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D-xylose is the second most abundant monosaccharide found in lignocellulose and is of biotechnological importance for producing second-generation ethanol and other high-value chemical compounds. D-xylose conversion to ethanol is promoted by microbial fermentation, mainly by bacteria, yeasts, or filamentous fungi. Among yeasts, species belonging to the CTG(Ser1) or CTG(Ala) clade display a remarkable ability to ferment D-xylose to ethanol and other compounds; however, these yeasts are not employed on an industrial scale given their poor fermentative performance compared to that of conventional yeasts, such as Saccharomyces cerevisiae, and because of the lack of a molecular toolbox for the development of new strains tailored to fermentation stress tolerance and performance. Thus, the purpose of this review was to evaluate the major genetic engineering tools (e.g., transformation markers and techniques, vectors, regulatory sequences, and gene editing techniques) available for the most studied yeasts of the CTG(Ser1) clade, such as Scheffersomyces , Spathaspora , Candida , and Yamadazyma species, and the CTG(Ala) clade, representative Pachysolen tannophilus. Furthermore, we systematized state-of-the-art molecular developments and perspectives to design D-xylose-fermenting yeast strains. • Review of genetic tools in CTG(Ser1) and CTG(Ala) yeasts for bioethanol production. • CTG codon reassignment impairs the use of conventional genetic engineering tools. • CTG(Ser1) yeasts can use genetic tools from clinically important Candida strains for research. [ABSTRACT FROM AUTHOR]

The production of cellulolytic enzymes in response to inducible carbon sources is mainly regulated at the transcriptional level in filamentous fungi. We have identified a cellobiose-response regulator (ClbR) controlling the expression of cellulolytic enzyme-encoding genes in Aspergillus aculeatus. However, the engineering potential of combining the deletion of transcriptional repressors with the overexpression of transcriptional activators to enhance enzyme production has not been analyzed. Here, we investigated the effect of the deletion of the transcriptional repressor creA and the overexpression of the transcriptional activator clbR in enzyme production in A. aculeatus. Here, we verified that a combination of creA deletion and clbR overexpression (Δc&OE) improved cellulase, β-1,4-xylanase, and β-glucosidase production. Cellulase and β-1,4-xylanase production increased 3.4- and 8.0-fold in Δc&OE compared with the host strain (MR12) at 96-h incubation, respectively. β-Glucosidase production in Δ creA and Δc&OE increased approximately 5.0-fold compared with that in MR12 at 240-h incubation. Transcriptional analysis revealed that the increase in enzyme production was due to increased expression of cellobiohydrolase, endo-β-1,4-glucanase, β-1,4-xylanase, and β-glucosidase 1 (bgl1). Interestingly, bgl1 expression in Δ creA increased in a dose-dependent manner in response to glucose. Thus, combinational manipulation of transcription factors improved cellulase, xylanase, and β-glucosidase production in A. aculeatus. [ABSTRACT FROM AUTHOR]

Mutations have underpinned research into gene characterization across all domains of life. This includes the discovery of the genes involved in the development of asexual spores in filamentous fungi. Mutants in the ascomycete Paecilomyces variotii were isolated with impaired biosynthesis of the characteristic yellow pigment produced by this filamentous fungus. The affected genes were identified as pvpP , encoding the polyketide synthase that is required for synthesis of the pigment YWA1, and abaA and wetA that are two genes that encode components of the AbaA-BrlA-WetA module required for the development of asexual spores in species in the Eurotiales order. WetA was further characterized. A strain expressing a functional WetA-GFP fusion was created and used to find that WetA is expressed primarily in spores and concentrated in their nuclei, providing evidence that this conserved protein likely functions as a regulator of transcription in conidia. Analysis of the phenotypes of the P. variotii wetA mutant suggests that how this three-protein module impacts fungal biology will vary from species-to-species, despite being conserved amongst filamentous Ascomycete species. • Insertional mutants of Paecilomyces variotii without pigments were isolated. • Genes impaired include two in the core abaA-brlA-wetA module for conidiation. • The WetA protein is predominantly produced in conidia and localized to nuclei. [ABSTRACT FROM AUTHOR]

Filamentous fungi are well-known for their efficiency in producing valuable molecules of industrial significance, but applications of fungal biomass remain relatively less explored despite its abundant and diverse opportunities in biotechnology. One promising application of mycelial biomass is as a platform to immobilize different cell types such as animal, plant, and microbial cells. Filamentous fungal biomass with little to no treatment is a sustainable biomaterial which can also be food safe compared to other immobilization supports which may otherwise be synthetic or heavily processed. Because of these features, the fungal-cell combination can be tailored towards the targeted application and be applied in a variety of fields from bioremediation to biomedicine. Optimization efforts to improve cell loading on the mycelium has led to advancements both in the applied and basic sciences to understand the inter- and intra-kingdom interactions. This comprehensive review compiles for the first time the current state of the art of the immobilization of animal, yeast, microalgae, bacteria, and plant cells in filamentous fungal supports and presents outlook of applications in intensified fermentations, food and biofuel production, and wastewater treatment. Opportunities for further research and development were identified to include elucidation of the physical, chemical, and biological bases of the immobilization mechanisms and co-culture dynamics; expansion of the cell-fungus combinations investigated; exploration of previously unconsidered applications; and demonstration of scaled-up operations. It is concluded that the potential exists to leverage the unique qualities of filamentous fungus as a cellular support in the creation of novel materials and products in support of the circular bioeconomy. [ABSTRACT FROM AUTHOR]

Fungal biomass is as a cost-effective and sustainable biosorbent utilized in both active and inactive forms. This study investigated the efficacy of inactivated and dried biomass of Fusarium sp. in adsorbing Ni2+ and Pb2+ from aqueous solutions. The strain underwent sequential cultivation and was recovered by filtration. Then, the biomass was dried in an oven at 80 ± 2 °C and sieved using a 0.1-cm mesh. The biosorbent was thoroughly characterized, including BET surface area analysis, morphology examination (SEM), chemical composition (XRF and FT-IR), thermal behavior (TGA), and surface charge determination (pH-PZC and zeta potential). The biosorption mechanism was elucidated by fitting equilibrium models of kinetics, isotherm, and thermodynamic to the data. The biosorbent exhibited a neutral charge, a rough surface, a relatively modest surface area, appropriate functional groups for adsorption, and thermal stability above 200 °C. Optimal biosorption was achieved at 25 ± 2 °C, using 0.05 g of adsorbent per 50 mL of metallic ion solution at initial concentrations ranging from 0.5 to 2.0 mg L−1 and at pH 4.5 for Pb2+ and Ni2+. Biosorption equilibrium was achieved after 240 min for Ni2+ and 1440 min for Pb2+. The process was spontaneous, mainly through chemisorption, in monolayer for Ni2+ and multilayer for Pb2+, with efficiencies of over 85% for both metallic ion removal. These findings underscore the potential of inactive and dry Fusarium sp. biomass (IDFB) as a promising material for the biosorption of Ni2+ and Pb2+. [ABSTRACT FROM AUTHOR]

Indoleamine 2,3‐dioxygenase (IDO) is a monomeric heme enzyme that catalyzes the oxidative cleavage of tryptophan (L‐Trp) to form N‐formyl‐kynurenine. Similar to other heme proteins, IDO only binds to O2 when the heme iron is ferrous (FeII), thereby rendering the enzyme active. Thus, ascorbate (Asc, a reducing agent) and methylene blue (MB, an electron carrier) are commonly added to in vitro IDO assay systems. However, Asc and MB have been recently reported to significantly impact the measurement of the enzymatic parameters of vertebrate IDO. Aspergillus fumigatus is a filamentous fungus and the most common cause of invasive aspergillosis; it has three IDO genes (IDOα, IDOβ, and IDOγ). The FeII–O2 IDOs of A. fumigatus, particularly FeII–O2 IDOγ, have relatively long half‐lives in their autoxidation; however, the autoxidation was accelerated by Asc. Similar to vertebrate IDOs, Asc acted as a competitive (or mixed‐competitive) inhibitor of the IDOs of A. fumigatus. A positive correlation (in the order of IDOγ > IDOβ > IDOα) was observed between the inhibitory sensitivity of the IDOs to Asc and the facilitation of their autoxidation by Asc. The FeII–O2 IDO can repeat the dioxygenase reaction as long as it reacts with L‐Trp; however, substrate‐free FeII–O2 IDO is converted into inactive FeIII–IDO by autoxidation. Thus, L‐Trp (which keeps the IDO active) competes with Asc (which inactivates IDO by accelerating autoxidation). This is probably why Asc, which is structurally quite different from L‐Trp, appears to function as a competitive (or mixed‐competitive) inhibitor of IDOs. [ABSTRACT FROM AUTHOR]

