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Background Lignocellulosic biomass is considered as a promising alternative to fossil resources for the production of fuels, materials and chemicals. Efficient enzymatic systems are needed to degrade the plant cell wall and overcome its recalcitrance. A widely used producer of cellulolytic cocktails is the ascomycete Trichoderma reesei, but this organism secretes a limited set of enzymes. To improve the saccharification yields, one strategy is to upgrade the T. reesei enzyme cocktail with enzymes produced by other biomass-degrading filamentous fungi isolated from biodiversity. Results In this study, the enzymatic cocktails secreted by five strains from the genus Aspergillus (Aspergillus japonicus strains BRFM 405, 1487, 1489, 1490 and Aspergillus niger strain BRFM 430) were tested for their ability to boost a T. reesei reference cocktail for the saccharification of pretreated biomass. Proteomic analysis of fungal secretomes that significantly improved biomass degradation showed that the presence of proteins belonging to a putative LPMO family previously identified by genome analysis and awaiting experimental demonstration of activity. Members of this novel LPMO family, named AA16, are encountered in fungi and oomycetes with life styles oriented toward interactions with plant biomass. One AA16 protein from Aspergillus aculeatus (AaAA16) was produced to high level in Pichia pastoris. LPMO-type enzyme activity was demonstrated on cellulose with oxidative cleavage at the C1 position of the glucose unit. AaAA16 LPMO was found to significantly improve the activity of T. reesei CBHI on cellulosic substrates. Conclusions Although Aspergillus spp. has been investigated for decades for their CAZymes diversity, we identified members of a new fungal LPMO family using secretomics and functional assays. Properties of the founding member of the AA16 family characterized herein could be of interest for use in biorefineries.

Xyloglucan is widely believed to function as a tether between cellulose microfibrils in the primary cell wall, limiting cell enlargement by restricting the ability of microfibrils to separate laterally. To test the biomechanical predictions of this \"tethered network\" model, we assessed the ability of cucumber (Cucumis sativus) hypocotyl walls to undergo creep (long-term, irreversible extension) in response to three family-12 endo-β-1, 4-glucanases that can specifically hydrolyze xyloglucan, cellulose, or both. Xyloglucan-specific endoglucanase (XEG from Aspergillus aculeatus) failed to induce cell wall creep, whereas an endoglucanase that hydrolyzes both xyloglucan and cellulose (Cell2A from Hypocrea jecorina) induced a high creep rate. A cellulose-specific endoglucanase (CEG from Aspergillus niger) did not cause cell wall creep, either by itself or in combination with XEG. Tests with additional enzymes, including a family-5 endoglucanase, confirmed the conclusion that to cause creep, endoglucanases must cut both xyloglucan and cellulose. Similar results were obtained with measurements of elastic and plastic compliance. Both XEG and Cell2A hydrolyzed xyloglucan in intact walls, but Cell2A could hydrolyze a minor xyloglucan compartment recalcitrant to XEG digestion. Xyloglucan involvement in these enzyme responses was confirmed by experiments with Arabidopsis (Arabidopsis thaliana) hypocotyls, where Cell2A induced creep in wild-type but not in xyloglucan-deficient (xxt1/xxt2) walls. Our results are incompatible with the common depiction of xyloglucan as a load-bearing tether spanning the 20-to 40-nm spacing between cellulose microfibrils, but they do implicate a minor xyloglucan component in wall mechanics. The structurally important xyloglucan may be located in limited regions of tight contact between microfibrils.

Rapidly increasing industry has resulted in greater discharge of hazardous chemicals in the soil. In the current study, soil samples were collected from Nanjing mine (32°09′19.29″ N 118°56′57.04″ E) and subjected to heavy metal analysis and microbe isolation. A total of 460 fungi were isolated, and five of these were yeast strains. Most of the strains exhibited tolerance to one metal. Five multimetal tolerant strains were selected and identified as Aspergillus sclerotiorum, Aspergillus aculeatus, Komagataella phaffii, Trichoderma harzianum, and Aspergillus niger. Isolated strains were grown in high concentrations of cadmium (Cd), chromium (Cr) and lead (Pb), for induced-tolerance training. The tolerance index (TI) revealed the highest Cd tolerance of novel K. phaffii strain at 5500 ppm (TI: 0.2). K. phaffii also displayed resistance at 4000 ppm against Cr (TI: 0.32) and Pb (TI: 0.32). In contrast, tolerance training for A. niger was not that successful. K. phaffii also displayed the highest bioaccumulation capacity for Cd (25.23 mg/g), Cu (21.63 mg/g), and Pb (20.63 mg/g) at 200 ppm. Scanning electron microscopy (SEM) explored the morphological changes in the mycelia of stressed fungi. Results of this study describe this delicate approach to be species and metal dependent and suggest a potential utilization of this fungal strain for the bioremediation of contaminated soils.

