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Objectives This study aimed to evaluate endophytic fungi isolated from Tocoyena bullata and Humiria balsamifera plant species for their antimycobacterial and anti-inflammatory activities, focusing on severe pulmonary tuberculosis cases which are often associated with exacerbated inflammation. Methods Mycobacterium suspensions were incubated with the samples for 5 days. RAW 264.7 macrophages stimulated with LPS were also incubated with them for 24 h to assess the inhibition of inflammatory mediator production and cytotoxicity. C57BL/6 mice were infected with Mtb M299 and treated for 15 days with lasiodiplodin (Lasio). Key findings Endophytic fungus Sordaria tamaensis, obtained from T. bullata, was the most promising. Its ethanolic extract impaired mycobacterial growth with MIC50 (µg/ml): 1.5 ± 0.6 (BCG), 66.8 ± 0.1 (H37Rv) and 80.0 ± 0.1 (M299). (R)-(+)-Lasio showed MIC50 92.2 ± 1.8 µg/ml (M299). In addition, Lasio was able to inhibit NO, IL-1β and TNF-α production and was not cytotoxic for macrophages. M. tuberculosis-infected C57BL/6 animals treated by Lasio reduced the number of acid-fast bacilli, lung pathology, leucocyte influx and proinflammatory cytokine production in the lungs. The class IIa fructose 1,6-bisphosphate aldolase was the predicted hypothetical target of Lasio. Conclusions (R)-(+)-Lasio stood out as a promising anti-TB compound, exhibiting anti-inflammatory and antimycobacterial effects, as well as low cytotoxicity.

Sporothrix (Sordariales, Ascomycota) is a well-supported monophyletic lineage within the Ophiostomatales, species of which occur in a diverse range of habitats including on forest trees, in the soil, associated with bark beetles and mites as well as on the fruiting bodies of some Basidiomycota. Several species have also been reported as important human and animal pathogens. During surveys of insect- and wound-associated Ophiostomatales from hardwood trees in Poland, many isolates with affinity to Sporothrix were recovered. In the present study, six undescribed Sporothrix spp. collected during these surveys are characterized based on their morphological characteristics and multi-locus phylogenenetic inference. They are described as Sporothrix cavum, Sporothrix cracoviensis, S. cryptarchum, S. fraxini, S. resoviensis, and S. undulata. Two of the Sporothrix spp. reside in the S. gossypina-complex, while one forms part of the S. stenoceras-complex. One Sporothrix sp. is a member of lineage F, and two other species grouped outside any of the currently defined species complexes. All the newly described species were recovered from hardwood habitats in association with sub-cortical insects, wounds or woodpecker cavities. These species were morphologically similar, with predominantly asexual states having hyaline or lightly pigmented conidia, which produce holoblastically on denticulate conidiogenous cells. Five of the new taxa produce ascomata with necks terminating in long ostiolar hyphae and allantoid ascospores without sheaths. The results suggest that Sporothrix species are common members of the Ophiostomatales in hardwood ecosystems of Poland.

β-Galactosidases are key enzymes in the food industry. Apart from the hydrolysis of the saccharide bond of lactose, they also catalyze transgalactosylation reactions, producing galactooligosaccharides (GOS) with prebiotic activity. Here we report the heterologous production in Pichia pastoris of a novel β-galactosidase from the fungus Thermothielavioides terrestris. The enzyme (TtbGal1) was purified and characterized, showing optimal activity at 60 °C and pH 4. TtbGal1 is thermostable, retaining almost full activity for 24 h at 50 °C. It was applied to the production of GOS from defined lactose solutions and acid whey, a liquid waste from the Greek yoghurt industry, reaching yields of 19.4 % and 14.8 %, respectively. HILIC-ESI-QTOF-MS analysis revealed the production of GOS with up to 4 saccharide monomers. The results demonstrate efficient GOS production catalyzed by TtbGal1, valorizing acid whey, a waste with a heavy polluting load from the dairy industry.

Fungi are eukaryotic organisms that have the ability to synthesize an enormous number of metabolites with a vast diversity of chemical structures and bioactivities. Chaetomiaceae species are a wealthy source of enzymes with diverse biotechnological and industrial applications such as PMO (polysaccharide monooxygenase), L-methioninase, β-1,3-glucanase, laccase, dextranase, lipolytic, pectinolytic, amylolytic, chitinolytic, and proteolytic enzymes. Different classes of secondary metabolites have been reported from this family are derived from various biosynthetic pathways such as alkaloids, polyketides, peptides, terpenes, and polyketide-amino acid hybrid secondary metabolites. These metabolites have attracted research interest due to their fascinating structural frameworks and bioactivities. Therefore, these compounds can be taken into account as candidates for the development of effective and novel lead compounds for medicine, as well as for plant protection. The current review represents the relevant information for Chaetomiaceae species, in particular, its new and first reported secondary metabolites and their pharmacological activities, as well as the biotechnological applications published from 2016 until the beginning of 2021.

Background: Molecular phylogenetic algorithms frequently disagree with the approaches considering reproductive compatibility and morphological criteria for species delimitation. The question stems if the resulting species boundaries from molecular, reproductive and/or morphological data are definitively not reconcilable; or if the existing phylogenetic methods are not sensitive enough to agree morphological and genetic variation in species delimitation. Objectives : We propose DISTATIS as an integrative framework to combine alignment-based (AB) and alignment-free (AF) distance matrices from ITS2 sequences/structures to shed light whether Gelasinospora and Neurospora are sister but independent genera. Methods: We aimed at addressing this standing issue by harmonizing genus-specific classification based on their ascospore morphology and ITS2 molecular data. To validate our proposal, three phylogenetic approaches: i) traditional alignment-based, ii) alignment-free and iii) novel distance integrative (DI)-based were comparatively evaluated on a set of Gelasinospora and Neurospora species. All considered species have been extensively characterized at both the morphological and reproductive levels and there are known incongruences between their ascospore morphology and molecular data that hampers genus-specific delimitation. Results: Traditional AB phylogenetic analyses fail at resolving the Gelasinospora and Neurospora genera into independent monophyletic clades following ascospore morphology criteria. In contrast, AF and DI approaches produced phylogenetic trees that could properly delimit the expected monophyletic clades. Conclusions: The DI approach outperformed the AF one in the sense that it could also divide the Neurospora species according to their reproduction mode.

The production of fuels and chemicals from renewable plant biomass has been proposed as a feasible strategy for global sustainable development. However, the economic efficiency of biorefineries is low. Here, through metabolic engineering, Myceliophthora thermophila, a cellulolytic thermophilic fungus, was constructed into a platform that can efficiently convert lignocellulose into important bulk chemicals—four carbon 1, 4-diacids (malic and succinic acid), building blocks for biopolymers—without the need for extra hydrolytic enzymes. Titers of >200 g/L from crystalline cellulose and 110 g/L from plant biomass (corncob) were achieved during fed-batch fermentation. Our study represents a milestone in consolidated bioprocessing technology and offers a new and promising system for the cost-effective production of chemicals and fuels from biomass.

Made available in DSpace on 2020-12-12T01:35:12Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-10-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Abstract: Arabinanases from glycoside hydrolase family GH93 are enzymes with exo-activity that hydrolyze the α-1,5 bonds between arabinose residues present on arabinan. Currently, several initiatives aiming to use byproducts rich in arabinan such as pectin and sugar beet pulp as raw material to produce various compounds of interest are being developed. However, it is necessary to use robust enzymes that have an optimal performance under pH and temperature conditions used in the industrial processes. In this work, the first GH93 from the thermophilic fungus Thermothielavioides terrestris (Abn93T) was heterologously expressed in Aspergillus nidulans, purified and biochemically characterized. The enzyme is a thermophilic glycoprotein (optimum activity at 70 °C) with prolonged stability in acid pHs (4.0 to 6.5). The presence of glycosylation affected slightly the hydrolytic capacity of the enzyme, which was further increased by 34% in the presence of 1 mM CoCl2. Small-angle X-ray scattering results show that Abn93T is a globular-like-shaped protein with a slight bulge at one end. The hydrolytic mechanism of the enzyme was elucidated using capillary zone electrophoresis and molecular docking calculations. Abn93T has an ability to produce (in synergism with arabinofuranosidases) arabinose and arabinobiose from sugar beet arabinan, which can be explored as fermentable sugars and prebiotics. Key points: • Thermophilic exo-arabinanase from family GH93 • Molecular basis of arabinan depolymerization. Department of Biotechnology Lorena School of Engineering University of São Paulo Departamento de Física e Ciências Aplicadas Instituto de Física de São Carlos Universidade de São Paulo Departamento de Bioquímica Instituto de Biologia Universidade Estadual de Campinas Programa de Processos Tecnológicos e Ambientais Universidade de Sorocaba Instituto de Pesquisas Tecnológicas do Estado de São Paulo Diretoria de Operações e Negócios Núcleo de Bionanomanufatura Department of Physics – Institute of Biosciences Humanities and Exact Sciences São Paulo State University (UNESP) Department of Physics – Institute of Biosciences Humanities and Exact Sciences São Paulo State University (UNESP) FAPESP: 2014/06923-6 FAPESP: 2014/18714-2 FAPESP: 2017/00525-0 FAPESP: 2019/01165-0 CNPq: 2019/06663-8 FAPESP: 2019/06663-8 FAPESP: 2019/22284-7 CNPq: 302627/2018-9 CNPq: 443916/2014-4

