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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]

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]

Abstract 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.

Most enzymes used in industry originate from microorganisms, such as endoglucanases that degrade cellulose. In this context, Thermothelomyces heterothallicus is a thermophilic filamentous fungus capable of producing substantial amounts of this enzyme. This study aimed to produce, purify, and characterize an endoglucanase from the novel strain of T. heterothallicus PA2S4T, utilizing orange peel as a carbon source, and applying this enzyme to denim fabric. Optimization of enzymatic production through experimental design yielded the best results for endoglucanases at the lowest temperature (28 °C) and shortest cultivation time (60 h), using the highest concentration of orange peel (4.2%), resulting in an endoglucanase activity of 61 U.mL−1. Following purification steps, sample isolation was confirmed by observing a single band on SDS-PAGE at a molecular weight of 36.3 kDa. The enzymatic reaction exhibited an optimal pH of 7.0, with stability over a wide pH range (from 3.0 to 8.0). The enzyme showed optimal activity at 50 °C and remained thermally stable up to this temperature. Application of the enzyme in the bio-polishing step of denim jeans manufacturing, at concentrations of 1–5 U.mL−1, resulted in weight losses of 2–5%. The best weight loss results were found after 12 h of treatment, at 50 ºC and pH 5.0. Additionally, the scanning electron microscopy showed that 2 and 5 U.mL−1 of enzyme improved appearance of denim fabric by removing protuberances and irregularities compared to untreated fabric. [ABSTRACT FROM AUTHOR]

Abstract Amylases, crucial in various industries such as food, textile, and biofuels, require optimized production and biochemical characterization. Thus, this study aimed to produce amylases from Rhizopus sp. I 1.2.1, characterize the cultivation conditions and the synthesized enzyme. The fungus was molecularly identified with 99.06% homology in the LSU gene, 98.08% identity in the ITS region, and placed phylogenetically closer to Rhizopus arrhizus CBS 112,07. The LSU gene and ITS region sequences were deposited in GenBank. The CP medium was optimal for amylase production by Rhizopus arrhizus, with peak activity on the 6th day. Supplementation with urea significantly increased amylolytic activity by 110-fold. CP salts outperformed other salts in enzyme production, achieving a maximum amylase activity of 15.5 U/ml. The pH 6.0 was optimal for amylase production, with higher specific activity at pH 5.0. Pumpkin peel and pineapple peel were the best carbon sources for amylase production with an amylolytic activity of 18 and 16 U/mL, respectively. The amylase production showed a significant increase when pineapple peel was used in the presence of glucose 0.5% showing an activity of 22.4 U/mL, representing a 1.5- and 1.6-fold increase over the control. The optimal reaction of the amylase was observed at pH 6.0 and 60 °C. The enzyme remained stable for 240 minutes at 50-55 °C and at pH 4.0-7.0. Amylase was inhibited by glucose concentration and certain salts, but EDTA and K2SO4 increased activity by 25%. These results suggest industrial potential based on residual carbon source use, cultivation conditions, and crude enzyme stability.

The growth and activity of adherent cells can be enabled or enhanced through attachment to a solid surface. For food and beverage production processes, these solid supports should be food-grade, low-cost, and biocompatible with the cell of interest. Solid supports that are edible can be a part of the final product, thus simplifying downstream operations in the production of fermented beverages and lab grown meat. We provide proof of concept that edible filamentous fungal pellets can function as a solid support by assessing the attachment and growth of two model cell types: yeast, and myoblast cells. The filamentous fungus Aspergillus oryzae was cultured to produce pellets with 0.9 mm diameter. These fugal pellets were inactivated by heat or chemical methods and characterized physicochemically. Chemically inactivated pellets had the lowest dry mass and were the most hydrophobic. Scanning electron microscope images showed that both yeast and myoblast cells naturally adhered to the fungal pellets. Over 48 h of incubation, immobilized yeast increased five-fold on active pellets and six-fold on heat-inactivated pellets. Myoblast cells proliferated best on heat-treated pellets, where viable cell activity increased almost two-fold, whereas on chemically inactivated pellets myoblasts did not increase in the cell mass. These results support the use of filamentous fungi as a novel cell immobilization biomaterial for food technology applications.

Abstract Background The well-known industrial fungus Trichoderma reesei has an excellent capability of secreting a large amount of cellulases and xylanases. The induced expression of cellulase and xylanase genes is tightly controlled at the transcriptional level. However, compared to the intensive studies on the intricate regulatory mechanism of cellulase genes, efforts to understand how xylanase genes are regulated are relatively limited, which impedes the further improvement of xylanase production by T. reesei via rational strain engineering. Results To identify transcription factors involved in regulating xylanase gene expression in T. reesei, yeast one-hybrid screen was performed based on the promoters of two major extracellular xylanase genes xyn1 and xyn2. A putative transcription factor named XTR1 showing significant binding capability to the xyn1 promoter but not that of xyn2, was successfully isolated. Deletion of xtr1 significantly increased the transcriptional level of xyn1, but only exerted a minor promoting effect on that of xyn2. The xylanase activity was increased by ~ 50% with XTR1 elimination but the cellulase activity was hardly affected. Subcellular localization analysis of XTR1 fused to a green fluorescence protein demonstrated that XTR1 is a nuclear protein. Further analyses revealed the precise binding site of XTR1 and nucleotides critical for the binding within the xyn1 promoter. Moreover, competitive EMSAs indicated that XTR1 competes with the essential transactivator XYR1 for binding to the xyn1 promoter. Conclusions XTR1 represents a new transcriptional repressor specific for controlling xylanase gene expression. Isolation and functional characterization of this new factor not only contribute to further understanding the stringent regulatory network of xylanase genes, but also provide important clues for boosting xylanase biosynthesis in T. reesei.

Honey bees are threatened by many fungal, parasitic and bacterial diseases. This study was aimed at identifying filamentous fungi colonizing the intestinal region of dead adult honey bees and determining resultant mycotoxin activity and its potential adverse implications for bee and human health by the PCR and UHPLC-Orbitrap- HRMS methods. For this purpose, dead bees were collected from the ground in front of 95 hives displaying mass mortality in the Hatay, Iğdır and Bingöl provinces, and 22 filamentous and 6 yeast-like fungi were isolated and identified from the intestinal region of these bees. Of the filamentous fungal isolates, 8 were identified as Aspergillus spp., 3 as Fusarium spp., 2 as Alternaria spp. 5 as Penicillium spp., 4 as Mucor spp., and 2 as Rhizopus spp.. The PCR analysis of the filamentous fungi using primers targeting the aflatoxin and ochratoxin A genes revealed the presence of aflatoxin in only 1 out of the 22 samples. Aflatoxin and ochratoxin were not detected in any of the other samples. The UHPLC-Orbitrap-HRMS method revealed the presence of aflatoxin B1 in 17, both B1 and B2 in 6, B1, B2 and G1 in 5, and aflatoxin G2 in 1 of the filamentous fungal samples. The results obtained in the present study suggest that filamentous fungi may produce mycotoxins in the intestinal region of honey bees, and thereby, honey bees may distribute mycotoxins into the environment and cause indirect adverse effects on human and animal health.

