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.

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.

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.

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.

Replacement of Pregastric Lipases in Cheese Production: Identification and Heterologous Expression of a Lipase from Pleurotus citrinopileatus.

Traditionally produced piquant cheeses such as Feta or Provolone rely on pregastric lipolytic enzymes of animal origin to intensify flavor formation during ripening. Herein, we report a novel fungal lipase, derived from the phylum Basidiomycota to replace animal-derived products. A screening of 31 strains for the desired hydrolytic activities was performed, which revealed a promising fungal species. The secretome of an edible golden oyster mushroom, Pleurotus citrinopileatus, provided suitable enzymatic activity, and the coding sequence of the corresponding enzyme was identified by combining transcriptome and liquid chromatography high-resolution electrospray tandem mass spectroscopy (LC-HR-ESI-MS/MS) data. Recombinant expression in Escherichia coli BL21 (DE3) using chaperones GroES-GroEL and DnaK-DnaJ-GrpE was established. The recombinant lipolytic enzyme was purified and biochemically characterized in terms of thermal and pH stability, optimal reaction conditions, and kinetic data toward p-nitrophenyl esters. An application in the microscale production of Feta-type brine cheese revealed promising sensory properties, which were confirmed by headspace solid-phase microextraction gas chromatography mass spectrometry (HS-SPME-GC-MS) analyses in comparison with the reference enzyme opti-zym z10uc from goat origin. Supplementation with 2.3 U of the heterologously expressed fungal lipase produced the most comparable free fatty acid profile after 30 days of ripening. The flavor and texture formed during the application of the new lipase from P. citrinopileatus proved to be competitive to the use of pregastric lipases and could therefore replace the products of animal origin.

Isolation of Bacterial and Fungal Microbiota Associated with Hermetia illucens Larvae Reveals Novel Insights into Entomopathogenicity.

Larvae of the black soldier fly (BSF) Hermetia illucens are polyphagous feeders and show tremendous bioconversion capabilities of organic matter into high-quality insect biomass. However, the digestion of lignocellulose-rich palm oil side streams such as palm kernel meal (PKM) is a particular challenge, as these compounds are exceptionally stable and are mainly degraded by microbes. This study aimed to investigate the suitability of BSF larvae as bioconversion agents of PKM. Since the intestinal microbiota is considered to play a key role in dietary breakdown and in increasing digestibility, the bacterial and fungal communities of BSF larvae were characterized in a culture-dependent approach and screened for their putative entomopathogenicity. The lethality of six putative candidates was investigated using intracoelomal injection. In total, 93 isolates were obtained with a bacterial share of 74% that were assigned to the four phyla Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria. Members of the genera Klebsiella, Enterococcus, and Sphingobacterium are part of the core microbiome, as they were frequently described in the gut of Hermetia larvae regardless of diet, nutritional composition, or rearing conditions. With 75%, a majority of the fungal isolates belonged to the phylum Ascomycota. We identified several taxa already published to be able to degrade lignocelluloses, including Enterococcus, Cellulomonas, Pichia yeasts, or filamentous Fusarium species. The injection assays revealed pronounced differences in pathogenicity against the larvae. While Alcaligenes faecalis caused no, Diutina rugosa weak (23.3%), Microbacterium thalassium moderate (53.3%), and Pseudomonas aeruginosa and Klebsiella pneumoniae high (≥80%) lethality, Fusarium solani injection resulted in 100% lethality.

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.

Wild Strawberry-like Flavor Produced by the Fungus Wolfiporia cocos─Identification of Character Impact Compounds by Aroma Dilution Analysis after Dynamic Headspace Extraction.

Brown-rot fungi are particularly suitable for the sustainable and cost-efficient biotechnological production of natural flavors. In this study, Wolfiporia cocos was employed for the fermentation of European black currant pomace supplemented with aspartate in surface cultures to produce a flavor reminiscent of wild strawberries. Aroma dilution analysis (ADA) by means of dynamic headspace extraction was developed as a suitable technique for solid samples. The character impact compounds were quantified by stable isotope dilution analysis and standard addition and validated by recombination experiments. (R)-Linalool (1879 µg kg-1, ADA 211), methyl anthranilate (2206 µg kg-1, 210), 2-aminobenzaldehyde (771 µg kg-1, 25), and geraniol (138 µg kg-1, 25) were identified as key aroma compounds. Recombination experiments demonstrated that the combination of the four analyzed compounds was responsible for the odor impression reminiscent of wild strawberries.

