Selective isolation of Arctic marine actinobacteria and a down-scaled fermentation and extraction strategy for identifying bioactive compounds

Actinobacteria are among the most prolific producers of bioactive secondary metabolites. In order to collect Arctic marine bacteria for the discovery of new bioactive metabolites, actinobacteria were selectively isolated during a research cruise in the Greenland Sea, Norwegian Sea and the Barents Sea. In the frame of the isolation campaign, it was investigated how different sample treatments, isolation media and sample-sources, such as animals and sediments, affected the yield of actinobacterial isolates to aid further isolation campaigns. Special attention was given to sediments, where we expected spores of spore forming bacteria to enrich. Beside actinobacteria a high share of bacilli was obtained which was not desired. An experimental protocol for down-scaled cultivation and extraction was tested and compared with an established low-throughput cultivation and extraction protocol. The heat-shock method proved suitable to enrich spore-, or endospore forming bacteria such as bacilli. Finally, a group bioactive compounds could be tentatively identified using UHPLC–MS/MS analysis of the active fractions.publishedVersio

Bioprospecting of marine fungi from the High Arctic: A study of high latitude marine fungi from understudied taxa; bioactivity potential, taxonomy and genomics

Marine fungi comprise a group of organisms that have been overlooked for a long time. Research interest has increased with the realization of the important ecological role and rich chemistry of marine fungi. Marine fungi have yielded thousands of new natural products the last decade, but many taxa remain unstudied. Marine fungi from the Arctic have not been reported in literature in regard to bioprospecting campaigns and represent a novel source of natural products. The aim of this thesis is to assess the potential of Arctic marine fungi to produce bioactive secondary metabolites by fermentation and genome analysis. This was achieved in three steps. First, fungi were isolated from the Svalbard archipelago. The 20 isolates obtained were characterized based on molecular markers and their antibacterial activity was tested using an agar diffusion assay (Paper 1). Secondly, three distinct marine fungi were whole genome sequenced and characterized. One of the fungi represented a putatively novel species which was circumscribed based on morphology and phylogenetic inference (Paper 2). Finally, a metabolite from one fungus among the 20 obtained around Svalbard was isolated and the bioactivities characterized (Paper 3). In Paper 1, half of the fungal isolates showed activity against pathogenic bacteria and every third isolate represents potentially new species of fungi. Five of these isolates are strictly marine fungi belonging to the order of Lulworthiales. The study showed that the Arctic can yield novel marine fungal diversity that can be utilized in bioprospecting. For Paper 2, three marine fungi were whole genome sequenced and their biosynthetic gene clusters were characterized. Mapping of the biosynthetic gene clusters (BGCs) within the Emericellopsis genome confirmed the detection of the secondary metabolite helvolic acid produced during fermentation. The study revealed numerous unknown biosynthetic gene clusters and a range of carbohydrate active enzymes. Each of the three genomes provides the first genome of their respective taxa and can contribute to understanding their evolutionary adaption to the marine environment. In Paper 3, a novel compound from the fermentation broth of Mytilinidion sp. was isolated and its bioactivity was characterized using seven different bioactivity assays. The compound turned out to be a modified medium component with IC50 of 43 µM in an ACE-inhibitory assay. The compound was novel and this is the first report of its bioactivity. Molecular networking could perhaps have provided early indications that the compound was a modified medium component

Cultivable marine fungi from the Arctic Archipelago of Svalbard and their antibacterial activity

During a research cruise in 2016, we isolated fungi from sediments, seawater, driftwood, fruiting bodies, and macroalgae using three different media to assess species richness and potential bioactivity of cultivable marine fungi in the High Arctic region. Ten stations from the Svalbard archipelago (73–80 °N, 18–31 °E) were investigated and 33 fungal isolates were obtained. These grouped into 22 operational taxonomic units (OTUs) using nuc rDNA internal transcribed spacer regions (ITS1-5.8S-ITS2 = ITS) with acut-off set at 98% similarity. The taxonomic analysis showed that 17 OTUs belonged to Ascomycota, one to Basidiomycota, two to Mucoromycota and two were fungal-like organisms. The nuc rDNA V1-V5 regions of 18S (18S) and D1-D3 regions of 28S (28S) were sequenced from representative isolates of each OTU for comparison to GenBank sequences. Isolates of Lulworthiales and Eurotiales were the most abundant, with seven isolates each. Among the 22 OTUs, nine represent potentially undescribed species based on low similarity to GenBank sequences and 10 isolates showed inhibitory activity against Gram-positive bacteria in an agar diffusion plug assay. These results show promise for the Arctic region as asource of novel marine fungi with the ability to produce bioactive secondary metabolites with antibacterial properties

Genomic characterization of three marine fungi, including Emericellopsis atlantica sp. nov. with signatures of a generalist lifestyle and marine biomass degradation.