The filamentous fungus Cunninghamella echinulata is a model of mammalian xenobiotic metabolism. Under certain conditions it grows as a biofilm, which is a natural form of immobilisation and enables the fungus to catalyse repeated biotransformations. Putative signalling molecules produced by other Cunninghamella spp., such as 3-hydroxytyrosol and tyrosol, do not affect the biofilm growth of C. echinulata , suggesting that it employs a different molecule to regulate biofilm growth. In this paper we report that 2-phenylethanol is produced in higher concentrations in planktonic cultures of C. echinulata than when the fungus is grown as a biofilm. We demonstrate that exogenously added 2-phenylethanol inhibits biofilm growth of C. echinulata but has no effect on planktonic growth. Furthermore, we show that addition of 2-phenylethanol to established C. echinulata biofilm causes detachment. Therefore, we conclude that this molecule is produced by the fungus to regulate biofilm growth. • 2-Phenylethanol regulates biofilm growth in the fungus Cunninghamella echinulata. • Planktonic cultures produce higher concentrations of the metabolite. • C. echinulata biofilm growth is specifically inhibited by 2-phenylethanol. • 2-Phenylethanol causes detachment of established C. echinulata biofilm. [ABSTRACT FROM AUTHOR]

Honey bees are threatened by many fungal, parasitic and bacterial diseases. This study was aimed at identifying filamentous fungi colonizing the intestinal region of dead adult honey bees and determining resultant mycotoxin activity and its potential adverse implications for bee and human health by the PCR and UHPLC-Orbitrap-HRMS methods. For this purpose, dead bees were collected from the ground in front of 95 hives displaying mass mortality in the Hatay, Iğdır and Bingöl provinces, and 22 filamentous and 6 yeast-like fungi were isolated and identified from the intestinal region of these bees. Of the filamentous fungal isolates, 8 were identified as Aspergillus spp., 3 as Fusarium spp., 2 as Alternaria spp. 5 as Penicillium spp., 4 as Mucor spp., and 2 as Rhizopus spp. The PCR analysis of the filamentous fungi using primers targeting the aflatoxin and ochratoxin A genes revealed the presence of aflatoxin in only 1 out of the 22 samples. Aflatoxin and ochratoxin were not detected in any of the other samples. The UHPLC-Orbitrap-HRMS method revealed the presence of aflatoxin B1 in 17, both B1 and B2 in 6, B1, B2 and G1 in 5, and aflatoxin G2 in 1 of the filamentous fungal samples. The results obtained in the present study suggest that filamentous fungi may produce mycotoxins in the intestinal region of honey bees, and thereby, honey bees may distribute mycotoxins into the environment and cause indirect adverse effects on human and animal health. [ABSTRACT FROM AUTHOR]

The filamentous fungus Colletotrichum gloeosporioides is the causative agent of one of the most serious diseases that damage plant fruit. In this study, we discovered and experimentally characterized a new gene in Colletotrichum gloeosporioides named CgloRPCYG. The CRISPR/Cas9 knockout mutant and complementary strain of CgloRPCYG were then obtained by polyethylene glycol (PEG)-mediated protoplast transformation to determine the related phenotypes. Compared with the wild-type strain and complementary mutant, the pathogenicity of the CRISPR/Cas9 knockout mutant was significantly decreased, the conidial yield was significantly reduced, and conidial germination was significantly delayed. These data indicate that CgloRPCYG contributes to pathogenicity, conidial yield, and germination in C. gloeosporioides. The successful application of the CRISPR/Cas9 system in C. gloeosporioides also confirms its utility in filamentous fungi for fundamental research and practical application. Furthermore, CgloRPCYG is a potential target gene for use in the development of plant protection technologies, such as spray-induced gene silencing, with the aim of controlling plant anthracnose disease caused by C. gloeosporioides. [ABSTRACT FROM AUTHOR]

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Introduction: The emergency use of vaccines has been the most efficient way to control the coronavirus disease 19 (COVID-19) pandemic. However, the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern has reduced the efficacy of currently used vaccines. The receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein is the main target for virus neutralizing (VN) antibodies. Methods: A SARS-CoV-2 RBD vaccine candidate was produced in the Thermothelomyces heterothallica (formerly, Myceliophthora thermophila) C1 protein expression system and coupled to a nanoparticle. Immunogenicity and efficacy of this vaccine candidate was tested using the Syrian golden hamster (Mesocricetus auratus) infection model. Results: One dose of 10-μg RBD vaccine based on SARS-CoV-2 Wuhan strain, coupled to a nanoparticle in combination with aluminum hydroxide as adjuvant, efficiently induced VN antibodies and reduced viral load and lung damage upon SARS-CoV-2 challenge infection. The VN antibodies neutralized SARS-CoV-2 variants of concern: D614G, Alpha, Beta, Gamma, and Delta. Discussion: Our results support the use of the Thermothelomyces heterothallica C1 protein expression system to produce recombinant vaccines against SARS-CoV-2 and other virus infections to help overcome limitations associated with the use of mammalian expression system. [ABSTRACT FROM AUTHOR]

Insects have adapted to a multitude of environmental conditions, including the presence of xenobiotic noxious substances. Environmental microorganisms, particularly rich on ephemeral resources, employ these noxious chemicals in a chemical warfare against predators and competitors, driving co‐evolutionary adaptations. In order to analyse how environmental microbes may be driving such evolutionary adaptations, we experimentally evolved Drosophila melanogaster populations by exposing larvae to the toxin‐producing mould Aspergillus nidulans that infests the flies' breeding substrate. To disentangle the effects of the mycotoxin Sterigmatocystin from other substrate modifications inflicted by the mould, we used the following four selection regimes: (i) control without fungus, (ii) A. nidulans wild type, (iii) a mutant of A. nidulans ΔlaeA with impaired toxin production, (iv) synthetic Sterigmatocystin. Experimental evolution was carried out in five independent D. melanogaster populations each, for a total of 11 generations. We further combined our evolution experiment with transcriptome analysis to identify evolutionary shifts in gene expression due to the selection regimes and mould confrontation. Populations that evolved in presence of the toxin‐producing mould or the pure mycotoxin rapidly adapted to the respective conditions and showed higher viability in subsequent confrontations. Yet, mycotoxin‐selected populations had no advantage in A. nidulans wild type confrontation. Moreover, distinctive changes in gene expression related to the selection‐regime contrast were only associated with the toxin‐producing‐fungus regime and comprised a narrow set of genes. Thus, it needs the specific conditions of the selection agent to enable adaptation to the fungus. [ABSTRACT FROM AUTHOR]

