different metabolite profile including the appearance of a co‐culture specific cluster, the co‐culture of Plenodomus influorescens (strong) and Pyrenochaeta nobilis (weak) was prioritized for chemical investigation. Large‐scale co‐cultivation resulted in isolation of five polyketide type compounds: two 12‐membered macrolides, dendrodolide E (1) and its new analog dendrodolide N (2), as well as two rare azaphilones spiciferinone (3) and its new analog 8a-hydroxy-spiciferinone (4). A well‐known bis‐naphtho‐γ‐pyrone type mycotoxin, cephalochromin (5), whose production was specifically enhanced in the co-culture, was also isolated. Chemical structures of compounds 1–5 were elucidated by NMR, HRMS and [] 20/D analyses. Compound 5 showed the strongest anti‐phytopathogenic activity against Xanthomonas campestris and Phytophthora infestans with IC50 values of 0.9 and 1.7 µg/mL, respectively.

Description: Sea foam harbors a diverse range of fungal spores with biological and ecological relevance in marine environments. Fungi are known as the producers of secondary metabolites that are used in health and agricultural sectors, however the potentials of sea foam-derived fungi have remained unexplored. In this study, organic extracts of six foam-derived fungal isolates belonging to the genera Penicillium, Cladosporium, Emericellopsis and Plectosphaerella were investigated for their antimicrobial activity against plant and human pathogens and anticancer activity. In parallel, an untargeted metabolomics study using UPLC-QToF–MS/MS-based molecular networking (MN) was performed to unlock their chemical inventory. Penicillium strains were identified as the most prolific producers of compounds with an average of 165 parent ions per strain. In total, 49 known mycotoxins and functional metabolites were annotated to specific and ubiquitous parent ions, revealing considerable chemical diversity. This allowed the identification of putative new derivatives, such as a new analog of the antimicrobial tetrapeptide, fungisporin. Regarding bioactivity, the Penicillium sp. isolate 31.68F1B showed a strong and broad-spectrum activity against seven plant and human pathogens, with the phytopathogen Magnaporthe oryzae and the human pathogen Candida albicans being the most susceptible (IC50 values 2.2 and 6.3 µg/mL, respectively). This is the first study mining the metabolome of the sea foam-derived fungi by MS/MS-based molecular networking, and assessing their biological activities against phytopathogens

Description: It is widely accepted that the commensal gut microbiota contributes to the health and well-being of its host. The solitary tunicate Ciona intestinalis emerges as a model organism for studying host–microbe interactions taking place in the gut, however, the potential of its gut-associated microbiota for marine biodiscovery remains unexploited. In this study, we set out to investigate the diversity, chemical space, and pharmacological potential of the gut-associated microbiota of C. intestinalis collected from the Baltic and North Seas. In a culture-based approach, we isolated 61 bacterial and 40 fungal strains affiliated to 33 different microbial genera, indicating a rich and diverse gut microbiota dominated by Gammaproteobacteria. In vitro screening of the crude microbial extracts indicated their antibacterial (64% of extracts), anticancer (22%), and/or antifungal (11%) potential. Nine microbial crude extracts were prioritized for in-depth metabolome mining by a bioactivity- and chemical diversity-based selection procedure. UPLC-MS/MS-based metabolomics combining automated (feature-based molecular networking and in silico dereplication) and manual approaches significantly improved the annotation rates. A high chemical diversity was detected where peptides and polyketides were the predominant classes. Many compounds remained unknown, including two putatively novel lipopeptides produced by a Trichoderma sp. strain. This is the first study assessing the chemical and pharmacological profile of the cultivable gut microbiota of C. intestinalis

Description: The brown algal genus Fucus provides essential ecosystem services crucial for marine environments. Macroalgae (seaweeds) release dissolved organic matter, hence, are under strong settlement pressure from micro- and macrofoulers. Seaweeds are able to control surface epibionts directly by releasing antimicrobial compounds onto their surfaces, and indirectly by recruiting beneficial microorganisms that produce antimicrobial/antifouling metabolites. In the Kiel Fjord, in the German Baltic Sea, three distinct Fucus species coexist: F. vesiculosus, F. serratus, and F. distichus subsp. evanescens. Despite sharing the same habitat, they show varying fouling levels; F. distichus subsp. evanescens is the least fouled, while F. vesiculosus is the most fouled. The present study explored the surface metabolomes and epiphytic microbiota of these three Fucus spp., aiming to uncover the factors that contribute to the differences in the fouling intensity on their surfaces. Towards this aim, algal surface metabolomes were analyzed using comparative untargeted LC-MS/MS-based metabolomics, to identify the marker metabolites influencing surface fouling. Their epiphytic microbial communities were also comparatively characterized using high-throughput amplicon sequencing, to pinpoint the differences in the surface microbiomes of the algae. Our results show that the surface of the least fouling species, F. distichus subsp. evanescens, is enriched with bioactive compounds, such as betaine lipids MGTA, 4-pyridoxic acid, and ulvaline, which are absent from the other species. Additionally, it exhibits a high abundance of the fungal genera Mucor and Alternaria, along with the bacterial genus Yoonia-Loktanella. These taxa are known for producing antimicrobial/antifouling compounds, suggesting their potential role in the observed fouling resistance on the surface of the F. distichus subsp. evanescens compared to F. serratus and F. vesiculosus. These findings provide valuable clues on the differential surface fouling intensity of Fucus spp., and their importance in marine chemical defense and fouling dynamics.