Alpha-single chains of collagen type VI inhibit the fibrogenic effects of triple helical collagen VI in hepatic stellate cells.

The interaction of extracellular matrix (ECM) components with hepatic stellate cells (HSCs) is thought to perpetuate fibrosis by stimulating signaling pathways that drive HSC activation, survival and proliferation. Consequently, disrupting the interaction between ECM and HSCs is considered a therapeutical avenue although respective targets and underlying mechanisms remain to be established. Here we have interrogated the interaction between type VI collagen (CVI) and HSCs based on the observation that CVI is 10-fold upregulated during fibrosis, closely associates with HSCs in vivo and promotes cell proliferation and cell survival in cancer cell lines. We exposed primary rat HSCs and a rat hepatic stellate cell line (CFSC) to soluble CVI and determined the rate of proliferation, apoptosis and fibrogenesis in the absence of any additional growth factors. We find that CVI in nanomolar concentrations prevents serum starvation-induced apoptosis. This potent anti-apoptotic effect is accompanied by induction of proliferation and acquisition of a pronounced pro-fibrogenic phenotype characterized by increased α-smooth muscle actin, TGF-ß, collagen type I and TIMP-1 expression and diminished proteolytic MMP-13 expression. The CVI-HSC interaction can be disrupted with the monomeric α2(VI) and α3(VI) chains and abrogates the activating CVI effects. Further, functional relevant α3(VI)-derived 30 amino acid peptides lead to near-complete inhibition of the CVI effect. In conclusion, CVI serves as a potent mitogen and activating factor for HSCs. The antagonistic effects of the CVI monomeric chains and peptides point to linear peptide sequences that prevent activation of CVI receptors which may allow a targeted antifibrotic therapy.

Genome and Secretome Analysis of Staphylotrichum longicolleum DSM105789 Cultured on Agro-Residual and Chitinous Biomass.

Staphylotrichum longicolleum FW57 (DSM105789) is a prolific chitinolytic fungus isolated from wood, with a chitinase activity of 0.11 ± 0.01 U/mg. We selected this strain for genome sequencing and annotation, and compiled its growth characteristics on four different chitinous substrates as well as two agro-industrial waste products. We found that the enzymatic mixture secreted by FW57 was not only able to digest pre-treated sugarcane bagasse, but also untreated sugarcane bagasse and maize leaves. The efficiency was comparable to a commercial enzymatic cocktail, highlighting the potential of the S. longicolleum enzyme mixture as an alternative pretreatment method. To further characterize the enzymes, which efficiently digested polymers such as cellulose, hemicellulose, pectin, starch, and lignin, we performed in-depth mass spectrometry-based secretome analysis using tryptic peptides from in-gel and in-solution digestions. Depending on the growth conditions, we were able to detect from 442 to 1092 proteins, which were annotated to identify from 134 to 224 putative carbohydrate-active enzymes (CAZymes) in five different families: glycoside hydrolases, auxiliary activities, carbohydrate esterases, polysaccharide lyases, glycosyl transferases, and proteins containing a carbohydrate-binding module, as well as combinations thereof. The FW57 enzyme mixture could be used to replace commercial enzyme cocktails for the digestion of agro-residual substrates.

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.

Insights into the genome and secretome of Fusarium metavorans DSM105788 by cultivation on agro-residual biomass and synthetic nutrient sources.