The number of fully sequenced fungal genomes is rapidly increasing. Since genetic tools are poorly developed for most filamentous fungi, it is currently difficult to employ genetic engineering for understanding the biology of these fungi and to fully exploit them industrially. For that reason there is a demand for developing versatile methods that can be used to genetically manipulate non-model filamentous fungi. To facilitate this, we have developed a CRISPR-Cas9 based system adapted for use in filamentous fungi. The system is simple and versatile, as RNA guided mutagenesis can be achieved by transforming a target fungus with a single plasmid. The system currently contains four CRISPR-Cas9 vectors, which are equipped with commonly used fungal markers allowing for selection in a broad range of fungi. Moreover, we have developed a script that allows identification of protospacers that target gene homologs in multiple species to facilitate introduction of common mutations in different filamentous fungi. With these tools we have performed RNA-guided mutagenesis in six species of which one has not previously been genetically engineered. Moreover, for a wild-type Aspergillus aculeatus strain, we have used our CRISPR Cas9 system to generate a strain that contains an AACU_pyrG marker and demonstrated that the resulting strain can be used for iterative gene targeting.

Cashew is a key economic crop in coastal Kenya, yet its production faces significant challenges due to post-harvest fungal contamination, particularly by Aspergillus flavus and Aspergillus aculeatus . These fungi are known producers of mycotoxins, including aflatoxins, potent carcinogenic and mutagenic compounds that pose serious food safety and public health risks. Despite growing concerns, the genomic architecture, metabolic potential, and ecological adaptations of these fungi in cashew-growing regions remain poorly understood. This study investigated the genetic diversity, aflatoxin biosynthetic gene clusters (BGCs), secondary metabolite profiles, and carbohydrate-active enzyme (CAZyme) repertoires of aflatoxigenic A. flavus and compared them with non-aflatoxigenic A. aculeatus isolates obtained from cashew nuts collected in Kilifi, Kwale, and Lamu counties. A total of 18 fungal isolates (16 A. flavus , 2 A. aculeatus ) were cultured and subjected to whole-genome sequencing using the Illumina platform. Genome mining and comparative analyses were performed using OrthoFinder, antiSMASH, and dbCAN. Phylogenomic analysis revealed five distinct clades among A. flavus isolates, suggesting substantial intraspecific diversity and potential regional adaptation, while A. aculeatus formed a single monophyletic clade. Comparative analysis of aflatoxin BGCs in A. flavus revealed notable structural variation, including gene deletions ( aflT , omtA ), insertions, and rearrangements, with conservation observed for core genes such as ordB , moxY , avfA , and adhA ; however, the regulatory gene aflR was not conserved across all isolates. All the 2 A. aculeatus isolates lacked aflatoxin biosynthetic genes, indicating they are not aflatoxigenic. Both species harbored diverse SMBGCs, including PKS, NRPS, and strain-specific clusters such as YWA1, fusarin, and aspergillic acid, suggesting potential for co-production of multiple bioactive compounds. CAZyme profiling revealed abundant glycoside hydrolases and auxiliary activity enzymes, underscoring adaptation to pectin-rich cashew substrates. Despite similar genome sizes and GC content, species-specific differences in carbohydrate metabolism and secondary metabolite pathways indicate ecological plasticity. Our findings demonstrate that A. flavus and A. aculeatus populations in coastal Kenya are genetically diverse, metabolically versatile, and shaped by local environmental and anthropogenic pressures. The study provides essential genomic insights for region-specific aflatoxin risk assessment and highlights the need for careful screening of atoxigenic strains in biocontrol applications, as well as further exploration of cryptic BGCs and CAZyme functions in post-harvest fungal ecology.