Figs (Ficus carica L.) are widely cultivated throughout the world owing to their excellent medical and edible value. In October 2021, we discovered a new leaf target-spot disease associated with the common fig in China. It is necessary to identify the causal agent, as it seriously affects the normal growth of figs and reduces their economic value. The initially infected leaves displayed light yellow lesions between the leaf veins. Under conditions of suitable temperature and humidity, the lesions expanded rapidly with light brown in the middle and dark brown at the margin accompanied by a yellow halo, and typical target holes appeared in the centre of the lesions. Three filamentous fungi were isolated from infected leaves, among which the isolate WHG-3 exerted the strongest virulence in vitro. Its newly grown mycelia were white and then gradually turned to dark grey. The conidia were hyaline, spindle-shaped or oval, with thin cell walls. Phylogenetic analyses based on the ITS, tef-1 and tub2 gene sequences verified that the isolates WHG-3, WHG-4 and WHG-5 were Neofusicoccum parvum. To the best of our knowledge, this is the first report that N. parvum causes leaf target-spot on Ficus carica L. in China. [ABSTRACT FROM AUTHOR]

While assessing the filamentous fungi associated with woody tissues of stem collar rots, necroses, and lesions of European ash trees (Fraxinus excelsior) presenting symptoms of ash dieback in Germany, Cryptostroma corticale was recovered from three different ash trees. These isolated strains were the first report of C. corticale on ash and the first proof of an association of this plant pathogen with woody tissues of other tree species than Acer spp. in Germany. To test the pathogenicity of C. corticale against F. excelsior and to fulfil Koch's postulates, inoculation tests in planta with strains isolated from Acer pseudoplatanus and F. excelsior were conducted according to Henle–Koch's postulates in a greenhouse located in Göttingen. The pathogenicity tests were performed with apparently healthy ash saplings from June 2021 until January 2022. After three and seven months, neither necroses or lesions due to C. corticale nor disease symptoms were observed. Mostly, the inoculation wounds healed over, and C. corticale could not be re-isolated from the ash woody tissue. In an attempt to re-isolate the inoculated strains, the filamentous fungal endophytes of the ash woody tissues were isolated and identified. A total of 32 taxa of the Ascomycota were found, where the most common species was Boeremia exigua. Most frequently observed orders were Pleosporales (58.4%), followed by Sordariales (13.5%), Hypocreales (9.4%), and Diaporthales (8.7%). On average, 3.7 endophytic species were recorded on each sapling. [ABSTRACT FROM AUTHOR]

Background New antifungal agents are required to mitigate against azole-resistant Aspergillus and drug-resistant non- Aspergillus moulds. The novel orotomide, olorofim (F2G, Manchester, UK), has potent fungicidal activity against Aspergillus including azole-resistant Aspergillus fumigatus, Lomentospora prolificans and Scedosporium spp. Development of olorofim-specific clinical breakpoints/epidemiological cut-off values requires reliable MIC data. Objectives Determine the in vitro activity of olorofim compared with standard antifungals against mould pathogens at an Australian hospital. Materials and methods Olorofim MICs were determined for 507 clinical mould isolates using the CLSI M38-A3 standard. MICs of amphotericin B, anidulafungin, posaconazole, voriconazole and isavuconazole were obtained using Sensititre™ YeastOne YO10 and AUSNMRCI panels (Thermo-Fisher Scientific). Results A. fumigatus sensu stricto was the commonest species (33.3%) followed by L. prolificans (18.3%), Scedosporium (11.4%) and Fusarium (6%) species. Olorofim modal MICs were ≤0.25 mg/L (MIC90 0.25 mg/L) for all Aspergillus except Aspergillus Section Usti (1 mg/L); MICs for nine azole-resistant/non-wild-type A. fumigatus ranged from 0.008 to 0.125 mg/L. The MIC90 of olorofim for L. prolificans was 0.5 mg/L, 0.25–0.5 mg/L for Scedosporium spp. and 8 mg/L for the F. solani complex but with modal MICs of 0.25 and 0.008 mg/L for F. oxysporum and F. proliferatum complexes, respectively. For Verruconis gallopava (n  = 10), the olorofim MIC90 was 0.06 mg/L (voriconazole MIC90 2 mg/L, isavuconazole MICs of 4–>8 mg/L). Olorofim had little activity against other dematiaceous moulds including Exophiala species. Conclusions Olorofim was highly active against Aspergillus spp. including azole-resistant A. fumigatus , L. prolificans , Scedosporium spp. and some Fusarium species with the new finding of potent activity against V. gallopava. [ABSTRACT FROM AUTHOR]

Xanthohumol (1) is a major prenylated flavonoid in hops (Humulus lupulus L.) which exhibits a broad spectrum of health-promoting and therapeutic activities, including anti-inflammatory, antioxidant, antimicrobial, and anticancer effects. However, due to its lipophilic nature, it is poorly soluble in water and barely absorbed from the gastrointestinal tract, which greatly limits its therapeutic potential. One method of increasing the solubility of active compounds is their conjugation to polar molecules, such as sugars. Sugar moiety introduced into the flavonoid molecule significantly increases polarity, which results in better water solubility and often leads to greater bioavailability. Entomopathogenic fungi are well known for their ability to catalyze O-glycosylation reactions. Therefore, we investigated the ability of selected entomopathogenic filamentous fungi to biotransform xanthohumol (1). As a result of the experiments, one aglycone (2) and five glycosides (3–7) were obtained. The obtained (2″E)-4″-hydroxyxanthohumol 4′-O-β-D-(4‴-O-methyl)-glucopyranoside (5) has never been described in the literature so far. Interestingly, in addition to the expected glycosylation reactions, the tested fungi also catalyzed chalcone–flavanone cyclization reactions, which demonstrates chalcone isomerase-like activity, an enzyme typically found in plants. All these findings undoubtedly indicate that entomopathogenic filamentous fungi are still an underexploited pool of novel enzymes. [ABSTRACT FROM AUTHOR]

'Beimei' is a hybrid cultivar of Vitis vinifera and wild Vitis amurensis with fungus- and cold-resistant characteristics that is widely grown in many regions of China. However, studies on the yeast diversity and aroma compound composition of this cultivar remain scarce. Here, indigenous yeast diversity, aroma compound composition, and their dynamic changes during the spontaneous fermentation of 'Beimei' were investigated and monitored using high-throughput sequencing and gas chromatography-mass spectrometry. The result indicated that non-Saccharomyces yeasts were predominant at the initial stages of 'Beimei' spontaneous fermentation and then sharply decreased. Cladosporium herbarum and Hanseniaspora uvarum were identified as the dominant species of filamentous fungi and yeast, respectively, at the beginning and on the first day of fermentation, whereas unclassified_f_Saccharomycetacease was dominant throughout fermentation, starting from the third day until the end of fermentation. The main volatile aroma chemicals were esters and alcohols, which accumulated abundantly at the late stage of fermentation. Taken together, this study represents the first step in exploring untapped yeast species and aroma compounds, which is helpful for the screening of native yeasts from 'Beimei' and producing distinctive aroma wines. [ABSTRACT FROM AUTHOR]

