Bioprospecting of Arctic marine microorganisms. Exploring microbial secondary metabolite production using the one strain-many compounds approach: isolation and characterization of secondary metabolites

Natural products have been used by humans since ancient times as benefactors for improved health. Prior to modern medicine and chemistry, these compounds remained hidden in the plants, animals and other organisms used to heal inflammation, wounds, headache and stomachache among other conditions. Since the start of modern drug discovery with isolation of morphine in 1805, numerous natural products have been isolated from plants, animals, macroorganisms and microorganisms. Today, natural products, or their derivatives, are used as pharmaceuticals within a wide range of therapeutic areas, including cancer, pathogenic infections, inflammation and pain. Microbial natural products have played a particularly important role in the field of antibiotics. The discovery of penicillin from the Pencillium rubens fungus by Alexander Fleming in 1928 marked the beginning of the “Golden Age” of antibiotics that lasted until 1962, where most antibiotic classes in clinical use today were discovered. Several marketed drugs originate from marine microorganisms. Marine microorganisms are underexplored, thus representing a potential source for discovering novel bioactive compounds. In this project, Arctic marine microorganisms were fermented under different conditions based on the OSMAC approach and evaluated for their production of antibacterial and cytotoxic compounds. In paper I, a Pseudomonas sp. bacterium was cultivated in different growth media. The fermentation extracts were fractionated and tested for bioactivity, revealing different bioactivity profiles of the fractions from the different media. Dereplication of the active fractions by UHPLC-HR-MS and molecular networking led to identification of six rhamnolipid compounds, including one novel mono-rhamnolipid. All six compounds had antimicrobial activities, while three had cytotoxic activities. In paper II, a fractionated extract of the bacterium Lacinutrix sp. displayed antibacterial activity. Dereplication of the active fraction resulted in identification of two lyso-ornithine lipids, 1 and 2. The compounds were isolated and their structures were elucidated with UHPLC-HR-MS and NMR. Bioactivity screening showed that 1 had antibacterial activity, while 2 had cytotoxic activity. In paper III, the fungus Digitatispora marina was fermented under different cultivation conditions. Fermentation extracts were fractionated and bioactivity screening of the fractions revealed antibacterial and cytotoxic activities. UHPLC-HR-MS analysis of the fractions showed a compound with an isotope distribution pattern for an ion with a single chlorine atom. The compound was isolated, and structure elucidation with NMR identified it as chlovalicin B. Its bioactive properties were broadly evaluated, revealing it had weak cytotoxic activity but no antimicrobial activities

Two Novel Lyso-Ornithine Lipids Isolated from an Arctic Marine Lacinutrix sp. Bacterium

The Lacinutrix genus was discovered in 2005 and includes 12 Gram-negative bacterial species. To the best of our knowledge, the secondary metabolite production potential of this genus has not been explored before, and examination of Lacinutrix species may reveal novel chemistry. As part of a screening project of Arctic marine bacteria, the Lacinutrix sp. strain M09B143 was cultivated, extracted, fractionated and tested for antibacterial and cytotoxic activities. One fraction had antibacterial activity and was subjected to mass spectrometry analysis, which revealed two compounds with elemental composition that did not match any known compounds in databases. This resulted in the identification and isolation of two novel isobranched lyso-ornithine lipids, whose structures were elucidated by mass spectrometry and NMR spectroscopy. Lyso-ornithine lipids consist of a 3-hydroxy fatty acid linked to the alpha amino group of an ornithine amino acid through an amide bond. The fatty acid chains were determined to be iso-C15:0 (1) and iso-C16:0 (2). Compound 1 was active against the Gram-positive S. agalactiae, while 2 showed cytotoxic activity against A2058 human melanoma cells

Pseudochelin A, a siderophore of Pseudoalteromonas piscicida S2040

A new siderophore containing a 4,5-dihydroimidazole moiety was isolated from Pseudoalteromonas piscicida S2040 together with myxochelins A and B, alteramide A and its cycloaddition product, and bromo- and dibromoalterochromides. The structure of pseudochelin A was established by spectroscopic techniques including 2D NMR and MS/MS fragmentation data. In bioassays selected fractions of the crude extract of S2040 inhibited the opportunistic pathogen Pseudomonas aeruginosa. Pseudochelin A displayed siderophore activity in the chrome azurol S assay at concentrations higher than 50 μM, and showed weak activity against the fungus Aspergillus fumigatus, but did not display antibacterial, anti-inflammatory or anticonvulsant activity

Bioprospecting of Arctic marine microorganisms. Exploring microbial secondary metabolite production using the one strain-many compounds approach: isolation and characterization of secondary metabolites

