5'-Methoxyarmillane, a Bioactive Sesquiterpenoid Aryl Ester from the Fungus Armillaria ostoyae.

Higher fungi of the genus Armillaria belonging to the phylum Basidiomycota produce bioactive sesquiterpenoid aryl esters called melleolides. A bioactivity-guided discovery process led to the identification of the new melleolide 5'­methoxyarmillane (1) in organic extracts from the mycelium of Armillaria ostoyae. Remarkably, supplementation of rapeseed oil to the culture medium potato dextrose broth increased the production of 1 by a factor of six during the course of the 35 days fermentation. Compound 1 was isolated and its structure elucidated by UHPLC-QTOF-HR-MS/MS and NMR spectroscopy. It showed toxicity against Madin-Darby canine kidney II (MDCK II, IC50 19.2 mg/mL, 44.1 mM) and human lung cancer Calu-3 cells (IC50 15.2 mg/mL, 34.9 mM) as well as moderate bioactivity against Mycobacterium tuberculosis (MIC 8 mg/mL, 18.4 mM) and Mycobacterium smegmatis (MIC 16 mg/mL, 36.8 mM), but not against Staphylococcus aureus, Escherichia coli, Candida albicans, and Septoria tritici. No inhibitory effects of 1 against the influenza viruses H3N2, H1N1pdm, B/Malaysia, and B/Massachusetts were observed.

Carotenoids and retinoids in the gonad of brood-stock pikeperch: accumulation during vitellogenesis and influence on egg quality in farmed pikeperch Sander lucioperca.

Carotenoids are determinants of reproductive fitness and egg quality. Here we studied the accumulation of astaxanthin (AX), canthaxanthin (CA) zeaxanthin (ZX), lutein (LU), retinol (RX) and dehydroretinol (DR) during vitellogenesis comparing previtellogenic and vitellogenic pikeperch (Sander lucioperca) eggs (n = 5 each), as well as selected tissues (liver, fat and muscles) in first süawning females (1176-1450 g). Futhermore, we compared egg batches with high (88-99% hatching rate, n = 5) or low (40-67% hatching rate, n= 5) egg quality. Vitellogenic follicles revealed higher concentrations of DR, RX, ZX and LU compared to previtellogenic follicles. Neither CA nor AX was detectable. In parallel, DR and RX were mobilized in the liver. In adipose and muscle tissue, comparing previtellogenic and vitellogenic females, no significant differences in carotenoid/retinoid content were observed. In high quality egg batches, both DR and RX were increased. LU was lower in high quality than in low quality eggs. In a conclusion, the amount of retinoids seems suboptimal in low quality egg batches and increased DR and RX are desirable in pikeperch. Since hypervitaminosis of retinoids can be problematic though, supplementation of the food with carotenoids, which can serve as precursors for retinoids, has to be carried out carefully.

Effect of bacterial volatiles on the mycelial growth of mushrooms.

Bacteria play an important role in the life cycle of fungi by influencing positively or negatively morphological features, mycelial growth and/or fruiting body induction. However, little is known about the underlying mechanisms and their species-dependence, especially among fungi of the phylum Basidiomycota. Hence, we analyzed the effects of seven bacterial isolates, that were previously obtained from Pleurotus ostreatus HK35, on the mycelial growth of P. ostreatus HK35, Pleurotus eryngii DSMZ 8264, Pleurotus sapidus DSMZ 8266, Pleurotus citrinopileatus DSMZ 5341, Cyclocybe aegerita AAE-3, Lentinula edodes CBS 389.89 and Kuehneromyces mutabilis DSMZ 1013 during eight days. Notably, the bacterial isolates only showed significant mycelial growth-promoting effects when co-cultivated on Petri dishes with Pleurotus species, except for P. citrinopileatus. In particular, Paenibacillus peoriae strain M48F induced remarkably the mycelial growth in P. ostreatus (∼47 %), P. eryngii (∼32 %) and P. sapidus (∼27 %) during the early cultivation stages, but with ongoing cultivation this strain inhibited the growth of all fungi. To investigate the impact of bacterial volatile organic compounds (VOCs) on the mycelial growth, P. ostreatus and P. eryngii were co-cultivated with the bacteria on bi-plates. No growth inhibition on bi-plates was observed while bacterial isolates and mycelia were separated by a physical barrier, assuring that late mycelial growth inhibition was not caused by bacterial volatile compounds. VOCs from strain M48F induced the strongest growth of P. ostreatus (∼50 %) and P. eryngii (∼20 %) mycelia compared to controls. Furthermore, we analyzed the VOCs of strain M48F alone and in combination with P. ostreatus, P. eryngii, P. sapidus and L. edodes using bi-plates and SPME-GC-MS. Strain M48F triggered the formation of ß-bisabolene when co-cultivated with P. ostreatus or P. eryngii, which may indicate a fungal defense reaction. Additionally, 2,5-diisopropylpyrazine dominated the volatilome of strain M48F on all eight sampling days. In samples of strain M48F, alone and co-cultivated with L. edodes, the amount of 2,5-diisopropylpyrazine remained quite constant. In contrast, the quantity of this substance declined substantially in co-cultures with P. ostreatus. Interestingly, 2,5-diisopropylpyrazine enhanced P. ostreatus mycelial growth significantly although the growth-promoting effect was not as pronounced as during co-cultivation with strain M48F. Our results show that the mycelial growth-promoting effects of bacteria are remarkably species-dependent, and that bacterial VOCs such as 2,5-diisopropylpyrazine can enhance mycelial growth.