Microbial cultures repurposing organic industrial residues for value-added metabolite production is pivotal for sustainable resource use. Highlighting polyunsaturated fatty acids (PUFAs), particularly gamma-linolenic acid (GLA), renowned for their nutritional and therapeutic value. Notably, Zygomycetes' filamentous fungi harbor abundant GLA-rich lipid content, furthering their relevance in this approach. In this study, the strain C. elegans NRRL Y-1392 was evaluated for its capability to metabolize glycerol and produce lipids rich in GLA under different culture conditions. Various carbon-to-nitrogen ratios (C/N = 11.0, 110.0, and 220.0 mol/mol) were tested in batch-flask cultivations. The highest GLA production of 224.0 mg/L (productivity equal to 2.0 mg/L/h) was observed under nitrogen excess conditions, while low nitrogen content promoted lipid accumulation (0.59 g of lipids per g of dry biomass) without yielding more PUFAs and GLA. After improving the C/N ratio at 18.3 mol/mol, even higher PUFA (600 mg/L) and GLA (243 mg/L) production values were recorded. GLA content increased when the fungus was cultivated at 12 °C (15.5% w/w compared to 12.8% w/w at 28 °C), but productivity values decreased significantly due to prolonged cultivation duration. An attempt to improve productivity by increasing the initial spore population did not yield the expected results. The successful scale-up of fungal cultivations is evidenced by achieving consistent results (compared to flask experiments under corresponding conditions) in both laboratory-scale (Working Volume—Vw = 1.8 L; C/N = 18.3 mol/mol) and semi-pilot-scale (Vw = 15.0 L; C/N = 110.0 mol/mol) bioreactor experiments. To the best of our knowledge, cultivation of the fungus Cunninghamella elegans in glycerol-based substrates, especially in 20 L bioreactor experiments, has never been previously reported in the international literature. The successful scale-up of the process in a semi-pilot-scale bioreactor illustrates the potential for industrializing the bioprocess. [ABSTRACT FROM AUTHOR]

Information on the detection of the presence and potential for degradation of synthetic polymers (SPs) under various environmental conditions is of increasing interest and concern to a wide range of specialists. At this stage, there is a need to understand the relationship between the main participants in the processes of (bio)degradation of SPs in various ecosystems (reservoirs with fresh and sea water, soils, etc.), namely the polymers themselves, the cells of microorganisms (MOs) participating in their degradation, and humic substances (HSs). HSs constitute a macrocomponent of natural non-living organic matter of aquatic and soil ecosystems, formed and transformed in the processes of mineralization of bio-organic substances in environmental conditions. Analysis of the main mechanisms of their influence on each other and the effects produced that accelerate or inhibit polymer degradation can create the basis for scientifically based approaches to the most effective solution to the problem of degradation of SPs, including in the form of microplastics. This review is aimed at comparing various aspects of interactions of SPs, MOs, and HSs in laboratory experiments (in vitro) and environmental investigations (in situ) aimed at the biodegradation of polymers, as well as pollutants (antibiotics and pesticides) that they absorb. Comparative calculations of the degradation velocity of different SPs in different environments are presented. A special place in the analysis is given to the elemental chemical composition of HSs, which are most successfully involved in the biodegradation of SPs. In addition, the role of photo-oxidation and photoaging of polymers under the influence of the ultraviolet spectrum of solar radiation under environmental conditions on the (bio)degradation of SPs in the presence of HSs is discussed. [ABSTRACT FROM AUTHOR]

Remediation of hydrocarbon contaminations requires much attention nowadays since it causes detrimental effects on land and even worse impacts on aquatic environments. Tools of bioremediation especially filamentous fungi permissible for cleaning up as much as conceivable, at least they turn into non-toxic residues with less consumed periods. Inorganic chemicals, CO2, H2O, and cell biomass are produced as a result of the breakdown and mineralization of petroleum hydrocarbon pollutants. This paper presents a detailed overview of three strategic rules of filamentous fungi in remediating the various aliphatic, and aromatic hydrocarbon compounds: utilizing carbons from hydrocarbons as sole energy, Co-metabolism manners (Enzymatic and Non-enzymatic theories), and Biosorption approaches. Upliftment in the degradation rate of complex hydrocarbon by the Filamentous Fungi in consortia scenario we can say, "Fungal Talk", which includes a variety of cellular mechanisms, including biosurfactant production, biomineralization, and precipitation, etc., This review not only displays its efficiency but showcases the field applications – cost-effective, reliable, eco-friendly, easy to culture as biomass, applicable in both land and any water bodies in operational environment cleanups. Nevertheless, the potentiality of fungi-human interaction has not been fully understood, henceforth further studies are highly endorsed with spore pathogenicity of the fungal species capable of high remediation rate, and the gene knockout study, if the specific peptides cause toxicity to any living matters via Genomics and Proteomics approaches, before application of any in situ or ex situ environments. [ABSTRACT FROM AUTHOR]

Controlling the morphology of filamentous fungi is crucial to improve the performance of fungal bioprocesses. Microparticle‐enhanced cultivation (MPEC) increases productivity, most likely by changing the fungal morphology. However, due to a lack of appropriate methods, the exact impact of the added microparticles on the structural development of fungal pellets is mostly unexplored. In this study synchrotron radiation‐based microcomputed tomography and three‐dimensional (3D) image analysis were applied to unveil the detailed 3D incorporation of glass microparticles in nondestructed pellets of Aspergillus niger from MPEC. The developed method enabled the 3D analysis based on 375 pellets from various MPEC experiments. The total and locally resolved volume fractions of glass microparticles and hyphae were quantified for the first time. At increasing microparticle concentrations in the culture medium, pellets with lower hyphal fraction were obtained. However, the total volume of incorporated glass microparticles within the pellets did not necessarily increase. Furthermore, larger microparticles were less effective than smaller ones in reducing pellet density. However, the total volume of incorporated glass was larger for large microparticles. In addition, analysis of MPEC pellets from different times of cultivation indicated that spore agglomeration is decisive for the development of MPEC pellets. The developed 3D morphometric analysis method and the presented results will promote the general understanding and further development of MPEC for industrial application. [ABSTRACT FROM AUTHOR]

Genetic code expansion technology allows the incorporation of unnatural amino acids (UAAs) into proteins, which is useful in protein engineering, synthetic biology, and gene therapy. Despite its potential applications in various species, filamentous fungi remain unexplored. This study aims to address this gap by developing these techniques in Aspergillus nidulans. We introduced an amber stop codon into a specific sequence within the reporter gene expressed in A. nidulans and replaced the anticodon of the fungal tRNATyr with CUA. This resulted in the synthesis of the target protein, confirming the occurrence of amber suppression in the fungus. When exogenous E. coli tRNATyr CUA (Ec. tRNATyr CUA) and E. coli tyrosyl-tRNA (Ec.TyrRS) were introduced into A. nidulans , they successfully synthesized the target protein via amber suppression and were shown to be orthogonal to the fungal translation system. By replacing the wild-type Ec.TyrRS with a mutant with a higher affinity for the UAA O -methyl-L-tyrosine, the fungal system was able to initiate the synthesis of the UAA-labeled protein (UAA-protein). We further increased the expression level of the UAA-protein through several rational modifications. The successful development of a genetic code expansion technique for A. nidulans has introduced a potentially valuable approach to the study of fungal protein structure and function. • Amber suppression in A. nidulans requires the exogenous amber suppressor. • E. coli tRNATyr CUA /Tyr-tRNA synthetase incorporates unnatural amino acids in fungus. • Increasing tRNA transcription improves unnatural amino acid incorporation efficiency in fungus. [ABSTRACT FROM AUTHOR]

Ozone is a molecule that has gained increasing interest in recent years by food industries for sanitization of food-grade surfaces. Compared to chemical sanitizers such as chlorine, hydrogen peroxide, or peracetic acid, ozone shows undeniable advantages, such as the absence of by-products that should affect human health or the possibility of generating it when needed. Therefore, the aim of this paper was the assessment of the resistance to ozonized water of two pathogenic bacteria (Listeria monocytogenes, Salmonella) and of three airborne food-spoiling fungi (Aspergillus brasiliensis, Hyphopichia burtonii, and Penicillium nordicum) inoculated on stainless steel tiles and treated in static conditions with 1 to 6 mg L−1 (pathogens) or 8.5 mg L−1 (filamentous fungi). Ozonized water gave different results based on the tested microorganisms: pathogenic bacteria proved markedly more sensible to ozone than filamentous fungi, even if great differences were observed at inter- and intra-specific levels for both categories of microorganisms. Nevertheless, the non-linear inactivation kinetics of the studied strains made the calculation of a punctual F-value difficult, so in industrial practice, adequate tailoring of the treatments to be applied, based on the registered extrinsic factors and the industrial bio-burden, would be appropriate. [ABSTRACT FROM AUTHOR]