Marine fungi remain poorly covered in global genome sequencing campaigns; the 1000 fungal genomes (1KFG) project attempts to shed light on the diversity, ecology and potential industrial use of overlooked and poorly resolved fungal taxa. This study characterizes the genomes of three marine fungi: Emericellopsis sp. TS7, wood-associated Amylocarpus encephaloides and algae-associated Calycina marina. These species were genome sequenced to study their genomic features, biosynthetic potential and phylogenetic placement using multilocus data. Amylocarpus encephaloides and C. marina were placed in the Helotiaceae and Pezizellaceae (Helotiales), respectively, based on a 15-gene phylogenetic analysis. These two genomes had fewer biosynthetic gene clusters (BGCs) and carbohydrate active enzymes (CAZymes) than Emericellopsis sp. TS7 isolate. Emericellopsis sp. TS7 (Hypocreales, Ascomycota) was isolated from the sponge Stelletta normani. A six-gene phylogenetic analysis placed the isolate in the marine Emericellopsis clade and morphological examination confirmed that the isolate represents a new species, which is described here as E. atlantica. Analysis of its CAZyme repertoire and a culturing experiment on three marine and one terrestrial substrates indicated that E. atlantica is a psychrotrophic generalist fungus that is able to degrade several types of marine biomass. FungiSMASH analysis revealed the presence of 35 BGCs including, eight non-ribosomal peptide synthases (NRPSs), six NRPS-like, six polyketide synthases, nine terpenes and six hybrid, mixed or other clusters. Of these BGCs, only five were homologous with characterized BGCs. The presence of unknown BGCs sets and large CAZyme repertoire set stage for further investigations of E. atlantica. The Pezizellaceae genome and the genome of the monotypic Amylocarpus genus represent the first published genomes of filamentous fungi that are restricted in their occurrence to the marine habitat and form thus a valuable resource for the community that can be used in studying ecological adaptions of fungi using comparative genomics

Genomic characterization of three marine fungi, including Emericellopsis atlantica sp. nov. with signatures of a generalist lifestyle and marine biomass degradation

Marine fungi remain poorly covered in global genome sequencing campaigns; the 1000 fungal genomes (1KFG) project attempts to shed light on the diversity, ecology and potential industrial use of overlooked and poorly resolved fungal taxa. This study characterizes the genomes of three marine fungi: Emericellopsis sp. TS7, wood-associated Amylocarpus encephaloides and algae-associated Calycina marina. These species were genome sequenced to study their genomic features, biosynthetic potential and phylogenetic placement using multilocus data. Amylocarpus encephaloides and C. marina were placed in the Helotiaceae and Pezizellaceae (Helotiales), respectively, based on a 15-gene phylogenetic analysis. These two genomes had fewer biosynthetic gene clusters (BGCs) and carbohydrate active enzymes (CAZymes) than Emericellopsis sp. TS7 isolate. Emericellopsis sp. TS7 (Hypocreales, Ascomycota) was isolated from the sponge Stelletta normani. A six-gene phylogenetic analysis placed the isolate in the marine Emericellopsis clade and morphological examination confirmed that the isolate represents a new species, which is described here as E. atlantica. Analysis of its CAZyme repertoire and a culturing experiment on three marine and one terrestrial substrates indicated that E. atlantica is a psychrotrophic generalist fungus that is able to degrade several types of marine biomass. FungiSMASH analysis revealed the presence of 35 BGCs including, eight non-ribosomal peptide synthases (NRPSs), six NRPS-like, six polyketide synthases, nine terpenes and six hybrid, mixed or other clusters. Of these BGCs, only five were homologous with characterized BGCs. The presence of unknown BGCs sets and large CAZyme repertoire set stage for further investigations of E. atlantica. The Pezizellaceae genome and the genome of the monotypic Amylocarpus genus represent the first published genomes of filamentous fungi that are restricted in their occurrence to the marine habitat and form thus a valuable resource for the community that can be used in studying ecological adaptions of fungi using comparative genomics