Bunch rot in grapes is an aggressive disease and needs to be controlled during the postharvest period. We investigate the antifungal potential of Zanthoxylum bungeanum Maxim., Zanthoxylum rhetsa , Cuminum cyminum , Coriandrum sativum , and Zingiber montanum (J. Koenig) Link ex A. Dietr. essential oils against Aspergillus aculeatus that cause bunch rot disease on postharvest grapes. C . cyminum essential oil exhibited stronger significantly inhibition percentage of 95.08% than other treatments in in vitro assay. Cumin aldehyde (33.94%) and α-terpinen-7-al (32.20%) were identified as major volatile compounds in C . cyminum oil. Antifungal potential of C . cyminum oil was then tested in conidia germination and in vitro tests compared to cumin aldehyde and α-terpinen-7-al. Their EC 50 values against the conidial germination were also estimated. Significant reduction of conidia germination was also detected in C . cyminum essential oil and cumin aldehyde at a concentration of 1,000 and 100 μg/mL, respectively. EC 50 values of the C . cyminum essential oil, cumin aldehyde, and α-terpinen-7-al were 67.28 μg/mL, 9.31 μg/mL, and 13.23 μg/mL, respectively. In vivo assay, the decrease of the disease severity (0.69%) and incidence (1.48%) percentage of A . aculeatus on grape berries treated at 1,000 μg/mL of C . cyminum essential oil was significantly greater than that obtained from other treatments after 10 days incubation. In addition, grape berries treated with C . cyminum essential oil decreased weight loss and retained fruit firmness. The changing of total soluble solids, total phenolic content, and antioxidant activity are also delayed in treated fruits. Therefore, essential oil of C . cyminum may be applied as a biological antifungal agent to control A . aculeatus in postharvest grapes without any negative effects on its quality.

The expression of functional proteins on the cell surface using glycosylphosphatidylinositol (GPI)-anchoring technology is a promising approach for constructing yeast cells with special functions. The functionality of surface-engineered yeast strains strongly depends on the amount of functional proteins displayed on their cell surface. On the other hand, since the yeast cell wall space is finite, heterologous protein carrying capacity of the cell wall is limited. Here, we report the effect of CCW12 and CCW14 knockout, which encode major nonenzymatic GPI-anchored cell wall proteins (GPI-CWPs) involved in the cell wall organization, on the heterologous protein carrying capacity of yeast cell wall. Aspergillus aculeatus β-glucosidase (BGL) was used as a reporter to evaluate the protein carrying capacity in Saccharomyces cerevisiae. No significant difference in the amount of cell wall–associated BGL and cell-surface BGL activity was observed between CCW12 and CCW14 knockout strains and their control strain. In contrast, in the CCW12 and CCW14 co-knockout strains, the amount of cell wall–associated BGL and its activity were approximately 1.4-fold higher than those of the control strain and CCW12 or CCW14 knockout strains. Electron microscopic observation revealed that the total cell wall thickness of the CCW12 and CCW14 co-knockout strains was increased compared to the parental strain, suggesting a potential increase in heterologous protein carrying capacity of the cell wall. These results indicate that the CCW12 and CCW14 co-knockout strains are a promising host for the construction of highly functional recombinant yeast strains using cell-surface display technology.Key points• CCW12 and/or CCW14 of a BGL-displaying S. cerevisiae strain were knocked out.• CCW12 and CCW14 co-disruption improved the display efficiency of BGL.• The thickness of the yeast cell wall was increased upon CCW12 and CCW14 knockout.

Chemical epigenetic regulation (CER) is an effective method to activate the silent pathway of fungal secondary metabolite synthesis. However, conventional methods for CER study are laborious and time-consuming. In the meantime, the overall profile of the secondary metabolites in the fungi treated by the CER reagent is not well characterized. In this study, suberohydroxamic acid (SBHA), a histone deacetylase inhibitor, was added to a culture of Aspergillus aculeatus DL1011 and a new strategy based on LC-MS/MS analysis integrated with various metabolomic tools (MetaboAnalyst, MS-DIAL, SIRIUS and GNPS) was developed to characterize the profile of induced metabolites. As a result, 13.6%, 29.5% and 27.2% of metabolites were identified as newly biosynthesized, increasing and decreasing in abundance by CER, respectively. The structures of the 18 newly induced secondary metabolites were further identified by the new strategy to demonstrate that 72.2% of them (1 novel compound and 12 known compounds) were first discovered in A. aculeatus upon SBHA treatment. The accuracy of the new approach was confirmed by purification and NMR data analysis of major newly biosynthesized secondary metabolites. The bioassay showed that the newly biosynthesized compounds, roseopurpurin analogues, showed selective activities against DPPH scavenging, cytotoxicity and SHP1 inhibition. Our research demonstrated that CER was beneficial for changing the secondary metabolic profile of fungi and was an effective means of increasing the diversity of active metabolites. Our work also supplied a metabolomic strategy to characterize the profile changes and determine the newly induced compounds in the secondary metabolites of fungi treated with the chemical epigenetic regulator.

