Bioprospecting Chemical Diversity and Bioactivity in a Marine Derived Aspergillus terreus

A comparative metabolomic study of a marine derived fungus (Aspergillus terreus) grown under various culture conditions is presented. The fungus was grown in eleven different culture conditions using solid agar, broth cultures, or grain based media (OSMAC). Multivariate analysis of LC/MS data from the organic extracts revealed drastic differences in the metabolic profiles and guided our subsequent isolation efforts. The compound 7‐desmethylcitreoviridin was isolated and identified, and is fully described for the first time. In addition, 16 known fungal metabolites were also isolated and identified. All compounds were elucidated by detailed spectroscopic analysis and tested for antibacterial activities against five human pathogens and tested for cytotoxicity. This study demonstrates that LC/MS based multivariate analysis provides a simple yet powerful tool to analyze the metabolome of a single fungal strain grown under various conditions. This approach allows environmentally‐induced changes in metabolite expression to be rapidly visualized, and uses these differences to guide the discovery of new bioactive molecules.     

新型抗结核药物研发：微生物非常规培养与沉默基因激活策略

由结核分枝杆菌感染引起的结核病是人类重要传染病之一。临床上结核菌耐药性日趋严重,不断出现的耐多药及广泛耐药结核病患者,使现有的一线至五线药物不能满足结核病防控需求。微生物来源的天然产物是药物先导化合物的重要来源。环境中存在大量常规培养条件下未培养微生物,同时微生物基因组中也存在大量未被表达的"沉默代谢途径"。运用各种方法对未培养微生物进行再培养,同时激活微生物的沉默代谢途径,进而获得潜在的新型抗生素药物已成为目前研究热点。文中系统阐述了近年来获取天然化合物所采用的微生物非常规培养技术及沉默代谢途径激活策略,同时总结了利用这两种方法获得的新型抗结核天然产物,并展望了这些方法在抗结核药物进一步研发中的应用前景。     

Metabolite diversification by cultivation of the endophytic fungus Dothideomycete sp. in halogen containing media: Cultivation of terrestrial fungus in seawater

The endophytic fungus, Dothideomycete sp. CRI7, isolated from the terrestrial plant, Tiliacora triandra, was salt tolerant, capable of growing in the culture medium prepared from seawater; salts in seawater did not have any effects on the fungal growth. Metabolite productions of the fungus CRI7 cultivated in media prepared from seawater (MSW), prepared from deionized water supplemented with potassium bromide (MKBr) or potassium iodide (MKI), and prepared from deionized water (MDW) were investigated. It was found that the cultivation of the fungus CRI7 in MKBr and MSW enabled the fungus to produce nine new metabolites (1–9). The production of an azaphilone, austdiol (10), of the fungus CRI7 grown in MDW was 0.04 g/L, which was much lower than that grown in MSW, MKBr, and MKI media which provided the yields of 0.5, 0.9, and 1.2 g/L, respectively, indicating that halogen salts significantly enhanced the production of the polyketide 10. The cultivation of terrestrial fungi in media containing halogen salts could therefore be useful for the metabolite diversification by one strain-many compounds (OSMAC) approach. Moreover, the isolated polyketides had significant biosynthetic relationship, suggesting that the cultivation of fungi in halogen containing media could provide the insights into certain polyketide biosynthesis. One of the isolated compounds exhibited antibacterial activity with the MIC value of 100 μg/mL.     

Heat shock and iron limitation modulate the metabolic profile of the cyanobacterium Microcystis aeruginosa NIES-88

The freshwater cyanobacterium Microcystis aeruginosa NIES-88, which can produce microcystins, micropeptins, and argicyclamides, was subjected to a one strain many compounds (OSMAC) analysis. We report its response to two environmental stressors, temperature and iron limitation, by means of untargeted and targeted metabolomics. The results demonstrated a slower specific growth rate of 0.20 per day and 0.16 per day in adverse conditions of 37°C and iron limitation, respectively. The metabolic signature of M. aeruginosa was highly dependent on incubation temperatures. Production of microcystins LR and RR was severely downregulated while that of argicyclamide B was significantly upregulated, with a highest 10-fold increase on day 14 of heat shock treatment. M. aeruginosa NIES-88 was found to produce a new compound, argicyclamide D (1), in iron limited medium, which has the same macrocyclic structure as the previously reported analogs. Hence, it is proposed that acclimation of M. aeruginosa to environmental stressors might be mediated by a change in the metabolic pathways as well as modulation of the levels of their expressed metabolites.     

