Novel bioreactor internals for the cultivation of spore-forming fungi in pellet form.

This study introduced an automated long-term fermentation process for fungals grown in pellet form. The goal was to reduce the overgrowth of bioreactor internals and sensors while better rheological properties in the fermentation broth, such as oxygen transfer and mixing time, can be achieved. Because this could not be accomplished with continuous culture and fed-batch fermentation, repeated-batch fermentation was implemented with the help of additional bioreactor internals ("sporulation supports"). This should capture some biomass during fermentation. After harvesting the suspended biomass, intermediate cleaning was performed using a cleaning device. The biomass retained on the sporulation support went through the sporulation phase. The spores were subsequently used as inocula for the next batch. The reason for this approach was that the retained pellets could otherwise cause problems (e.g., overgrowth on sensors) in subsequent batches because the fungus would then show undesirable hyphal growth. Various sporulation supports were tested for sufficient biomass fixation to start the next batch. A reproducible spore concentration within the range of the requirements could be achieved by adjusting the sporulation support (design and construction material), and an intermediate cleaning adapted to this.

Sesquiterpene Lactones from Vernonia tufnelliae: Structural Characterization and Biological Evaluation.

The genus Vernonia is an extremely rich source of biologically active sesquiterpene lactones. The present report describes the spectroscopic structure elucidation and the cytotoxic and antimicrobial properties of five hitherto unknown germacranolide-like sesquiterpenoids and several known compounds. These new derivatives include a compound (1) with an unprecedented 10/5/5/6 tetracyclic framework featuring a hexahydro-1H,3H,7H-furo[3',4':3,4]furo[3,2-c]pyridin-1-one core resulting from an intramolecular cyclization cascade involving a methacrylate substituent and a low molecular weight amine. Furthermore, an elemane-germacranolide hybrid (2) and three amino acid-derived lactones (3-5) were characterized. A plausible biosynthetic pathway to the key alkaloid is presented, while shielding tensor calculations using DFT in combination with the DP4+ method were applied to elucidate its stereostructure. The newly characterized compounds along with ten known sesquiterpene lactones and phenolic compounds have been isolated from Vernonia tufnelliae, a medicinal plant from the western region of Cameroon. Their structures were consistent with spectroscopic and spectrometric data recorded. The present report is the first investigation of the chemistry and biology of V. tufnelliae.

Myristic Acid Inhibits the Activity of the Bacterial ABC Transporter BmrA.

ATP-binding cassette (ABC) transporters are conserved in all kingdoms of life, where they transport substrates against a concentration gradient across membranes. Some ABC transporters are known to cause multidrug resistances in humans and are able to transport chemotherapeutics across cellular membranes. Similarly, BmrA, the ABC transporter of Bacillus subtilis, is involved in excretion of certain antibiotics out of bacterial cells. Screening of extract libraries isolated from fungi revealed that the C14 fatty acid myristic acid has an inhibitory effect on the BmrA ATPase as well as the transport activity. Thus, a natural membrane constituent inhibits the BmrA activity, a finding with physiological consequences as to the activity and regulation of ABC transporter activities in biological membranes.

Xylochemical Synthesis and Biological Evaluation of Shancigusin C and Bletistrin G.

The biological activities of shancigusin C (1) and bletistrin G (2), natural products isolated from orchids, are reported along with their first total syntheses. The total synthesis of shancigusin C (1) was conducted by employing the Perkin reaction to forge the central stilbene core, whereas the synthesis of bletistrin G (2) was achieved by the Wittig olefination followed by several regioselective aromatic substitution reactions. Both syntheses were completed by applying only renewable starting materials according to the principles of xylochemistry. The cytotoxic properties of shancigusin C (1) and bletistrin G (2) against tumor cells suggest suitability as a starting point for further structural variation.

Anti-phytopathogen terpenoid glycosides from the root bark of Chytranthus macrobotrys and Radlkofera calodendron.