This study evaluated an innovative method to improve soil erosion resistance by fungi. An edible filamentous fungus, Pleurotus ostreatus, was included in the study. The result showed that Pleurotus ostreatus is able to grow and completely cover the circular surface of 60.20 mm diameter in 15 days when inoculated on the top surface and in 20 days when inoculated 5 mm under the soil surface. The erosion resistance of fungi mediated soil samples was evaluated by a Simplified Scour Resistance Test (SSRT). The results showed that fungi-mediated sand samples demonstrated much higher erosion resistance compared with control sand sample, both in terms of increasing the critical flow velocity and reducing the slope of erosion rate versus flow velocity curve. The mechanism of improved erosion resistance was investigated. The microstructure measured by SEM test showed that the sand particles were entangled by fungal fibers. Chemical precipitations were observed on sand surface and attached to fungal hyphae, which were identified as calcite, calcium oxalate monohydrate and calcium oxalate dihydrate by FTIR spectra and XRD analysis. The fungi mycelium surface showed hydrophobic behaviors with an average measured contact angle of 116.42°. The hydrophobicity generated slip along the interface. Analyses with computational fluid dynamics (CFD) model indicated that the slip due to the hydrophobicity of fungi reduced the shear stress at the water-soil interface, which also improved the erosion resistance by fungi-mediated sand. [ABSTRACT FROM AUTHOR]

Chitosan is a non-cytotoxic polysaccharide that, upon hydrolysis, releases oligomers of different sizes that may have antioxidant, antimicrobial activity and the inhibition of cancer cell growth, among other applications. It is, therefore, a hydrolysis process with great biotechnological relevance. Thus, this study aims to use a crude enzyme concentrate (CEC) produced by a filamentous fungus to obtain oligomers with different molecular weights. The microorganism was cultivated in a liquid medium (modified Czapeck—with carboxymethylcellulose as enzyme inducer). The enzymes present in the CEC were identified by LC-MS/MS, with an emphasis on cellobiohydrolase (E.C 3.2.1.91). The fungus of the Aspergillus genus was identified by amplifying the ITS1-5.8S-ITS2 rDNA region and metaproteomic analysis, where the excreted enzymes were identified with sequence coverage greater than 84% to A. nidulans. Chitosan hydrolysis assays compared the CEC with the commercial enzyme (Celluclast 1.5 L®). The ability to reduce the initial molecular mass of chitosan by 47.80, 75.24, and 93.26% after 2.0, 5.0, and 24 h of reaction, respectively, was observed. FTIR analyses revealed lower absorbance of chitosan oligomers' spectral signals, and their crystallinity was reduced after 3 h of hydrolysis. Based on these results, we can conclude that the crude enzyme concentrate showed a significant technological potential for obtaining chitosan oligomers of different sizes. [ABSTRACT FROM AUTHOR]

Background: Camels are known for their survival under harsh and nutrient-deficient climates. Camel rumen ecosystem presents a unique opportunity to study the ruminal microbes helping the camel in this task. The genus Aspergillus is the extensively studied filamentous fungus due to its ability to secret industrially important enzymes. The present study was aimed to isolate and characterize microbes with lignocellulolytic capacity from camel rumen. Results: The fungal isolate Aspergillus sydowii C6d, isolated from camel rumen, was sequenced and analysed for its CAZyme content responsible for lignocellulose degradation. The C6d isolate was evaluated for its capacity to produce cellulase, pectinase, xylanase, and amylase with their respective assays and further evaluated for their optimum pH. The genome sequencing and assembly resulted in 32.27 Mb of genome size with a GC % of 50.59. The CAZyme analysis revealed that the C6d produced 543 polysaccharide-degrading CAZymes amongst which, 148 CAZymes were potentially involved in lignocellulose degradation. The genomic comparison of the C6d with 30 commonly used lignocellulolytic fungi (white rot, brown rot, and soft rot fungus) showed the enrichment of cellulolytic and pectinolytic CAZymes in C6d genome as compared to others. The saccharification of lignocellulosic substrate wheat straw resulted in the release of 50.85% of reducing sugars. Conclusions: The study provides important insights into the CAZymes responsible for lignocellulolytic ability in the novel fungus Aspergillus sydowii C6d isolated from camel rumen and presents a valuable source of CAZymes to be further evaluated for potential biotechnological applications.How to cite: Tulsani NJ, Jakhesara SJ, Hinsu AT, et al. Genome analysis and CAZy repertoire of a novel fungus Aspergillus sydowii C6d with lignocellulolytic ability isolated from camel rumen. Electron J Biotechnol 2022;59. https://doi.org/10.1016/j.ejbt.2022.06.004.

The disaccharide trehalose has long been recognized for its role as a stress solute, but in recent years some of the protective effects previously ascribed to trehalose have been suggested to arise from a function of the trehalose biosynthesis enzyme trehalose-6-phosphate (T6P) synthase that is distinct from its catalytic activity. In this study, we use the maize pathogenic fungus Fusarium verticillioides as a model to explore the relative contributions of trehalose itself and a putative secondary function of T6P synthase in protection against stress as well as to understand why, as shown in a previous study, deletion of the TPS1 gene coding for T6P synthase reduces pathogenicity against maize. We report that a TPS1 -deletion mutant of F. verticillioides is compromised in its ability to withstand exposure to oxidative stress meant to simulate the oxidative burst phase of maize defense and experiences more ROS-induced lipid damage than the wild-type strain. Eliminating T6P synthase expression also reduces resistance to desiccation, but not resistance to phenolic acids. Expression of catalytically-inactive T6P synthase in the TPS1 -deletion mutant leads to a partial rescue of the oxidative and desiccation stress-sensitive phenotypes, suggesting the importance of a T6P synthase function that is independent of its role in trehalose synthesis. • T6P synthase expression protects against oxidative stress and lipid damage. • T6P synthase expression protects against desiccation stress. • Protective effect of T6P synthase due to role distinct from transferase activity. [ABSTRACT FROM AUTHOR]

Fungi possess extraordinary strength in attachment to biotic and abiotic surfaces. This review focuses on adhesion mechanisms of yeast and filamentous fungi and the proposed combination of the adhesive forces of both organisms in an immobilization system called yeast biocapsules, whereby Saccharomyces cerevisiae cells are attached to the hyphae of Penicillium chrysogenum. The natural adherent properties of each organism, one multicellular and another unicellular, allow yeast to be fixated securely on the filamentous fungi and complete alcoholic fermentation. Following alcoholic fermentation, the hyphae become an inert support for yeast cells while maintaining shape and integrity. Biocapsules have been used successfully in both wine and bioethanol production. Investigation of the potential genes involved in fungal-yeast fusion suggests that natural hydrophobic interactions of both organisms play a major role. Analysis of the possible mechanisms involved in fungus and yeast adhesion, future perspectives on improving yeast immobilization, and proposed applications of the biocapsules are explored.