BACKGROUND: The transition to a biobased economy involving the depolymerization and fermentation of renewable agro-industrial sources is a challenge that can only be met by achieving the efficient hydrolysis of biomass to monosaccharides. In nature, lignocellulosic biomass is mainly decomposed by fungi. We recently identified six efficient cellulose degraders by screening fungi from Vietnam. RESULTS: We characterized a high-performance cellulase-producing strain, with an activity of 0.06 U/mg, which was identified as a member of the Fusarium solani species complex linkage 6 (Fusarium metavorans), isolated from mangrove wood (FW16.1, deposited as DSM105788). The genome, representing nine potential chromosomes, was sequenced using PacBio and Illumina technology. In-depth secretome analysis using six different synthetic and artificial cellulose substrates and two agro-industrial waste products identified 500 proteins, including 135 enzymes assigned to five different carbohydrate-active enzyme (CAZyme) classes. The F. metavorans enzyme cocktail was tested for saccharification activity on pre-treated sugarcane bagasse, as well as untreated sugarcane bagasse and maize leaves, where it was complemented with the commercial enzyme mixture Accellerase 1500. In the untreated sugarcane bagasse and maize leaves, initial cell wall degradation was observed in the presence of at least 196 µg/mL of the in-house cocktail. Increasing the dose to 336 µg/mL facilitated the saccharification of untreated sugarcane biomass, but had no further effect on the pre-treated biomass. CONCLUSION: Our results show that F. metavorans DSM105788 is a promising alternative pre-treatment for the degradation of agro-industrial lignocellulosic materials. The enzyme cocktail promotes the debranching of biopolymers surrounding the cellulose fibers and releases reduced sugars without process disadvantages or loss of carbohydrates.

Engineering a lipoxygenase from cyclocybe aegerita towards long chain polyunsaturated fatty acids.

The basidiomycetous lipoxygenase Lox1 from Cyclocybe aegerita catalyzes the oxygenation of polyunsaturated fatty acids (PUFAs) with a high preference towards the C18-PUFA linoleic acid (C18:2 (ω-6)). In contrast, longer PUFAs, generally not present in the fungal cell such as eicosatrienoic acid (C20:3(ω-3)) and docosatrienoic acid (C22:3 (ω-3)), are converted with drastically lower activities. With site-directed mutagenesis, we were able to create two variants with enhanced activities towards longer chain PUFAs. The W330L variant showed a ~ 20 % increased specific activity towards C20:3(ω-3), while a ~ 2.5-fold increased activity against C22:3 (ω-3) was accomplished by the V581 variant.

Bioinformatics-aided identification, characterization and applications of mushroom linalool synthases.

Enzymes empower chemical industries and are the keystone for metabolic engineering. For example, linalool synthases are indispensable for the biosynthesis of linalool, an important fragrance used in 60-80% cosmetic and personal care products. However, plant linalool synthases have low activities while expressed in microbes. Aided by bioinformatics analysis, four linalool/nerolidol synthases (LNSs) from various Agaricomycetes were accurately predicted and validated experimentally. Furthermore, we discovered a linalool synthase (Ap.LS) with exceptionally high levels of selectivity and activity from Agrocybe pediades, ideal for linalool bioproduction. It effectively converted glucose into enantiopure (R)-linalool in Escherichia coli, 44-fold and 287-fold more efficient than its bacterial and plant counterparts, respectively. Phylogenetic analysis indicated the divergent evolution paths for plant, bacterial and fungal linalool synthases. More critically, structural comparison provided catalytic insights into Ap.LS superior specificity and activity, and mutational experiments validated the key residues responsible for the specificity.

Enzymatic Oxidation of Ca-Lignosulfonate and Kraft Lignin in Different Lignin-Laccase-Mediator-Systems and MDF Production.

Laccase-mediator-oxidized lignin offers replacement for conventional chemical binders to produce fiberboards. Compared to the previously reported laccase-mediator system (LMS), a lignin-laccase-mediator-system (LLMS) has an advantage in that it requires much shorter fiber-enzyme incubation time due to significantly increased redox reactions. However, the cost of regularly applying laccase on an industrial scale is currently too high. We have employed CcLcc5 from cultures of the basidiomycete Coprinopsis cinerea as a novel basi-laccase (a CAZy subfamily AA1_1 laccase) in medium-density fiberboard (MDF) production, in comparison to the commercial formulation Novozym 51003 with recombinantly produced asco-laccase MtL (a CAZy subfamily AA1_3 laccase-like multicopper oxidase from the ascomycete Myceliophthora thermophila). With the best-performing natural mediator 2,6-dimethoxyphenol (DMP), unpurified CcLcc5 was almost as good as formulated Novozym 51003 in increasing the molecular weight (MW) of the technical lignins tested, the hydrophilic high-MW Ca-lignosulfonate and the hydrophobic low-MW kraft lignin (Indulin AT). Oxygen consumption rates of the two distantly related, poorly conserved enzymes (31% sequence identity) with different mediators and lignosulfonate were also comparable, but Indulin AT significantly reduced the oxidative activity of Novozym 51003 unlike CcLcc5, regardless of the mediator used, either DMP or guaiacol. Oxygen uptake by both laccases was much faster with both technical lignins with DMP than with guaiacol. In case of lignosulfonate and DMP, 20-30 min of incubation was sufficient for full oxygen consumption, which fits in well in time with the usual binder application steps in industrial MDF production processes. LLMS-bonded MDF was thus produced on a pilot-plant scale with either crude CcLcc5 or Novozym 51003 at reduced enzyme levels of 5 kU/kg absolutely dry wood fiber with lignosulfonate and mediator DMP. Boards produced with CcLcc5 were comparably good as those made with Novozym 51003. Boards reached nearly standard specifications in internal bond strength (IB) and modulus of rupture (MOR), while thickness swelling (TS) was less good based on the hydrophilic character of lignosulfonate. LLMS-bonded MDF with Indulin AT and DMP performed better in TS but showed reduced IB and MOR values.