Description: Microbial co-cultivation is a promising approach for the activation of biosynthetic gene clusters (BGCs) that remain transcriptionally silent under artificial culture conditions. As part of our project aiming at the discovery of marine-derived fungal agrochemicals, we previously used four phytopathogens as model competitors in the co-cultivation of 21 marine fungal strains. Based on comparative untargeted metabolomics analyses and anti-phytopathogenic activities of the co-cultures, we selected the co-culture of marine Cosmospora sp. with the phytopathogen Magnaporthe oryzae for in-depth chemical studies. UPLC-MS/MS-based molecular networking (MN) of the co-culture extract revealed an enhanced diversity of compounds in several molecular families, including isochromanones, specifically induced in the co-culture. Large scale co-cultivation of Cosmospora sp. and M. oryzae resulted in the isolation of five isochromanones from the whole co-culture extract, namely the known soudanones A, E, D (1-3) and their two new derivatives, soudanones H-I (4-5), the known isochromans, pseudoanguillosporins A and B (6, 7), naphtho-γ-pyrones, cephalochromin and ustilaginoidin G (8, 9), and ergosterol (10). Their structures were established by NMR, HR-ESIMS, FT-IR, electronic circular dichroism (ECD) spectroscopy, polarimetry ([α]D), and Mosher’s ester reaction. Bioactivity assays revealed antimicrobial activity of compounds 2 and 3 against the phytopathogens M. oryzae and Phytophthora infestans, while pseudoanguillosporin A (6) showed the broadest and strongest anti-phytopathogenic activity against Pseudomonas syringae, Xanthomonas campestris, M. oryzae and P. infestans. This is the first study assessing the anti-phytopathogenic activities of soudanones.

Description: The marine mesopelagic zone extends from water depths of 200 m to 1000 m and is home to a vast number and diversity of species. It is one of the least understood regions of the marine environment with untapped resources of pharmaceutical relevance. The mesopelagic jellyfish Periphylla periphylla is a well-known and widely distributed species in the mesopelagic zone; however, the diversity or the pharmaceutical potential of its cultivable microbiota has not been explored. In this study, we isolated microorganisms associated with the inner and outer umbrella of P. periphylla collected in Irminger Sea by a culture-dependent approach, and profiled their chemical composition and biological activities. Sixteen mostly gram-negative bacterial isolates were selected and subjected to an OSMAC cultivation regime approach using liquid and solid marine broth (MB) and glucose–yeast–malt (GYM) media. Their ethyl acetate (EtOAc) extracts were assessed for cytotoxicity and antimicrobial activity against fish and human pathogens. All, except one extract, displayed diverse levels of antimicrobial activities. Based on low IC50 values, four most bioactive gram-negative strains; Polaribacter sp. SU124, Shewanella sp. SU126, Psychrobacter sp. SU143 and Psychrobacter sp. SU137, were prioritized for an in-depth comparative and untargeted metabolomics analysis using feature-based molecular networking. Various chemical classes such as diketopiperazines, polyhydroxybutyrates (PHBs), bile acids and other lipids were putatively annotated, highlighting the biotechnological potential in P. periphylla-associated microbiota as well as gram-negative bacteria. This is the first study providing an insight into the cultivable bacterial community associated with the mesopelagic jellyfish P. periphylla and, indeed, the first to mine the metabolome and antimicrobial activities of these microorganisms.

Description: Co-cultivation, coupled with the OSMAC approach, is considered an efficient method for expanding microbial chemical diversity through the activation of cryptic biosynthetic gene clusters (BGCs). As part of our project aiming to discover new fungal metabolites for crop protection, we previously reported five polyketides, the macrolides dendrodolides E (1) and N (2), the azaphilones spiciferinone (3) and 8α-hydroxy-spiciferinone (4), and the bis-naphtho-γ-pyrone cephalochromin (5) from the solid Potato Dextrose Agar (PDA) co-culture of two marine sediment-derived fungi, Plenodomus influorescens and Pyrenochaeta nobilis. However, some of the purified metabolites could not be tested due to their minute quantities. Here we cultivated these fungi (both axenic and co-cultures) in liquid regime using three different media, Potato Dextrose Broth (PDB), Sabouraud Dextrose Broth (SDB), and Czapek-Dox Broth (CDB), with or without shaking. The aim was to determine the most ideal co-cultivation conditions to enhance the titers of the previously isolated compounds and to produce extracts with stronger anti-phytopathogenic activity as a basis for future upscaled fermentation. Comparative metabolomics by UPLC-MS/MS-based molecular networking and manual dereplication was employed for chemical profiling and compound annotations. Liquid co-cultivation in PDB under shaking led to the strongest activity against the phytopathogen Phytophthora infestans. Except for compound 1, all target compounds were detected in the co-culture in PDB. Compounds 2 and 5 were produced in lower titers, whereas the azaphilones (3 and 4) were overexpressed in PDB compared to PDA. Notably, liquid PDB co-cultures contained meroterpenoids and depside clusters that were absent in the solid PDA co-cultures. This study demonstrates the importance of culture regime in BGC regulation and chemical diversity of fungal strains in co-culture studies.