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.

Fortifying a meal with oyster mushroom powder beneficially affects postprandial glucagon-like peptide-1, non-esterified free fatty acids and hunger sensation in adults with impaired glucose tolerance: a double-blind randomized controlled crossover trial.

PURPOSE: Impaired glucose tolerance (IGT) is a pathophysiological condition characterized by insulin resistance with known metabolic consequences such as postprandial hyperglycemia and hypertriglyceridemia. We hypothesized that fortifying a meal with mushrooms rich in ß-glucans may diminish glucose and triglyceride responses by improving postprandial gastrointestinal hormone release. METHODS: In a randomized controlled crossover study, 22 subjects with IGT ingested a meal either enriched with 20 g powder (8.1 g ß-glucans) of oven-dried Pleurotus ostreatus (enriched meal, EN) or without enrichment (control meal, CON). Blood was collected before and repeatedly within 4 h after the meal to determine AUC of glucose (primary outcome), insulin, triglycerides, non-esterified free fatty acids (NEFAs), glucagon-like peptide-1 (GLP-1), gastric inhibitory polypeptide (GIP) and ghrelin. Appetite sensations (hunger, satiety, fullness, and desire to eat) were assessed before and after meal consumption by visual analog scales. RESULTS: Postprandial glucose, insulin, triglycerides, GIP and ghrelin concentrations as well as the corresponding AUCs did not differ between EN and CON. NEFAs-AUC was 14% lower (P = 0.026) and GLP-1-AUC 17% higher (P = 0.001) after EN compared to CON. Appetite ratings did not differ between treatments, except for hunger (AUC 22% lower after EN vs. CON; P = 0.031). CONCLUSION: The observed immediate postprandial metabolic changes indicate that an easily manageable fortification of a single meal with powder from dried oyster mushrooms as ß-glucan source may improve postprandial metabolism. If the effect is preserved long term, this measure can diminish the risk for further development of overweight/obesity and type 2 diabetes in subjects with IGT. CLINICAL TRIAL REGISTRATION: German Clinical Trial Register on 09/08/2018; trial-ID: DRKS00015244.

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.

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.

Heterologous Production and Functional Characterization of Ageritin, a Novel Type of Ribotoxin Highly Expressed during Fruiting of the Edible Mushroom Agrocybe aegerita.

Fungi produce various defense proteins against antagonists, including ribotoxins. These toxins cleave a single phosphodiester bond within the universally conserved sarcin-ricin loop of ribosomes and inhibit protein biosynthesis. Here, we report on the structure and function of ageritin, a previously reported ribotoxin from the edible mushroom Agrocybe aegerita The amino acid sequence of ageritin was derived from cDNA isolated from the dikaryon A. aegerita AAE-3 and lacks, according to in silico prediction, a signal peptide for classical secretion, predicting a cytoplasmic localization of the protein. The calculated molecular weight of the protein is slightly higher than the one reported for native ageritin. The A. aegerita ageritin-encoding gene, AaeAGT1, is highly induced during fruiting, and toxicity assays with AaeAGT1 heterologously expressed in Escherichia coli showed a strong toxicity against Aedes aegypti larvae yet not against nematodes. The activity of recombinant A. aegerita ageritin toward rabbit ribosomes was confirmed in vitro Mutagenesis studies revealed a correlation between in vivo and in vitro activities, indicating that entomotoxicity is mediated by ribonucleolytic cleavage. The strong larvicidal activity of ageritin makes this protein a promising candidate for novel biopesticide development.IMPORTANCE Our results suggest a pronounced organismal specificity of a protein toxin with a very conserved intracellular molecular target. The molecular details of the toxin-target interaction will provide important insight into the mechanism of action of protein toxins and the ribosome. This insight might be exploited to develop novel bioinsecticides.

New terpenoids from the fermentation broth of the edible mushroom Cyclocybe aegerita.

The strophariaceous basidiomycete Cyclocybe aegerita (synonyms Agrocybe aegerita and A. cylindracea) is one of the most praised cultivated edible mushrooms and is being cultivated at large scale for food production. Furthermore, the fungus serves as a model organism to study fruiting body formation and the production of secondary metabolites during the life cycle of Basidiomycota. By studying the secondary metabolite profiles of C. aegerita, we found several terpenoids in submerged cultures. Aside from the main metabolite, bovistol (1), two new bovistol derivatives B and C (2, 3) and pasteurestin C as a new protoilludane (4) were isolated by preparative HPLC. Their structures were elucidated by mass spectrometry and NMR spectroscopy. The relative configurations of 2-4 were assigned by ROESY correlations, and 3 J H,H coupling constants in the case of 4. Applying quantitative PCR for gene expression validation, we linked the production of bovistol and its derivatives to the respective biosynthesis gene clusters.