Organismal interactions within microbial consortia and their responses to harmful intruders remain largely understudied. An important step toward the goal of understanding functional ecological interactions and their evolutionary selection is the study of increasingly complex microbial interaction systems. Here, we discovered a tripartite biosystem consisting of the fungus Aspergillus nidulans, the unicellular green alga Chlamydomonas reinhardtii, and the algicidal bacterium Streptomyces iranensis. Genetic analyses and MALDI-IMS demonstrate that the bacterium secretes the algicidal compound azalomycin F upon contact with C. reinhardtii. In co-culture, A. nidulans attracts the motile alga C. reinhardtii, which becomes embedded and surrounded by fungal mycelium and is shielded from the algicide. The filamentous fungus Sordaria macrospora was susceptible to azalomycin F and failed to protect C. reinhardtii despite chemotactically attracting the alga. Because S. macrospora was susceptible to azalomycin F, this data imply that for protection the fungus needs to be resistant. Formation of the lichen-like association between C. reinhardtii and A. nidulans increased algal growth. The protection depends on the increased amounts of membrane lipids provided by resistant fungi, thereby generating a protective shelter against the bacterial toxin. Our findings reveal a strategy whereby algae survive lethal environmental algicides through cooperation with fungi.

Fruiting bodies are among the most complex multicellular structures formed by fungi, and the molecular mechanisms that regulate their development are far from understood. However, studies with a number of fungal model organisms have started to shed light on this developmental process. One of these model organisms isSordaria macrospora, a filamentous ascomycete from the orderSordariales. This fungus has been a genetic model organism since the 1950s, but its career as a model organism for molecular genetics really took off in the 1990s, when the establishment of a transformation protocol, a mutant collection, and an indexed cosmid library provided the methods and resources to start revealing the molecular mechanisms of fruiting body development. In the 2000s, “omics” methods were added to theS. macrosporatool box, and by 2020, 58 developmental genes have been identified in this fungus. This review gives a brief overview of major method developments forS. macrospora, and then focuses on recent results characterizing different processes involved in regulating development including several regulatory protein complexes, autophagy, transcriptional and chromatin regulation, and RNA editing.Key points•Sordaria macrospora is a model system for analyzing fungal fruiting body development.•More than 100 developmental mutants are available for S. macrospora.•More than 50 developmental genes have been characterized in S. macrospora.

Trehalase catalyzes the hydrolysis of the non-reducing disaccharide trehalose. The highly active trehalase MthT from Myceliophthora thermophila was screened from the trehalase genes of six species of filamentous fungi. An ingenious multi-copy knock-in expression strategy mediated by the CRISPR/Cas9 tool and medium optimization were used to improve MthT production in Aspergillus niger, up to 1698.83 U/mL. The protein background was dramatically abated due to insertion. The recombinant MthT showed optimal activity at pH 5.5 and 60 °C, and exhibited prominent thermal stability between 50 and 60 °C under acid conditions (pH 4.5–6.5). The ethanol conversion rate (ethanol yield/total glucose) was significantly improved by addition of MthT (51.88%) compared with MthT absence (34.38%), using 30% starch saccharification liquid. The results of this study provided an effective strategy, established a convenient platform for heterologous expression in A. niger and showed a potential strategy to decrease production costs in industrial ethanol production.

The economical production of pectin oligosaccharides with a specific degree of polymerization and structure from agro-food waste is an industrially important process. This study identified a novel pectate lyase gene (plhy1) from the thermophilic cellulolytic fungus H. insolens Y1 and tested its ability to produce pectin oligosaccharides. The recombinant PLHY1 produced in Pichia pastoris was superior to other similar enzymes due to its high thermal and pH stability. PLHY1 demonstrated optimal enzymatic activity at 55°C and pH 10.0 in the presence of 0.4 mM Ca2+, and preferred methyl esterified substrates for digestion. High performance anion exchange chromatography-pulsed amperometric detector and ultra high performance liquid chromatography in combination with electrospray ionization tandem mass spectrometry analysis showed that galacturonic acid-oligosaccharides with a small degree of polymerization (4–6) were the major hydrolysates produced by the degradation of apple peel pectin by PLHY1. The properties of PLHY1 make it valuable for application in the agro-food industry for the production of pectin oligosaccharides.

Cellulolytic fungi usually have multiple genes for C1-oxidizing auxiliary activity 9 (AA9) lytic polysaccharide monooxygenases (LPMOs) in their genomes, but their potential functional differences are less understood. In this study, two C1-oxidizing AA9 LPMOs, SbLPMO9A and SbLPMO9B, were identified from Sordaria brevicollis, and their differences, particularly in terms of thermostability, reducing agent specificity, and synergy with cellulase, were explored. The two enzymes exhibited weak binding to cellulose and intolerance to hydrogen peroxide. Their oxidative activity was influenced by cellulose crystallinity and surface morphology, and both enzymes tended to oxidize celluloses of lower crystallinity and high surface area. Comparably, SbLPMO9A had much better thermostability than SbLPMO9B, which may be attributed to the presence of a carbohydrate binding module 1 (CBM1)-like sequence at its C-terminus. In addition, the two enzymes exhibited different specificities and responsivities toward electron donors. SbLPMO9A and SbLPMO9B were able to boost the catalytic efficiency of endoglucanase I (EGI) on physically and chemically pretreated substrates but with different degrees of synergy. Substrate- and enzyme-specific synergism was observed by comparing the synergistic action of SbLPMO9A or SbLPMO9B with commercial Celluclast 1.5L on three kinds of cellulosic substrates. On regenerated amorphous cellulose and PFI (Papirindustriens Forskningsinstitut)-fibrillated bleached eucalyptus pulp, SbLPMO9B showed a higher synergistic effect than SbLPMO9A, while on delignified wheat straw, the synergistic effect of SbLPMO9A was higher than that of SbLPMO9B. On account of its excellent thermostability and boosting effect on the enzymatic hydrolysis of delignified wheat straw, SbLPMO9A may have high application potential in biorefineries for lignocellulosic biomass. KEY POINTS: • C1-oxidizing SbLPMO9A displayed higher thermostability than SbLPMO9B, probably due to the presence of a CBM1-like module. • The oxidative activity of the two SbLPMO9s on celluloses increased with decreasing cellulose crystallinity or increasing beating degree. • The two SbLPMO9s boosted the catalytic efficiency of cellulase, but the synergistic effect was substrate- and enzyme-specific.

In our continuous search for antibacterial agents against Pseudomonas syringae pv. actinidiae (Psa) from kiwi-associated fungi, two pairs of epimeric cytochalasins, zopfiellasins A–D (1–4), were characterized from the fungus Zopfiella sp. The structures were established on the basis of spectroscopic data analysis, while the absolute configurations were determined by single-crystal X-ray diffraction. Compounds 1 and 3 exhibited antibacterial activity against Psa with MIC values of 25 and 50 μg/mL, respectively. This is the first report of anti-Psa activity of cytochalasin derivatives.