Considering an ever-growing global population, which hit 8 billion people in the fall of 2022, it is essential to find solutions to avoid croplands competition between human food and animal feed. Agricultural co-products such as soybean meals have become important components of the circular economy thanks to their use in animal feed. Their implementation was made possible by the addition of exogenous enzymes in the diet of monogastric animals, especially fungal carbohydrate-active enzymes (CAZymes). Here, we describe a time-course production and analysis of Aspergillus terreus secretomes for the identification of CAZymes able to enhance the digestibility of soybean meals. Functional assays revealed that the release of nutrients and the degradation of pectins in soybean meals can be tightly interconnected. Using a comparative proteomics approach, we identified several fungal pectin-degrading enzymes leading to increased assimilable nutrients in the soluble fraction of soybean meals. Our results reinforce the importance of deconstructing pectic polysaccharides in feedstuffs and contribute to sharpen our understanding of the fungal enzymatic interplays involved in pectin hydrolysis. [ABSTRACT FROM AUTHOR]

Acremonium and the recently separated acremonium-like genera, such as Sarocladium, are emerging causes of opportunistic disease in humans, mainly post-traumatic infections in immunocompetent hosts, but also invasive infections in immunocompromised patients, such as those undergoing transplantation. Acremonium egyptiacum has emerged as the major pathogenic Acremonium species in humans, implicated mainly in nail but also in disseminated and organ specific infections. In this first study of acremonium-like clinical isolates in Greece, 34 isolates were identified and typed by sequencing the internal transcribed spacer, and their antifungal susceptibility was determined by a modified CLSI standard M38 3rd Edition method for filamentous fungi. A. egyptiacum was the primary species (18 isolates) followed by Sarocladium kiliense (8), Acremonium charticola, Gliomastix polychroma, Proxiovicillium blochii, Sarocladium terricola, Sarocladium zeae, and Stanjemonium dichromosporum (all with one isolate). Two isolates, each with a novel ITS sequence, possibly represent undescribed species with an affinity to Emericellopsis. All three A. egyptiacum ITS barcode types described to date were identified, with 3 being the major type. Flutrimazole, lanoconazole, and luliconazole presented the lower minimum inhibitory concentration (MIC) values against A. egyptiacum, with a geometric mean (GM) MIC of 2.50, 1.92, and 1.57 μg/mL, respectively. Amphotericin B, itraconazole, posaconazole, voriconazole, terbinafine, amorolfine, and griseofulvin MICs were overall high (GM 12.79–29.49 μg/mL). An analysis of variance performed on absolute values showed that flutrimazole, lanoconazole, and luliconazole were equivalent and notably lower than those of all the other drugs tested against A. egyptiacum. Antifungal susceptibility of the three different A. egyptiacum genotypes was homogeneous. Overall, the high MICs recorded for all systemically administered drugs, and for some topical antifungals against the tested A. egyptiacum and other acremonium-like clinical isolates, justify the routine susceptibility testing of clinical isolates. [ABSTRACT FROM AUTHOR]

Colletotrichum lindemuthianum is the most frequent pathogenic fungus of the common bean Phaseolus vulgaris. This filamentous fungus employs a hemibiotrophic nutrition/infection strategy, which is characteristic of many Colletotrichum species. Due to host–pathogen coevolution, C. lindemuthianum includes pathotypes with a diversity of virulence against differential common bean varieties. In this study, we performed comparative genomic analyses on three pathotypes with different virulence levels and a non-pathogenic pathotype, isolated from different geographical areas in Mexico. Our results revealed large genomes with high transposable element contents that have undergone expansions, generating intraspecific diversity. All the pathotypes exhibited a similar number of clusters of orthologous genes (COGs) and Gene Ontology (GO) terms. TFomes contain families that are typical in fungal genomes; however, they show different contents between pathotypes, mainly in transcription factors with the fungal-specific TF and Zn2Cys6 domains. Peptidase families mainly contain abundant serine peptidases, metallopeptidases, and cysteine peptidases. In the secretomes, the number of genes differed between the pathotypes, with a high percentage of candidate effectors. Both the virulence gene and CAZyme gene content for each pathotype was abundant and diverse, and the latter was enriched in hemicellulolytic enzymes. We provide new insights into the nature of intraspecific diversity among C. lindemuthianum pathotypes and the origin of their ability to rapidly adapt to genetic changes in its host and environmental conditions. [ABSTRACT FROM AUTHOR]

Light is a key environmental factor affecting conidiation in filamentous fungi. The cryptochrome/photolyase CryA, a blue-light receptor, is involved in fungal development. In the present study, a homologous CryA (AoCryA) was identified from the widely occurring nematode-trapping (NT) fungus Arthrobotrys oligospora, and its roles in the mycelial growth and development of A. oligospora were characterized using gene knockout, phenotypic comparison, staining technique, and metabolome analysis. The inactivation of AocryA caused a substantial decrease in spore yields in dark conditions but did not affect spore yields in the wild-type (WT) and ∆AocryA mutant strains in light conditions. Corresponding to the decrease in spore production, the transcription of sporulation-related genes was also significantly downregulated in dark conditions. Contrarily, the ∆AocryA mutants showed a substantial increase in trap formation in dark conditions, while the trap production and nematode-trapping abilities of the WT and mutant strains significantly decreased in light conditions. In addition, lipid droplet accumulation increased in the ∆AocryA mutant in dark conditions, and the mutants showed an increased tolerance to sorbitol, while light contributed to the synthesis of carotenoids. Finally, AoCryA was found to affect secondary metabolic processes. These results reveal, for the first time, the function of a homologous cryptochrome in NT fungi. [ABSTRACT FROM AUTHOR]

Epiphytic yeasts represent an important source for the development of novel strategies aiming to combat food microbial contamination. The present study deals with the characterization of nine yeast strains belonging to Starmerella, Candida, Metschinikowia, Lachancea, Kodamaea and Pichia genera, isolated from the surface of plants from the Botanical Garden "Dimitrie Brandza" (Bucharest, Romania) for use as antimicrobial and probiotic agents. The tests involved the determination of the safe status, cell growth under stress conditions, and activity against pathogenic Candida and bacteria strains, respectively, as well as phytopathogenic filamentous fungi and lipolytic activity. None of the nine strains showed all the characteristics for virulence and pathogenicity, with the rare positive results being explained rather by their adaptability to the habitats of origin. The strains Lachancea thermotolerans CMGB-ST12 and Kodamaea ohmeri CMGB-ST19 grew at 37 °C; Metschnikowia reukaufii CMGB-ST21.2, M. reukaufii CMGB-ST.8.1 and M. reukaufii CMGB ST10 grew in the presence of 10% NaCl, while L. thermotolerans CMGB-ST12 and K. ohmeri CMGB-ST19 tolerated both acidic and alkaline pH values well (3.0 to 12.0). The studied yeast strains showed good antimicrobial activity against Candida krusei, Candida albicans and Gram-negative bacterial strains, with K. ohmeri CMGB-ST19 and Pichia membranaefaciens CMGB-ST53 inhibiting up to 100% the development of filamentous fungi. All the strains produced lipases for tributyrin hydrolysis, the best producer being Starmerella bombi CMGB-ST1, and only Candida magnoliae CMGB-ST8.2 tested positive against other probiotic yeasts. Overall, our nine yeast strains show high potential for industrial applications, for obtaining probiotic products and for preventing the development of a wide range of microbial food contaminants. [ABSTRACT FROM AUTHOR]