Natural products have been used by humans since ancient times as benefactors for improved health. Prior to modern medicine and chemistry, these compounds remained hidden in the plants, animals and other organisms used to heal inflammation, wounds, headache and stomachache among other conditions. Since the start of modern drug discovery with isolation of morphine in 1805, numerous natural products have been isolated from plants, animals, macroorganisms and microorganisms. Today, natural products, or their derivatives, are used as pharmaceuticals within a wide range of therapeutic areas, including cancer, pathogenic infections, inflammation and pain. Microbial natural products have played a particularly important role in the field of antibiotics. The discovery of penicillin from the Pencillium rubens fungus by Alexander Fleming in 1928 marked the beginning of the “Golden Age” of antibiotics that lasted until 1962, where most antibiotic classes in clinical use today were discovered. Several marketed drugs originate from marine microorganisms. Marine microorganisms are underexplored, thus representing a potential source for discovering novel bioactive compounds. In this project, Arctic marine microorganisms were fermented under different conditions based on the OSMAC approach and evaluated for their production of antibacterial and cytotoxic compounds. In paper I, a Pseudomonas sp. bacterium was cultivated in different growth media. The fermentation extracts were fractionated and tested for bioactivity, revealing different bioactivity profiles of the fractions from the different media. Dereplication of the active fractions by UHPLC-HR-MS and molecular networking led to identification of six rhamnolipid compounds, including one novel mono-rhamnolipid. All six compounds had antimicrobial activities, while three had cytotoxic activities. In paper II, a fractionated extract of the bacterium Lacinutrix sp. displayed antibacterial activity. Dereplication of the active fraction resulted in identification of two lyso-ornithine lipids, 1 and 2. The compounds were isolated and their structures were elucidated with UHPLC-HR-MS and NMR. Bioactivity screening showed that 1 had antibacterial activity, while 2 had cytotoxic activity. In paper III, the fungus Digitatispora marina was fermented under different cultivation conditions. Fermentation extracts were fractionated and bioactivity screening of the fractions revealed antibacterial and cytotoxic activities. UHPLC-HR-MS analysis of the fractions showed a compound with an isotope distribution pattern for an ion with a single chlorine atom. The compound was isolated, and structure elucidation with NMR identified it as chlovalicin B. Its bioactive properties were broadly evaluated, revealing it had weak cytotoxic activity but no antimicrobial activities

Characterization of Rhamnolipids Produced by an Arctic Marine Bacterium from the Pseudomonas fluorescence Group

The marine environment is a rich source of biodiversity, including microorganisms that have proven to be prolific producers of bioactive secondary metabolites. Arctic seas are less explored than warmer, more accessible areas, providing a promising starting point to search for novel bioactive compounds. In the present work, an Arctic marine Pseudomonas sp. belonging to the Pseudomonas (P.) fluorescence group was cultivated in four different media in an attempt to activate biosynthetic pathways leading to the production of antibacterial and anticancer compounds. Culture extracts were pre-fractionated and screened for antibacterial and anticancer activities. One fraction from three of the four growth conditions showed inhibitory activity towards bacteria and cancer cells. The active fractions were dereplicated using molecular networking based on MS/MS fragmentation data, indicating the presence of a cluster of related rhamnolipids. Six compounds were isolated using HPLC and mass-guided fractionation, and by interpreting data from NMR and high-resolution MS/MS analysis; the structures of the compounds were determined to be five mono-rhamnolipids and the lipid moiety of one of the rhamnolipids. Molecular networking proved to be a valuable tool for dereplication of these related compounds, and for the first time, five mono-rhamnolipids from a bacterium within the P. fluorescence group were characterized, including one new mono-rhamnolipid

Characterization of Rhamnolipids Produced by an Arctic Marine Bacterium from the Pseudomonas fluorescence Group

The marine environment is a rich source of biodiversity, including microorganisms that have proven to be prolific producers of bioactive secondary metabolites. Arctic seas are less explored than warmer, more accessible areas, providing a promising starting point to search for novel bioactive compounds. In the present work, an Arctic marine Pseudomonas sp. belonging to the Pseudomonas (P.) fluorescence group was cultivated in four different media in an attempt to activate biosynthetic pathways leading to the production of antibacterial and anticancer compounds. Culture extracts were pre-fractionated and screened for antibacterial and anticancer activities. One fraction from three of the four growth conditions showed inhibitory activity towards bacteria and cancer cells. The active fractions were dereplicated using molecular networking based on MS/MS fragmentation data, indicating the presence of a cluster of related rhamnolipids. Six compounds were isolated using HPLC and mass-guided fractionation, and by interpreting data from NMR and high-resolution MS/MS analysis; the structures of the compounds were determined to be five mono-rhamnolipids and the lipid moiety of one of the rhamnolipids. Molecular networking proved to be a valuable tool for dereplication of these related compounds, and for the first time, five mono-rhamnolipids from a bacterium within the P. fluorescence group were characterized, including one new mono-rhamnolipid

Chlovalicin B, a Chlorinated Sesquiterpene Isolated from the Marine Mushroom Digitatispora marina

As part of our search for bioactive metabolites from understudied marine microorganisms, the new chlorinated metabolite chlovalicin B (1) was isolated from liquid cultures of the marine basidiomycete Digitatispora marina, which was collected and isolated from driftwood found at Vannøya, Norway. The structure of the novel compound was elucidated by spectroscopic methods including 1D and 2D NMR and analysis of HRMS data, revealing that 1 shares its molecular scaffold with a previously isolated compound, chlovalicin. This represents the first compound isolated from the Digitatispora genus, and the first reported fumagillin/ovalicin-like compound isolated from Basidiomycota. Compound 1 was evaluated for antibacterial activities against a panel of five bacteria, its ability to inhibit bacterial biofilm formation, for antifungal activity against Candida albicans, and for cytotoxic activities against malignant and non-malignant human cell lines. Compound 1 displayed weak cytotoxic activity against the human melanoma cell line A2058 (~50% survival at 50 µM). No activity was detected against biofilm formation or C. albicans at 50 µM, or against bacterial growth at 100 µM nor against the production of cytokines by the human acute monocytic leukemia cell line THP-1 at 50 µ