BACKGROUND A growing number of non-dermatophytic moulds and yeasts with the ability to act as human pathogens are reported every year. Dematiaceous fungi cause phaeohyphomycosis which encompasses a broad spectrum of diseases ranging from superficial (cutaneous and subcutaneous) to disseminated infections. Such fungal infections are responsible for causing significant morbidity and mortality, frequently in immunocompromised patients and rarely in immunocompetent patients. OBJECTIVES To investigate the prevalence of cutaneous mycosis in Jammu district (India) and to isolate and identify the recovered causal agents from the affected skin of the patients. METHODS For direct microscopy, 10% KOH was used. Skin samples were collected carefully from the affected areas of suspected patients, followed by the isolation and identification of the causal agents by cultural examination, morphological examination and ITS sequencing. RESULTS Herein, we report and describe three new cases of cutaneous phaeohyphomycosis from District Jammu of Union Territory Jammu and Kashmir, India. The age of the patients under study ranged from 17 to 42 years and the duration of infection from 1 to 2 years. The etiological agents that were recovered from the patients under study were Alternaria alstromeriae, Epicoccum tritici and Phialemonium obovatum. These dematiaceous fungal species were isolated from the skin specimen of immunocompetent hosts. CONCLUSION Among the three isolated etiological agents, two (Alternaria alstromeriae, Epicoccum tritici) represent new global records and one (Phialemonium obovatum) new record to India as causal agents of cutaneous phaeohyphomycosis. Careful microscopic and mycological examination form the basis of correct diagnosis of such fungal infections in the absence of simple and reliable laboratory tests (serologic or antigen tests).

The formation of fruiting bodies is one of the most complex developmental processes in filamentous ascomycetes. It requires the development of sexual structures that give rise to meiosporangia (asci) and meiotic spores (ascospores) as well as surrounding structures for protection and dispersal of the spores. Previous studies have shown that these developmental processes are accompanied by significant changes of the transcriptome, and comparative transcriptomics of different fungi as well as the analysis of transcriptome changes in developmental mutants have aided in the identification of differentially regulated genes that are themselves involved in regulating fruiting body development. In previous analyses, we used transcriptomics to identify the genes asm2 and spt3, which result in developmental phenotypes when deleted in Sordaria macrospora. In this study, we identified another gene, asm3, required for fruiting body formation, and performed transcriptomics analyses of Δasm2, Δasm3, and Δspt3. Deletion of spt3, which encodes a subunit of the SAGA complex, results in a block at an early stage of development and drastic changes in the transcriptome. Deletion mutants of asm2 and asm3 are able to form fruiting bodies, but have defects in ascospore maturation. Transcriptomics analysis of fruiting bodies revealed a large overlap in differentially regulated genes in Δasm2 and Δasm3 compared to the wild type. Analysis of nuclear distribution during ascus development showed that both mutants undergo meiosis and postmeiotic divisions, suggesting that the transcriptomic and morphological changes might be related to defects in the morphogenesis of structural features of the developing asci and ascospores.

Lytic Polysaccharide Monooxygenases (LPMOs) are powerful redox enzymes able to oxidatively cleave recalcitrant polysaccharides. Widely conserved across biological kingdoms, LPMOs of the AA9 family are deployed by phytopathogens to deconstruct cellulose polymers. In response, plants have evolved sophisticated mechanisms to sense cell wall damage and thus self-triggering Damage Triggered Immunity responses. Here, we show that Arabidopsis plants exposed to LPMO products triggered the innate immunity ultimately leading to increased resistance to the necrotrophic fungus Botrytis cinerea. We demonstrated that plants undergo a deep transcriptional reprogramming upon elicitation with AA9 derived cellulose- or cello-oligosaccharides (AA9_COS). To decipher the specific effects of native and oxidized LPMO-generated AA9_COS, a pairwise comparison with cellobiose, the smallest non-oxidized unit constituting cellulose, is presented. Moreover, we identified two leucine-rich repeat receptor-like kinases, namely STRESS INDUCED FACTOR 2 and 4, playing a crucial role in signaling the AA9_COS-dependent responses such as camalexin production. Furthermore, increased levels of ethylene, jasmonic and salicylic acid hormones, along with deposition of callose in the cell wall was observed. Collectively, our data reveal that LPMOs might play a crucial role in plant-pathogen interactions., Zarattini et al. confirm the capacity of Lytic Polysaccharide Monooxygenases (LPMO) active on cellulose to trigger immune responses in Arabidopsis. These results bring insight to the field of cell wall modifying enzymes and their roles in plant defense mechanisms.

Structure-selective endonucleases cleave branched DNA substrates. Slx1 is unique among structure-selective nucleases because it can cleave all branched DNA structures at multiple sites near the branch point. The mechanism behind this broad range of activity is unknown. The present study structurally and biochemically investigated fungal Slx1 to define a new protein interface that binds the non-cleaved arm of branched DNAs. The DNA arm bound at this new site was positioned at a sharp angle relative to the arm that was modeled to interact with the active site, implying that Slx1 uses DNA bending to localize the branch point as a flexible discontinuity in DNA. DNA binding at the new interface promoted a disorder-order transition in a region of the protein that was located in the vicinity of the active site, potentially participating in its formation. This appears to be a safety mechanism that ensures that DNA cleavage occurs only when the new interface is occupied by the non-cleaved DNA arm. Models of Slx1 that interacted with various branched DNA substrates were prepared. These models explain the way in which Slx1 cuts DNA toward the 3′ end away from the branch point and elucidate the unique ability of Slx1 to cleave various DNA structures.

Lytic polysaccharide monooxygenases (LPMOs) have attracted vast attention because of their unique mechanism of oxidative degradation of carbohydrate polymers and the potential application in biorefineries. This study characterized a novel LPMO from Myceliophthora thermophila, denoted MtLPMO9L. The structure model of the enzyme indicated that it belongs to the C1-oxidizing LPMO, which has neither an extra helix in the L3 loop nor extra loop region in the L2 loop. This was confirmed subsequently by the enzymatic assays since MtLPMO9L only acts on cellulose and generates C1-oxidized cello-oligosaccharides. Moreover, synergetic experiments showed that MtLPMO9L significantly improves the efficiency of cellobiohydrolase (CBH) II. In contrast, the inhibitory rather than synergetic effect was observed when combining used MtLPMO9L and CBHI. Changing the incubation time and concentration ratio of MtLPMO9L and CBHI could attenuate the inhibitory effects. This discovery suggests a different synergy detail between MtLPMO9L and two CBHs, which implies that the composition of cellulase cocktails may need reconsideration.

D-glucuronic acid (GlcUA) is an important intermediate with numerous applications in the food, cosmetics, and pharmaceutical industries. Its biological production routes which employ myo-inositol oxygenase (MIOX) as the key enzyme are attractive. In this study, five diverse MIOX-encoding genes, from Cryptococcus neoformans, Chaetomium thermophilum, Arabidopsis thaliana, Thermothelomyces thermophila, and Mus musculus were overexpressed in Escherichia coli, respectively. A novel MIOX from Thermothelomyces thermophila (TtMIOX) exhibited high specific activity, and efficiently converted myo-inositol to GlcUA. Meanwhile, the degradation of GlcUA was inhibited by inactivation of uxaC from the Escherichia coli genome. Finally, the BWΔuxaC whole-cell biocatalyst harboring TtMIOX resulted in the production of 106 g/L GlcUA within 12 h in a 1-L bioreactor, corresponding to a conversion of 91% and productivity of 8.83 g/L/h. This study provides a feasible method for the industrial production of GlcUA.

The eye arose during the Cambrian explosion from pre-existing proteins that would have been recruited for the formation of the specialized components of this organ, such as the transparent lens. Proteins suitable for the role of lens crystallins would need to possess unusual physical properties and the study of such earliest analogs of ocular crystallins would add to our understanding of the nature of recruitment of proteins as lens/corneal crystallins. We show that the Abundant Perithecial Protein (APP) of the fungi Neurospora and Sordaria fulfils the criteria for an early crystallin analog. The perithecia in these fungal species are phototropic, and APP accumulates at a high concentration in the neck of the pitcher-shaped perithecium. Spores are formed at the base of the perithecium, and light contributes to their maturation. The hydrodynamic properties of APP appear to exclude dimer formation or aggregation at high protein concentrations. APP is also deficient in Ca2+ binding, a property seen in its close homolog, the calcium-binding cell adhesion molecule (DdCAD-1) from Dictyostelium discoidum. Comparable to crystallins, APP occurs in high concentrations and seems to have dispensed with Ca2+ binding in exchange for greater stability. These crystallin-like attributes of APP lead us to demonstrate that it is a primitive form of ocular crystallins.