Genomic characterization of three marine fungi, including Emericellopsis atlantica sp. nov. with signatures of a generalist lifestyle and marine biomass degradation

Marine fungi remain poorly covered in global genome sequencing campaigns; the 1000 fungal genomes (1KFG) project attempts to shed light on the diversity, ecology and potential industrial use of overlooked and poorly resolved fungal taxa. This study characterizes the genomes of three marine fungi: Emericellopsis sp. TS7, wood-associated Amylocarpus encephaloides and algae-associated Calycina marina. These species were genome sequenced to study their genomic features, biosynthetic potential and phylogenetic placement using multilocus data. Amylocarpus encephaloides and C. marina were placed in the Helotiaceae and Pezizellaceae (Helotiales), respectively, based on a 15-gene phylogenetic analysis. These two genomes had fewer biosynthetic gene clusters (BGCs) and carbohydrate active enzymes (CAZymes) than Emericellopsis sp. TS7 isolate. Emericellopsis sp. TS7 (Hypocreales, Ascomycota) was isolated from the sponge Stelletta normani. A six-gene phylogenetic analysis placed the isolate in the marine Emericellopsis clade and morphological examination confirmed that the isolate represents a new species, which is described here as E. atlantica. Analysis of its CAZyme repertoire and a culturing experiment on three marine and one terrestrial substrates indicated that E. atlantica is a psychrotrophic generalist fungus that is able to degrade several types of marine biomass. FungiSMASH analysis revealed the presence of 35 BGCs including, eight non-ribosomal peptide synthases (NRPSs), six NRPS-like, six polyketide synthases, nine terpenes and six hybrid, mixed or other clusters. Of these BGCs, only five were homologous with characterized BGCs. The presence of unknown BGCs sets and large CAZyme repertoire set stage for further investigations of E. atlantica. The Pezizellaceae genome and the genome of the monotypic Amylocarpus genus represent the first published genomes of filamentous fungi that are restricted in their occurrence to the marine habitat and form thus a valuable resource for the community that can be used in studying ecological adaptions of fungi using comparative genomics

Genomic characterization of three marine fungi, including Emericellopsis atlantica sp. nov. with signatures of a generalist lifestyle and marine biomass degradation

Marine fungi remain poorly covered in global genome sequencing campaigns; the 1000 fungal genomes (1KFG) project attempts to shed light on the diversity, ecology and potential industrial use of overlooked and poorly resolved fungal taxa. This study characterizes the genomes of three marine fungi: Emericellopsis sp. TS7, wood-associated Amylocarpus encephaloides and algae-associated Calycina marina. These species were genome sequenced to study their genomic features, biosynthetic potential and phylogenetic placement using multilocus data. Amylocarpus encephaloides and C. marina were placed in the Helotiaceae and Pezizellaceae (Helotiales), respectively, based on a 15-gene phylogenetic analysis. These two genomes had fewer biosynthetic gene clusters (BGCs) and carbohydrate active enzymes (CAZymes) than Emericellopsis sp. TS7 isolate. Emericellopsis sp. TS7 (Hypocreales, Ascomycota) was isolated from the sponge Stelletta normani. A six-gene phylogenetic analysis placed the isolate in the marine Emericellopsis clade and morphological examination confirmed that the isolate represents a new species, which is described here as E. atlantica. Analysis of its CAZyme repertoire and a culturing experiment on three marine and one terrestrial substrates indicated that E. atlantica is a psychrotrophic generalist fungus that is able to degrade several types of marine biomass. FungiSMASH analysis revealed the presence of 35 BGCs including, eight non-ribosomal peptide synthases (NRPSs), six NRPS-like, six polyketide synthases, nine terpenes and six hybrid, mixed or other clusters. Of these BGCs, only five were homologous with characterized BGCs. The presence of unknown BGCs sets and large CAZyme repertoire set stage for further investigations of E. atlantica. The Pezizellaceae genome and the genome of the monotypic Amylocarpus genus represent the first published genomes of filamentous fungi that are restricted in their occurrence to the marine habitat and form thus a valuable resource for the community that can be used in studying ecological adaptions of fungi using comparative genomics