Chytranthus macrobotrys and Radlkofera calodendron are two Sapindaceae characterized by a lack of phytochemical data. Both root barks from the two Sapindaceae species were processed by ethanol extraction followed by the isolation of their primary constituents by liquid chromatography. This process yielded four previously undescribed terpenoid glycosides together with eight known analogues. Extracts and isolated compounds from C. macrobotrys and R. calodendron were then screened for antimicrobial activity against fifteen phytopathogens. The biological screening also involved extracts and pure compounds from Blighia unijugata and Blighia welwitschii, two Sapindaceae previously studied by our group. Phytopathogens were chosen based on their economic impact on agriculture worldwide. The selection was composed primarily of fungal species including; Pyricularia oryzae, Gaeumannomyces graminis var. tritici, Zymoseptoria tritici, Fusarium oxysporum, Botrytis cinerea, Pythium spp., Trichoderma spp. and Rhizoctonia solani. Furthermore, pure terpenoid glycosides were tested for the first time against wood-inhabiting phytopathogens such as; Phaeomoniella chlamydospora, Phaeoacremonium minimum, Fomitiporia mediterranea, Eutype lata and Xylella fastidiosa. Raw extracts exhibited different levels of activity dependent on the organism. Some pure compounds, including 3-O-α-L-arabinopyranosyl-(1 â†’ 4)-ß-D-xylopyranosyl-(1 â†’ 3)-α-L-rhamnopyranosyl-(1 â†’ 2)-α-L-arabinopyranosylhederagenin, 3-O-α-L-rhamnopyranosyl-(1 â†’ 2)-α-L-arabinopyranosylhederagenin (α-hederin), 3-O-ß-D-glucopyranosyl-(1 â†’ 3)-α-L-rhamnopyranosyl-(1 â†’ 2)-α-L-arabinopyranosylhederagenin (macranthoside A) and 3-O-α-L-arabinopyranosyl-(1 â†’ 3)-α-L-rhamnopyranosyl-(1 â†’ 2)-α-L-arabinopyranosylhederagenin (clemontanoside C), exhibited significant growth inhibitions on Pyricularia oryzae, Gaeumannomyces graminis var. tritici, Fomitiporia mediterranea and Zymoseptoria tritici. Monodesmoside triterpene saponins, in particular, exhibited MIC (IC100) values as low as 25 µg/ml and IC50 values as low as 10 µg/ml against these phytopathogens. Structure-activity relationships, as well as plant-microbe interactions, were discussed.

Applicability of a single-use bioreactor compared to a glass bioreactor for the fermentation of filamentous fungi and evaluation of the reproducibility of growth in pellet form.

The implementation of single-use technologies offers several major advantages, e.g. prevention of cross-contamination, especially when spore-forming microorganisms are present. This study investigated the application of a single-use bioreactor in batch fermentation of filamentous fungus Penicillium sp. (IBWF 040-09) from the Institute of Biotechnology and Drug Research (IBWF), which is capable of intracellular production of a protease inhibitor against parasitic proteases as a secondary metabolite. Several modifications to the SU bioreactor were suggested in this study to allow the fermentation in which the fungus forms pellets. Simultaneously, fermentations in conventional glass bioreactor were also conducted as reference. Although there are significant differences in the construction material and gassing system, the similarity of the two types of bioreactors in terms of fungal metabolic activity and the reproducibility of fermentations could be demonstrated using statistic methods. Under the selected cultivation conditions, growth rate, yield coefficient, substrate uptake rate, and formation of intracellular protease-inhibiting substance in the single-use bioreactor were similar to those in the glass bioreactor.

Neo-clerodane diterpenoids from Conyza pyrrhopappa Sch.Bip. ex A.Rich.