Aspergillus sydowii: Genome Analysis and Characterization of Two Heterologous Expressed, Non-redundant Xylanases.

A prerequisite for the transition toward a biobased economy is the identification and development of efficient enzymes for the usage of renewable resources as raw material. Therefore, different xylanolytic enzymes are important for efficient enzymatic hydrolysis of xylan-heteropolymers. A powerful tool to overcome the limited enzymatic toolbox lies in exhausting the potential of unexplored habitats. By screening a Vietnamese fungal culture collection of 295 undiscovered fungal isolates, 12 highly active xylan degraders were identified. One of the best xylanase producing strains proved to be an Aspergillus sydowii strain from shrimp shell (Fsh102), showing a specific activity of 0.6 U/mg. Illumina dye sequencing was used to identify our Fsh102 strain and determine differences to the A. sydowii CBS 593.65 reference strain. With activity based in-gel zymography and subsequent mass spectrometric identification, three potential proteins responsible for xylan degradation were identified. Two of these proteins were cloned from the cDNA and, furthermore, expressed heterologously in Escherichia coli and characterized. Both xylanases, were entirely different from each other, including glycoside hydrolases (GH) families, folds, substrate specificity, and inhibition patterns. The specific enzyme activity applying 0.1% birch xylan of both purified enzymes were determined with 181.1 ± 37.8 or 121.5 ± 10.9 U/mg for xylanase I and xylanase II, respectively. Xylanase I belongs to the GH11 family, while xylanase II is member of the GH10 family. Both enzymes showed typical endo-xylanase activity, the main products of xylanase I are xylobiose, xylotriose, and xylohexose, while xylobiose, xylotriose, and xylopentose are formed by xylanase II. Additionally, xylanase II showed remarkable activity toward xylotriose. Xylanase I is stable when stored up to 30°C and pH value of 9, while xylanase II started to lose significant activity stored at pH 9 after exceeding 3 days of storage. Xylanase II displayed about 40% activity when stored at 50°C for 24 h. The enzymes are tolerant toward mesophilic temperatures, while acting in a broad pH range. With site directed mutagenesis, the active site residues in both enzymes were confirmed. The presented activity and stability justify the classification of both xylanases as highly interesting for further development.

Does Light Color Temperature Influence Aspects of Oviposition by the Black Soldier Fly (Diptera: Stratiomyidae)?

In recent years, black soldier fly, Hermetia illucens (L.), larvae have attracted increasing attention because of their high capacity for bioconversion of diverse organic material into high-quality protein and lipids. Although previous studies have focused on optimization of breeding conditions, such as the acceptance of substrates, and temperatures and moisture contents, little is known about light-dependent adult development. Artificial light sources are important to commercial H. illucens breeding, especially at latitudes with short days in autumn and winter months. We examined how 3,000, 4,000, and 6,500 K color temperatures affect aspects of oviposition. Mating occurred under all of the broad spectrum light-emitting diode panels, resulting in fertilized egg clusters. Oviposition lasted up to 15 d, while the shortest oviposition period, in the 3,000 K light treatment, was 2 d. Total oviposition performance and oviposition period were not affected by the light treatments. Oviposition peaked 1-7 d after eggs were first deposited. The time until oviposition peaked was positively correlated with increasing color temperature.

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.