One Strain Many Compounds Approach Research Articles

Potential Antiviral Xanthones from a Coastal Saline Soil Fungus Aspergillus iizukae

Five new (1–5) and two known xanthones (6 and 7), one of the latter (6) obtained for the first time as a natural product, together with three known anthraquinones, questin, penipurdin A, and questinol, were isolated from the coastal saline soil-derived Aspergillus iizukae by application of an OSMAC (one strain many compounds) approach. Their structures were determined by interpretation of nuclear magnetic resonance (NMR) and high-resolution electrospray ionization mass spectroscopy (HRESIMS) data, as well as comparison of these data with those of related known compounds. Antiviral activity of xanthones 1−7 was evaluated through the cytopathic effect (CPE) inhibition assay, and compound 2 exhibited distinctly strong activity towards influenza virus (H1N1), herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) with IC50 values of 44.6, 21.4, and 76.7 μM, respectively, which indicated that it was worth to further investigate it as a potential lead compound. The preliminary structure-activity relationship of the xanthones is discussed.

Spatial-temporal profiling of prodiginines and serratamolides produced by endophytic Serratia marcescens harbored in Maytenus serrata

An endophytic bacterium, Serratia marcescens MSRBB2, isolated from inner bark of a Cameroonian Maytenus serrata plant, was subjected to the OSMAC (One Strain Many Compounds) approach and metabolic profiling using HPLC-HRMSn. We identified 7 prodiginines along with 26 serratamolides. Their biosynthetic pathways were elucidated by feeding with labeled precursors in combination with HRMSn. Dual-culture confrontation/restriction assays of the bacterial endophyte were devised with coexisting fungal endophytes (Pestalotiopsis virgatula, Aspergillus caesiellus and Pichia spp.) as well as with unrelated, non-endophytic fungi belonging to the same genera. The assays were combined with scanning electron microscopy (SEM) as well as matrix-assisted laser desorption ionization imaging high-resolution mass spectrometry (MALDI-imaging-HRMS) for visualizing, both in high spatial and temporal resolution, the distribution and interplay of the compounds during microbial interactions. We demonstrated the effect of prodigiosin produced by endophytic S. marcescens MSRBB2 as an allelochemical that specifically inhibits coexisting endophytic fungi. Our results provide new insights into the physiological and ecological relevance of prodiginines and serratamolides within the context of allelopathy and chemical defense interaction occurring between coexisting endophytes harbored in M. serrata.

Secondary Metabolites from Alternaria sp., a Fungal Endophyte Isolated from the Seeds of Ziziphus jujuba

From the seeds of the traditional medicinal plant Ziziphus jujuba growing in Uzbekistan, the fungal endophyte Alternaria sp. was isolated. Extracts of this fungus grown on solid rice and liquid Wickerham culture media yielded 21 natural products. These natural products include the new phthalide natural product (±)7-methoxyphthalide-3-acetic acid (1) in addition to 20 known compounds including 19 polyketides and one alkaloid. Moreover, the OSMAC (One Strain MAny Compounds) approach is applied in this study. The unambiguous structure of the new compound was elucidated on the basis of one- and two-dimensional NMR spectroscopy in addition to HR-ESI-MS. Compounds 2, 3, 4, and 7 exhibited a cytotoxic activity against mouse lymphoma cell lines (L5178Y) with IC50 values of 1.7, 7.8, 6.8, and 6.2 μM, respectively when compared with the positive control kahalalide F (IC50 4.3 μM).

Antibacterial secondary metabolites from an endophytic fungus, Fusarium solani JK10

Extensive chemical investigation of the endophytic fungus, Fusarium solani JK10, harbored in the root of the Ghanaian medicinal plant Chlorophora regia, using the OSMAC (One Strain Many Compounds) approach resulted in the isolation of seven new 7–desmethyl fusarin C derivatives (1–7), together with five known compounds (8–12). The structures of the new compounds were elucidated by analysis of their spectroscopic data including 1D, 2D NMR, HRESI–MSn and IR data. The relative configuration of compounds 1/2 was deduced by comparison of their experimental electronic circular dichroism (ECD) and optical rotation data with those reported in literature. The absolute configuration of solaniol (10), a known compound with undefined absolute stereochemistry, was established for the first time by X–ray diffraction analysis of a single–crystal structure using Cu–Kα radiation. The antibacterial activities of the crude fungal extract and the compounds isolated from the fungus were evaluated against some clinically important bacterial strains such as Staphylococcus aureus and Bacillus subtilis, as well as an environmental strain of Escherichia coli and the soil bacterium Acinetobacter sp. BD4. Compounds 3/4 and 6 exhibited antibacterial efficacies against the soil bacterium Acinetobacter sp., comparable to the reference standard streptomycin. All the tested compounds (1–9) demonstrated antibacterial activity against the environmental strain of E. coli, whereas no antibacterial activity was observed against S. aureus and B. subtilis. The antibacterial activity of the isolated compounds typically against E. coli and Acinetobacter sp. provides further insight into the possible involvement of root-borne endophytes in chemical defense of their host plants in selected ecological niches.

