OSMAC Approach Research Articles

Naphthalene derivatives and halogenate quinoline from the coral-derived fungus Trichoderma harzianum (XS-20090075) through OSMAC approach

OSMAC approach was performed on the soft coral-derived fungus Trichoderma harzianum (XS-20090075) leading to the significant changes of its secondary metabolites by using two different cultures. A new naphthalene derivative, trichoharzin B (1) and a new natural product, methyl-trichoharzin (2) were isolated by using rice medium. Whereas, a new natural product, ethyl 2-bromo-4-chloroquinoline-3-carboxylate (9) was obtained by using Czapek’s medium. Their structures were established by extensive spectroscopic investigation. The absolute configuration of 5 was determined by single-crystal X-ray diffraction. Compound 9 was the first halogenate quinoline derivative isolated from the genus of Trichoderma.

Marine Fungi from the Sponge Grantia compressa: Biodiversity, Chemodiversity, and Biotechnological Potential.

The emergence of antibiotic resistance and viruses with high epidemic potential made unexplored marine environments an appealing target source for new metabolites. Marine fungi represent one of the most suitable sources for the discovery of new compounds. Thus, the aim of this work was (i) to isolate and identify fungi associated with the Atlantic sponge Grantia compressa; (ii) to study the fungal metabolites by applying the OSMAC approach (one strain; many compounds); (iii) to test fungal compounds for their antimicrobial activities. Twenty-one fungal strains (17 taxa) were isolated from G. compressa. The OSMAC approach revealed an astonishing metabolic diversity in the marine fungus Eurotium chevalieri MUT 2316, from which 10 compounds were extracted, isolated, and characterized. All metabolites were tested against viruses and bacteria (reference and multidrug-resistant strains). Dihydroauroglaucin completely inhibited the replication of influenza A virus; as for herpes simplex virus 1, total inhibition of replication was observed for both physcion and neoechinulin D. Six out of 10 compounds were active against Gram-positive bacteria with isodihydroauroglaucin being the most promising compound (minimal inhibitory concentration (MIC) 4–64 µg/mL) with bactericidal activity. Overall, G. compressa proved to be an outstanding source of fungal diversity. Marine fungi were capable of producing different metabolites; in particular, the compounds isolated from E. chevalieri showed promising bioactivity against well-known and emerging pathogens.

Expanding the chemical diversity of an endophytic fungus Bulgaria inquinans, an ascomycete associated with mistletoe, through an OSMAC approach.

An endophytic fungus Bulgaria inquinans (isolate MSp3-1), isolated from mistletoe (Viscum album), was subjected to fermentation on solid Czapek medium. Chromatographic workup of the crude EtOAc extract yielded five new natural products (1–5). Subsequent application of the “One Strain, MAny Compounds” (OSMAC) strategy on this strain by the addition of a mixture of salts (MgSO4, NaNO3 and NaCl) to solid Czapek medium induced the accumulation of nine additional new secondary metabolites (6–13, 16), with most of them (8, 10–12) not detectable in cultures lacking the salt mixture. The structures of the new compounds were established on the basis of the 1D/2D NMR and HRESIMS data. The TDDFT-ECD method was applied to determine the absolute configurations of the new compounds 1, 4 and 6 as well as of the previously reported bulgarialactone B (14), for which the absolute configuration was unknown so far. The modified Mosher's method was performed to assign the absolute configurations of 12 and 13. TDDFT-ECD analysis also allowed determining the absolute configuration of (+)-epicocconone, which had an enantiomeric absolute configuration in the tricyclic moiety compared to that of bulgarialactone B (14). All the isolated metabolites were evaluated for their cytotoxic activity. Compound 2 was found to possess strong cytotoxic activity against the murine lymphoma cell line L5178Y with an IC50 value of 1.8 μM, while the remaining metabolites were shown to be inactive.