Two hitherto unknown neo-clerodane-type diterpenoids along with twelve known compounds have been isolated from Conyza pyrrhopappa Sch.Bip. ex A.Rich, a medicinal plant traditionally used across tropical Africa to relieve fever. The structures of isolates have been elucidated by a combination of spectroscopic techniques. The crude extract and the isolated compounds were evaluated in the Hela-S3 cell line and in a panel of microorganisms (bacteria and fungi) at concentrations up to 50 µg/mL. The new compounds were inactive while the pentamethylated flavonoids showed low to significant activity against the cancer cell line used. However, none of the samples showed any activity against the tested microorganisms at this concentration. The present manuscript is the first investigation of the cytotoxicity of phytochemicals and extract from C. pyrrhopappa.

Boshramycinones A-C: New anthracyclinones produced by a marine-derived Streptomyces sp.: isolation, structure elucidation and biological activities.

Boshramycinones A-C (1-3), three new anthracyclinones, were isolated from the culture broth of the marine-derived Streptomyces sp. Mei 16-1,2 together with 2-acetyl-1,8-dihydroxy-3-methyl-anthraquinone (4) and bafilomycins B1, B2, and C1-amide. The isolated compounds were identified by NMR spectroscopy and mass spectrometry, the absolute configuration of 3 was determined by comparison of experimental and ab initio-calculated chiroptical data. The antimicrobial activity of the bacterial extract and the isolated compounds were assayed using a set of microorganisms, and cytotoxic activities were determined against 36 human cancer cell lines.

New Alkenylresorcinols with Cytotoxic and Antimicrobial Activities from the Leaves of Embelia schimperi.

A phytochemical study of the methanol extract of the leaves of Embelia schimperi resulted in the isolation of three new alkenylresorcinols, 1:  - 3: , together with the known analogs 4:  - 7: . Their structures were established by a combination of spectroscopic techniques. Compounds 1:  - 7: exhibited moderate cytotoxic activity against human cervical cancer cells HeLa-S3 and more pronounced antimicrobial properties towards bacteria and filamentous fungi. The present study falls into an ongoing research project on the characterization of bioactive phenolic lipids from plants of the family Primulaceae.

Phosphorylation and activation of endothelial nitric oxide synthase by red fruit (Pandanus conoideus Lam) oil and its fractions.

ETHNOPHARMACOLOGICAL RELEVANCE: Red fruit (Pandanus conoideus Lam) oil (RFO) is utilized by inhabitants of the Papua Island to treat diseases such as infections, cancer, and cardiovascular disease, but the mechanism of action is unknown. AIM OF THE STUDY: We have recently shown that RFO stimulates nitric oxide (NO) production in endothelial cells. The present study was conducted to investigate the molecular mechanism of endothelial NO synthase (eNOS) activation by RFO. MATERIALS AND METHODS: NO production by endothelial cells was determined with electron paramagnetic resonance. The vascular function of isolated mouse aorta was examined using a wire myograph system. Phosphorylation of eNOS was studied with Western blot analyses. RESULTS: RFO induced concentration-dependent vasodilation in isolated mouse aorta. The vasodilator effect of RFO was lost in endothelium-denuded aorta and in aorta from mice deficient in eNOS. Treatment of human EA.hy 926 endothelial cells with RFO led to an enhancement of eNOS phosphorylation at serine 1177 and NO production. The RFO-induced eNOS phosphorylation and NO production were reduced by inhibitors of Akt or AMPK, but not by an inhibitor of CaMKII. The effects of RFO were decreased by pharmacological inhibition of PI3K, indicating an involvement of the PI3K-Akt pathway. Moreover, acetone-soluble fractions and oily fractions of RFO showed higher efficacies than the RFO polar fraction in activating eNOS. CONCLUSIONS: RFO contains highly active compounds that enhance NO production through Akt- or AMPK-mediated eNOS phosphorylation. The increase in endothelial NO production is likely to represent one of the molecular mechanisms responsible for the therapeutic effects of RFO.

Promoter Activation in Δhfq Mutants as an Efficient Tool for Specialized Metabolite Production Enabling Direct Bioactivity Testing.