Episomal AMA1‐based plasmids are increasingly used for expressing biosynthetic pathways and CRISPR/Cas systems in filamentous fungi cell factories due to their high transformation efficiency and multicopy nature. However, the gene expression from AMA1 plasmids has been observed to be highly heterogeneous in growing mycelia. To overcome this limitation, here we developed next‐generation AMA1‐based plasmids that ensure homogeneous and strong expression. We achieved this by evaluating various degradation tags fused to the auxotrophic marker gene on the AMA1 plasmid, which introduces a more stringent selection pressure throughout multicellular fungal growth. With these improved plasmids, we observed in Aspergillus nidulans a 5‐fold increase in the expression of a fluorescent reporter, a doubling in the efficiency of a CRISPRa system for genome mining, and a up to a 10‐fold increase in the production of heterologous natural product metabolites. This strategy has the potential to be applied to diverse filamentous fungi. [ABSTRACT FROM AUTHOR]

Filamentous fungi with their diverse inventory of carbohydrate‐active enzymes promise a holistic usage of lignocellulosic residues. A major challenge for application is the inherent repression of enzyme production by carbon catabolite repression (CCR). In the presence of preferred carbon sources, the transcription factor CreA/CRE‐1 binds to specific but conserved motifs in promoters of genes involved in sugar metabolism, but the status of CCR is notoriously difficult to quantify. To allow for a real‐time evaluation of CreA/CRE‐1‐mediated CCR at the transcriptional level, we developed a luciferase‐based construct, representing a dynamic, highly responsive reporter system that is inhibited by monosaccharides in a quantitative fashion. Using this tool, CreA/CRE‐1‐dependent CCR triggered by several monosaccharides could be measured in Neurospora crassa, Aspergillus niger and Aspergillus nidulans over the course of hours, demonstrating distinct and dynamic regulatory processes. Furthermore, we used the reporter to visualize the direct impacts of multiple CreA truncations on CCR induction. Our reporter thus offers a widely applicable quantitative approach to evaluate CreA/CRE‐1‐mediated CCR across diverse fungal species and will help to elucidate the multifaceted effects of CCR on fungal physiology for both basic research and industrial strain engineering endeavours. [ABSTRACT FROM AUTHOR]

Background: Cystic fibrosis (CF), an inherited autosomal recessive disorder, is linked with high morbidity and mortality rates due to bacteria, filamentous, yeast and black yeast‐like fungi colonisation in the upper respiratory tract. Although Candida species are the most common fungi isolated from CF patients, azole‐resistant Aspergillus fumigatus (ARAf) is a big concern for invasive aspergillosis. Notably, the exact prevalences of Aspergillus species and the prevalence of ARAf isolates among Iranian CF patients have yet to be previously reported and are unknown. We aimed to investigate the prevalence of ARAf isolates in CF patients among Iranian populations by focusing on molecular mechanisms of the mutations in the target gene. Methods: The 1 year prospective study recovered 120 sputum samples from 103 CF patients. Of these, 55.1% (86/156) yielded Aspergillus species, screened for ARAf using plates containing itraconazole (4 mg/L) and voriconazole (1 mg/L). According to the CLSI‐M38 guidelines, antifungal susceptibility testing was performed using the broth microdilution method. In all phenotypically resistant isolates, the target of azole agents, the cyp51A gene, was sequenced to detect any possible single nucleotide polymorphisms (SNP) mediating resistance. Results: Of 120 samples, 101 (84.2%) were positive for filamentous fungi and yeast‐like relatives, with 156 fungal isolates. The most common colonising fungi were Aspergillus species (55.1%, 86/156), followed by Candida species (39.8%, 62/156), Exophiala species (3.8%, 6/156) and Scedosporium species (1.3%, 2/156). Forty out of 86 (46.5%) were identified for section Fumigati, 36 (41.9%) for section Flavi, 6 (7%) for section Nigri and 4 (4.6%) for section Terrei. Fourteen out of 40 A. fumigatus isolates were phenotypically resistant. The overall proportion of ARAf in total fungal isolates was 9% (14/156). cyp51A gene analysis in resistant isolates revealed that 13 isolates harboured G448S, G432C, T289F, D255E, M220I, M172V, G138C, G54E and F46Y mutations and one isolate carried G448S, G432C, T289F, D255E, M220I, G138C, G54E and F46Y mutations. Additionally, this study detects two novel cyp51A single‐nucleotide polymorphisms (I242V and D490E). Conclusions: This study first investigated ARAf isolates in Iranian CF patients. Due to a resistance rate of up to 9%, it is recommended that susceptibility testing of Aspergillus isolates from CF patients receiving antifungal treatment be a part of the routine diagnostic workup. However, extensive multicentre studies with a high volume of CF patients are highly warranted to determine the impact of ARAf on CF patients. [ABSTRACT FROM AUTHOR]

Fungi occupy important environmental, cultural, and socioeconomic roles. However, biological research of this diverse kingdom has lagged behind that of other phylogenetic groups. This is partially the result of the notorious difficulty in culturing a diverse array of filamentous fungal species due to their (i) often unpredictable growth, (ii) unknown preferences for culturing conditions, and (iii) long incubation times compared with other microorganisms such as bacteria and yeasts. Given the complexity associated with concurrently culturing diverse fungal species, developing practical methods for preserving as many species as possible for future research is vital. The widely accepted best practice for preserving fungal tissue is the use of cryogenic biobanking at −165 C, allowing for the preservation and documentation of stable genetic lineages, thus enabling long-term diversity-centered research. Despite the extensive literature on fungal cryopreservation, substantial barriers remain for implementation of cryogenic biobanks in smaller mycological laboratories. In this work, we present practical considerations for the establishment of a fungal culture biobank, as well as provide evidence for the viability of 61 fungal genera in cryogenic storage. By providing a pragmatic methodology for cryogenically preserving and managing many filamentous fungi, we show that creating a biobank can be economical for independently owned and operated mycology laboratories, which can serve as a long-term resource for biodiversity, conservation, and strain maintenance. [ABSTRACT FROM AUTHOR]

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The accumulation of microplastics (MPs) in the environment has been a bottleneck for scientific society. Several approaches have been described as possibilities for reducing MPs in aquatic and terrestrial ecosystems; however, most of them are not environmentally friendly. Filamentous fungi (Ff) cells are currently considered a promising solution as a treatment for MPs. Therefore, the present study reports the potential ability of Ff isolated from mangrove sediments to biodegrade low-density polyethylene MPs (LDPEMPs). Six Ff strains were grown in batch cultures for 28 days, and one of them, Aspergillus sp. (AQ3A), showed the most prominent profile to biodegrade polymeric compounds. After morphological and molecular analysis, all strains were identified as belonging to the genera Aspergillus (MQ1C, AQ2A and AQ3A), Penicillium (MQ1A), and Trichoderma (MQ1B and MQ2A). The strain Aspergillus sp. (AQ3A) showed the most promising results with a LDPEMPs reduction rate of 47% and biomass formation of 0.0890 g·mL−1. Complementary studies with Aspergillus sp. (AQ3A) using Fourier-transform infrared spectroscopy (FTIR) highlighted changes in the molecular structure of LDPEMPs. These results indicate that Ff can contribute to the biodegradation of LDPEMPs. However, other parameters, mainly associated with the enzymes that are involved in this biodegradation process, need to be explored. [ABSTRACT FROM AUTHOR]

The biosynthesis of antibiotics and other secondary metabolites (also named special metabolites) is regulated by multiple regulatory networks and cascades that act by binding transcriptional factors to the promoter regions of different biosynthetic gene clusters. The binding affinity of transcriptional factors is frequently modulated by their interaction with specific ligand molecules. In the last decades, it was found that the biosynthesis of penicillin is induced by two different molecules, 1,3-diaminopropane and spermidine, but not by putrescine (1,4-diaminobutane) or spermine. 1,3-diaminopropane and spermidine induce the expression of penicillin biosynthetic genes in Penicillium chrysogenum. Proteomic studies clearly identified two different proteins that respond to the addition to cultures of these inducers and are involved in β-alanine and pantothenic acid biosynthesis. These compounds are intermediates in the biosynthesis of phosphopantetheine that is required for the activation of non-ribosomal peptide synthetases, polyketide synthases, and fatty acid synthases. These large-size multidomain enzymes are inactive in the "apo" form and are activated by covalent addition of the phosphopantetheine prosthetic group by phosphopantetheinyl transferases. Both 1,3-diaminopropane and spermidine have a similar effect on the biosynthesis of cephalosporin by Acremonium chrysogenum and lovastatin by Aspergillus terreus, suggesting that this is a common regulatory mechanism in the biosynthesis of bioactive secondary metabolites/natural products. [ABSTRACT FROM AUTHOR]