Corn bins in the midwestern United States can reach temperatures up to 52 C. High temperatures combined with sufficient moisture and humidity in bins provide the perfect environment to promote the growth of thermophilic and thermotolerant fungi. In this article, we characterize for the first time thermophilic and thermotolerant fungi in corn grain bins using culture-based methods and pyrosequencing techniques. Corn samples were collected from local farms in western Illinois. Samples were plated and incubated at 50 C using a variety of approaches. Of several hundred kernels examined, more than 90% showed colonization. Species identified using culture methods included Thermomyces lanuginosus, Thermomyces dupontii, Aspergillus fumigatus, Thermoascus crustaceus, and Rhizomucor pusillus. Pyrosequencing was also performed directly on corn grain using fungal-specific primers to determine whether thermophilic fungi could be detected using this technique. Sequences were dominated by pathogenic fungi, and thermophiles were represented by less than 2% of the sequences despite being isolated from 90% of the grain samples using culturing techniques. The high abundance of previously undocumented viable fungi in corn could have negative implications for grain quality and pose a potential risk for workers and consumers of corn-derived products in the food industry. Members of the Sordariales were absent among thermophile isolates and were not represented in nuc rDNA internal transcribed spacer (ITS) sequences. This is in striking contrast with results obtained with other substrates such as litter, dung, and soils, where mesophilic and thermophilic members of the Sordariaceae and Chaetomiaceae are common. This absence appears to reflect an important difference between the ecology of Sordariales and other orders within the Ascomycota in terms of their ability to compete in microhabitats rich in sugars and living tissues.

There are many diseases linked to oxidative stress, including cancer. Importantly, endogenous antioxidants are insufficient to protect against this process. Peptides derived from food proteins produced by hydrolysis have been investigated as exogenous antioxidants. The present study aimed to identify novel peptides with antioxidant potential produced from egg and milk proteins hydrolysis with two new fungal proteases isolated from

Glucuronoyl esterases (CE15 family) enable targeted cleavage of ester linkages in lignin-carbohydrate complexes (LCCs), particularly those linking lignin and glucuronoyl residues in xylan. A substantial challenge in characterization and kinetic analysis of CE15 enzymes has been the lack of proper substrates. Here, we present an assay using an insoluble LCC-rich lignin fraction from birch; lignin-rich pellet (LRP). The assay employs quantification of enzyme reaction products by LC-MS. The kinetics of four fungal CE15 enzymes, PsGE, CuGE, TtGE, and AfuGE originating from lignocellulose-degrading fungi Punctularia strigosozonata, Cerrena unicolor, Thielavia terrestris, and Armillaria fuscipes respectively were characterized and compared using this new assay. All four enzymes had activity on LRP and showed a clear preference for the insoluble substrate compared with smaller soluble LCC mimicking esters. End-product profiles were near identical for the four enzymes but differences in kinetic parameters were observed. TtGE possesses an alternative active site compared with the three other enzymes as it has the position of the catalytic glutamic acid occupied by a serine. TtGE performed poorly compared with the other enzymes. We speculate that glucuronoyl LCCs are not the preferred substrate of TtGE. Removal of an N-terminal CBM on CuGE affected the catalytic efficiently of the enzyme by reducing K cat by more than 30%. Reaction products were detected from all four CE15s on a similar substrate from spruce indicating a more generic GE activity not limited to the hardwood. The assay with natural substrate represents a novel tool to study the natural function and kinetics of CE15s.

Thermophilic fungi can represent a rich source of industrially relevant enzymes. Here, 105 fungal strains capable of growing at 50 °C and pH 2.0 were isolated from compost and decaying plant matter. Maximum growth temperatures of the strains were in the range 50 °C to 60 °C. Sequencing of the internal transcribed spacer (ITS) regions indicated that 78 fungi belonged to 12 species of Ascomycota and 3 species of Zygomycota, while no fungus of Basidiomycota was detected. The remaining 27 strains could not be reliably assigned to any known species. Phylogenetically, they belonged to the genus Thielavia, but they represented 23 highly divergent genetic groups different from each other and from the closest known species by 12 to 152 nucleotides in the ITS region. Fungal secretomes of all 105 strains produced during growth on untreated rice straw were studied for lignocellulolytic activity at different pH and temperatures. The endoglucanase and xylanase activities differed substantially between the different species and strains, but in general, the enzymes produced by the novel Thielavia spp. strains exhibited both higher thermal stability and tolerance to acidic conditions. The study highlights the vast potential of an untapped diversity of thermophilic fungi in the tropics.

Laccase-like multicopper oxidases (LMCOs) are a heterogeneous group of oxidases, acting mainly on phenolic compounds and which are widespread among many microorganisms, including Basidiomycetes and Ascomycetes. Here, we report the cloning, heterologous expression, purification and characterization of a novel LMCO from the thermophilic fungus Thermothelomyces thermophila. The 1953 bp lmco gene sequence comprises of 3 exons interrupted by 2 introns and according to the LccED database the translated sequence belongs to superfamily 6 of multicopper oxidases. After removal of the introns, the gene was transformed into Pichia pastoris, under the control of the alcohol oxidase (AOX1) promoter. The heterologous enzyme was purified with an apparent molecular weight of 80 kDa. TtLMCO1 displayed optimum activity at pH 4 and 50 °C and appeared thermostable up to 50 °C. A variety of phenolic compounds were oxidized by TtLMCO1, including standard laccase substrates such as ABTS and 2,6 dimethoxyphenol. The UV/Vis spectrum of purified TtLMCO1 indicates that it belongs to yellow laccase-like oxidases. The enzyme was used for the bioconversion of 2′,3,4-trihydroxychalcone to 3′,4′-dihydroxy-aurone, a bioactive aurone recently shown to possess inhibitory activity against several isoforms of the histone deacetylase complex (HDAC). Overall, the thermophilic yellow LMCO TtLMCO1 presents a number of superior properties with potential use in industrial biocatalysis.

Lipidic cubic phase systems (LCPs) are excellent carriers for immobilized enzymes due to their biocompatibility and well-defined nanoporous structure. Lipidic cubic phases act as a convenient matrix to incorporate enzymes and hold them in the vicinity of electrode surfaces in their fully active forms. Corynascus thermophilus cellobiose dehydrogenase (CtCDH) was trapped in a monoolein cubic phase, which increased not only its stability, but also its catalytic performance with both enhanced mediated and direct electron transfer with electrodes. For studies of mediated electron transfer, three mediators with different formal potentials (E°') were employed: horse-heart cytochrome c (cyt c), electron acceptor active with the cytochrome domain of CtCDH, and 2,6-dichlorophenolindophenol (DCPIP) as well as hexaammineruthenium(II) chloride [Ru(NH3)Cl2] both electron acceptors with the dehydrogenase domain. Ru(NH3)Cl2, having the most negative E°' of -0.138V vs. Ag|AgCl at pH7.5, gave a catalytic current for lactose oxidation of 32.10μAcm-2 in MOPS buffer at pH7.5. The process carried out in the same solution but under direct electron conditions transfer resulted in a catalytic current of 9.22μAcm-2. Electrodes covered with CtCDH in a LCP film retained their catalytic activity after 28days showing a slightly increased current density after 6days.

Sordariomycetes is an earlier and one of the widely distributed class of Ascomycota. The class was initially classified based on morphology in having inoperculate and unitunicate asci. With the development of DNA based phylogenetic analysis, several undetermined or polyphyletic members of Sordariomycetes were reclassified. However, not all species belonging to this class have been sequenced and analyzed. There are a number of species, especially those old and poorly studied ones which have never been sequenced before and not even recollected again for further taxonomic verification. One of the main objective in this study is to revise and update the taxonomy of several well-known old and poorly studied species whose classification are still obscure. Herein, we re-examined the type materials and/or authentic specimens together to explore 74 relatively poorly-studied genera, which mainly belong to Boliniales, Calosphaeriales, Chaetosphaeriales, Jobellisiales, and Sordariales classified under Diaporthomycetidae and Sordariomycetidae. We provide descriptions, notes, figures and/or drawings and discussed their phylogenetic relationships. As a result, the monotypic Jobellisiales is transferred from Hypocreomycetidae to Diaporthomycetidae. Based on phylogenetic analysis, the polyphyletic Lasiosphaeriaceae is divided into five families, Bombardiaceae (Apodospora, Bombardia, Bombardioidea and Fimetariella), Lasiosphaeriaceae (Anopodium, Bellojisia, Corylomyces, Lasiosphaeria, Mammaria and Zopfiella), Lasiosphaeridaceae (Lasiosphaeris), Strattoniaceae (Strattonia) and Zygospermellaceae (Episternus and Zygospermella). In addition, a new family Neoschizotheciaceae is established based on Neoschizothecium. Analysis of the type species of Boothiella, Stellatospora, Sulcatistroma and Tengiomyces placed them in Sordariaceae, Chaetomiaceae, Hypocreales and Coronophorales, respectively. We classify the genera lacking molecular data based on their morphology and expect them to be recollected; that is, Kacosphaeria in Calosphaeriales; Arnium, Biconiosporella, Camptosphaeria, Diffractella, Emblemospora, Eosphaeria, Periamphispora, Ramophialophora, Synaptospora and Tripterosporella in Sordariales; Conidiotheca in Sordariomycetes; Copromyces, Effetia, Endophragmiella and Tulipispora are accommodated in Ascomycota. Besides, we establish a new genus Neoschizothecium based on phylogenetic analysis. New combinations proposed include: Camaropella amorpha, Cam. microspora, Cam. plana, Cladorrhinum grandiusculum, Cla. leucotrichum, Cla. terricola, Cla. olerum, Helminthosphaeria plumbea, Immersiella hirta, Jugulospora minor, Lasiosphaeris arenicola, Neoschizothecium aloides, Neo. carpinicola, Neo. conicum, Neo. curvisporum, Neo. fimbriatum, Neo. glutinans, Neo. inaequale, Neo. minicaudum, Neo. selenosporum, Neo. tetrasporum, Neurospora autosteira, Podospora brunnescens, P. flexuosa, P. jamaicensis, P. hamata, P. macrospora, P. spinosa, Strattonia petrogale and Triangularia microsclerotigena, T. nannopodalis, T. praecox, T. samala, T. tarvisina, T. unicaudata, T. yaeyamensis. New epithets are proposed for Apiorhynchostoma apiosporum and Podospora dacryoidea.