Identification of volatile producing enzymes in higher fungi: Combining analytical and bioinformatic methods.

Filamentous fungi harbor the genetic potential for the biosynthesis of several secondary metabolites including various volatile organic compounds (VOCs). Nonetheless, under standard laboratory conditions, many of these VOCs are not formed. Furthermore, little is known about enzymes involved in the production of fungal VOCs. To tap these interesting topics, we developed an approach to identify enzymes putatively involved in the fungal VOC biosynthesis. In this chapter, we highlight different fungal cultivation methods and techniques for the extraction of VOCs, including a method that allows the noninvasive analysis of VOCs. In addition using terpene synthases as an example, it is depicted how enzymes putatively involved in VOC synthesis can be identified by means of bioinformatic approaches. Transcriptomic data of chosen genes combined with volatilome data obtained during different developmental stages is demonstrated as a powerful tool to identify enzymes putatively involved in fungal VOC biosynthesis. Especially with regard to subsequent enzyme characterization, this procedure is a target-oriented way to save time and efforts by considering only the most important enzymes.

Transcriptome of different fruiting stages in the cultivated mushroom Cyclocybe aegerita suggests a complex regulation of fruiting and reveals enzymes putatively involved in fungal oxylipin biosynthesis.

BACKGROUND: Cyclocybe aegerita (syn. Agrocybe aegerita) is a commercially cultivated mushroom. Its archetypal agaric morphology and its ability to undergo its whole life cycle under laboratory conditions makes this fungus a well-suited model for studying fruiting body (basidiome, basidiocarp) development. To elucidate the so far barely understood biosynthesis of fungal volatiles, alterations in the transcriptome during different developmental stages of C. aegerita were analyzed and combined with changes in the volatile profile during its different fruiting stages. RESULTS: A transcriptomic study at seven points in time during fruiting body development of C. aegerita with seven mycelial and five fruiting body stages was conducted. Differential gene expression was observed for genes involved in fungal fruiting body formation showing interesting transcriptional patterns and correlations of these fruiting-related genes with the developmental stages. Combining transcriptome and volatilome data, enzymes putatively involved in the biosynthesis of C8 oxylipins in C. aegerita including lipoxygenases (LOXs), dioxygenases (DOXs), hydroperoxide lyases (HPLs), alcohol dehydrogenases (ADHs) and ene-reductases could be identified. Furthermore, we were able to localize the mycelium as the main source for sesquiterpenes predominant during sporulation in the headspace of C. aegerita cultures. In contrast, changes in the C8 profile detected in late stages of development are probably due to the activity of enzymes located in the fruiting bodies. CONCLUSIONS: In this study, the combination of volatilome and transcriptome data of C. aegerita revealed interesting candidates both for functional genetics-based analysis of fruiting-related genes and for prospective enzyme characterization studies to further elucidate the so far barely understood biosynthesis of fungal C8 oxylipins.

Agrocybe aegerita Serves As a Gateway for Identifying Sesquiterpene Biosynthetic Enzymes in Higher Fungi.