The genome sequence of the commercially cultivated mushroom Agrocybe aegerita reveals a conserved repertoire of fruiting-related genes and a versatile suite of biopolymer-degrading enzymes.

BACKGROUND: Agrocybe aegerita is an agaricomycete fungus with typical mushroom features, which is commercially cultivated for its culinary use. In nature, it is a saprotrophic or facultative pathogenic fungus causing a white-rot of hardwood in forests of warm and mild climate. The ease of cultivation and fructification on solidified media as well as its archetypal mushroom fruit body morphology render A. aegerita a well-suited model for investigating mushroom developmental biology. RESULTS: Here, the genome of the species is reported and analysed with respect to carbohydrate active genes and genes known to play a role during fruit body formation. In terms of fruit body development, our analyses revealed a conserved repertoire of fruiting-related genes, which corresponds well to the archetypal fruit body morphology of this mushroom. For some genes involved in fruit body formation, paralogisation was observed, but not all fruit body maturation-associated genes known from other agaricomycetes seem to be conserved in the genome sequence of A. aegerita. In terms of lytic enzymes, our analyses suggest a versatile arsenal of biopolymer-degrading enzymes that likely account for the flexible life style of this species. Regarding the amount of genes encoding CAZymes relevant for lignin degradation, A. aegerita shows more similarity to white-rot fungi than to litter decomposers, including 18 genes coding for unspecific peroxygenases and three dye-decolourising peroxidase genes expanding its lignocellulolytic machinery. CONCLUSIONS: The genome resource will be useful for developing strategies towards genetic manipulation of A. aegerita, which will subsequently allow functional genetics approaches to elucidate fundamentals of fruiting and vegetative growth including lignocellulolysis.

Laccase production and pellet morphology of Coprinopsis cinerea transformants in liquid shake flask cultures.

Laccase production and pellet formation of transformants of Coprinopsis cinerea strain FA2222 of C. cinerea laccase gene lcc1 subcloned behind the gpdII-promoter from Agaricus bisporus were compared with a control transformant carrying no extra laccase gene. At the optimum growth temperature of 37 °C, maximal laccase yields of 2.9 U/ml were obtained by the best lcc1 transformant pYSK7-26 in liquid shake flask cultures. Reduction in temperature to 25 °C increased laccase yields up to 9.2 U/ml. The control transformant had no laccase activities at 37 °C but native activity at 25 °C (3.5 U/ml). Changing the temperature had severe effects on the morphology of the mycelial pellets formed during cultivation, but links of distinct pellet morphologies to native or recombinant laccase production could not be established. Automated image analysis was used to characterise pellet formation and morphological parameters (pellet area, diameter, convexity and mycelial structure). Cross sections of selected pellets showed that they differentiated in an outer rind and an inner medulla of loosened hyphae. Pellets at 25 °C had a small and dense outer zone and adopted with time a smooth surface. Pellets at 37 °C had a broader outer zone and a fringy surface due to generation of more and larger protuberances in the rind that when released can serve for production of further pellets.

Enrichment of vitamin D2 in mycelium from submerged cultures of the agaric mushroom Pleurotus sapidus.

Ergosterol, a precursor of vitamin D2, is present in the cell membrane of all fungi. It can be transformed into vitamin D2 by UV-B exposure. In this study, a basidiomycete, Pleurotus sapidus, cultivated in liquid malt extract medium was exposed to UV-B light. In addition, autoclaved, abiotic mycelium was put through UV-B exposure for the first time. The effects of different UV-B exposure times, surface areas and temperatures on vitamin D2 formation were analyzed. The conversion of ergosterol to vitamin D2 at ambient temperature almost reached completion within 10 min resulting in vitamin D2 concentrations of 365 µg (g dry matter)-1. Prolonged exposure of the biotic mycelium for up to 60 min resulted in a reduced vitamin D2 concentration with stagnation at about 280 µg (g dry matter)-1. Exposure of the abiotic mycelium showed a slower increase but also leveled off at the same concentration. Furthermore, it could be shown that vitamin D2 formation depends on the temperature and the exposed surface area. The vitamin D2 concentration augmented after increasing the exposed surface from 65.0 to 298.6 cm2. The ergosterol content of P. sapidus was analyzed in a way similar to vitamin D2 and resulted in 3.75 ± 0.06 mg (g dry matter)-1 ergosterol.

