ALBERT

Description: This study aimed at isolating microorganisms associated with the mesopelagic jellyfish Periphylla periphylla collected in Irminger Sea at a depth of 325 m in July 2020. Three different solid cultivation media; Hastings, Marine agar and Wickerham media were used for the isolation of the associated microorganisms. A total of 43 bacteria were isolated from the inner and outer surfaces of the umbrella of P. periphylla, but unfortunately, no fungal strain was isolated. Isolates were further identified by Sanger sequencing of the 16S rRNA gene, and based on phylogenetic distinctiveness (differences in closest relative species according to the nucleotide BLAST), 16 bacteria belonging to 8 different genera were selected and subjected to an OSMAC cultivation regime approach using liquid and solid marine broth and glucose– yeast–malt media. After 7 days of cultivation, cultures were extracted with ethyl acetate and assessed for antimicrobial activity against fish and human pathogens. Based on antimicrobial activity assessment, four most bioactive strains; Polaribacter sp. SU124, Shewanella sp. SU126, Psychrobacter sp. SU143 and Psychrobacter sp. SU137, were prioritized for a comparative and untargeted metabolomics analysis using feature-based molecular networking. These findings highlight the biotechnological potential of P. periphylla-associated microbiota.

Description: Identification of 16 selected bacteria associated to the umbrella of Periphylla periphylla collected in the Irminger Sea based on sequencing of the 16S ribosomal RNA gene and BLAST against the NCBI nucleotide database. The table displays the first 3 hits from two blastn searches, one against all database entries and the second against type strains only. Nucleotide identities (%) with database hits and NCBI accession numbers assigned to the isolated bacteria are also displayed. Colonies were cultured either in marine broth, Hastings medium or Wickerham medium for comparison.

Description: Putative annotation of metabolites produced by the Psychrobacter sp. SU143, Psychrobacter sp. SU137, Shewanella sp. SU126 and Polaribacter sp. SU124 in the liquid and solid regime of Marine Broth and Glucose-Yeast-Malt media. Annotation was based on the m/z [M+H]+ or other adducts, retention time, predicted molecular formula, fragmentation pattern and source of the hit. The source of the ion is indicated by the area under a peak (peak area count). Confidence level of annotation are given based on the reporting standards proposed by Sumner, et al. 2007, i.e. identified compound, is putative annotation without reference standards, is putative characterized compound class, and is unknown compound. The mass spectrometry data used for the molecular networking analysis were deposited in the MassIVE Public GNPS database under the accession number MSV000091067.

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: 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 [α] D 20 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: 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: Fungi represent a rich source of bioactive metabolites and some are marketed as alternatives to synthetic agrochemicals against plant pathogens. However, the culturability of fungal strains in artificial laboratory conditions is still limited and the standard mono-cultures do not reflect their full spectrum chemical diversity. Phytopathogenic fungi and bacteria have successfully been used in the activation of cryptic biosynthetic pathways to promote the production of new secondary metabolites in co-culture experiments. The aim of this study was to map the fungal diversity of Windebyer Noor, a brackish lake connected to Baltic Sea (Germany), to induce the chemical space of the isolated marine-adapted fungi by co-culturing with phytopathogens, and to assess their inhibitory potential against six commercially important phytopathogens. Out of 123 marine-adapted fungal isolates obtained, 21 were selected based on their phylogenetic and metabolite diversity. They were challenged with two phytopathogenic bacteria (Pseudomonas syringae and Ralstonia solanacearum) and two phytopathogenic fungi (Magnaporthe oryzae and Botrytis cinerea) on solid agar. An in-depth untargeted metabolomics approach incorporating UPLC–QToF–HRMS/MS-based molecular networking (MN), in silico MS/MS databases, and manual dereplication was employed for comparative analysis of the extracts belonging to nine most bioactive co-cultures and their respective mono-cultures. The phytopathogens triggered interspecies chemical communications with marine-adapted fungi, leading to the production of new compounds and enhanced expression of known metabolites in co-cultures. MN successfully generated a detailed map of the chemical inventory of both mono- and co-cultures. We annotated overall 18 molecular clusters (belonging to terpenes, alkaloids, peptides, and polyketides), 9 of which were exclusively produced in co-cultures. Several clusters contained compounds, which could not be annotated to any known compounds, suggesting that they are putatively new metabolites. Direct antagonistic effects of the marine-adapted fungi on the phytopathogens were observed and anti-phytopathogenic activity was demonstrated.The untargeted metabolomics approach combined with bioactivity testing allowed prioritization of two co-cultures for purification and characterization of marine fungal metabolites with crop-protective activity. To our knowledge, this is the first study employing plant pathogens to challenge marine-adapted fungi.

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