Antimicrobial alkaloids produced by the mangrove endophyte Penicillium brocae MA-231 using the OSMAC approach

The OSMAC (one strain-many compounds) protocol was used in this study to obtain various metabolites from the mangrove-derived endophytic fungus Penicillium brocae MA-231.

Effects of Media on the Fungal Metabolome

Variation in the growing environment (e.g. temperature, pH, nutrition) can have significant impacts on the quantity and diversity of fungal secondary metabolites. In the industrial setting, optimization of growing conditions can lead to significantly increased production of a compound of interest. Such optimization becomes much more difficult in a drug-discovery screening situation, as the ideal conditions for one organism may induce poor metabolic diversity for a different organism. The manipulation of culture growing conditions for the purpose of augmenting the natural metabolic diversity of microorganisms is often referred to as the OSMAC (one strain, many compounds) approach. Here, the impact of different media types on the secondary metabolite production of several fungal strains is examined in the context of the drug-discovery screening process.

Multifaceted (Mecsus) Protocol for Microbial Metabolites Studies

The advent of new chemical genetic tools and high-throughput screening technologies and platforms have already led and will continue to lead in accessing the natural product diversity of microorganisms. This paper represents a continuation of the work on the investigation into means of making better use of the advantages of new technologies and approaches in drug discovery that the Atta-ur-Rahman Research Institute of Natural Product Discovery (RiND) at MARA University of Technology has engaged in. MECSUS (Microtiter plate, Elicitors, Combination, Solid phase extraction, UHPLC, Statistical analysis) is a protocol for microbial metabolites studies that involves miniaturized parallel fermentations in 96-well microtiter plate with up to ninety six different media, parallel extraction and data analysis.The aforementioned protocol introduces elements of incremental novelty in the natural product screening program by means of combining and harnessing existing ideas, techniques, and technologies into a protocol for the implementation of the OSMAC (one strain many compounds) approach at micro-scale. Its main advantage is the decrease of the scale of operation with the use of the 96-well microtiter plates. It’s benefits include the possibility of overcoming few issues such as processing time and human resources that have somewhat hampered the implementation of the OSMAC approach and/or the systematic study of a large library of microorganisms.

Studies on Enantioselective Total Synthesis of Ascospiroketal B (I)

Ascospiroketal B (1) was isolated from the Ascochyta slicorniae, a marine-derived fungus known as rich sources of secondary metabolites, by OSMAC (One Strain-Many Compounds) approach. Although the relative structure of ascospiroketal B (1) was elucidated by G. M. Konig in 2007 by use of H NMR, C NMR, IR, 2D HSQC, 2D NOSEY and so on, the relative configurations at C2’, C3’ and C15 still need to be determined, probably due to the flexibility of the side chain. Key structural features include a tricyclic spiro skeleton and eight stereogenic centers with one quaternary carbon center at C2 position. We report herein the first synthesis of C1-C10 fragment 20 of ascospiroketal B (1). Retrosynthetic strategy is summarized in Figure 1. The C11C12 bond would be introduced by Stille coupling of the vinyl tin 2 and C1-C11 fragmenet 3. Fragment 3 should be derived from the intermediate 4 and the intermediate 4 could be synthesized from NHK reaction between vinyl iodide 5 and aldehyde 6. We envisioned that installation of the quaternary chiral center at C2 of ascospiroketal B (1) would be accomplished by asymmetric alkylation protocol. Key intermediate 10 was prepared from the commercially available 1,4-butanediol (7) (Scheme 1). After mono-protection of the diol 7 using NaH, PMBCl, and TBAI in THF in 64% yield, the primary hydroxyl group was converted quantitatively to the aldehyde by Swern oxidation. Aldehyde was subsequently converted to α,β-unsaturated ester using stabilized Wittig reagent in benzene, and hydrolysis using 2 N NaOH in MeOH provided the carboxylic acid 8 in 81% two-step yield. Pivaloylation of the acid moiety in 8 by pivaloyl chloride afforded the mixed anhydride, which was immediately treated with the lithium salt of (S)-4-isopropyloxazolidin-2-one to give the intermediate 9 in 73% yield. Quaternary center at C2 of the intermediate 9 was constructed with NaHMDS and BOMCl to provide the desired C-alkylation product 10(59%) along with the O-alkylated product 11(11%). Use of HMPA as additive gave the O-alkylation product 11 only. The chiral auxiliary in 10 was removed using LiOH and H2O2 in aqueous THF in 83% yield (Scheme 2). Although Sharpless asymmetric dihydroxylation of carboxylic acid 12 took 2 days using AD-mix-α and methanesulfonamide in tBuOH and H2O at 0 C, the resulting 1,2-diol was cyclized