An aryl-alcohol oxidase of Pleurotus sapidus: heterologous expression, characterization, and application in a 2-enzyme system.

Aryl-alcohol oxidases (AAOs) are enzymes supporting the degradation of lignin by fungal derived class II peroxidases produced by white-rot fungi. AAOs are able to generate H2O2 as a by-product via oxidation of an aryl-alcohol into its correspondent aldehyde. In this study, an AAO was heterologously expressed in a basidiomycete host for the first time. The gene for an AAO of the white-rot fungus Pleurotus sapidus, a close relative to the oyster mushroom Pleurotus ostreatus, was cloned into an expression vector and put under control of the promotor of the glyceraldehyde-3-phosphate dehydrogenase gene 2 (gpdII) of the button mushroom Agaricus bisporus. The expression vector was transformed into the model basidiomycete Coprinopsis cinerea, and several positive transformants were obtained. The best producing transformants were grown in shake-flasks and in a stirred tank reactor reaching enzymatic activities of up to 125 U L(-1) using veratryl alcohol as a substrate. The purified AAO was biochemically characterized and compared to the previously described native and recombinant AAOs from other Pleurotus species. In addition, a two-enzyme system comprising a dye-decolorizing peroxidase (DyP) from Mycetinis scorodonius and the P. sapidus AAO was successfully employed to bleach the anthraquinone dye Reactive Blue 5.

Non-growing Rhodopseudomonas palustris increases the hydrogen gas yield from acetate by shifting from the glyoxylate shunt to the tricarboxylic acid cycle.

When starved for nitrogen, non-growing cells of the photosynthetic bacterium Rhodopseudomonas palustris continue to metabolize acetate and produce H2, an important industrial chemical and potential biofuel. The enzyme nitrogenase catalyzes H2 formation. The highest H2 yields are obtained when cells are deprived of N2 and thus use available electrons to synthesize H2 as the exclusive product of nitrogenase. To understand how R. palustris responds metabolically to increase H2 yields when it is starved for N2, and thus not growing, we tracked changes in biomass composition and global transcript levels. In addition to a 3.5-fold higher H2 yield by non-growing cells we also observed an accumulation of polyhydroxybutyrate to over 30% of the dry cell weight. The transcriptome of R. palustris showed down-regulation of biosynthetic processes and up-regulation of nitrogen scavenging mechanisms in response to N2 starvation but gene expression changes did not point to metabolic activities that could generate the reductant necessary to explain the high H2 yield. We therefore tracked (13)C-labeled acetate through central metabolic pathways. We found that non-growing cells shifted their metabolism to use the tricarboxylic acid cycle to metabolize acetate in contrast to growing cells, which used the glyoxylate cycle exclusively. This shift enabled cells to more fully oxidize acetate, providing the necessary reducing power to explain the high H2 yield.

Prolyl-specific peptidases for applications in food protein hydrolysis.

Various food proteins including, e.g. gluten, collagen and casein are rich in L-proline residues. Due to the cyclic structure of proline, these proteins are well protected from enzymatic degradation by typical digestive enzymes. Proline-specific peptidases (PsP) belong to different families of hydrolases acting on peptide bonds (EC 3.4.x.x). They occur in various organisms including bacteria, fungi, plants and insects. Based on their biochemical characteristics, PsP type enzymes are further grouped into different subclasses of which prolyl aminopeptidases (EC 3.4.11.5, PAP), prolyl carboxypeptidases (EC 3.4.17.16, PCP) and prolyl oligopeptidases/prolyl endopeptidases (EC 3.4.21.26, POP/PEP) are of major interest for applications in food biotechnology. This mini review summarises the biochemical assays employed for these subclasses of PsP and their structural properties and the reaction mechanisms. A special focus was set on PsP derived from fungi and insects and important industrial applications in the field of food biotechnology. The degradation of gluten and collagen as well as the hydrolysis of bitter peptides are discussed.

Effects of a biotechnologically produced Pleurotus sapidus mycelium on gut microbiome, liver transcriptome and plasma metabolome of broilers.