Melanins are pigments used by fungi to withstand various stresses and to strengthen vegetative and reproductive structures. In Sordariales fungi, their biosynthesis starts with a condensation step catalyzed by an evolutionary-conserved polyketide synthase. Here we show that complete inactivation of this enzyme in the model ascomycete Podospora anserina through targeted deletion of the PaPks1 gene results in reduced female fertility, in contrast to a previously analyzed nonsense mutation in the same gene that retains full fertility. We also show the utility of PaPks1 mutants for detecting rare genetic events in P. anserina, such as parasexuality and possible fertilization and/or apomixis of nuclei devoid of mating-type gene.

Thermophilic fungi have several biotechnological and industrial applications such as thermostable enzyme production, biodegradation, and tobacco processing, etc. Thermophilic fungi cannot survive at temperatures below 20 °C. Owing to their inability to grow at low temperatures, they are not stable, so stocking is very difficult. Although a large number of different storage methods are available and described, no method can be universally applied to all fungi. Thermophilic fungi present “heat-loving” characteristics, and therefore a new challenge for its preservation and there is no universal protocol for the preservation of thermophilic fungi. The aim of this study was to evaluate the viability, contamination and stability of thermophilic fungi stored under different preservation methods. In this work, 25 thermophilic fungal isolates of species Thermomyces thermophilus, Rhizomucor pusillus, Trichocladium griseum, Melanocarpus albomyces, Malbranchea cinnamomea, Thermothelomyces thermophilus, Thermothelomyces hinnuleus, Thermothielavioides terrestris, Mycothermus thermophilus, Humicola insolens maintained constant sub-culturing at room temperature, +4 °C and +20 °C, lyophilization at +4 °C, freezing at −20 °C, freezing block at −20 °C and a new technique liquid preservation at room temperature for the periods ranging 5 years. We evaluated the effect of preservation methods by sub-culturing onto either sabouraud dextrose agar (SDA) or yeast extract soluble starch agar (YpSs) on growth, production and viability of spores and macro- and micromorphology. In this study, preservation methods for thermophilic fungi were investigated extensively for the first time and it is clearly shown that freezing block at −20 °C method and lyophilization were better methods for long-term preservation up to 5 years.

Triangularia mangenotti was analyzed for the production of secondary metabolites, resulting in the isolation of known zopfinol (1) and its new derivatives zopfinol B–C (2–4), the 10-membered lactones 7-O-acetylmultiplolide A (5) and 8-O-acetylmultiplolide A (6), together with sordarin (7), sordarin B (8), and hypoxysordarin (9). The absolute configuration of 1 was elucidated by the synthesis of MPTA-esters. Compound 1 showed antimicrobial activity against the Gram-positive bacteria Bacillus subtilis and Staphylococcus aureus and the fungus Mucor hiemalis. While 4 was weakly antibacterial, 3 showed stronger antibiotic activity against the Gram-positive bacteria and weak antifungal activity against M. hiemalis and Rhodotorula glutinis. We furthermore observed the cytotoxicity of 1, 3 and 4 against the mammalian cell lines KB3.1 and L929. Moreover, the new genus Pseudorhypophila is introduced herein to accommodate Triangularia mangenotii together with several species of Zopfiella—Z. marina, Z. pilifera, and Z. submersa. These taxa formed a well-supported monophyletic clade in the recently introduced family Navicularisporaceae, located far from the type species of the respective original genera, in a phylogram based on the combined dataset sequences of the internal transcribed spacer region (ITS), the nuclear rDNA large subunit (LSU), and fragments of the ribosomal polymerase II subunit 2 (rpb2) and β-tubulin (tub2) genes. Zopfiella submersa is synonymized with P. marina due to the phylogenetic and morphological similarity. The isolation of zopfinols 1–4 and sordarins 7–9 confirms the potential of this fungal order as producers of bioactive compounds and suggests these compounds as potential chemotaxonomic markers.

Polyphenol oxidases (PPOs) are an industrially relevant family of enzymes, being involved in the postharvest browning of fruits and vegetables, as well as in human melanogenesis. Their involvement lies in their ability to oxidize phenolic or polyphenolic compounds, which subsequently form pigments. The PPO family includes tyrosinases and catechol oxidases, which, in spite of their high structural similarity, exhibit different catalytic activities. Long-standing research efforts have not yet managed to decipher the structural determinants responsible for this differentiation, as every new theory is disproved by a more recent study. In the present work, we combined biochemical along with structural data in order to better understand the function of a previously characterized PPO from Thermothelomyces thermophila (TtPPO). The crystal structure of a TtPPO variant, determined at 1.55 A resolution, represents the second known structure of an ascomycete PPO. Kinetic data for structure-guided mutants prove the implication of “gate” residue L306, residue HB1+1 (G292), and HB2+1 (Y296) in TtPPO function against various substrates. Our findings demonstrate the role of L306 in the accommodation of bulky substrates and show that residue HB1+1 is unlikely to determine monophenolase activity, as was suggested from previous studies. IMPORTANCE PPOs are enzymes of biotechnological interest. They have been extensively studied both biochemically and structurally, with a special focus on the plant-derived counterparts. Even so, explicit description of the molecular determinants of their substrate specificity is still pending. For ascomycete PPOs, only one crystal structure has been determined so far, thus limiting our knowledge on this tree branch of the family. In the present study, we report the second crystal structure of an ascomycete PPO. Combined with site-directed mutagenesis and biochemical studies, we depict the amino acids in the vicinity of the active site that affect enzyme activity and perform a detailed analysis on a variety of substrates. Our findings improve current understanding of structure-function relations of microbial PPOs, which is a prerequisite for the engineering of biocatalysts of desired properties.

The modification of gum Arabic with ferulic acid oxidation products was performed in aqueous medium, at 30 °C and pH 7.5, in the presence of Myceliophthora thermophila laccase as biocatalyst. First, this study aimed to investigate the structures of the oxidation products of ferulic acid that could possibly be covalently grafted onto gum Arabic. HPLC analyses revealed that this reaction produced several oxidation products, whose structures were investigated using LC-MS/MS analyses (liquid chromatography-mass spectrometry with mass fragmentation analyses) and NMR experiments. The chemical structure of one intermediate reaction product was fully elucidated as the 2-(4-hydroxy-3-methoxyphenyl)-4-[(4-hydroxy-3-methoxyphenyl) methylidene] cyclobutane-1, 3-dione, called by the authors cyclobutadiferulone. Secondly, this study aimed to locate the grafting of the oxidation products onto gum Arabic by performing several NMR experiments. This study did not determine how much and specifically which oxidation products were grafted but some of them were undeniably present onto modified gum Arabic, close to the glucuronic acid C5 carbon or close to the galactose C6 carbon.