Natural products (NPs) from microorganisms have been important sources for discovering new therapeutic and chemical entities. While their corresponding biosynthetic gene clusters (BGCs) can be easily identified by gene-sequence-similarity-based bioinformatics strategies, the actual access to these NPs for structure elucidation and bioactivity testing remains difficult. Deletion of the gene encoding the RNA chaperone, Hfq, results in strains losing the production of most NPs. By exchanging the native promoter of a desired BGC against an inducible promoter in Δhfq mutants, almost exclusive production of the corresponding NP from the targeted BGC in Photorhabdus, Xenorhabdus and Pseudomonas was observed including the production of several new NPs derived from previously uncharacterized non-ribosomal peptide synthetases (NRPS). This easyPACId approach (easy Promoter Activated Compound Identification) facilitates NP identification due to low interference from other NPs. Moreover, it allows direct bioactivity testing of supernatants containing secreted NPs, without laborious purification.

Identification of Patulin from Penicillium coprobium as a Toxin for Enteric Neurons.

The identification and characterization of fungal commensals of the human gut (the mycobiota) is ongoing, and the effects of their various secondary metabolites on the health and disease of the host is a matter of current research. While the neurons of the central nervous system might be affected indirectly by compounds from gut microorganisms, the largest peripheral neuronal network (the enteric nervous system) is located within the gut and is exposed directly to such metabolites. We analyzed 320 fungal extracts and their effect on the viability of a human neuronal cell line (SH-SY5Y), as well as their effects on the viability and functionality of the most effective compound on primary enteric neurons of murine origin. An extract from P. coprobium was identified to decrease viability with an EC50 of 0.23 ng/µL in SH-SY5Y cells and an EC50 of 1 ng/µL in enteric neurons. Further spectral analysis revealed that the effective compound was patulin, and that this polyketide lactone is not only capable of evoking ROS production in SH-SY5Y cells, but also diverse functional disabilities in primary enteric neurons such as altered calcium signaling. As patulin can be found as a common contaminant on fruit and vegetables and causes intestinal injury, deciphering its specific impact on enteric neurons might help in the elaboration of preventive strategies.

Georatusin, a Specific Antiparasitic Polyketide-Peptide Hybrid from the Fungus Geomyces auratus.

Georatusin (1), featuring a highly reduced, methylated polyketide moiety fused to a tryptophan by an amide and ester bond forming a 13-membered ring, was produced by the soil fungus Geomyces auratus. An HMQC-COSY spectrum was measured to build up the connectivities despite the overlapping proton signals. DQF-COSY, HETLOC, J-HMBC, and ROESY were implemented to determine the relative configuration of the flexible moiety. Georatusin (1) shows specific antiparasitic activities against Leishmania donovani and Plasmodium falciparum without obvious cytotoxicity. The biosynthesis of 1 was also proposed.

The Chinese herbal formula Free and Easy Wanderer ameliorates oxidative stress through KEAP1-NRF2/HO-1 pathway.

Posttraumatic stress disorder (PTSD) gains a lot of attention due to high prevalence and strong psychological upset, but the etiology remains undefined and effective treatment is quite limited. Growing studies demonstrated the involvement of oxidative stress in various psychiatry diseases, suggesting anti-oxidation therapy might be a strategy for PTSD treatment. Free and Easy Wanderer (FAEW) is a poly-herbal drug clinically used in China for hundreds of years in the treatment of psychiatric disorder. We hypothesized that FAEW exerts clinical effects through the activity against oxidative stress with fluoxetine as antidepressant control drug. Our results revealed that FAEW significantly reduced both endogenous and H2O2-induced exogenous ROS levels in the human glioblastoma T98G and neuroblastoma SH-SY5Y cell lines. Transcriptome-wide microarray analysis indicated NRF2/HO-1 as the common target of FAEW and fluoxetine. Western blotting assay proved that the two drugs promoted NRF2 release from KEAP1 in the cytoplasm and translocation to the nuclei in a KEAP1-dependent manner, the expression of the protein HO-1 increased accordingly, suggesting the participation of KEAP1-NRF2/HO-1 pathway. The chemical constituents of FAEW (i.e. paeoniflorin, baicalin) bound to KEAP1 in silico, which hence might be the effective substances of FAEW. In conclusion, FAEW counteracted H2O2-induced oxidative stress through KEAP1-NRF2/HO-1 pathway.