In a previous study, raw cashew kernels were assayed for the fungal contamination focusing on strains belonging to the genus Aspergillus and on aflatoxins producers. These samples showed high contamination with Aspergillus section Nigri species and absence of aflatoxins. To investigate the diversity of secondary metabolites, including mycotoxins, the species of A. section Nigri may produce and thus threaten to contaminate the raw cashew kernels, 150 strains were isolated from cashew samples and assayed for their production of secondary metabolites using liquid chromatography high resolution mass spectrometry (LC-HRMS). Seven species of black Aspergilli were isolated based on morphological and chemical identification: A. tubingensis (44%), A. niger (32%), A. brasiliensis (10%), A. carbonarius (8.7%), A. luchuensis (2.7%), A. aculeatus (2%) and A. aculeatinus (0.7%). From these, 45 metabolites and their isomers were identified. Aurasperone and pyranonigrin A, produced by all species excluding A. aculeatus and A. aculeatinus, were most prevalent and were encountered in 146 (97.3%) and 145 (95.7%) isolates, respectively. Three mycotoxins groups were detected: fumonisins (B2 and B4) (2.7%) ochratoxin A (13.3%), and secalonic acids (2%), indicating that these mycotoxins could occur in raw cashew nuts. Thirty strains of black Aspergilli were randomly sampled for verification of species identity based on sequences of β-tubulin and calmodulin genes. Among them, 27 isolates were positive to the primers used and 11 were identified as A. niger, 7 as A. tubingensis, 6 as A. carbonarius, 2 as A. luchuensis and 1 as A. welwitschiae confirming the species names as based on morphology and chemical features. These strains clustered in 5 clades in A. section Nigri. Chemical profile clustering also showed also 5 groups confirming the species specific metabolites production.

In order to figure out the induction mechanisms of glycoside hydrolase genes in Aspergillus aculeatus, we screened approximately 9,000 transfer DNA (T-DNA)-inserted mutants for positive regulators involved in the induction. Since the mutants possess the orotidine 5′-monophosphate decarboxylase gene as a reporter gene to monitor the cellulose-responsive expression of the cellobiohydrolase I gene (cbhI), candidate strains were isolated by counterselection against 5-fluoroorotic acid (5-FOA). One 5-FOA-resistant mutant harboring the T-DNA at the uge5 locus showed reduced cellulose utilization and cbhI expression. A. aculeatus Uge5 is homologous to Aspergillus fumigatus uge5 (Afu5g10780; E-value, 0.0; identities, 93%), which catalyzes the conversion of uridine diphosphate (UDP)-glucose to UDP-galactopyranose. The uge5 deletion mutant in A. aculeatus (Δuge5) showed reduced conidium formation on minimal media supplemented with galactose, locust bean gum (LBG), and guar gum as a carbon source. β-1,4-Endoglucanase and β-1,4-mannanase production in submerged culture containing LBG was reduced to 10% and 6% of the control strain at day 5, respectively, but no difference was observed in cultures containing wheat bran. The expression of major cellulolytic and mannolytic genes in the presence of mannobiose in Δuge5 was reduced to less than 15% of the control strain, while cellobiose-responsive expression was only modestly reduced at early inducing time points. Since all test genes were controlled by a transcription factor ManR, these data demonstrate that Uge5 is involved in inducer-dependent selective expression of genes controlled via ManR.Key points• UDP-glucose 4-epimerase (Uge5) regulates expression of glycosyl hydrolase genes.• ManR regulates both cellobiose- and mannobiose-responsive expression.• Uge5 plays a key role in mannobiose-responsive expression.