Abstract Background A total of 11 β-glucosidases are predicted in the genome of Trichoderma reesei, which are of great importance for regulating cellulase biosynthesis. Nevertheless, the relevant function and regulation mechanism of each β-glucosidase remained unknown. Results We evidenced that overexpression of cel1b dramatically decreased cellulase synthesis in T. reesei RUT-C30 both at the protein level and the mRNA level. In contrast, the deletion of cel1b did not noticeably affect cellulase production. Protein CEL1B was identified to be intracellular, being located in vacuole and cell membrane. The overexpression of cel1b reduced the intracellular pNPGase activity and intracellular/extracellular glucose concentration without inducing carbon catabolite repression. On the other hand, RNA-sequencing analysis showed the transmembrane transport process and endoplasmic reticulum function were affected noticeably by overexpressing cel1b. In particular, some important sugar transporters were notably downregulated, leading to a compromised cellular uptake of sugars including glucose and cellobiose. Conclusions Our data suggests that the cellulase inhibition by cel1b overexpression was not due to the β-glucosidase activity, but probably the dysfunction of the cellular transport process (particularly sugar transport) and endoplasmic reticulum (ER). These findings advance the knowledge of regulation mechanism of cellulase synthesis in filamentous fungi, which is the basis for rationally engineering T. reesei strains to improve cellulase production in industry.

ABSTRACT Many filamentous fungi develop a conidiation process as an essential mechanism for their dispersal and survival in natural ecosystems. However, the mechanisms underlying conidial persistence in environments are still not fully understood. Here, we report that autophagy is crucial for conidial lifespans (i.e., viability) and vitality (e.g., stress responses and virulence) in the filamentous mycopathogen Beauveria bassiana. Specifically, Atg11-mediated selective autophagy played an important, but not dominant, role in the total autophagic flux. Furthermore, the aspartyl aminopeptidase Ape4 was found to be involved in conidial vitality during dormancy. Notably, the vacuolar translocation of Ape4 was dependent on its physical interaction with autophagy-related protein 8 (Atg8) and associated with the autophagic role of Atg8, as determined through a truncation assay of a critical carboxyl-tripeptide. These observations revealed that autophagy acted as a subcellular mechanism for conidial recovery during dormancy in environments. In addition, a novel Atg8-dependent targeting route for vacuolar hydrolase was identified, which is essential for conidial exit from a long-term dormancy. These new insights improved our understanding of the roles of autophagy in the physiological ecology of filamentous fungi as well as the molecular mechanisms involved in selective autophagy. IMPORTANCE Conidial environmental persistence is essential for fungal dispersal in ecosystems while also serving as a determinant for the biocontrol efficacy of entomopathogenic fungi during integrated pest management. This study identified autophagy as a mechanism to safeguard conidial lifespans and vitality postmaturation. In this mechanism, the aspartyl aminopeptidase Ape4 translocates into vacuoles via its physical interaction with autophagy-related protein 8 (Atg8) and is involved in conidial vitality during survival. The study revealed that autophagy acted as a subcellular mechanism for maintaining conidial persistence during dormancy, while also documenting an Atg8-dependent targeting route for vacuolar hydrolase during conidial recovery from dormancy. Thus, these observations provided new insight into the roles of autophagy in the physiological ecology of filamentous fungi and documented novel molecular mechanisms involved in selective autophagy.

The development of engineered living materials (ELMs) has recently attracted significant attention from researchers across multiple disciplines. Fungi-derived ELMs represent a new type of macroscale, cost-effective, environmentally sustainable materials. However, current fungi-based ELMs either have to undergo a final process to heat-kill the living cells or rely on the co-culture with a model organism for functional modification, which hinders the engineerability and versatility of these materials. In this study, we report a new type of ELMs – grown from programmable Aspergillus niger mycelial pellets – by a simple filtration step under ambient conditions. We demonstrate that A. Niger pellets can provide sufficient cohesion to maintain large-area self-supporting structures even under low pH conditions. Subsequently, by tuning the inducible expression of genes involved in melanin biosynthesis, we verified the fabrication of self-supporting living membrane materials with tunable colors in response to xylose concentration in the surroundings, which can be further explored as a potential biosensor for detecting xylose level in industrial wastewater. Notably, the living materials remain alive, self-regenerative, and functional even after 3-month storage. Thus, beyond reporting a new engineerable fungi chassis for constructing ELMs, our study provides new opportunities for developing bulk living materials for real-world applications such as the production of fabrics, packaging materials, and biosensors.

Talaromyces albobiverticillius 30548, a marine-derived fungus, produces Monascus-like azaphilone red/orange pigments which have the potential for various industrial applications. The objective of this study was to scale up pigment production in a 2 L bioreactor with a working volume of 1.3 L media and to compare its biomass growth and pigment production against small volume (500 mL) shake flasks with 200 mL working volume. Additionally, fungal morphology, pigment intensity, fermentation length and duration of pigment production were also compared. Experiments were carried out at laboratory scale in 200 mL shake flasks without controlling pH and oxygen. In parallel, fermentation was performed in a 2 L bioreactor as an initial scale-up to investigate the influence of dissolved oxygen, agitation speed and controlled pH on pigment production and biomass growth of T. albobiverticillius 30548. The highest orange and red pigment production in bioreactor at 24 °C was noticed after 160 h of fermentation (70% pO2) with 25.95 AU 470 nm for orange pigments and 22.79 AU 500 nm for red pigments, at pH set point 5.0. Meanwhile, the fermentation using 200 mL shake flasks effectively produced orange pigments with 22.39 AU 470 nm and red pigments with 14.84 AU 500 nm at 192 h under the same experimental conditions (24 °C, pH 5.0, 150 rpm). Regarding fungal morphology, growth of fungus in the bioreactor was in the form of pellets, whereas in the shake flasks it grew in the form of filaments. From the observed differences in shake flasks and closed bioreactor, it is known that the bioprocess was significantly influenced by dissolved oxygen saturation and agitation speed in scale-up. Thus, oxygen transfer appears to be the rate-limiting factor, which highly influences overall growth and production of pigments in Talaromyces albobiverticillius 30548 liquid culture. [ABSTRACT FROM AUTHOR]

Like many hemibiotrophic plant pathogens, the root-infecting vascular wilt fungus Fusarium oxysporum induces an increase in the pH of the surrounding host tissue. How alkalinization promotes fungal infection is not fully understood, but recent studies point towards the role of cytosolic pH (pHc) and mitogen-activated protein kinase (MAPK) signaling. In fungi, pHc is mainly controlled by the essential plasma membrane H+-ATPase Pma1. Here we created mutants of F. oxysporum lacking casein kinase 1 (Ck1), a known negative regulator of Pma1. We found that the ck1Δ mutants have constitutively high Pma1 activity and exhibit reduced alkalinization of the surrounding medium as well as decreased hyphal growth and conidiation. Importantly, the ck1Δ mutants exhibit defects in hyphal chemotropism towards plant roots and in pathogenicity on tomato plants. Thus, Ck1 is a key regulator of the development and virulence of F. oxysporum. [ABSTRACT FROM AUTHOR]