Lytic polysaccharide monooxygenases (LPMOs) are known to act synergistically with glycoside hydrolases in industrial cellulolytic cocktails. However, a few studies have reported severe impeding effects of C1-oxidizing LPMOs on the activity of reducing-end cellobiohydrolases. The mechanism for this effect remains unknown, but it may have important implications as reducing-end cellobiohydrolases make up a significant part of such cocktails. To elucidate whether the impeding effect is general for different reducing-end cellobiohydrolases and study the underlying mechanism, we conducted a comparative biochemical investigation of the cooperation between a C1-oxidizing LPMO from Thielavia terrestris and three reducing-end cellobiohydrolases; Trichoderma reesei (TrCel7A), Thielavia terrestris (TtCel7A), and Myceliophthora heterothallica (MhCel7A). The enzymes were heterologously expressed in the same organism and thoroughly characterized biochemically. The data showed distinct differences in synergistic effects between the LPMO and the cellobiohydrolases; TrCel7A was severely impeded, TtCel7A was moderately impeded, while MhCel7A was slightly boosted by the LPMO. We investigated effects of C1-oxidations on cellulose chains on the activity of the cellobiohydrolases and found reduced activity against oxidized cellulose in steady-state and pre-steady-state experiments. The oxidations led to reduced maximal velocity of the cellobiohydrolases and reduced rates of substrate-complexation. The extend of these effects differed for the cellobiohydrolases and scaled with the extent of the impeding effect observed in the synergy experiments. Based on these results, we suggest that C1-oxidized chain ends are poor attack-sites for reducing-end cellobiohydrolases. The severity of the impeding effects varied considerably among the cellobiohydrolases, which may be relevant to consider for optimization of industrial cocktails.

Agaricus bisporus cultivation is based on a selective substrate prepared by a meticulous composting process where thermophilic and/or thermotolerant fungi might play an important role in straw biomass depolymerization. Since fungi have physiological limitations to survive and grow in high-temperature environments, we set out different pasteurization regimes (57 °C/6 h, 60 °C/2 h, and 68 °C/2 h) to evaluate the impact on the fungal community assembly. The fungal community profile generated by high-throughput sequencing showed shifts in community diversity and composition under different pasteurization regimes. Most of the recovered sequences belong to the Ascomycota phylum. Among 73 species detected, Mycothermus thermophilus, Talaromyces thermophilus, and Thermomyces lanuginosus were the most abundant. In the current study, we outlined that pasteurization regimes can reshape the fungal community in compost which can potentially impact the A. bisporus development.

Thermophilic fungi constitute an ecologically well-defined group, commonly found in environments wherever decomposition of organic matter takes place, making them self-heating. The importance of thermophilic fungus in ecosystems contrasts with the incompleteness of our understanding of the group’s biogeography patterns, phylogenies and coevolution relationships. Actually, the lack of data about thermophilic fungi from the Brazil is a limiting factor that also contributes for this scenario. In order to reduce this gap of knowledge, we aimed to characterize thermophilic filamentous fungi in Araucaria Forest, Atlantic Forest biome. Species identification was achieved by using internal transcribed spacers (ITS) as molecular ribosomal markers. In total, 240 heat-tolerant fungal strains were isolated and identified as Thermothielavioides terrestris, Thielavia sp., Thermoascus crustaceus, Aspergillus fumigatus, Rhizomucor miehei, Rhizomucor pusillus, and Rhizopus microsporus. All thermophilic strains exhibited optimal growth at 45 °C. T. crustaceus, T. miehei e R. pusillus were the dominant species, with the frequencies of occurrence of 35.00%, 28.33% and 23.33%, respectively. Our data reveals the apparent diversity of the Neotropical realm and may serve as reference to future studies that will try to elucidate important aspects of group.

The cloning of plasmids can be time-consuming or expensive. Yet, cloning is a prerequisite for many standard experiments for the functional analysis of genes, including the generation of deletion mutants and the localization of gene products. Here, we provide Golden Gate vectors for fast and easy cloning of gene fusion as well as gene deletion vectors applicable to diverse fungi. In Golden Gate cloning, restriction and ligation occur simultaneously in a one-pot reaction. Our vector set contains recognition sites for the commonly used type IIS restriction endonuclease BsaI. We generated plasmids for C- as well as N-terminal tagging with GFP, mRFP and 3xFLAG. For gene deletion, we provide five different donor vectors for selection marker cassettes. These include standard cassettes for hygromycin B, nourseothricin and phleomycin resistance genes as well as FLP/FRT-based marker recycling cassettes for hygromycin B and nourseothricin resistance genes. To make cloning most feasible, we provide robust protocols, namely (1) an overview of cloning procedures described in this paper, (2) specific Golden Gate reaction protocols and (3) standard primers for cloning and sequencing of plasmids and generation of deletion cassettes by PCR and split-marker PCR. We show that our vector set is applicable for the biotechnologically relevant Penicillium chrysogenum and the developmental model system Sordaria macrospora. We thus expect these vectors to be beneficial for other fungi as well. Finally, the vectors can easily be adapted to organisms beyond the kingdom fungi.

Aims Root rot caused by a group of fungi is a serious disease in mulberry. This study aims to identify and characterize Rhizopus oryzae and other fungal species associated with root rot of mulberry in India. Methods and results Rotted root samples were collected from the mulberry gardens from four states of Southern India. The majority of the isolates identified were R. oryzae, and others were saprophytic fungi, less abundant to occasional. Two methods of inoculations were tested to confirm the pathogenicity of the selected isolates and R. oryzae was found to be pathogenic on susceptible mulberry genotypes RC2 and SRDC-1. Multi gene phylogenetic analyses using the internal transcribed spacer region (ITS), actin (ACT) and translation elongation factor 1-α (TEF), identified the isolates as R. oryzae. Additionally, Ovatospora brasiliensis, Amesia nigricolor, Gongronella butleri, Myrmecridium schulzeri, Scedosporium boydii, Graphium euwallacea, Clonostachys rosea andTalaromyces spp. were also identified. Conclusion This study revealed the existence of eleven species of fungi including the first report of R. oryzae and the occurrence of weak pathogens or saprophytes that are associated with the root rot of mulberry in India. Significance and impact of the study This is the first report of R. oryzae causing Rhizopus rot of mulberry in India. Moreover, the occurrence of saprophytes associated with root rot of mulberry was identified. Further studies should focus more on the ability of these species to generate secondary metabolites and extracellular lytic enzymes as they are beneficial for the management of root rot disease.

The striatin-interacting phosphatase and kinase (STRIPAK) multi-subunit signaling complex is highly conserved within eukaryotes. In fungi, STRIPAK controls multicellular development, morphogenesis, pathogenicity, and cell-cell recognition, while in humans, certain diseases are related to this signaling complex. To date, phosphorylation and dephosphorylation targets of STRIPAK are still widely unknown in microbial as well as animal systems. Here, we provide an extended global proteome and phosphoproteome study using the wild type as well as STRIPAK single and double deletion mutants (Δpro11, Δpro11Δpro22, Δpp2Ac1Δpro22) from the filamentous fungus Sordaria macrospora. Notably, in the deletion mutants, we identified the differential phosphorylation of 129 proteins, of which 70 phosphorylation sites were previously unknown. Included in the list of STRIPAK targets are eight proteins with RNA recognition motifs (RRMs) including GUL1. Knockout mutants and complemented transformants clearly show that GUL1 affects hyphal growth and sexual development. To assess the role of GUL1 phosphorylation on fungal development, we constructed phospho-mimetic and -deficient mutants of GUL1 residues. While S180 was dephosphorylated in a STRIPAK-dependent manner, S216, and S1343 served as non-regulated phosphorylation sites. While the S1343 mutants were indistinguishable from wild type, phospho-deficiency of S180 and S216 resulted in a drastic reduction in hyphal growth, and phospho-deficiency of S216 also affects sexual fertility. These results thus suggest that differential phosphorylation of GUL1 regulates developmental processes such as fruiting body maturation and hyphal morphogenesis. Moreover, genetic interaction studies provide strong evidence that GUL1 is not an integral subunit of STRIPAK. Finally, fluorescence microscopy revealed that GUL1 co-localizes with endosomal marker proteins and shuttles on endosomes. Here, we provide a new mechanistic model that explains how STRIPAK-dependent and -independent phosphorylation of GUL1 regulates sexual development and asexual growth., Author summary In eukaryotes, the striatin-interacting phosphatase and kinase (STRIPAK) multi-subunit signaling complex controls a variety of developmental processes, and the lack of single STRIPAK subunits is associated with severe developmental defects and diseases. However, in humans, animals, as well as fungal microbes, the phosphorylation and dephosphorylation targets of STRIPAK are still largely unknown. The filamentous fungus Sordaria macrospora is a well-established model system used to study the function of STRIPAK, since a collection of STRIPAK mutants is experimentally accessible. We previously established an isobaric tag for relative and absolute quantification (iTRAQ)-based proteomic and phosphoproteomic analysis to identify targets of STRIPAK. Here, we investigate mutants that lack one or two STRIPAK subunits. Our analysis resulted in the identification of 129 putative phosphorylation targets of STRIPAK including GUL1, a homolog of the RNA-binding protein Ssd1 from yeast. Using fluorescence microscopy, we demonstrate that GUL1 shuttles on endosomes. We also investigated deletion, phospho-mimetic, and -deletion mutants and revealed that GUL1 regulates sexual and asexual development in a phosphorylation-dependent manner. Collectively, our comprehensive genetic and cellular analysis provides new fundamental insights into the mechanism of how GUL1, as a STRIPAK target, controls multiple cellular functions.