Emericelactones A-D: Four novel polyketides produced by Emericella sp. XL 029, a fungus associated the leaves of Panax notoginseng

The application of an OSMAC approach on an endophytic fungus Emericella sp. XL 029 has yielded four novel polyketides, emericelactones A-D (1–4).The structures and stereochemistry of 1–4 were elucidated by NMR and MS data analyses, computational methods, as well as by comparison with known compounds. Compound 1 contained an unprecedented linear pentaene substructure ending in an oxabicyclo[2.2.1]heptane moiety, while compounds 2–4 are epimers possessing an unprecedented linear triene structures ending in two cyclic moieties of an oxabicyclo[2.2.1]heptane and a cyclopentan-1-one. Compounds 1–4 showed moderate antimicrobial activities against three agricultural pathogenic fungi (Verticillium dahliae Kleb, Rhizoctonia solani and Gibberella saubinetii) and two human pathogenic bacteria (Micrococcus lysodeikticus and Salmonella typhi) with MIC values of 25–50 μg/mL.

Metabolomics-Driven Discovery of Meroterpenoids from a Mussel-Derived Penicillium ubiquetum.

Penicillium ubiquetum MMS330 isolated from the blue mussel Mytilus edulis collected on the Loire estuary in France was here investigated. As very few secondary metabolites have been documented for this species, its metabolome was studied following the OSMAC approach to enhance as many biosynthetic pathways as possible. Interestingly, HPLC-HRMS based hierarchical clustering analysis together with MS/MS molecular networking highlighted the selective overproduction of some structurally related compounds when the culture was performed on seawater CYA (Czapek Yeast extract Agar) medium. Mass-guided purification from large scale cultivation on this medium led to the isolation of nine meroterpenoids including two new analogues, 22-deoxyminiolutelide A (1) and 4-hydroxy-22-deoxyminiolutelide B (2), together with seven known compounds (3-9). The structures of 1 and 2 were elucidated on the basis of HR-ESIMS and NMR spectroscopic data analysis. Furthermore, NMR signals of 22-deoxyminiolutelide B (3) were reassigned.

Induction of new metabolites from sponge-associated fungus Aspergillus carneus by OSMAC approach

A comparative study on the metabolic profile of the sponge-associated fungus Aspergillus carneus using the OSMAC approach was conducted. The fungal strain was fermented on three different media including solid rice medium with or without sea salt and modified Czapek medium. Three new natural products, isopropylchaetominine (1), isoterrelumamide A (2) and 5′-epi-averufanin (3), together with fourteen known compounds (4–17) were isolated. The structures of the new compounds were established by 1D and 2D NMR spectroscopic analysis as well as by HRESIMS. Compound 2 was only found when the fungus was cultivated on modified Czapek medium, whereas compounds 4, 7, 11, 12, and 14 were only detected in fungal extracts from solid rice media lacking sea salt. Compounds 8 and 13 on the other hand were only found when A. carneus was cultured on solid rice with sea salt. The cytotoxic and antimicrobial activities of all isolated compounds were evaluated. Compounds 1, 9 and 17 showed strong cytotoxicity against the mouse lymphoma cell line L5178Y with IC50 values of 0.4, 0.3 and 0.2 μM, respectively. In addition, compounds 3, 5 and 6 showed inhibitory activity against different Gram-positive bacterial strains with MIC values ranging from 2.3 to 18.4 μg/mL.

Extending the "One Strain Many Compounds" (OSMAC) Principle to Marine Microorganisms.