Physcion is a characteristic component of the traditional herb rhubarb with diverse pharmacological activities that has been commercially approved as an herbal fungicide. Nevertheless, its extremely low contents, costly purification procedure and geographically restricted planting severely hinder its application. Here, a cell factory was constructed in the filamentous fungus Aspergillus terreus for physcion production via microbial fermentation by integrating a pathway-modified emodin accumulation module and a position-selective emodin methylation module. Specifically, 1.71 g/L emodin accumulated when the transcriptional activator GedR and the emodin-1-OH- O -methyltransferase GedA in the geodin biosynthetic pathway were overexpressed and knocked out, respectively. Subsequently, potential emodin-3-OH- O -methyltransferase candidates were enzymatically screened in vitro and introduced into the emodin-accumulating mutant in vivo to generate a physcion-producing strain showing the highest titre of 6.3 g/L in fed-batch fermentation. Thus, our study provides an alternative strategy for the highly efficient, economical production of physcion and a representative example for microbial synthetic biology. • A microbial cell factory was built to achieve highly efficient physcion production. • The emodin titre reached its highest level of 1.71 g/L by shake flasks cultivation. • The highest titre of physcion reached 6.3 g/L in fed-batch fermentation. • This is the first study to achieve physcion production by microbes. [ABSTRACT FROM AUTHOR]

The mhg1 (NCU02695/ ada-23) gene encodes the mitochondrial high-mobility group box (HMG-box or HMGB) protein in Neurospora crassa. The mhg1 KO strain (mhg1 KO) has mitochondrial DNA (mtDNA) instability and a short lifespan; however, the function of MHG1 remains unclear. To investigate the role of this protein in the maintenance of mtDNA, domain deleted MHG1 proteins were expressed in the mhg1 KO strain, and their effects were analyzed. We identified two putative HMG-domains, HMGBI and HMGBII. Although deletion of the HMG-box did not abolish MHG1's mitochondrial localization, the mhg1 KO phenotype of a severe growth defect and a high sensitivity to mutagens could not be restored by introduction of HMG-box deleted mhg1 gene into the KO strain. It was indicated that recombinant full-length MHG1, i.e., mitochondrial targeting sequence (MTS) containing protein, did not exhibit explicit DNA binding, whereas the MHG1 protein truncated for the MTS sequence did in vitro by an electrophoretic mobility shift assay. Furthermore, recombinant MHG1 protein lacking MTS and HMG-domains, either HMGBI or HMGBII, had DNA affinity and an altered band shift pattern compared with MTS-truncated MHG1 protein. These results suggest that cleavage of MTS and appropriate DNA binding via HMG-domains are indispensable for maintaining mtDNA in N. crassa. • Two high-mobility group domains are required for the function of MHG1. • The two high-mobility group domains have different DNA-binding properties. • Neither high-mobility group domain recapitulates the DNA-binding properties of MHG1. • Cleavage of the MTS motif is necessary for the interaction between MHG1 and DNA. [ABSTRACT FROM AUTHOR]

Objectives:To present the demographic, etiological, clinical, and mycological characteristics and treatment results of fungal keratitis patients admitted to our clinic.Materials and Methods:The medical records of patients diagnosed with fungal keratitis between October 2012 and 2018 were reviewed. The diagnosis of fungal keratitis was confirmed mycologically and/or cytologically. Treatment response was defined as complete infiltrate resolution and re-epithelization with medical treatment and minor surgical interventions. Patients who underwent penetrating keratoplasty or evisceration due to clinical deterioration despite treatment were classified as treatment nonresponders and were compared with responders in terms of demographic, etiological, and clinical characteristics.Results:Seventy-two (12.8%) of 559 patients diagnosed with microbial keratitis in the 6-year period were fungal keratitis. Of these, 38 cases (38 eyes) without polymicrobial etiology were included in the study. The patients’ mean age was 44.9±19.0 years (range: 2-80) and males predominated (14 females [36.8%], 24 males [63.2%]). Trauma (63.6%) was the most common predisposing factor in patients younger than 40 years old, whereas pathologies impairing ocular surface immunity were the leading risk factor (48.1%) in patients older than 40 years. Filamentous fungi were detected in 34 (89.5%) cases, while yeasts were found in 4 (10.5%) cases. Among 26 cases with positive cultures, Aspergillus species were the most common pathogens (42.3%). Infiltrate size before treatment was larger in nonresponders (14/38, 36.8%) compared to treatment responders (19/38, 50%) (p=0.049). In addition, rates of treatment response were higher in cases in which the infiltrate was located paracentrally compared to other cases (p=0.036).Conclusion:Fungal keratitis is an important public health problem in our region. Ocular trauma is a leading etiology in men under the age of 40 years. In the 6-year period, we observed that the main causes of fungal keratitis were filamentous fungi, and most commonly Aspergillus species. In cases presenting with large and central lesions, aggressive treatment options should be considered and these patients should be followed up more closely.

The cellulolytic filamentous fungus Trichoderma reesei has a strong capability in protein synthesis and secretion and is increasingly used as a fungal chassis for the production of heterologous proteins or secondary metabolites. However, bidirectional promoters that would significantly facilitate multiple genes' expression have not been characterized in T. reesei. Herein, we show that a 767-bp intergenic region between two polyketide synthase encoding genes that were involved in the biosynthesis of the typical yellow pigment served as a bidirectional promoter in T. reesei. This region was shown to be able to drive the simultaneous expression of two fluorescence reporter genes when fused to each end. Quantitative RT-PCR analysis demonstrated that the driving strength of this bidirectional promoter from each direction reached about half of that of the commonly used promoter PgpdA. Moreover, the co-expression of two cellulase genes driven by this bidirectional promoter enabled T. reesei to produce cellulases on glucose and improved the total cellulase activities with cellulose Avicel as the carbon source. Our work identified the first bidirectional promoter in T. reesei, which would facilitate gene co-expression and find applications in synthetic biology using fungal systems. [ABSTRACT FROM AUTHOR]

Background: Camels are known for their survival under harsh and nutrient-deficient climates. Camel rumen ecosystem presents a unique opportunity to study the ruminal microbes helping the camel in this task. The genus Aspergillus is the extensively studied filamentous fungus due to its ability to secret industrially important enzymes. The present study was aimed to isolate and characterize microbes with lignocellulolytic capacity from camel rumen. Results: The fungal isolate Aspergillus sydowii C6d, isolated from camel rumen, was sequenced and analysed for its CAZyme content responsible for lignocellulose degradation. The C6d isolate was evaluated for its capacity to produce cellulase, pectinase, xylanase, and amylase with their respective assays and further evaluated for their optimum pH. The genome sequencing and assembly resulted in 32.27 Mb of genome size with a GC % of 50.59. The CAZyme analysis revealed that the C6d produced 543 polysaccharidedegrading CAZymes amongst which, 148 CAZymes were potentially involved in lignocellulose degradation. The genomic comparison of the C6d with 30 commonly used lignocellulolytic fungi (white rot, brown rot, and soft rot fungus) showed the enrichment of cellulolytic and pectinolytic CAZymes in C6d genome as compared to others. The saccharification of lignocellulosic substrate wheat straw resulted in the release of 50.85% of reducing sugars. Conclusions: The study provides important insights into the CAZymes responsible for lignocellulolytic ability in the novel fungus Aspergillus sydowii C6d isolated from camel rumen and presents a valuable source of CAZymes to be further evaluated for potential biotechnological applications. [ABSTRACT FROM AUTHOR]