The filamentous fungus Talaromyces liani (Kamyschko) Yilmaz, Frisvad & Samson, 2014, has attracted considerable interest in biotechnology due to its diverse industrial applications and physiological characteristics. However, the mitochondrial genome of T. liani remains uncharacterized. Here, we present the complete mitochondrial genome of T. liani, comprising 38,000 bp with a GC content of 24.61%. This genome includes 15 core protein-coding genes, 4 independent ORFs, 6 intronic ORFs, 26 tRNAs, and 2 rRNA genes. Phylogenetic analysis using Bayesian inference (BI) revealed the evolutionary relationships among 15 fungi from Eurotiales, strongly supporting distinct clades and indicating that T. liani most closely related to T. pinophilus. [ABSTRACT FROM AUTHOR]

Filamentous fungi associated with disease symptoms in non-native giant sequoia (Sequoiadendron giganteum) in Germany were investigated in ten cases of disease in Northwest Germany. During the study period from 2018 to 2023, a total of 81 species of Dikaria were isolated from woody tissue and needles of giant sequoia and morphotyped. Morphotypes were assigned to species designations based on ITS-sequence comparison and, in part, multi-locus phylogenetic analyses. Nine species were recognised as new reports for Germany or on giant sequoia: Amycosphaerella africana, Botryosphaeria parva, Coniochaeta acaciae, C. velutina, Muriformistrickeria rubi, Pestalotiopsis australis, P. monochaeta, Phacidiopycnis washingtonensis, and Rhizosphaera minteri. The threat posed to giant sequoia and other forest trees in Germany by certain, especially newly reported, fungal species is being discussed. The detection of a considerable number of new fungal records in the trees studied suggests that giant sequoia cultivation may be a gateway for alien fungal species in Germany. [ABSTRACT FROM AUTHOR]

Fermentation is a powerful tool for enhancing flavor, improving sustainability, and expanding creative possibilities in the kitchen. However, most fermentations done in gastronomic contexts are restricted to a small set of readily available microbial species. Expanding beyond this limited biological diversity holds promise to unlock new gastronomic innovation. Here, we explore novel culinary applications of Neurospora intermedia, an edible fungus traditionally used in Java, Indonesia to produce the fermented food oncom. Our work demonstrates that N. intermedia can be readily used in the production of oncom-like meat alternatives with non-traditional substrates, as well as in the production of enzymes for starch-to-sugar conversion. As an example, we harness secreted N. intermedia starch-degrading enzymes to produce an amazake that has a distinct volatile aroma composition compared to traditional amazake produced with Aspergillus oryzae. In addition to providing texture and flavor, N. intermedia can be used to add an orange color to dishes due to the development of brightly colored spores and aerial mycelia. This property, along with its secreted enzymes, was utilized to create a dish for fine-dining restaurant Alchemist in Copenhagen, Denmark. Overall, the novel foods produced in this study were rated favorably in consumer trials, indicating a broad sensory appeal of N. intermedia across different culinary applications and cultural contexts. The protocols and approaches presented in this study represent a new addition to the chef's toolbox that hold promise to expand gastronomic possibilities with fermentation in restaurants and beyond.

Secondary metabolites (SM) produced by filamentous fungi have been extensively applied in biomedicine, biological control, food, and other fields. The biosynthesis of most SMs is closely associated with the catalysis of non-ribosome peptide synthetase (NRPS), therefore the expression regulation of NRPS is crucial for SM biosynthesis. In order to further understand the expression regulation patterns of NRPS gene clusters in filamentous fungi, herein, the research progresses on the transcription factors regulating NRPS-mediated SM production are reviewed, of which both pathway-specific transcription factors and global transcription factors are individually descripted. The summarization is hoped to deepen the comprehension of NRPS-mediated SMs production regulated by transcription factors and provide a reference for development of natural products in filamentous fungi in the future.

The public health problem caused by mycotoxins contamination has received a great deal of attention worldwide. Mycotoxins produced by filamentous fungi widely distributed in foodstuffs can cause adverse impacts on humans and livestock, posing serious health threats. Particularly worth mentioning is that mycotoxins can accumulate in organisms and be enriched through the food chain. Improving early trace detection and control from the source is a more desirable approach than the contaminated food disposal process to ensure food safety. Conventional sensors are susceptible to interference from various components in intricate food matrices when detecting trace mycotoxins. The application of ratiometric sensors avoids signal fluctuations, and reduce background influences, which casts new light on developing sensors with superior performance. This work is the first to provide an overview of the recent progress of ratiometric sensors in the detection of mycotoxins in intricate food matrices, and highlight the output types of ratiometric signal with respect to accurate quantitative analysis. The prospects of this field are also included in this paper and are intended to have key ramifications on the development of sensing detection conducive to food safety. [ABSTRACT FROM AUTHOR]

Abstract Background Recent advancements in the collaboration between two scientific disciplines—fungal biotechnology and materials sciences—underscore the potential of fungal mycelium as renewable resource for sustainable biomaterials that can be harnessed in different industries. As fungal mycelium can be biotechnologically obtained from different filamentous fungi and is as a material very versatile, respective research and commercial application should be thriving. However, some granted patents in the field of fungal mycelium-based materials have caused uncertainty in the community as to which subject matter is patent-protected and for how long the protection is expected to last. Results This opinion paper therefore maps the patent landscape of fungal mycelium-based materials with a specific focus on technical applications including building construction, insulation, packaging, and the like. We provide an overview of granted patents (73) and pending applications (34) related to granted patents, the dominant patent portfolios (five, with the number of patents and/or applications per owner between six and 44), the patent owners, and highlight the key claims formulated to protect the inventions. Additionally, we outline various options towards an increased activity in the field. Conclusion Patent developments in the field leave the impression that fungal materials, despite their high potential as renewable and biodegradable materials, have been held back due to patent over-protection. Considering the need for replacing current petroleum-based materials with renewable biomaterials, coordinated efforts may be called for to intensify efforts in the field.

Abstract Background Renewable materials made using environmentally friendly processes are in high demand as a solution to reduce the pollution created by the fashion industry. In recent years, there has been a growing trend in research on renewable materials focused on bio-based materials derived from fungi. Results Recently, fungal cell wall material of a chitosan producing fungus has been wet spun to monofilaments. This paper presents a modification for the fungal monofilament spinning process, by the development of a benign method, dry gel spinning, to produce continuous monofilaments and twisted multifilament yarns, from fungal cell wall, that can be used in textile applications. The fungal biomass of Rhizopus delemar, grown using bread waste as a substrate, was subjected to alkali treatment with a dilute sodium hydroxide solution to isolate alkali-insoluble material (AIM), which mainly consists of the fungal cell wall. The treatment of AIM with dilute lactic acid resulted in hydrogel formation. The morphology of the hydrogels was pH dependent, and they exhibited shear thinning viscoelastic behavior. Dry gel spinning of the fungal hydrogels was first conducted using a simple lab-scale syringe pump to inject the hydrogels through a needle to form a monofilament, which was directly placed on a rotating receiver and left to dry at room temperature. The resulting monofilament was used to make twisted multifilament yarns. The process was then improved by incorporating a heated chamber for the quicker drying of the monofilaments (at 30⁰C). Finally, the spinning process was scaled up using a twin-screw microcompounder instead of the syringe pump. The monofilaments were several meters long and reached a tensile strength of 63 MPa with a % elongation at break of 14. When spinning was performed in the heated chamber, the tensile strength increased to 80 MPa and further increased to 103 MPa when a micro-compounder was used for spinning. Conclusion The developed dry gel spinning method shows promising results in scalability and demonstrates the potential for renewable material production using fungi. This novel approach produces materials with mechanical properties comparable to those of conventional textile fibers.