Genomic data often highlights an inconsistency between the number of gene clusters identified using bioinformatic approaches as potentially producing secondary metabolites and the actual number of chemically characterized secondary metabolites produced by any given microorganism. Such gene clusters are generally considered as “silent”, meaning that they are not expressed under laboratory conditions. Triggering expression of these “silent” clusters could result in unlocking the chemical diversity they control, allowing the discovery of novel molecules of both medical and biotechnological interest. Therefore, both genetic and cultivation-based techniques have been developed aimed at stimulating expression of these “silent” genes. The principles behind the cultivation based approaches have been conceptualized in the “one strain many compounds” (OSMAC) framework, which underlines how a single strain can produce different molecules when grown under different environmental conditions. Parameters such as, nutrient content, temperature, and rate of aeration can be easily changed, altering the global physiology of a microbial strain and in turn significantly affecting its secondary metabolism. As a direct extension of such approaches, co-cultivation strategies and the addition of chemical elicitors have also been used as cues to activate “silent” clusters. In this review, we aim to provide a focused and comprehensive overview of these strategies as they pertain to marine microbes. Moreover, we underline how changes in some parameters which have provided important results in terrestrial microbes, but which have rarely been considered in marine microorganisms, may represent additional strategies to awaken “silent” gene clusters in marine microbes. Unfortunately, the empirical nature of the OSMAC approach forces scientists to perform extensive laboratory experiments. Nevertheless, we believe that some computation and experimental based techniques which are used in other disciplines, and which we discuss; could be effectively employed to help streamline the OSMAC based approaches. We believe that natural products discovery in marine microorganisms would be greatly aided through the integration of basic microbiological approaches, computational methods, and technological innovations, thereby helping unearth much of the as yet untapped potential of these microorganisms.

Substituted l-tryptophan-l-phenyllactic acid conjugates produced by an endophytic fungus Aspergillus aculeatus using an OSMAC approach.

The endophytic fungus Aspergillus aculeatus isolated from leaves of the papaya plant Carica papaya was fermented on solid rice medium, yielding a new l-tryptophan-l-phenyllactic acid conjugate (1) and thirteen known compounds (11, 14–25). In addition, an OSMAC approach was employed by adding eight different sodium or ammonium salts to the rice medium. Addition of 3.5% NaNO3 caused a significant change of the metabolite pattern of the fungus as indicated by HPLC analysis. Subsequent isolation yielded several new substituted l-tryptophan-l-phenyllactic acid conjugates (1–10) in addition to three known compounds (11–13), among which compounds 2–10, 12–13 were not detected in the rice control culture. All structures were unambiguously elucidated by one and two dimensional NMR spectroscopy and by mass spectrometry. The absolute configuration of the new compounds was determined by Marfey's reaction and X-ray single crystal diffraction. Compounds 19–22 showed cytotoxicity against the L5178Y mouse lymphoma cell line with IC50 values of 3.4, 1.4, 7.3 and 23.7 μM, respectively.

Sugar Induced Modification in Glycolipid Production in Acremonium sp. Revealed by LC-MS Lipidomic Approach

Background: Marine-derived fungi are an underexploited source of lipids for health and nutrition. Little is known about the impact of sugar type on the lipid diversity produced by a marine-derived fungi. Keywords: Acremonium, fatty acid, glycolipid, lipid, lipidomic, oleaginous fungus, OSMAC approach.

4-Me-6E,8E-hexadecadienoic acid isolated from a marine-derived strain of Clonostachys rosea reduces viability of MCF-7 breast cancer cells and gene expression of lipogenic enzymes

Conjugated fatty acids (CFA) have attracted particular attention because of their remarkable biological activities such as anti-carcinogenic, anti-obesity, antithrombotic and anti-atherogenic [1]. The main natural sources of CFA correspond to ruminant milk, algae and seed oils of several plants [2], but fungi have been widely recognized for their production of essential FA such as arachidonic or docosahexaenoic acids which have a major interest in human health and nutrition. Our attention was focused on a marine-derived strain of Clonostachys rosea isolated from marine sediments of the Loire estuary (France), which produced high amounts of triglycerides and an original branched CFA, first identified as 4-Me-6,8-hexadecadienoic acid (4-Me-6,8 – 16:2). In order to optimize the production of this CFA to undergo pharmacological studies, an OSMAC approach has been performed on 7 culture media. Cultures on DCA medium showed the highest production with 19% of total FA. After fungal biomass extraction and saponification of the lipid crude extract, 4-Me-6,8 – 16:2 was purified using urea inclusion and liquid chromatography on AgNO3-impregnated silica gel. The CFA structure was deduced from 1H-NMR and IR analyses confirming the trans configuration of double bonds. When tested against MCF-7 breast cancer cell line, 4-Me-6E,8E-16:2 reduced the viability in a dose dependent manner (up to 75% inhibition after 48h at 100µM). Numerous studies have demonstrated that cell proliferation is associated with hyperactivity of lipogenesis [3, 4]. We have then assessed the effect of 4-Me-6E,8E-16:2 on gene expression of two lipogenic enzymes, the ACC (Acetyl coA Carboxylase) and the FAS (Fatty Acid Synthase). At 50µM and after 24h, expression of ACC and FAS were reduced of 50% and 35%, respectively. These results are promising and need to be completed in order to determine the mechanisms by which 4-Me-6E,8E-16:2 reduce cancer cell proliferation and inhibits the expression of lipogenic enzymes.