Malic acid, mainly used as acidulant and taste enhancer in the food industry, is currently produced from fossil resources. In this study, microbial L-malate production with the filamentous fungus A. oryzae using the carbon source acetate was evaluated. Acetate is for example contained in biomass-derived substrates such as lignocellulosic hydrolysates and condensates of fast pyrolysis, thus avoiding competition with food production. Since research on malic acid synthesis from acetate is limited and reported productivities and yields are low, this work aimed to improve the process. First, different cultivation temperatures were tested. This parameter was found to affect the ratio between malic and succinic acid, which is the major by-product of organic acid production with A. oryzae. At 32°C, the malate share was highest (53.7 ± 1.6%), while it was lowest at 38°C (43.3 ± 1.1%) whereas succinate represented the main product (51.5 ± 1.0%). Besides the temperature, the type of nitrogen source was also found to affect malate synthesis as well as biomass production. In the pre-culture, the biomass concentration was increased by a factor of 3.4–3.9, and germination started earlier with the complex nitrogen sources yeast extract, casein hydrolysate and peptone compared to the defined nitrogen source (NH4)2SO4. Especially with yeast extract, malate synthesis in the main culture was accelerated and the titer obtained after 48 h was about 2.6 times higher than that quantified with (NH4)2SO4. To reduce substrate inhibition in acetate medium, fed-batch and repeated-batch processes were evaluated using (NH4)2SO4 or yeast extract as nitrogen source. In the fed-batch process, the period of malate production was extended, and the maximum product concentration was increased to 11.49 ± 1.84 g/L with (NH4)2SO4 and 12.08 ± 1.25 g/L with yeast extract. In the repeated-batch process, the total acid production was highest within the first 240 h of fermentation, but optimization is required to maintain high production rates in later cycles. The lessons learned in this study will help in the development of further process strategies to maximize malate production using acetate as alternative substrate to the commonly used glucose.

The filamentous fungus Colletotrichum gloeosporioides is the causative agent of one of the most serious diseases that damage plant fruit. In this study, we discovered and experimentally characterized a new gene in Colletotrichum gloeosporioides named CgloRPCYG. The CRISPR/Cas9 knockout mutant and complementary strain of CgloRPCYG were then obtained by polyethylene glycol (PEG)-mediated protoplast transformation to determine the related phenotypes. Compared with the wild-type strain and complementary mutant, the pathogenicity of the CRISPR/Cas9 knockout mutant was significantly decreased, the conidial yield was significantly reduced, and conidial germination was significantly delayed. These data indicate that CgloRPCYG contributes to pathogenicity, conidial yield, and germination in C. gloeosporioides. The successful application of the CRISPR/Cas9 system in C. gloeosporioides also confirms its utility in filamentous fungi for fundamental research and practical application. Furthermore, CgloRPCYG is a potential target gene for use in the development of plant protection technologies, such as spray-induced gene silencing, with the aim of controlling plant anthracnose disease caused by C. gloeosporioides.

Aspergillus fumigatus is a ubiquitous and saprophytic filamentous fungus and the main etiologic agent of aspergillosis. Infections caused by A. fumigatus culminate in a strong inflammatory response that can evolve into respiratory failure and may be lethal in immunocompromised individuals. In the last decades, it has been demonstrated that extracellular vesicles (EVs) elicit a notable biological response in immune cells. EVs carry a variety of biomolecules, therefore are considered potential antigen delivery vehicles. The role of EVs as a strategy for modulating an effective response against infections caused by A. fumigatus remains unexplored. Here we investigate the use of EVs derived from A. fumigatus as an immunization tool to induce a more robust immune response to A. fumigatus pulmonary infection. In order to investigate that, male C57BL/6 mice were immunized with two doses of EVs and infected with A. fumigatus. Pre-exposure of mice to EVs was able to induce the production of specific IgG serum for fungal antigens. Besides that, the immunization with EVs reduced the neutrophilic infiltrate into the alveoli, as well as the extravasation of total proteins and the production of proinflammatory mediators IL-1β, IL-6, and CXCL-1. In addition, immunization prevented extensive lung tissue damage and also improved phagocytosis and fungus clearance. Noteworthy, immunization with EVs, associated with subclinical doses of Amphotericin B (AmB) treatment, rescued 50% of mice infected with A. fumigatus from lethal fungal pneumonia. Therefore, the present study shows a new role for A. fumigatus EVs as host inflammatory response modulators, suggesting their use as immunizing agents. [ABSTRACT FROM AUTHOR]

Background: A total of 11 β-glucosidases are predicted in the genome of Trichoderma reesei, which are of great importance for regulating cellulase biosynthesis. Nevertheless, the relevant function and regulation mechanism of each β-glucosidase remained unknown. Results: We evidenced that overexpression of cel1b dramatically decreased cellulase synthesis in T. reesei RUT-C30 both at the protein level and the mRNA level. In contrast, the deletion of cel1b did not noticeably affect cellulase production. Protein CEL1B was identified to be intracellular, being located in vacuole and cell membrane. The overexpression of cel1b reduced the intracellular pNPGase activity and intracellular/extracellular glucose concentration without inducing carbon catabolite repression. On the other hand, RNA-sequencing analysis showed the transmembrane transport process and endoplasmic reticulum function were affected noticeably by overexpressing cel1b. In particular, some important sugar transporters were notably downregulated, leading to a compromised cellular uptake of sugars including glucose and cellobiose. Conclusions: Our data suggests that the cellulase inhibition by cel1b overexpression was not due to the β-glucosidase activity, but probably the dysfunction of the cellular transport process (particularly sugar transport) and endoplasmic reticulum (ER). These findings advance the knowledge of regulation mechanism of cellulase synthesis in filamentous fungi, which is the basis for rationally engineering T. reesei strains to improve cellulase production in industry. [ABSTRACT FROM AUTHOR]

Aspergillus fumigatus is a ubiquitous and saprophytic filamentous fungus and the main etiologic agent of aspergillosis. Infections caused by A. fumigatus culminate in a strong inflammatory response that can evolve into respiratory failure and may be lethal in immunocompromised individuals. In the last decades, it has been demonstrated that extracellular vesicles (EVs) elicit a notable biological response in immune cells. EVs carry a variety of biomolecules, therefore are considered potential antigen delivery vehicles. The role of EVs as a strategy for modulating an effective response against infections caused by A. fumigatus remains unexplored. Here we investigate the use of EVs derived from A. fumigatus as an immunization tool to induce a more robust immune response to A. fumigatus pulmonary infection. In order to investigate that, male C57BL/6 mice were immunized with two doses of EVs and infected with A. fumigatus. Pre-exposure of mice to EVs was able to induce the production of specific IgG serum for fungal antigens. Besides that, the immunization with EVs reduced the neutrophilic infiltrate into the alveoli, as well as the extravasation of total proteins and the production of proinflammatory mediators IL-1β, IL-6, and CXCL-1. In addition, immunization prevented extensive lung tissue damage and also improved phagocytosis and fungus clearance. Noteworthy, immunization with EVs, associated with subclinical doses of Amphotericin B (AmB) treatment, rescued 50% of mice infected with A. fumigatus from lethal fungal pneumonia. Therefore, the present study shows a new role for A. fumigatus EVs as host inflammatory response modulators, suggesting their use as immunizing agents.