Abstract The black fungus Exophiala causes a wide range of infections from superficial to subcutaneous, but also invasive fungal infections in immunocompromised patients as well as healthy individuals. In addition, Exophiala, is a common colonizer of the air ways of patients with cystic fibrosis. However, the source of infection and mode of transmission is still unclear. The aim of this study was to investigate the presence of Exophiala in samples collected from Swedish indoor environments. We found that the Exophiala species were commonly found in dishwashers and that Exophiala dermatitidis was the most common Exophiala species, being identified in 70% (26 out of the 37) of samples. Almost all E. dermatitidis isolates had the ability to grow at 42 °C (P = 0.0002) and were catalase positive. Voriconazole and posaconazole exhibited the lowest MICs, while caspofungin and anidulafungin lack the antifungal activities in vitro. Future studies are needed to illuminate the transmission mode of the fungi.

Nucleolus is the most conspicuous sub-nuclear compartment that is well known as the site of RNA polymerase I-mediated rDNA transcription and assembly of ribosome subunits in eukaryotes. Recent studies on mammalian cells suggest that functions of nucleolus are not limited to ribosome biogenesis, and that nucleolus is involved in a diverse array of nuclear and cellular processes such as DNA repair, stress responses, and protein sequestration. In fungi, knowledge of nucleolus and its functions was primarily gleaned from the budding yeast. However, little is known about nucleolus of the filamentous fungi. Considering that the filamentous fungi are multi-cellular eukaryotes and thus distinct from the yeast in many aspects, researches on nucleoli of filamentous fungi would have the potential to uncover the evolution of nucleolus and its roles in the diverse cellular processes. Here we provide a brief up-to-date overview of nucleolus in general, and evidence suggesting their roles in fungal physiology and development.

Abstract Background Penicillium digitatum is a fungal plant pathogen that causes the green mold disease in harvested citrus fruits. Due to its economical relevance, many efforts have focused on the development of genetic engineering tools for this fungus. Adaptation of the CRISPR/Cas9 technology was previously accomplished with self-replicative AMA1-based plasmids for marker-free gene editing, but the resulting efficiency (10%) limited its practical implementation. In this study, we aimed to enhance the efficiency of the CRISPR/Cas9-mediated gene editing in P. digitatum to facilitate its practical use. Results Increasing the culture time by performing additional culture streaks under selection conditions in a medium that promotes slower growth rates significantly improved the gene editing efficiency in P. digitatum up to 54–83%. To prove this, we disrupted five candidate genes that were chosen based on our previous high-throughput gene expression studies aimed at elucidating the transcriptomic response of P. digitatum to the antifungal protein PdAfpB. Two of these genes lead to visual phenotypic changes (PDIG_53730/pksP, and PDIG_54100/arp2) and allowed to start the protocol optimization. The other three candidates (PDIG_56860, PDIG_33760/rodA and PDIG_68680/dfg5) had no visually associated phenotype and were targeted to confirm the high efficiency of the protocol. Conclusion Genome editing efficiency of P. digitatum was significantly increased from 10% to up to 83% through the modification of the selection methodology, which demonstrates the feasibility of the CRISPR/Cas9 system for gene disruption in this phytopathogenic fungus. Moreover, the approach described in this study might help increase CRISPR/Cas9 gene editing efficiencies in other economically relevant fungal species for which editing efficiency via CRISPR/Cas9 is still low.

Abstract Objectives Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is extensively employed for the identification of filamentous fungi on MALDI Biotyper (Bruker Daltonics) and Vitek MS (biomerieux), but the performance of fungi identification on new EXS2600 (Zybio) is still unknow. Our study aims to evaluate the new EXS2600 system's (Zybio) ability to rapidly identify filamentous fungi and determine its effect on turnaround time (TAT) in our laboratory. Methods We tested 117 filamentous fungi using two pretreatment methods: the formic acid sandwich (FA-sandwich) and a commercial mold extraction kit (MEK, Zybio). All isolates were confirmed via sequence analysis. Laboratory data were extracted from our laboratory information system over two 9-month periods: pre-EXS (April to December 2022) and post-EXS (April to December 2023), respectively. Results The total correct identification (at the species, genus, or complex/group level) rate of fungi was high, FA-sandwich (95.73%, 112/117), followed by MEK (94.02%, 110/117). Excluding 6 isolates not in the database, species-level identification accuracy was 92.79% (103/111) for FA-sandwich and 91.89% (102/111) for MEK; genus-level accuracy was 97.29% (108/111) and 96.39% (107/111), respectively. Both methods attained a 100% correct identification rate for Aspergillus, Lichtheimia, Rhizopus Mucor and Talaromyces species, and were able to differentiate between Fusarium verticillioides and Fusarium proliferatum within the Fusarium fujikuroi species complex. Notably, high confidence was observed in the species-level identification of uncommon fungi such as Trichothecium roseum and Geotrichum candidum. The TAT for all positive cultures decreased from pre EXS2600 to post (108.379 VS 102.438, P

Abstract In filamentous fungi, microtubules are important for polar growth and morphological maintenance and serve as rails for intracellular trafficking. The molecular mechanisms associated with microtubules have been analyzed. However, little is known about when and where tubulin, a component of microtubules, is biosynthesized in multinuclear and multicellular filamentous fungi. In this study, we visualized microtubules based on the enhanced green fluorescence protein (EGFP)-labeled α-tubulin and β-tubulin mRNA tagged by the EGFP-mediated MS2 system in living yellow Koji mold Aspergillus oryzae cells in order to understand the spatiotemporal production mechanism of tubulin. We found that mRNA of btuA, encoding for β-tubulin, localized at dot-like structures through the apical, middle and basal regions of the hyphal cells. In addition, some btuA mRNA dots showed microtubule-dependent motor protein-like dynamics in the cells. Furthermore, it was found that btuA mRNA dots were decreased in the cytoplasm just before mitosis but increased immediately after mitosis, followed by a gradual decrease. In summary, the localization and abundance of β-tubulin mRNA is spatiotemporally regulated in living A. oryzae hyphal cells.

During an ongoing survey on fungal diversity in Sichuan Province, China, a white filamentous fungus growing on decaying marbled bamboo (Chimonobambusa marmoreal) leaves was collected. Upon conducting the initial morphological diagnosis and BLASTn searches, the new collection was assigned to the genus Arthrobotrys. A multi-locus phylogeny, based on a concatenated dataset (SSU-ITS-LSU-TEF1-RPB2), revealed the new collection in close phylogenetic proximity to Arthrobotrys eryuanensis and A. hyrcanus. However, they differ in shape and size of conidia. The conidia of the new collection are comparatively shorter than those of A. hyrcanus and wider than those of A. eryuanensis. Based on the difference in morphology and molecular data, we proposed the new collection as a new species, Arthrobotrys chinensis sp. nov. Detailed morphological characteristics, illustrations, and an updated phylogeny is provided for the new species. [ABSTRACT FROM AUTHOR]

Most elements of filamentous fungi seen in human tissue by pathologists are hyphae, and encountering other elements may interfere with diagnosis. Sporangia and chlamydospores are such elements that have been described in only a few case reports. We present an autopsy case with the extremely rare coexistence of Mucorales sporangia and chlamydospores in the lung. These fungal elements must be recognized and identified accurately because they can easily be mistaken for other fungi, microorganisms, or degenerated tissue structures. [ABSTRACT FROM AUTHOR]

