Total biosynthesis of fungal tetraketide pyrones.

Fungal tetraketide pyrones possess important and potent bioactivities, but their detailed biosynthetic pathways are unknown and synthetic routes to their production are lengthy. Here we investigated the fungal pathways to the multiforisins and compounds related to islandic acid. Heterologous expression experiments yield high titres of these compounds and pathway intermediates. The results both elucidate the pathway and offer a platform for the total biosynthesis of this class of metabolites.

Purification and characterization of seven bioactive compounds from the newly isolated Streptomyces cavourensis TN638 strain via solid-state fermentation.

The strain TN638 was isolated from Tunisian soil contaminated with industrial wastewater and selected for its potent antimicrobial activity against the tested Gram positive bacteria: Staphylococcus aureus (S. aureus) ATCC 6538 and Listeria monocytogenes (L. monocytogenes) ATCCC 19117, and Gram negative bacteria: Agrobacterium tumefaciens (A. tumefaciens) ATCC 23308 and Salmonella typhimurium (S. typhimurium) ATCC 14028 and fungi: Candida albicans (C. albicans) ATCC 10231, Rhizoctonia solani (R. solani) ATCC 58938 and Fusarium sp. Solide-state fermentation (SSF) dry crude extract of the TN638 strain presents a strong inhibitory activity notably against the phytopathogenic microorganism A. tumefaciens ATCC 23308 and the two pathogenic bacteria S. aureus ATCC 6538 and L. monocytogenes ATCCC 19117 with a zone of inhibition of 48, 34 and 34 mm respectively. According to the morphological characteristic, the complete 16S rRNA gene nucleotide sequence determination [1492 bp deposited in National Center of Biotechnology Information (NCBI) database under the accession no. LN854629.1; https://www.ncbi.nlm.nih.gov/nuccore/LN854629.1/], and the phylogenetic analysis, we can deduce that our isolate is an actinomycete bacterium belonging to the genus Streptomyces and the most closely related strain was Streptomyces cavourensis (S. cavourensis) NRRL 2740T (99.9%). We propose the assignment of our strain as Streptomyces cavourensis (S. cavourensis) TN638 strain. Work-up and purification of the strain extract using different chromatographic techniques afforded seven bio-compounds namely: Cyclo-(Leu-Pro) (1), Cyclo-(Val-Pro) (2), Cyclo-(Phe-Pro) (3), nonactin (4), monactin (5), dinactin (6) and trinactin (7). The chemical structures of compounds 1-7 were confirmed by nuclear magnetic resonance (NMR) 1D and 2D spectroscopy, mass spectrometry, and comparison with literature data. The three purified diketopiperazine (DKP) derivatives (1-3), demonstrated significant antibacterial activity against A. tumefaciens ATCC 23308 and S. typhimurium ATCC 14028. The four pure macrotetrolides (4-7), exhibited strong inhibitory effect against all tested Gram positive and Gram negative bacteria notably against A. tumefaciens ATCC 23308 and S. typhimurium ATCC 14028 with a minimum inhibitory concentration (MIC) around 8 µg/mL quite similar to that of ampicillin. Thus, we propose the use of the (SSF) active extract of the S. cavourensis TN638 strain as safe biological product to control disease caused by plant pathogen A. tumefaciens. Also, the purified active molecules produced by this strain could be used in pharmaceutical field.

Impact of the Cultivation Technique on the Production of Secondary Metabolites by Chrysosporium lobatum TM-237-S5, Isolated from the Sponge Acanthella cavernosa.

The fungi Chrysosporium lobatum TM-237-S5 was isolated from the sponge Acanthella cavernosa, collected from the mesophotic coral ecosystem of the Red Sea. The strain was cultivated on a potato dextrose agar (PDA) medium, coupling solid-state fermentation and solid-state extraction (SSF/SSE) with a neutral macroreticular polymeric adsorbent XAD Amberlite resin (AMBERLITE XAD1600N). The SSF/SSE lead to high chemodiversity and productivity compared to classical submerged cultivation. Ten phenalenone related compounds were isolated and fully characterized by one-dimensional and two-dimensional NMR and HRMS. Among them, four were found to be new compounds corresponding to isoconiolactone, (-)-peniciphenalenin F, (+)-8-hydroxyscleroderodin, and (+)-8-hydroxysclerodin. It is concluded that SSF/SSE is a powerful strategy, opening a new era for the exploitation of microbial secondary metabolites.