Wood serves as a renewable resource; however, its sustainability is threatened by wood-rotting fungi, a type of filamentous fungi. Although its decay mechanism has been widely investigated, essential factors controlling fungal responses are poorly understood owing to the complex wood architecture mainly formed by cellulose microfibrils, hemicellulose, and lignin. This study aimed to explore the effects of wood structure and chemical components on wood-rotting fungi using chemically simplified wood substrates. Cellulose-based substrates were successfully prepared from wood sections of Cryptomeria japonica by removing non-cellulosic components while maintaining the inherent wood structure. Confocal microscopy analysis combined with Hough transformation after decay tests revealed a clear tendency of hyphae to elongate along tracheids on cellulosic substrates with wood structures. Furthermore, scanning electron microscopy demonstrated that hyphae passed through bordered pits without completely destroying the pit structure. These results indicate that wood structure is the dominant factor in hyphal responses and that the physical forces of tip growth and enzymatic degradation of components contribute simultaneously to fungal invasion in wood. This study demonstrates the usefulness of single-component substrates with preserved wood structures as new evaluation materials for studying wood-rotting, potentially facilitating research on decay for wood preservation. • The effects of wood structure and chemical components on wood-rotting fungi were studied. • Cellulosic substrates with wood anatomical features were successfully prepared. • Structured hyphal elongation was observed on the cellulosic substrates. • Hyphae passed through bordered pits without completely destroying the pit structure. • Physical substrate structure plays a crucial role in hyphal response during the invasion process. [ABSTRACT FROM AUTHOR]

Chitosan is a non-cytotoxic polysaccharide that, upon hydrolysis, releases oligomers of different sizes that may have antioxidant, antimicrobial activity and the inhibition of cancer cell growth, among other applications. It is, therefore, a hydrolysis process with great biotechnological relevance. Thus, this study aims to use a crude enzyme concentrate (CEC) produced by a filamentous fungus to obtain oligomers with different molecular weights. The microorganism was cultivated in a liquid medium (modified Czapeck—with carboxymethylcellulose as enzyme inducer). The enzymes present in the CEC were identified by LC-MS/MS, with an emphasis on cellobiohydrolase (E.C 3.2.1.91). The fungus of the Aspergillus genus was identified by amplifying the ITS1-5.8S-ITS2 rDNA region and metaproteomic analysis, where the excreted enzymes were identified with sequence coverage greater than 84% to A. nidulans. Chitosan hydrolysis assays compared the CEC with the commercial enzyme (Celluclast 1.5 L®). The ability to reduce the initial molecular mass of chitosan by 47.80, 75.24, and 93.26% after 2.0, 5.0, and 24 h of reaction, respectively, was observed. FTIR analyses revealed lower absorbance of chitosan oligomers’ spectral signals, and their crystallinity was reduced after 3 h of hydrolysis. Based on these results, we can conclude that the crude enzyme concentrate showed a significant technological potential for obtaining chitosan oligomers of different sizes.

Objectives: To present the demographic, etiological, clinical, and mycological characteristics and treatment results of fungal keratitis patients admitted to our clinic. Materials and Methods: The medical records of patients diagnosed with fungal keratitis between October 2012 and 2018 were reviewed. The diagnosis of fungal keratitis was confirmed mycologically and/or cytologically. Treatment response was defined as complete infiltrate resolution and re-epithelization with medical treatment and minor surgical interventions. Patients who underwent penetrating keratoplasty or evisceration due to clinical deterioration despite treatment were classified as treatment nonresponders and were compared with responders in terms of demographic, etiological, and clinical characteristics. Results: Seventy-two (12.8%) of 559 patients diagnosed with microbial keratitis in the 6-year period were fungal keratitis. Of these, 38 cases (38 eyes) without polymicrobial etiology were included in the study. The patients' mean age was 44.9±19.0 years (range: 2-80) and males predominated (14 females [36.8%], 24 males [63.2%]). Trauma (63.6%) was the most common predisposing factor in patients younger than 40 years old, whereas pathologies impairing ocular surface immunity were the leading risk factor (48.1%) in patients older than 40 years. Filamentous fungi were detected in 34 (89.5%) cases, while yeasts were found in 4 (10.5%) cases. Among 26 cases with positive cultures, Aspergillus species were the most common pathogens (42.3%). Infiltrate size before treatment was larger in nonresponders (14/38, 36.8%) compared to treatment responders (19/38, 50%) (p=0.049). In addition, rates of treatment response were higher in cases in which the infiltrate was located paracentrally compared to other cases (p=0.036). Conclusion: Fungal keratitis is an important public health problem in our region. Ocular trauma is a leading etiology in men under the age of 40 years. In the 6-year period, we observed that the main causes of fungal keratitis were filamentous fungi, and most commonly Aspergillus species. In cases presenting with large and central lesions, aggressive treatment options should be considered and these patients should be followed up more closely. [ABSTRACT FROM AUTHOR]

ABSTRACT Autophagy is a conserved intracellular degradation mechanism in eukaryotes and is initiated by the protein kinase autophagy-related protein 1 (Atg1). However, except for the autophosphorylation activity of Atg1, the target proteins phosphorylated by Atg1 are largely unknown in filamentous fungi. In Beauveria bassiana (a filamentous insect-pathogenic fungus), Atg1 is indispensable for autophagy and is associated with fungal development. Comparative omics-based analyses revealed that B. bassiana Atg1 (BbAtg1) has key influence on the proteome and phosphoproteome during conidiogenesis. In terms of its physiological functions, the BbAtg1-mediated phosphoproteome is primarily associated with metabolism, signal transduction, cell cycle, and autophagy. At the proteomic level, BbAtg1 mainly regulates genes involved in protein synthesis, protein fate, and protein with binding function. Furthermore, integrative analyses of phosphoproteomic and proteomic data led to the identification of several potential targets regulated by BbAtg1 phosphorylation activity. Notably, we demonstrated that BbAtg1 phosphorylated BbAtg3, an essential component of the ubiquitin-like conjugation system in autophagic progress. Our findings indicate that in addition to being a critical component of the autophagy initiation, Atg1 orchestrates autophagosome elongation via its phosphorylation activity. The data from our study will facilitate future studies on the noncanonical targets of Atg1 and help decipher the Atg1-mediated phosphorylation networks. IMPORTANCE Autophagy-related protein 1 (Atg1) is a serine/threonine protein kinase for autophagy initiation. In contrast to the unicellular yeast, the target proteins phosphorylated by Atg1 are largely unknown in filamentous fungi. In this study, the entomopathogenic fungus Beauveria bassiana was used as a representative of filamentous fungi due to its importance in the applied and fundamental research. We revealed that Atg1 mediates the comprehensive proteome and phosphoproteome, which differ from those revealed in yeast. Further investigation revealed that Atg1 directly phosphorylates the E2-like enzyme Atg3 of the ubiquitin-like conjugation system (ULCS), and the phosphorylation of Atg3 is indispensable for ULCS functionality. Interestingly, the phosphorylation site of Atg3 is conserved among a set of insect- and plant-pathogenic fungi but not in human-pathogenic fungi. This study reveals new regulatory mechanisms of autophagy and provides new insights into the evolutionary diversity of the Atg1 kinase signaling pathways among different pathogenic fungi.