During the course of a screening for novel biologically active secondary metabolites produced by the Sordariomycetes (Ascomycota, Fungi), the ex-type strain of Jugulospora vestita was found to produce seven novel xanthone-anthraquinone heterodimers, xanthoquinodin A11 (1) and xanthoquinodins B10&ndash, 15 (2&ndash, 7), together with the already known compound xanthoquinodin B4 (8). The structures of the xanthoquinodins were determined by analysis of the nuclear magnetic resonance (NMR) spectroscopic and mass spectrometric data. Moreover, the absolute configurations of these metabolites were established by analysis of the 1H&minus, 1H coupling constants, nuclear Overhauser effect spectroscopy (NOESY) correlations, and Electronic Circular Dichroism (ECD) spectroscopic data. Antifungal and antibacterial activities as well as cytotoxicity of all compounds were tested. Xanthoquinodin B11 showed fungicidal activities against Mucor hiemalis [minimum inhibitory concentration (MIC) 2.1 &micro, g/mL], Rhodotorula glutinis (MIC 2.1 &micro, g/mL), and Pichia anomala (MIC 8.3 &micro, g/mL). All the compounds 1&ndash, 8 displayed anti-Gram-positive bacteria activity (MIC 0.2&ndash, 8.3 &micro, g/mL). In addition, all these eight compounds showed cytotoxicity against KB 3.1, L929, A549, SK-OV-3, PC-3, A431, and MCF-7 mammalian cell lines. The six novel compounds (1&ndash, 3, 5&ndash, 7), together with xanthoquinodin B4, were also found in the screening of other strains belonging to Jugulospora rotula, revealing the potential chemotaxonomic significance of the compound class for the genus.

A recombinant strain producing a complex of extracellular enzymes including chitinase from Myceliophtora thermophila was created based on the fungus Penicillium verruculosum. The activity of the enzyme preparations obtained from the cultural fluid of the producer strain was 0.55, 0.53, and 0.66 U/mg protein with chitin and chitosans with the molecular weight of 200 and 1000 kDa, respectively. The temperature optimum for the recombinant chitinase was 52-65°C; the pH optimum was 4.5-6.2, which corresponded to the published data for this class of the enzymes. The content of heterologous chitinase in the obtained enzyme preparations was 47% of total protein content in the cultural fluid. Enzyme preparations produced by the recombinant P. verruculosum XT403 strain and containing heterologous chitinase were able to degrade the mycelium of micromycetes, including phytopathogenic ones, and were very efficient in the bioconversion of microbiological industry waste.

Background GH74 xyloglucanases are composed of two separate domains connected by two unstructured peptides. Previously, a hypothesis was made that the movement of domains may affect the enzyme mechanism of catalysis. Methods The molecular dynamics (MD) simulations of endo-processive xyloglucanases from Paenibacillus odorifer (PoGH74cat) and Myceliophthora thermophila (MtXeg74A) were carried out. Results MD simulations for both enzymes in complex with XXLG and XGXXLG oligosaccharides confirmed the possibility of domain movement. In the case of MtXeg74A, changes in the distances between Cα atoms of aromatic residues involved in xyloglucan binding in −3 and +3 subsites of the active site cleft and those of selected residues on the opposite side of the cleft reached values up to 10–12 A. For PoGH74cat the conformational changes were less pronounced. In MtXeg74A variants, the deletion of loop 1, which partially closes the entrance to the cleft, and the additional double mutation of two Trp residues in +3 and +5 subsites caused the enhanced mobility of the XGXXLG and also induced changes in topography of the cleft. Conclusions These findings demonstrate the possibility of existence of GH74 xyloglucanases in a more open and more closed enzyme conformation. The enzyme in an open conformation may more easily accommodate the branched polysaccharide, while its transition to the closed conformation, together with loop 1 function, should aid processivity. General significance Our results provide an insight into a mechanism of action of GH74 xyloglucanases and may be useful for discussing the catalytic mechanisms of glycoside hydrolases from other families.

In mitochondria, β-barrel outer membrane proteins mediate protein import, metabolite transport, lipid transport, and biogenesis. The Sorting and Assembly Machinery (SAM) complex consists of three proteins that assemble as a 1:1:1 complex to fold β-barrel proteins and insert them into the mitochondrial outer membrane. We report cryoEM structures of the SAM complex from Myceliophthora thermophila, which show that Sam50 forms a 16-stranded transmembrane β-barrel with a single polypeptide-transport-associated (POTRA) domain extending into the intermembrane space. Sam35 and Sam37 are located on the cytosolic side of the outer membrane, with Sam35 capping Sam50, and Sam37 interacting extensively with Sam35. Sam35 and Sam37 each adopt a GST-like fold, with no functional, structural, or sequence similarity to their bacterial counterparts. Structural analysis shows how the Sam50 β-barrel opens a lateral gate to accommodate its substrates., The Sorting and Assembly Machinery (SAM) complex folds beta-barrel proteins and inserts them into the mitochondrial outer membrane. Here authors report cryoEM structures of the SAM complex from Myceliophthora thermophila, which reveals a GST-like fold for Sam35 and Sam37 and sheds light on how the Sam50 beta-barrel opens a lateral gate to accommodate its substrates.

The Diaporthales (Sordariomycetes) includes many important families of plant pathogenic fungi, such as the notorious Cryphonectriaceae. The aim of the present study was to reevaluate this family, along with other families in Diaporthales. Based on phylogenetic analyses using combined sequence data of the internal transcribed spacer (ITS) region, large subunit of the nrDNA (28S), and the translation elongation factor 1-alpha (tef1-α) and DNA-directed RNA polymerase II second largest subunit (rpb2) genes, Cryphonectriaceae is separated into two subclades, comprising 21 genera and 55 species. Foliocryphiaceae, fam. nov., is morphologically and phylogenetically its closest relative but is distinct due to its phylogeny and dimorphic conidia. Mastigosporellaceae, fam. nov., is distinguished from other families in Diaporthales by owning apical conidial appendages. Neocryphonectria, gen. nov., within the family Foliocryphiaceae, with two species associated with Carpinus canker in China, is characterized by fusoid, aseptate macroconidia. Additionally, two new combinations are proposed, namely, Cryphonectria citrine, based on Chromendothia citrine, and Cytospora viridistroma, based on Endothia viridistoma. Based on results obtained in this study, 31 families are accepted into Diaporthales.

To construct a new thermophilic platform for glucoamylase production through 2A peptide strategy combined with CRISPR-Cas9 system using Myceliophthora thermophila as host, thermophilic filamentous fungus with industrial attractiveness to produce enzymes and chemicals from biomass. We adapted the viral 2A peptide approach for M. thermophila and constructed a bicistronic vector for co-expressing two heterologous genes MhglaA and egfp. We obtained positive transformants OE-MhglaA-gfp overexpressing MhGlaA-9 ×His-2A-eGFP through convenient fluorescence screening, western blotting and RT-qPCR. We purified and characterized the recombinant MhGlaA, which exhibited stability in a broader pH range of 3.0–9.0 and thermostable stability at 65 °C, suggesting its potential industrial application. Furthermore, to improve glucoamylase secretion, we genetically engineered the obtained strain OE-MhglaA-gfp through our efficient CRISPR/Cas9 system and generated the quintuple mutant OE-MhglaA-gfpOE-amyRΔalp-1Δres-1Δcre-1, in which protein productivity and amylase activity were increased by approximately 12.0- and 8.2-fold compared with WT. The 2A peptide approach worked well in M. thermophila and can be used to heterologously co-express two different proteins, and thus in combination with efficient CRISPR-Cas system will accelerate establishing hyper-secretion platforms for biotechnological applications.