Phytotoxic dioxolanones are potential virulence factors in the infection process of Guignardia bidwellii.

Phytotoxic dioxolanones from Guignardia bidwellii can be described as potential virulence factors which cause the formation of lesions upon an infection by G. bidwellii. The toxin guignardic acid was found in planta of G. bidwellii-infected Vitis vinifera leaves, whereas no phytotoxic dioxolanones were detected in uninfected leaf material. Secondary metabolism analyses of further phytopathogenic fungi from the genus Guignardia led to the observation that all species investigated can produce the phytotoxins known from G. bidwellii. In addition to these studies, it was demonstrated that phenguignardic acid is biosynthetically derived from two molecules of phenylalanine and that phenylalanine is a key precursor in the biosynthesis of the two other phytotoxins - alaguignardic acid and guignardic acid.

Identification of NF-κB as Determinant of Posttraumatic Stress Disorder and Its Inhibition by the Chinese Herbal Remedy Free and Easy Wanderer.

Posttraumatic stress disorder (PTSD) is a mental disorder developing after exposure to traumatic events. Although psychotherapy reveals some therapeutic effectiveness, clinically sustainable cure is still uncertain. Some Chinese herbal formulae are reported to work well clinically against mental diseases in Asian countries, but the safety and their mode of action are still unclear. In this study, we investigated the mechanisms of Chinese remedy free and easy wanderer (FAEW) on PTSD. We used a reverse pharmacology approach combining clinical data to search for mechanisms of PTSD with subsequent in vitro verification and bioinformatics techniques as follows: (1) by analyzing microarray-based transcriptome-wide mRNA expression profiling of PTSD patients; (2) by investigating the effect of FAEW and the antidepressant control drug fluoxetine on the transcription factor NF-κB using reporter cell assays and western blotting; (3) by performing molecular docking and literature data mining based on phytochemical constituents of FAEW. The results suggest an involvement of inflammatory processes mediated through NF-κB in the progression of PTSD. FAEW was non-cytotoxic in vitro and inhibited NF-κB activity and p65 protein expression. FAEW's anti-inflammatory compounds, i.e., paeoniflorin, isoliquiritin, isoliquiritin apioside and ononin were evaluated for binding to IκK and p65-RelA in a molecular docking approach. Paeoniflorin, albiflorin, baicalin, isoliquiritin and liquiritin have been reported to relieve depression in vivo or in clinical trials, which might be the active ingredients for FAEW against PTSD.

Discovery of an Extended Austinoid Biosynthetic Pathway in Aspergillus calidoustus.

Filamentous fungi produce a wide range of natural products that are commonly used in various industrial contexts (e.g., pharmaceuticals and insecticides). Meroterpenoids are natural products of interest because of their various biological activities. Among the meroterpenoids, there is a group of insecticidal compounds known as the austinoids. These compounds have also been studied because of their intriguing spiro-lactone ring formation along with various modifications. Here, we present an extension of the original austinol/dehydroaustinol biosynthesis pathway from Aspergillus nidulans in the recently identified filamentous fungus Aspergillus calidoustus. Besides the discovery and elucidation of further derivatives, genome mining led to the discovery of new putative biosynthetic genes. The genes involved in the biosynthesis of later austinoid products were characterized, and among them was a second polyketide synthase gene in the A. calidoustus cluster that was unusual because it was a noninterative polyketide synthase producing a diketide. This diketide product was then loaded onto the austinoid backbone, resulting in a new insecticidal derivative, calidodehydroaustin.

