Production of Induced Secondary Metabolites by a Co-Culture of Sponge-Associated Actinomycetes, Actinokineospora sp. EG49 and Nocardiopsis sp. RV163

Two sponge-derived actinomycetes, Actinokineospora sp. EG49 and Nocardiopsis sp. RV163, were grown in co-culture and the presence of induced metabolites monitored by ¹H NMR. Ten known compounds, including angucycline, diketopiperazine and β-carboline derivatives 1-10, were isolated from the EtOAc extracts of Actinokineospora sp. EG49 and Nocardiopsis sp. RV163. Co-cultivation of Actinokineospora sp. EG49 and Nocardiopsis sp. RV163 induced the biosynthesis of three natural products that were not detected in the single culture of either microorganism, namely N-(2-hydroxyphenyl)-acetamide (11), 1,6-dihydroxyphenazine (12) and 5a,6,11a,12-tetrahydro-5a,11a-dimethyl1,4benzoxazino3,2-b1,4benzoxazine (13a). When tested for biological activity against a range of bacteria and parasites, only the phenazine 12 was active against Bacillus sp. P25, Trypanosoma brucei and interestingly, against Actinokineospora sp. EG49. These findings highlight the co-cultivation approach as an effective strategy to access the bioactive secondary metabolites hidden in the genomes of marine actinomycete

Elicitation of secondary metabolism in actinomycetes

Genomic sequence data have revealed the presence of a large fraction of putatively silent biosynthetic gene clusters in the genomes of actinomycetes that encode for secondary metabolites, which are not detected under standard fermentation conditions. This review focuses on the effects of biological (co-cultivation), chemical, as well as molecular elicitation on secondary metabolism in actinomycetes. Our review covers the literature until June 2014 and exemplifies the diversity of natural products that have been recovered by such approaches from the phylum Actinobacteria

Dereplication strategies for targeted isolation of new antitrypanosomal actinosporins A and B from a marine sponge associated-Actinokineospora sp. EG49

High resolution Fourier transform mass spectrometry (HRFTMS) and nuclear magnetic resonance (NMR) spectroscopy were employed as complementary metabolomic tools to dereplicate the chemical profile of the new and antitrypanosomally active sponge-associated bacterium Actinokineospora sp. EG49 extract. Principal Component (PCA), hierarchical clustering (HCA), and orthogonal partial least square-discriminant analysis (OPLS-DA) were used to evaluate the HRFTMS and NMR data of crude extracts from four different fermentation approaches. Statistical analysis identified the best culture one-strain-many-compounds (OSMAC) condition and extraction procedure, which was used for the isolation of novel bioactive metabolites. As a result, two new O-glycosylated angucyclines, named actinosporins A (1) and B (2), were isolated from the broth culture of Actinokineospora sp. strain EG49, which was cultivated from the Red Sea sponge Spheciospongia vagabunda. The structures of actinosporins A and B were determined by 1D- and 2D-NMR techniques, as well as high resolution tandem mass spectrometry. Testing for antiparasitic properties showed that actinosporin A exhibited activity against Trypanosoma brucei brucei with an IC₅₀ value of 15 µM; however no activity was detected against Leishmania major and Plasmodium falciparum, therefore suggesting its selectivity against the parasite Trypanosoma brucei brucei; the causative agent of sleeping sickness

Trypanocidal Activity of Marine Natural Products

Marine natural products are a diverse, unique collection of compounds with immense therapeutic potential. This has resulted in these molecules being evaluated for a number of different disease indications including the neglected protozoan diseases, human African trypanosomiasis and Chagas disease, for which very few drugs are currently available. This article will review the marine natural products for which activity against the kinetoplastid parasites; Trypanosoma brucei brucei, T.b. rhodesiense and T. cruzi has been reported. As it is important to know the selectivity of a compound when evaluating its trypanocidal activity, this article will only cover molecules which have simultaneously been tested for cytotoxicity against a mammalian cell line. Compounds have been grouped according to their chemical structure and representative examples from each class were selected for detailed discussion