Terpenoids constitute a structurally diverse group of natural products with wide applications in the pharmaceutical, nutritional, flavor and fragrance industries. Fungi are known to produce a large variety of terpenoids, yet fungal terpene synthases remain largely unexploited. Here, we report the sesquiterpene network and gene clusters of the black poplar mushroom Agrocybe aegerita. Among 11 putative sesquiterpene synthases (STSs) identified in its genome, nine are functional, including two novel synthases producing viridiflorol and viridiflorene. On this basis, an additional 1133 STS homologues from higher fungi have been curated and used for a sequence similarity network to probe isofunctional STS groups. With the focus on two STS groups, one producing viridiflorene/viridiflorol and one Δ6-protoilludene, the isofunctionality was probed and verified. Three new Δ6-protoilludene synthases and two new viridflorene/viridiflorol synthases from five different fungi were correctly predicted. The study herein serves as a fundamental predictive framework for the discovery of fungal STSs and biosynthesis of novel terpenoids. Furthermore, it becomes clear that fungal STS function differs between the phyla Ascomycota and Basidiomycota with the latter phylum being more dominant in the overall number and variability. This study aims to encourage the scientific community to further work on fungal STS and the products, biological functions, and potential applications of this vast source of natural products.

Volatilomes of Cyclocybe aegerita during different stages of monokaryotic and dikaryotic fruiting.

Volatile organic compounds (VOC) are characteristic for different fungal species. However, little is known about VOC changes during development and their biological role. Therefore, we established a laboratory cultivation system in modified crystallizing dishes for analyzing VOC during fruiting body development of the dikaryotic strain Cyclocybe aegerita AAE-3 as well as four monokaryotic offspring siblings exhibiting different fruiting phenotypes. From these, VOC were extracted directly from the headspace (HS) and analyzed by means of gas chromatography-mass spectrometry (GC-MS). For all tested strains, alcohols and ketones, including oct-1-en-3-ol, 2-methylbutan-1-ol and cyclopentanone, were the dominant substances in the HS of early developmental stages. In the dikaryon, the composition of the VOC altered with ongoing fruiting body development and, even more drastically, during sporulation. At the latter stage, sesquiterpenes, especially Δ6-protoilludene, α-cubebene and δ-cadinene, were the dominant substances. After sporulation, the amount of sesquiterpenes decreased, while additional VOC, mainly octan-3-one, appeared. In the HS of the monokaryons, less VOC were present of which all were detectable in the HS of the dikaryon C. aegerita AAE-3. The results of the present study show that the volatilome of C. aegerita changes considerably depending on the developmental stage of the fruiting body.

Aroma Profile Analyses of Filamentous Fungi Cultivated on Solid Substrates.

Filamentous fungi have been used since centuries in the production of food by means of solid substrate fermentation (SSF). The most applied SSF involving fungi is the cultivation of mushrooms, e.g., on tree stumps or sawdust, for human consumption. However, filamentous fungi are also key players during manufacturing of several processed foods, like mold cheese, tempeh, soy sauce, and sake. In addition to their nutritive values, these foods are widely consumed due to their pleasant flavors. Based on the potentials of filamentous fungi to grow on solid substrates and to produce valuable aroma compounds, in recent decades, several studies concentrated on the production of aroma compounds with SSF, turning cheap agricultural wastes into valuable flavors. In this review, we focus on the presentation of common analytical methods for volatile substances and highlight various applications of SSF of filamentous fungi dealing with the production of aroma compounds. Graphical Abstract.

RNA Hairpin Folding in the Crowded Cell.

Precise secondary and tertiary structure formation is critically important for the cellular functionality of ribonucleic acids (RNAs). RNA folding studies were mainly conducted in vitro, without the possibility of validating these experiments inside cells. Here, we directly resolve the folding stability of a hairpin-structured RNA inside live mammalian cells. We find that the stability inside the cell is comparable to that in dilute physiological buffer. On the contrary, the addition of in vitro artificial crowding agents, with the exception of high-molecular-weight PEG, leads to a destabilization of the hairpin structure through surface interactions and reduction in water activity. We further show that RNA stability is highly variable within cell populations as well as within subcellular regions of the cytosol and nucleus. We conclude that inside cells the RNA is subject to (localized) stabilizing and destabilizing effects that lead to an on average only marginal modulation compared to diluted buffer.