Metabolites from the Endophytic Fungus Curvularia sp. M12 Act as Motility Inhibitors against Phytophthora capsici Zoospores.

The endophytic fungus Curvularia sp., strain M12, was isolated from a leaf of the medicinal plant Murraya koenigii and cultured on rice medium followed by chemical screening of the culture extract. Chromatographic analysis led to the isolation of four new compounds, murranofuran A (1), murranolide A (2), murranopyrone (3a), and murranoic acid A (4a), along with six known metabolites, N-(2-hydroxy-6-methoxyphenyl)acetamide (5), curvularin (6), (S)-dehydrocurvularin (7), pyrenolide A (8), modiolide A (9), and 8-hydroxy-6-methoxy-3-methylisocoumarin (10). The structures of the known compounds were confirmed by comparing ESI HR mass spectra, 1H and 13C NMR, and optical rotation data with values reported in the literature. The planar structures of the new compounds were elucidated by extensive analysis of 1D and 2D NMR and mass data. The absolute configurations of the new compounds were established by coupling constant analysis, modified Mosher's method, and CD data. Compound 8 showed a strong motility impairing activity against Phytophthora capsici zoospores at a low concentration (100% at 0.5 µg/mL) in a short time (30 min). Compounds 2, 3a, 6, 7, 9, and 10 exhibited zoospore motility impairment activity at higher concentrations (IC50: 50-100 µg/mL).

Unravelling the biosynthesis of pyriculol in the rice blast fungus Magnaporthe oryzae.

Pyriculol was isolated from the rice blast fungus Magnaporthe oryzae and found to induce lesion formation on rice leaves. These findings suggest that it could be involved in virulence. The gene MoPKS19 was identified to encode a polyketide synthase essential for the production of the polyketide pyriculol in the rice blast fungus M. oryzae. The transcript abundance of MoPKS19 correlates with the biosynthesis rate of pyriculol in a time-dependent manner. Furthermore, gene inactivation of MoPKS19 resulted in a mutant unable to produce pyriculol, pyriculariol and their dihydro derivatives. Inactivation of a putative oxidase-encoding gene MoC19OXR1, which was found to be located in the genome close to MoPKS19, resulted in a mutant exclusively producing dihydropyriculol and dihydropyriculariol. By contrast, overexpression of MoC19OXR1 resulted in a mutant strain only producing pyriculol. The MoPKS19 cluster, furthermore, comprises two transcription factors MoC19TRF1 and MoC19TRF2, which were both found individually to act as negative regulators repressing gene expression of MoPKS19. Additionally, extracts of ΔMopks19 and ΔMoC19oxr1 made from axenic cultures failed to induce lesions on rice leaves compared to extracts of the wild-type strain. Consequently, pyriculol and its isomer pyriculariol appear to be the only lesion-inducing secondary metabolites produced by M. oryzae wild-type (MoWT) under these culture conditions. Interestingly, the mutants unable to produce pyriculol and pyriculariol were as pathogenic as MoWT, demonstrating that pyriculol is not required for infection.

Terphenyl Derivatives from Allantophomopsis lycopodina.

Three secondary fungal metabolites 1-3 with a benzo[b]naphtho[2,1-d]furan skeleton were isolated from submerged cultures of the ascomycete Allantophomopsis lycopodina. The NMR-based structure elucidation was challenging due to a low H/C ratio of only 0.64 and 0.68, respectively. NMR measurements in two different solvents and the use of NMR experiments such as HSQC-TOCSY and LR-HSQMBC proved to be helpful in this respect. The proposed structures obtained from the comprehensive analysis of the NMR data were verified by comparison of recorded and computed NMR chemical shifts from quantum chemical calculations of several constitutional isomers and were further analyzed with the aid of the DP4 and DP4+ probabilities.