Production of polyketides with anthelmintic activity by the fungus Talaromyces wortmannii using one strain-many compounds (OSMAC) method

Three new polyketides, wortmannilactones I1-I3, were purified from Talaromyces wortmannii using the One Strain Many Compounds (OSMAC) strategy. The polyketides’ structures were established using IR, 1D and 2D NMR, and HRESIMS analyses and the absolute configurations were identified by comparison of experimental and calculated ECDs. These polyketides exhibit selective inhibitory activity against NADH-fumarate reductase.     

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

Aims: 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. Methods and Results: 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. Conclusions: 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. Significance and Impact of the Study: 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.     

Metabolite induction via microorganism co-culture: A potential way to enhance chemical diversity for drug discovery

Microorganisms have a long track record as important sources of novel bioactive natural products, particularly in the field of drug discovery. While microbes have been shown to biosynthesize a wide array of molecules, recent advances in genome sequencing have revealed that such organisms have the potential to yield even more structurally diverse secondary metabolites. Thus, many microbial gene clusters may be silent under standard laboratory growth conditions. In the last ten years, several methods have been developed to aid in the activation of these cryptic biosynthetic pathways. In addition to the techniques that demand prior knowledge of the genome sequences of the studied microorganisms, several genome sequence-independent tools have been developed. One of these approaches is microorganism co-culture, involving the cultivation of two or more microorganisms in the same confined environment. Microorganism co-culture is inspired by the natural microbe communities that are omnipresent in nature. Within these communities, microbes interact through signaling or defense molecules. Such compounds, produced dynamically, are of potential interest as new leads for drug discovery. Microorganism co-culture can be achieved in either solid or liquid media and has recently been used increasingly extensively to study natural interactions and discover new bioactive metabolites. Because of the complexity of microbial extracts, advanced analytical methods (e.g., mass spectrometry methods and metabolomics) are key for the successful detection and identification of co-culture-induced metabolites.     

Exploration of the Production of Three Thiodiketopiperazines by an Endophytic Fungal Strain of Cophinforma mamane

Endophytic fungi possess a versatile metabolism which is related to their ability to live in diverse ecological niches. While culturing under laboratory conditions, their metabolism is mainly influenced by the culture media, time of incubation and other physicochemical factors. In this study, we focused on the production of 3 thiodiketopiperazines (TDKPs) botryosulfuranols A−C produced by an endophytic strain of Cophinforma mamane isolated from the leaves of Bixa orellana L collected in the Peruvian Amazon. We studied the time-course production of botryosulfuranols A−C during 28 days and evaluated the variations in the production of secondary metabolites, including the TDKPs, produced by C. mamane in response to different culture media, light versus dark conditions and different incubation times. We observed a short time-frame production of botryosulfuranol C while its production was significantly affected by the light conditions and nutrients of the culture media. Botryosulfuranols A and B showed a similar production pattern and a similar response to culturing conditions. Molecular networking allowed us to detect three compounds related to TDKPs that will be the focus of future experiments.     

Derivatives of Holomycin and Cyclopropaneacetic Acid from Streptomyces sp. DT‐A37

On the basis of the one strain–many compounds strategy, five compounds including two new holomycin derivatives 2 – 3 , two new cyclopropaneacetic acid derivatives 4 – 5 , together with one known compound holomycin ( 1 ) were isolated from a marine‐derived bacterium Streptomyces sp. DT‐A37. Their structures were elucidated using NMR and HR‐ESI‐MS analyses. All these compounds were evaluated for their antimicrobial activity, cytotoxic activity, and inhibitory activity against BRD4 protein. Compound 1 exhibited potent cytotoxicity against H1975 cells with IC₅₀ value of 1 μm , and its minimal inhibitory concentration values against Escherichia coli and Staphylococcus aureus were both 64 μm .