Submerged cultivation using low-value agro-industrial side streams allows large-scale and efficient production of fungal mycelia, which has a high nutritional value. As the dietary properties of fungal mycelia in poultry are largely unknown, the present study aimed to investigate the effect of feeding a Pleurotus sapidus (PSA) mycelium as a feed supplement on growth performance, composition of the cecal microbiota and several physiological traits including gut integrity, nutrient digestibility, liver lipids, liver transcriptome and plasma metabolome in broilers. 72 males, 1-day-old Cobb 500 broilers were randomly assigned to 3 different groups and fed 3 different adequate diets containing either 0% (PSA-0), 2.5% (PSA-2.5) and 5% (PSA-5.0) P. sapidus mycelium in a 3-phase feeding system for 35 d. Each group consisted of 6 cages (replicates) with 4 broilers/cage. Body weight gain, feed intake and feed:gain ratio and apparent ileal digestibility of crude protein, ether extract and amino acids were not different between groups. Metagenomic analysis of the cecal microbiota revealed no differences between groups, except that one α-diversity metric (Shannon index) and the abundance of 2 low-abundance bacterial taxa (Clostridia UCG 014, Eubacteriales) differed between groups (P < 0.05). Concentrations of total and individual short-chain fatty acids in the cecal digesta and concentrations of plasma lipopolysaccharide and mRNA levels of proinflammatory genes, tight-junction proteins, and mucins in the cecum mucosa did not differ between groups. None of the plasma metabolites analyzed using targeted-metabolomics differed across the groups. Hepatic transcript profiling revealed a total of 144 transcripts to be differentially expressed between group PSA-5.0 and group PSA-0 but none of these genes was regulated greater 2-fold. Considering either the lack of effects or the very weak effects of feeding the P. sapidus mycelium in the broilers it can be concluded that inclusion of a sustainably produced fungal mycelium in broiler diets at the expense of other feed components has no negative consequences on broilers´ performance and metabolism.

13C-flux analysis reveals NADPH-balancing transhydrogenation cycles in stationary phase of nitrogen-starving Bacillus subtilis.

In their natural habitat, microorganisms are typically confronted with nutritional limitations that restrict growth and force them to persevere in a stationary phase. Despite the importance of this phase, little is known about the metabolic state(s) that sustains it. Here, we investigate metabolically active but non-growing Bacillus subtilis during nitrogen starvation. In the absence of biomass formation as the major NADPH sink, the intracellular flux distribution in these resting B. subtilis reveals a large apparent catabolic NADPH overproduction of 5.0 ± 0.6 mmol g(-1)h(-1) that was partly caused by high pentose phosphate pathway fluxes. Combining transcriptome analysis, stationary (13)C-flux analysis in metabolic deletion mutants, (2)H-labeling experiments, and kinetic flux profiling, we demonstrate that about half of the catabolic excess NADPH is oxidized by two transhydrogenation cycles, i.e. isoenzyme pairs of dehydrogenases with different cofactor specificities that operate in reverse directions. These transhydrogenation cycles were constituted by the combined activities of the glyceraldehyde 3-phosphate dehydrogenases GapA/GapB and the malic enzymes MalS/YtsJ. At least an additional 6% of the overproduced NADPH is reoxidized by continuous cycling between ana- and catabolism of glutamate. Furthermore, in vitro enzyme data show that a not yet identified transhydrogenase could potentially reoxidize ∼20% of the overproduced NADPH. Overall, we demonstrate the interplay between several metabolic mechanisms that concertedly enable network-wide NADPH homeostasis under conditions of high catabolic NADPH production in the absence of cell growth in B. subtilis.

Lcc1 and Lcc5 are the main laccases secreted in liquid cultures of Coprinopsis cinerea strains.

The litter-degrading dung fungus Coprinopsis cinerea has the high number of seventeen different laccase genes. In this work, ten different monokaryons were compared in their ability to produce laccases in two different complete media at different temperatures. Few strains showed laccase activity at the optimal growth temperature of 37 °C. Nine of the strains gave laccase activities between 0.2 and 5.9 U mL(-1) at the suboptimal temperature of 25 °C in mKjalke medium. Laccase activities in YMG/T medium were detected for only three strains (0.5-4.5 U mL(-1)). Zymograms of supernatants from mKjalke medium resulted in total in 10 different laccase bands but strains differed in distribution. LC-MS/MS analysis with Mascot searches of the annotated C. cinerea genome identified isoenzymes from five different genes (Lcc1, Lcc2, Lcc5, Lcc9 and Lcc10) and of Lcc1 three and of Lcc5 two distinct electrophoretical forms. Lcc1 and Lcc5 were expressed in all laccase positive strains, but not all forms were found in all of the strains. Lcc2, Lcc9 and Lcc10 occurred only in three strains as minor laccases, indicating that Lcc1 and Lcc5 are the main laccases of C. cinerea secreted in liquid mKjalke medium.