Soil fungi in desert ecosystems are adapted to harsh environmental conditions such as high soil surface temperatures and limited organic matter and water. Given limited carbon inputs from plant material, heterotrophic fungi likely use unconventional sources of carbon in these systems. A baiting method was used to culture keratinophilic fungi from biocrust and rhizosphere soils in an arid grassland in Utah, USA. Fungi were baited using llama and sheep wool, horsehair, and snakeskin on two media, and pure cultures were identified using ITS and LSU rRNA sequences. One hundred-eighteen fungal colonies were grown, representing a total of 32 Operational Taxonomic Units (OTUs) at 97 % similarity. Cultures were dominated by the phylum Ascomycota (88 %) followed by Mucoromycota (8.6 %) and Basidiomycota (3.4 %). The orders Pleosporales, Eurotiales, Hypocreales, and Sordariales were commonly isolated, with the dominant taxa Alternaria (27 %), Aspergillus (22 %), Fusarium (11 %), and Chaetomium (8%). Thirty percent of the fungi isolated have the capacity to degrade keratin in vitro using a keratin azure assay, with Penicillium showing the highest degradation followed by Geomyces, Alternaria, and Fusarium. Although keratin degraders can be infectious, dermatophytes associated with skin infections were not isolated in culture or detected in Illumina sequencing. Illumina sequencing was used to determine general patterns in seasonal variation and habitat preference of keratinophiles. Alternaria was the most abundant genus with >70 % of the sequences. The combination of Illumina data with culture-dependent approaches facilitated the characterization of a specialized community and confirmed the low abundance of dermatophytes in this arid site.

A total of 96 isolates were obtained from 375 segments, isolated from the healthy roots, stems, leaves, hypocotyls and flowers of Bruguiera sexangula (Lour.) Poir. collected at the Dong Zhai Gang Mangrove Garden on Hainan Island, and 20 independent representative isolates were identified using a combination of morphological and molecular approaches. The most frequent endophytic fungal species isolated were Diaporthe phaseolorum (relative frequency = 31.2%). The Shannon–Wiener diversity and Simpson’s diversity index both showed that stems possessed the highest diversity compared to the other tissues estimated. Ethyl acetate extracts and the isolated metabolites were tested for antimicrobial activity using the serial dilution technique and for antioxidant activity using 2,2′-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonicacid) (ABTS) radical-scavenging capacity assays, respectively. The fungal isolate HL18 (Gelasinospora endodonta) cultured on Czapek’s agar (CA) displayed a broad spectrum of antimicrobial activities and was significantly active against Escherichia coli (MIC = 0.0625 mg ml−1). Antioxidant assays showed that most of the fungal isolates (60.0%) exhibited some degree of antioxidant capacity (%RSA > 50%). The stain HL14 (Pestalotiopsis mangiferae) grown on potato dextrose agar (PDA) exhibited the highest DPPH and ABTS radical-scavenging capability with IC50 values of 0.717 ± 0.012 mg ml−1 and 0.787 ± 0.027 mg ml−1, respectively. Furthermore, five known secondary metabolites 1–5 were isolated and identified from HL-14. Compounds 1 and 5 exhibited weak antioxidant activity.

A novel phospholipase C gene (TtPLC) from Thielavia terrestris CAU709 was cloned and efficiently expressed in Pichia pastoris. The deduced protein sequence of TtPLC shared the highest identity of 33% with the characterized phospholipase C from Arabidopsis thaliana. The highest phospholipase C yield of 98, 970 U mL−1, with a protein concentration of 4.9 mg mL−1 was obtained by high-cell density fermentation in a 5-L fermentor. The recombinant enzyme (TtPLC) was purified to homogeneity with a recovery yield of 59.1% and a specific activity of 22, 910 U mg−1. TtPLC was most active at pH 6.5 and 55 °C, respectively. It was stable within the pH range of 4.5–8.0 and up to 45 °C. The enzyme exhibited excellent stability in different surfactants and organic solvents, including Tween 20 (147.6%), Tween 40 (180.6%), Tween 60 (205.4%), cyclohexane (160.0%), n-octane (178.2%), n-heptane (180.7%), acetone (187.5%) etc. The application of TtPLC in crude soybean oil degumming process significantly reduced the residual phosphorus content from 135.4 mg kg−1 to 7.9 mg kg−1 under the optimized conditions, which satisfied the requirement of environmental friendly physical refining process for oil refining industry. Therefore, TtPLC should be a good candidate in oil refining industry.

Biochemistry 59(36), 3347 - 3358 (2020). doi:10.1021/acs.biochem.0c00312, Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes which cleave polysaccharide substrates oxidatively. First discovered because of their action on recalcitrant crystalline substrates (chitin and cellulose) a number of LPMOs are now reported to act on soluble substrates including oligosaccharides. However, crystallographic complexes with oligosaccharides have only been reported for a single LPMO so far, an enzyme from the basidiomycete fungus Lentinus similis (LsAA9_A). Here we present a more detailed comparative study of LsAA9_A and an LPMO from the ascomycete fungus Collariella virescens (CvAA9_A) with which it shares 41.5% sequence identity. LsAA9_A is considerably more thermostable than CvAA9_A, and the structural basis for the difference has been investigated. We have compared the patterns of oligosaccharide cleavage and the patterns of binding in several new crystal structures explaining the basis for product preferences by the two enzymes. Obtaining structural information on complexes of LPMOs with carbohydrates has proven very difficult in general judging from the structures reported in the literature thus far and this can only partly be attributed to low affinity for small substrates. We have thus evaluated the use of differential scanning fluorimetry as a guide to obtaining complex structures. Furthermore, an analysis of crystal packing of LPMOs and glycoside hydrolases, corroborates the hypothesis that active site occlusion is a very significant problem for LPMO-substrate interaction analysis by crystallography, due to their relatively flat and extended substrate binding sites., Published by American Chemical Society, Columbus, Ohio

Mannanase 19287 enzyme is an engineered β-mannanase that can be added to diets for animals raised for human consumption to hydrolyze β-mannans. Established toxicological analyses were conducted with the enzyme preparation to ensure the safety of this product for the intended use. The mannanase 19287 preparation was produced with Thermothelomyces thermophilus strain DSM 33149. In vitro toxicity studies presented here used dosages of the mannanase 19287 test articles up to 5000 μg/plate. For in vivo toxicity studies in Wistar rats, test articles were administered at 5.1 mg/L for inhalation toxicity and up to 15,000 mg/kg rat feed for oral toxicity, based on the Total Organic Solids (TOS) content in each test article. No treatment related adverse effects were reported in any study. The No Observed Adverse Effect Levels in the high dose group of the subchronic oral toxicity study were calculated as 1117–1298 mg TOS/kg bw/day in rats. Comparing these values to an Estimated Daily Intake for poultry demonstrated safety factors larger than 5000. Our results confirm that T. thermophilus fulfills the recognized safety criteria for the manufacture of food enzyme preparations and represent the first peer-reviewed safety evaluation of an enzyme preparation by T. thermophilus. The results of the toxicity studies presented herein attest to the safety of the mannanase 19287 enzyme for its intended use.

The STRIPAK complex is involved in growth, cell fusion, development and signaling pathways, and thus malfunctions in the human STRIPAK complex often result in severe neuronal diseases and cancer. Despite the high degree of general conservation throughout the complex, several STRIPAK complex-associated small coiled-coil proteins of animals and yeasts are not conserved across species. As there are no data for filamentous ascomycetes, we addressed this through affinity purification with HA-tagged striatin ortholog PRO11 in Sordaria macrospora. Combining the method with liquid chromatography-mass spectrometry, we were able to co-purify STRIPAK complex interactor 1 (SCI1), the first STRIPAK-associated small coiled-coil protein in filamentous ascomycetes. Using yeast two-hybrid experiments, we identified SCI1 protein regions required for SCI1-PRO11 interaction, dimerization of SCI1 and interaction with other STRIPAK components. Further, both proteins PRO11 and SCI1 co-localize with the nuclear basket protein SmPOM152 at the nuclear envelope. Expression of the gene sci1 occurs during early developmental stages of S. macrospora, and the protein SCI1 in combination with PRO11 is required for cell fusion, vegetative growth and sexual development. The results of the present study will help to understand the underlying molecular mechanisms of STRIPAK signaling and function in cellular development and diseases in higher eukaryotes.