Actinomycete Metabolome Induction/Suppression with N-Acetylglucosamine

The metabolite profiles of three sponge-derived actinomycetes, namely, Micromonospora sp. RV43, Rhodococcus sp. RV157, and Actinokineospora sp. EG49 were investigated after elicitation with N-acetyl-d-glucosamine. 1H NMR fingerprint methodology was utilized to study the differences in the metabolic profiles of the bacterial extracts before and after elicitation. Our study found that the addition of N-acetyl-d-glucosamine modified the secondary metabolite profiles of the three investigated actinomycete isolates. N-Acetyl-d-glucosamine induced the production of 3-formylindole (11) and guaymasol (12) in Micromonospora sp. RV43, the siderophore bacillibactin 16, and surfactin antibiotic 17 in Rhodococcus sp. RV157 and increased the production of minor metabolites actinosporins E–H (21–24) in Actinokineospora sp. EG49. These results highlight the use of NMR fingerprinting to detect changes in metabolism following addition of N-acetyl-d-glucosamine. N-Acetyl-d-glucosamine was shown to have multiple effects including suppression of metabolites, induction of new metabolites, and increased production of minor compounds

A systems approach using OSMAC, Log P and NMR fingerprinting: An approach to novelty

The growing number of sequenced microbial genomes has revealed a remarkably large number of secondary metabolite biosynthetic clusters for which the compounds are still unknown. The aim of the present work was to apply a strategy to detect newly induced natural products by cultivating microorganisms in different fermentation conditions. The metabolomic analysis of 4160 fractions generated from 13 actinomycetes under 32 different culture conditions was carried out by 1H NMR spectroscopy and multivariate analysis. The principal component analysis (PCA) of the 1H NMR spectra showed a clear discrimination between those samples within PC1 and PC2. The fractions with induced metabolites that are only produced under specific growth conditions was identified by PCA analysis. This method allows an efficient differentiation within a large dataset with only one fractionation step. This work demonstrates the potential of NMR spectroscopy in combination with metabolomic data analysis for the screening of large sets of fractions

Metabolomic strategies in dereplication for targeted cultivation and isolation of new bioactive secondary metabolites from fungal endophytes and marine microbial symbionts

High resolution Fourier transform mass spectrometry (HRFTMS) and nuclear magnetic resonance (NMR) spectroscopy were employed as complimentary metabolomic tools to dereplicate chemical profiles of sponge-associated bacteria and fungal endophytes. The innovative strategy involved targeted cultivation and isolation of biologically active compounds. Principal Component (PCA), Hierarchical Clustering (HCA), and Orthogonal Partial Least Square-Discriminant (OPLS-DA) Analysis were used to analyze the HRFTMS and NMR data of culture extracts. The results of the statistical analysis identified and validated the best culture conditions and extraction procedure which optimized the isolation of novel bioactive metabolites. Sponge-derived actinomycetes have become a rich source of new natural products with interesting pharmacological activities. New O-glycosylated angucyclines, were isolated from the broth culture of Actinokineospora sp. strain EG49 which was cultivated from the Red Sea sponge Spheciospongia vagabunda. The structure of the compounds were determined by 1D- and 2D-NMR techniques as well as high resolution tandem mass spectrometry

Chemical Constituents of Kino Extract from Corymbia torelliana

Seven flavanones were identified from kino exudate of Corymbia torelliana by spectroscopic and spectrometric methods including UV, 1D and 2D NMR and UPLC-HR-MS. The study identified seven molecules, namely 3,4\u27,5,7-tetrahydroxyflavanone (1), 3\u27,4\u27,5,7-tetrahydroxyflavanone (2), 4\u27,5,7-trihydroxyflavanone (3), 3,4\u27,5-trihydroxy-7-methoxyflavanone (4), (+)-(2S)-4\u27,5,7-trihydroxy-6-methylflavanone (5), 4\u27,5,7-trihydroxy-6,8-dimethylflavanone (6) and 4\u27,5-dihydroxy-7-methoxyflavanone (7) from this eucalypt species. This is the first report of these natural products from C. torelliana kino exudate

Two new antioxidant actinosporin analogues from the calcium alginate beads culture of sponge-associated Actinokineospora sp. strain EG49

Marine sponge-associated actinomycetes represent an exciting new resource for the identification of new and novel natural products . Previously, we have reported the isolation and structural elucidation of actinosporins A (1) and B (2) from Actinokineospora sp. strain EG49 isolated from the marine sponge Spheciospongia vagabunda. Herein, by employing different fermentation conditions on the same microorganism, we report on the isolation and antioxidant activity of structurally related metabolites, actinosporins C (3) and D (4). The antioxidant potential of actinosporins C and D was demonstrated using the ferric reducing antioxidant power (FRAP) assay. Additionally, at 1.25 μM, actinosporins C and D showed a significant antioxidant and protective capacity from the genomic damage induced by hydrogen peroxide in the human promyelocytic (HL-60) cell line