Neuroprotective Effect of CR-777, a Glutathione Derivative of Withaferin A, Obtained through the Bioconversion of Withania somnifera (L.) Dunal Extract by the Fungus Beauveria bassiana.

The bioconversion of Withania somnifera extract by the fungus Beauveria bassiana leads to cysteine and glutathione derivatives of withaferin A at the C-6 position. The compounds were purified and fully characterized by 1D-NMR, 2D-NMR, and HRMS analysis. The glutathione derivative CR-777 was evaluated as a neuroprotective agent from damage caused by different neurotoxins mimicking molecular symptoms in Parkinson´s disease (PD), including 1-methyl-4-phenylpyridinium (MPP+), 6-hydroxydopamine (6-OHDA), and α-synuclein (α-Syn). CR-777, at nanomolar concentrations, protected dopaminergic and cortical neurons. In 6-OHDA-treated neurons, CR-777 increased cell survival and neurite network and decreased the expression of α-Syn. Using specific inhibitors of cell toxicity signaling pathways and specific staining experiments, the observed role of CR-777 seemed to involve the PI3K/mTOR pathway. CR-777 could be considered as a protective agent against a large panel of neuronal stressors and was engaged in further therapeutic development steps.

Biohydroxylation of (-)-ambrox®, (-)-sclareol, and (+)-sclareolide by whole cells of Brazilian marine-derived fungi.

A screening was performed using nine marine-derived fungi as biocatalysts and the natural products (-)-ambrox® (1), (-)-sclareol (2), and (+)-sclareolide (3) in order to select the microorganisms able to catalyze the biooxidation of these compounds. It was observed that only the Aspergillus sydowii CBMAI 934, Botryosphaeria sp., Eutypella sp., and Xylaria sp. presented active oxidoreductases and catalyzed the regioselective hydroxylation in the natural products. The hydroxylated metabolites obtained were 1ß-hydroxy-ambrox (1a) (14%, A. sydowii CBMAI 934); 3ß-hydroxy-ambrox (1b) (17%, Botryosphaeria sp.; 11%, Eutypella sp.); 3ß-hydroxy-sclareol (2a) (31%, Xylaria sp.; 69%, Botryosphaeria sp.; 55%, Eutypella sp.); 18-hydroxy-sclareol (2b) (10%, Xylaria sp.); and 3ß-hydroxy-sclareolide (3a) (34%, Botryosphaeria sp.; 7%, Eutypella sp.). This is the first report of biohydroxylation of (-)-ambrox® (1), (-)-sclareol (2), and (+)-sclareolide (3) by whole mycelia of marine-derived fungi.

Antiangiogenic-Like Properties of Fermented Extracts of Ayurvedic Medicinal Plants.

The three ayurvedic medicinal plants, Withania somnifera, Emblica officinalis, and Bacopa monnieri, were extracted by high-pressure static extraction using the Zippertex(®) technology. The extracts were mixed to reach quantifiable amounts of active compounds identified by high-pressure liquid chromatography-mass spectrometry (HPLC-MS) analysis. The mixture of extracts was incubated with resting cells of the fungus Beauveria bassiana ATCC 7159. The fermentation promoted the fluidization of the starting dense mixture, while HPLC monitoring evidenced the disappearance of glucogallin from E. officinalis extract and the concomitant increase in gallic acid content. Topical exposure of the chick embryo chorioallantoic membrane (CAM) to the nonfermented extract led to the extensive necrosis and destruction of the treated membrane. However, the fermented extract was shown to be free of any toxicity. Furthermore, compared with the untreated CAM, the fermented sample reduced CAM vascularization, suggesting its antiangiogenic potency. The innocuity of the fermented extract was demonstrated using the in vivo LD50 test, the morphological examination of internal organs of treated rats, as well as the evaluation of blood biomarkers of liver damage (aspartate aminotransferase and alanine aminotransferase). The fermented extract was developed as a nutraceutical antiangiogenic treatment of age-related macular degeneration and commercialized in an oral form named Ethnodyne-Visio™.

Bicyclic and tetracyclic diterpenes from a Trichoderma symbiont of Taxus baccata.

Trichoderma atroviridae UB-LMA is an endophytic fungus isolated from Taxus baccata trees. Liquid-state fermentation coupled to in situ solid phase extraction (SPE) was applied, and four compounds were discovered. Compounds 2-4 belong to the harziane tetracyclic diterpene family. Bicylic compound 1 may represent the biosynthetic precursor of this scarce family of compounds.