Protein- and Carbohydrate-Rich Supplements in Feeding Adult Black Soldier Flies (Hermetia illucens) Affect Life History Traits and Egg Productivity.

The black soldier fly, Hermetia illucens (BSF; Diptera: Stratiomyidae), has come into the focus of research over the past decade since its larvae are polyphagous feeders with an exceptional substrate range, making them a promising candidate for the bioconversion of various organic side streams into valuable insect protein. While larval nutritional requirements have been studied in detail, basic information on adult feeding is still lacking. The reproduction of adult flies is a bottleneck and key determinant in rearing BSF, which has extensive potential for improvement. In the present study, we examined the impact of different carbohydrate (honey and d-glucose) and protein sources (Spirulina and Chlorella powder) on a variety of life history traits using a highly standardized single pair approach. Feeding a 5% honey solution was shown to make females live 2.8 d longer, become more fecund (9 egg clutches per 10 females), lay more eggs (increasing 1.7-fold to 182.4 mg per 10 females), reduce the number of failed oviposition events 3-fold and increase multiple oviposition events from 2 to 15. Additionally, female longevity after oviposition improved 1.7-fold from 6.7 to 11.5 d. In order to further optimize adult feeding, mixtures of proteins and carbohydrates with varying ratios should be tested.

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.

Fatty acids derived from oviposition systems guide female black soldier flies (Hermetia illucens) toward egg deposition sites.

The black soldier fly, Hermetia illucens, comes with big promises for industrial purposes since its larvae feed polyphagously on a broad spectrum of organic substrates. However, research focusing on adult flies is scarce, which is inconsistent with their reproductive relevance within the rearing cycle. In particular, directed oviposition is a challenge in artificial systems. Currently, decomposing organic matter is commonly used as oviposition substrate, which has extensive potential for improvement in view of the lack of standardization and the risk of microbial contamination. Here, we identified three fatty acids and one fatty acid methyl ester derived from the surface of old oviposition sites and targeted to elucidate their effect on preference behavior and oviposition site selection using Y-olfactometry and prepared oviposition sites, respectively. Exposure to tetradecanoic acid attracted gravid females and stimulated oviposition most strongly, while decanoic acid demonstrated a repulsive effect. Females kept in mixed-sex populations were attracted by tetradecanoic acid, resulting in a higher egg mass found in the compound box (3.0-11.4 fold), a ≥ 2.3 fold reduction of nonspecifically deposited eggs, and the highest total egg mass. Conversely, decanoic and dodecanoic acid caused females to lay a greater proportion of eggs nonspecifically outside both boxes. Our data suggest that fatty acids, especially tetradecanoic acid, are important cues for oviposition site selection in black soldier flies. In order to achieve a directed oviposition behavior, the role of further short- and long-chain fatty acids as attractants should be examined.

Collisional fragmentation of central carbon metabolites in LC-MS/MS increases precision of ¹³C metabolic flux analysis.

Experimental determination of fluxes by (13)C-tracers relies on detection of (13)C-patterns in metabolites or by-products. In the field of (13)C metabolic flux analysis, the most recent developments point toward recording labeling patterns by liquid chromatography (LC)-mass spectrometry (MS)/MS directly in intermediates in central carbon metabolism (CCM) to increase temporal resolution. Surprisingly, the flux studies published so far with LC-MS measurements were based on intact metabolic intermediates-thus neglected the potential benefits of using positional information to improve flux estimation. For the first time, we exploit collisional fragmentation to obtain more fine-grained (13)C-data on intermediates of CCM and investigate their impact in (13)C metabolic flux analysis. For the case study of Bacillus subtilis grown in mineral medium with (13)C-labeled glucose, we compare the flux estimates obtained by iterative isotopologue balancing of (13)C-data obtained either by LC-MS/MS for solely intact intermediates or LC-MS/MS for intact and fragmented intermediates of CCM. We show that with LC-MS/MS data, fragment information leads to more precise estimates of fluxes in pentose phosphate pathway, glycolysis, and to the tricarboxylic acid cycle. Additionally, we present an efficient analytical strategy to rapidly acquire large sets of (13)C-patterns by tandem MS, and an in-depth analysis of the collisional fragmentation of primary intermediates. In the future, this catalogue will enable comprehensive in silico calculability analyses to identify the most sensitive measurements and direct experimental design.

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.