Bioeconomy goals for using biomass feedstock for biofuels and bio-based production has arisen the demand for fungal strains and enzymes for biomass processing. Despite well-known Trichoderma and Aspergillus commercial strains, continuous bioprospecting has revealed the fungal biodiversity potential for production of biomass degrading enzymes. The strain Aspergillus fumigatus LMB-35Aa has revealed a great potential as source of lignocellulose-degrading enzymes. Nevertheless, genetic improvement should be considered to increase its biotechnological potential. Molecular manipulation based on homologous direct recombination (HDR) in filamentous fungi poses a challenge since its low recombination rate. Currently, CRISPR/Cas9-mediated mutagenesis can enable precise and efficient editing of filamentous fungi genomes. In this study, a CRISPR/Cas9-mediated gene editing strategy for improving endoglucanase activity of A. fumigatus LMB-35Aa strain was successfully used, which constitutes the first report of heterologous cellulase production in filamentous fungi using this technology. For this, eglA gene from A. niger ATCC 10,864 was integrated into conidial melanin pksP gene locus, which facilitated the selection of edited events discerned by the emergence of albino colonies. Heterologous production of the EglA enzyme in a biofilm fermentation system resulted in a 40% improvement in endoglucanase activity of the mutant strain compared to the wild type. [ABSTRACT FROM AUTHOR]

Fusarium oxysporum is an asexual filamentous fungus that causes vascular wilt in hundreds of crop plants and poses a threat to public health through Fusariosis. F. oxysporum f. sp. conglutinans strain Fo5176, originally isolated from Brassica oleracea, is pathogenic to Arabidopsis, making it a model pathosystem for dissecting the molecular mechanisms underlying host-pathogen interactions. Assembling the F. oxysporum genome is notoriously challenging due to the presence of repeat-rich accessory chromosomes. Here, we report a gap-free genome assembly of Fo5176 using PacBio HiFi and Hi-C data. The 69.56 Mb assembly contained 18 complete chromosomes, including all centromeres and most telomeres (20/36), representing the first gap-free genome sequence of a pathogenic F. oxysporum strain. In total, 21,460 protein-coding genes were annotated, a 26.3% increase compared to the most recent assembly. This high-quality reference genome for F. oxysporum f. sp. conglutinans Fo5176 provides a valuable resource for further research into fungal pathobiology and evolution. [ABSTRACT FROM AUTHOR]

Background: Sclerotinia sclerotiorum is a highly destructive phytopathogenic fungus that poses a significant threat to a wide array of crops. The current constraints in genetic manipulation techniques impede a thorough comprehension of its pathogenic mechanisms and the development of effective control strategies. Results: Herein, we present a highly efficient genetic transformation system for S. sclerotiorum, leveraging the use of fusiform nanoparticles, which are synthesized with FeCl3 and 2,6-diaminopyrimidine (DAP). These nanoparticles, with an average longitude length of 59.00 nm and a positively charged surface, facilitate the direct delivery of exogenous DNA into the mycelial cells of S. sclerotiorum, as well as successful integration with stable expression. Notably, this system circumvents fungal protoplast preparation and tedious recovery processes, streamlining the transformation process considerably. Furthermore, we successfully employed this system to generate S. sclerotiorum strains with silenced oxaloacetate acetylhydrolase-encoding gene Ss-oah1. Conclusions: Our findings demonstrate the feasibility of using nanoparticle-mediated delivery as a rapid and reliable tool for genetic modification in S. sclerotiorum. Given its simplicity and high efficiency, it has the potential to significantly propel genetic research in filamentous fungi, offering new avenues for elucidating the intricacies of pathogenicity and developing innovative disease management strategies. [ABSTRACT FROM AUTHOR]

AbstractThis study aimed to identify proteolysis-resistant extracellular uricase by screening three hundred marine microorganisms, including bacteria, actinobacteria and filamentous fungi. The isolate, demonstrating resistance to proteolysis and producing extracellular uricase with relatively high activity was identified as Streptomyces rochei 6SM85. The enzyme was purified approximately 3.6 fold, yielding 1.23%, using ion exchange chromatography. The molecular weight of the purified uricase was estimated to be approximately 14 kDa by SDS-PAGE. The optimum temperature and pH for uricase were determined to be 35 °C and 8–9, respectively. It was demonstrated that uricase retained up to 46% of its activity after half an hour at 40 °C and exhibited higher activity at neutral and basic pH compared to acidic pH. Its activity increased in the presence of 1 mM Mg2+, Na+, and EDTA, while it decreased by 86% in the presence of Cu2+. This study represents the first report on the production and characterization of extracellular proteolysis-resistant uricase from marine-derived Streptomyces. [ABSTRACT FROM AUTHOR]

Simple Summary: Ruminant farming, which includes animals like cows and sheep, is resource-intensive and this is becoming more of an issue as the human population grows. People are also worried about the use of antibiotics in animal feed. Scientists are looking for new, sustainable ways to feed these animals to improve their health and production without antibiotics. One promising solution is using edible fungi, like Aspergillus oryzae. This fungus can provide important nutrients, such as proteins, which are usually obtained from plants or animals. By adding these fungi to animal feed, it can reduce the need for conventional protein sources and antibiotics, enhancing the quality of the feed and the well-being of the animals. This review paper explores the effects of including an Aspergillus oryzae fungal biomass in ruminant feed and explains how this fungus can be grown on organic residues. In summary, using Aspergillus oryzae in animal feed provides essential nutrients that can support animal health and productivity, all of which are valuable to society. The resource-intensive nature of the ruminant farming sector, which has been exacerbated by population growth and increasing pressure to reduce feed antibiotics and growth promoters, has sparked interest in looking for sustainable alternative feed sources to enhance ruminant production efficiency. Edible filamentous fungi, rich in macronutrients like proteins, offer promise in reducing the reliance on conventional protein sources and antimicrobials to improve feed quality and animal performance. The inclusion of single-cell proteins, particularly filamentous fungi, in ruminant feed has long been of scientific and industrial interest. This review focuses on the potential application of the extensively studied Aspergillus oryzae and its fermentation extracts in ruminant nutrition. It provides an overview of conventional ruminant feed ingredients, supplements, and efficiency. Additionally, this review analyzes the re-utilization of organic residues for A. oryzae cultivation and examines the effects of adding fungal extracts to ruminant feed on ruminal digestibility and animal performance, all within a circular bioeconomy framework. [ABSTRACT FROM AUTHOR]

Antifungal drug resistance in filamentous fungi, particularly Aspergillus species, is increasing worldwide. Therefore, new antifungal drugs or combinations of drugs are urgently required to overcome this public health situation. In the present study, we examined the antifungal activity of vancomycin-functionalized AuNPs. These functionalized AuNPs were characterized, and their antifungal activity and associated killing mechanism were investigated using conventional methodologies against the conidia of A. fumigatus and A. flavus. The differential antifungal activity of vancomycin-functionalized Au-NPs against the conidia of Aspergillus species is dependent on structural differences in the conidial cell wall. The results demonstrated potent fungicidal activity against A. fumigatus, with a MIC value of 4.68 µg/mL, 93% germination inhibition, and 38.4% killing rate within 8 h of exposure. However, the activity against A. flavus was fungistatic; a MIC value of 18.7 µg/mL and 35% conidial germination inhibition, followed by 28.4% killing rate, were noted under similar conditions. Furthermore, endogenous reactive oxygen species (ROS) accumulation was 37.4 and 23.1% in conidial populations of A. fumigatus and A. flavus, respectively. Raman spectroscopy analysis confirmed the possible (but not confirmed) binding of functionalized AuNPs with the chitin and galactomannan components of the cell wall. A potential strategy that involves the exploration of antibacterial drugs using AuNPs as efficient drug carriers may also be appropriate for countering emerging drug resistance in filamentous fungi. [ABSTRACT FROM AUTHOR]

This contribution aims to review the presence and the potential double role—positive or beneficial and negative or harmful—of fungi in fermented sausages as well as their use as starter cultures. Traditionally, studies have been focused on lactic acid bacteria; however, over the years, interest in the study of fungi has increased. The important contribution of yeasts and filamentous fungi to the quality and safety of fermented sausages has emerged from reviewing the literature regarding these fermented products. In conclusion, this review contributes to the existing literature by considering the double role of filamentous fungi and yeasts, the global fermented sausage market size, the role and use of starters, and the starters mainly present in the worldwide market, as well as the main factors to take into account to optimize production. Finally, some suggestions for future broadening of the sector are discussed. [ABSTRACT FROM AUTHOR]