Cladosins F and G, two new hybrid polyketides from the deep-sea-derived Cladosporium sphaerospermum 2005-01-E3

Two new fungal hybrid polyketides, cladosins F (1) and G (2), with rare 6(3)-enamino-8,10-dihydroxy-tetraketide system were discovered from the deep-sea-derived fungus Cladosporium sphaerospermum 2005-01-E3 guided by OSMAC approach. Their structures were elucidated on the basis of comprehensive spectroscopic analyses, and cytotoxicity, antitubercular, anti-influenza A H1N1 virus, and NF-κB inhibitory activities were evaluated.

Integrated Analytical Approaches Towards Fungal Natural Products Discovery, Pathway Elucidation and Synthetic Biology

At Center for Microbial Biotechnology we have studied the chemistry of filamentous fungi such as Aspergillus and Penicillium for more than twenty years. Fungi possess an advanced secondary metabolism that is regulated and coordinated in a complex manner depending on environmental challenges. We have demonstrated that the profiles of secondary metabolites (SM) are species specific and that phenotypic based microbial taxonomy can therefore be used in microbial drug discovery (1). This involves the use of multivariate methods for analysis and clustering of mass spectrometric data allowing selection of talented strains for cultivation on various media using the OSMAC approach before testing in anticancer and antibiotic bioassays (2). Single compound identification is done by dereplication, where we use state-of-the-art high resolution mass spectrometry (<1 ppm mass accuracy and accurate isotope pattern) in combination with comprehensive compound databases (3). When likely unknown compounds have been identified an explorative solid-phase extraction approach (4) is applied for micro-scale fractionation on a set of different types of columns (RP, ion-exchange, size, NP), in order to clarify the chemical properties of the bioactive(s). This further aids selection of a fast optimal purification strategy towards the pure bioactive prior to NMR characterization. As part of our dereplication procedure we also use a novel algorithm (X-hitting) that use automated search of full UV-spectra to target both known and novel compounds having characteristic UV chromophore systems (1). More recently we have focused our effort towards characterization of the chemistry of full genome sequenced aspergilli (2). Bioinformatics analysis has demonstrated that for the majority of genes that putatively encode enzymes for SM production, the product is not known or detected, calling for sophisticated molecular biological to trigger and detect the production of SMs from cryptic gene clusters. This talk will highlight our recent efforts towards discovery and linking of novel SMs to their respective gene clusters.

Diverse Metabolic Profiles of a Streptomyces Strain Isolated from a Hyper-arid Environment

The metabolic profile of Streptomyces sp. strain C34, isolated from the Chilean hyper-arid Atacama Desert soil, is dependent on the culture media used for its growth. The application of an OSMAC approach on this strain using a range of cultivation media resulted in the isolation and identification of three new compounds from the rare class of 22-membered macrolactone polyketides, named chaxalactins A-C (1-3). In addition, the known compounds deferroxamine E (4), hygromycin A (5), and 5″-dihydrohygromycin A (6) were detected. The isolated compounds were characterized by NMR spectroscopy and accurate mass spectrometric analysis. Compounds 1-3 displayed strong activity against Gram-positive but weak activity Gram-negative strains tested.