Three New Cytochalasins from the Marine‐Derived Fungus Spicaria elegans KLA03 by Supplementing the Cultures with L‐ and D‐Tryptophan

Three new cytochalasins Z(21) -Z(23) (1-3, resp.), together with five analogs, 4-8, were isolated from Spicaria elegans KLA03 by the OSMAC (one strain-many compounds) approach with adding L- and D-tryptophan during its cultivation. The structures of new cytochalasins were elucidated on the basis of comprehensive 1D- and 2D-NMR and HR-ESI-MS analyses. Cytochalasins Z(21) and Z(22) (1 and 2, resp.), and compound 5 showed cytotoxic activities against A-549 cell lines with IC(50) values of 8.2, 20.0, and 3.1 μM, respectively.

Spicochalasin A and New Aspochalasins from the Marine‐Derived Fungus Spicaria elegans

AbstractDifferent culture conditions directed by the OSMAC (one strain‐many compounds) approach drastically modified the metabolites of the fungus Spicaria elegans, which yielded the novel spicochalasin A (1), five new aspochalasins M–Q (2–6), and two known aspochalasins (7 and 8). The gross structures of 1–6 were elucidated by 1D and 2D NMR and MS methods, and their absolute configurations were determined by X‐ray diffraction and the Mosher ester method. Spicochalasin A (1) has a unique pentacyclic system and was found to be moderately cytotoxic towards human leukemic HL‐60 cells with an IC50 value of 19.9 μM.(© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009)

Enhancement of antibiotic and secondary metabolite detection from filamentous fungi by growth on nutritional arrays

We asked to what extent does the application of the OSMAC (one strain, many compounds) approach lead to enhanced detection of antibiotics and secondary metabolites in fungi? Protocols for bacterial microfermentations were adapted to grow fungi in nutritional arrays. Protocols for microfermentations of non-sporulating fungi were validated using known antifungal-producing fungi. Detection of antifungal activity was often medium dependent. The effects of medium arrays and numbers of strains on detection of antifungal signals were modelled by interpolation of rarefaction curves derived from matrices of positive and negative extracts. Increasing the number of fermentation media for any given strain increased the probability of detection of growth inhibition of Candida albicans. Increasing biodiversity increased detection of antifungal phenotypes, however, nutritional arrays could partly compensate for lost antibiotic phenotypes when biodiversity was limiting. Growth and extraction in microtiter plates can enable a discovery strategy emphasizing low-cost medium arrays that can better exploit the metabolic potential of strains. Increasing fermentation parameters raise the probability of detecting bioactive metabolites from strains. The protocols can be used to pre-select strains and their growth conditions for scale up that will most likely yield antibiotics and secondary metabolites.

Big effects from small changes: possible ways to explore nature's chemical diversity.

Fungi or bacteria that produce secondary metabolites often have the potential to bring up various compounds from a single strain. The molecular basis for this well-known observation was confirmed in the last few years by several sequencing projects of different microorganisms. Besides well-known examples about induction of a selected biosynthesis (for example, by high- or low-phosphate cultivation media), no overview about the potential in this field for finding natural products was given. We have investigated the systematic alteration of easily accessible cultivation parameters (for example, media composition, aeration, culture vessel, addition of enzyme inhibitors) in order to increase the number of secondary metabolites available from one microbial source. We termed this way of revealing nature's chemical diversity the 'OSMAC (One Strain-Many Compounds) approach' and by using it we were able to isolate up to 20 different metabolites in yields up to 2.6 g L(-1) from a single organism. These compounds cover nearly all major natural product families, and in some cases the high production titer opens new possibilities for semisynthetic methods to enhance even more the chemical diversity of selected compounds. The OSMAC approach offers a good alternative to industrial high-throughput screening that focuses on the active principle in a distinct bioassay. In consequence, the detection of additional compounds that might be of interest as lead structures in further bioassays is impossible and clearly demonstrates the deficiency of the industrial procedure. Furthermore, our approach seems to be a useful tool to detect those metabolites that are postulated to be the final products of an amazing number of typical secondary metabolite gene clusters identified in several microorganisms. If one assumes a (more or less) defined reservoir of genetic possibilities for several biosynthetic pathways in one strain that is used for a highly flexible production of secondary metabolites depending on the environment, the OSMAC approach might give more insight into the role of secondary metabolism in the microbial community or during the evolution of life itself.