Filamentous fungi are extensively used as an important expression host for the production of a variety of essential industrial proteins. They have significant promise as an expression system for protein synthesis due to their inherent superior secretory capabilities. The purpose of this study was to develop a novel expression system by utilizing a Penicillium oxalicum strain that possesses a high capacity for protein secretion. The expression of glycoside hydrolases in P. oxalicum was evaluated in a cleaner extracellular background where the formation of two major amylases was inhibited. Four glycoside hydrolases (CBHI, Amy15B, BGL1, and Cel12A) were expressed under the highly constitutive promoter PubiD. It was found that the proteins exhibited high purity in the culture supernatant after cultivation with starch. Two inducible promoters, Pamy15A and PempA, under the activation of the transcription factor AmyR were used as elements in the construction of versatile vectors. When using the cellobiohydrolase CBHI as the extracellular quantitative reporter, the empA promoter screened from the AmyR-overexpressing strain was shown to be superior to the amy15A promoter based on RNA-sequencing data. Therefore, we designed an expression system consisting of a cleaner background host strain and an adjustable promoter. This system enables rapid and high-throughput evaluation of glycoside hydrolases from filamentous fungi. Key points • A new protein expression system derived from Penicillium oxalicum has been developed. • The expression platform is capable of secreting recombinant proteins with high purity. • The adjustable promoter may allow for further optimization of recombinant protein synthesis. [ABSTRACT FROM AUTHOR]

High cost and low efficiency of lignocellulolytic enzymes are the main challenges that must be overcome to make second-generation ethanol more competitive in the fuel market. The cultivation of microorganisms using different agro-industrial wastes as substrates is one of the alternatives to reduce the process costs and obtain more efficient enzyme cocktails in the biomass hydrolysis. Since the fungus Chrysoporthe cubensis has proved to be a promising source of lignocellulolytic enzymes from wheat bran, this study aimed to evaluate the effect of different lignocellulosic materials in its enzyme production and the effectiveness of a new enzymatic cocktail on sugarcane bagasse saccharification. Primarily, this fungus was grown under solid-state fermentation, using wheat bran, elephant grass, or sugarcane bagasse as carbon sources. Afterwards, the wheat bran was combined with sugar beet flour in different ratios, and both were used as carbon sources. The enzymatic profiles were investigated and the most promising enzyme extract was applied to pretreated sugarcane bagasse saccharification. A new cocktail obtained from the combination of wheat bran and sugar beet flour in ratio 1:1 showed the highest activity for almost all enzymes tested and was more efficient than extract obtained with only wheat bran, especially in saccharification of alkaline pretreated sugarcane bagasse, releasing 18 g.L−1 of glucose and 14.8 g.L−1 of xylose, which correspond to 38.5% of cellulose and 61.6% of hemicellulose, respectively. Therefore, the combination of both substrates is an effective strategy to induce Chrysoporthe cubensis to produce a complex and efficient enzymatic cocktail in the biomass hydrolysis. [ABSTRACT FROM AUTHOR]

This manuscript deals with the bioremediation of aqueous media containing Cd2+ and Pb2+ by using dead biomass of the fungus Phialomyces macrosporus. This filamentous fungus with 19.1 m2 g−1 of surface area showed promising adsorption results, since more than 80 % of Cd2+ and Pb2+ were removed from synthetic aqueous media. Adsorption isotherms from this fungal biomass were satisfactorily linearized (R2 > 0.95) by the mathematical Langmuir model and maximum adsorption capacities (MAC) were 1.03 × 10−4 and 6.48 × 10−5 mol g−1 for Cd2+ and Pb2+, respectively. In turn, Gibbs free energy variations were about –25 kJ mol−1, thus highlighting predominantly physical adsorptions. Concomitant cations decreased Cd2+ and Pb2+ MAC values by up to 72.6 % and 70.4 %, respectively, although this limitation has been largely circumvented after increasing the amounts of fungal biomass. As an environmental application of the biosorption of Cd2+ and Pb2+ on Phialomyces macrosporus, real chemical laboratory wastewaters were successfully treated with this filamentous fungus, thus meeting the Brazilian legal standards for cadmium (≤ 1.8 × 10−6 mol L−1) and lead (≤ 2.4 × 10−6 mol L−1). Based on these results and considering that the use of Phialomyces macrosporus to adsorb cadmium and lead is being evaluated for the first time, the original contribution of this study was to broaden the understanding of bioremediation mechanisms. [ABSTRACT FROM AUTHOR]

Lectins are characterized of the carbohydrate-binding ability and play comprehensive roles in fungal physiology (e.g., defense response, development and host–pathogen interaction). Beauveria bassiana , a filamentous entomopathogenic fungus, has a lectin-like protein containing a Fruit Body_domain (BbLec1). BbLec1 could bind to chitobiose and chitin in fungal cell wall. BbLec1 proteins interacted with each other to form multimers, and translocated into eisosomes. Further, the interdependence between BbLec1 and the eisosome protein PliA was essential for stabilizing the eisosome architecture. To test the BbLec1 roles in B. bassiana , we constructed the gene disruption and complementation mutants. Notably, the BbLec1 loss resulted in the impaired cell wall in mycelia and conidia as well as conidial formation capacity. In addition, disruption of BbLec1 led to the reduced cytomembrane integrity and the enhanced sensitivity to osmotic stress. Finally, Δ BbLec1 mutant strain displayed the weakened virulence when compared with the wild-type strain. Taken together, BbLec1 traffics into eisosome and links the functionality of eisosome to development and virulence of B. bassiana. • A lectin with Fruit_Body domain exists in filamentous fungus Beauveria bassiana. • The lectin traffics into eisosome and maintains functional membrane and cell wall. • The lectin contributes to fungal development, stress response and virulence. [ABSTRACT FROM AUTHOR]

Background: The present study describes the production of biosurfactant (BS) and emulsifier (BE) by the filamentous fungus Mucor hiemalis UCP 0039, as well as the characterization and stability of the both biomolecules for environmental or industrial applications. Results: Biosurfactants and bioemulsifiers are amphiphilic compounds and are produced as extracellular molecules. The results showed that bioproduct obtained by shaker condition reduced the water surface tension of 72 to 32 mN/m and reached an emulsification index of 96%, while the static cultivation resulted in a biomolecule with a surface tension of 40 mN/m and an emulsification index of 96%, suggesting the production of a biosurfactant and bioemulsifier, respectively. The compounds showed glycolipid nature but the biosurfactant presented cationic charge, while the bioemulsifier, anionic charge. Thus, the results confirmed that M. hiemalis produced two distinct biomolecules under different parameters and in the same culture medium. Conclusions: It is the first time that biosurfactant and emulsifier production has been described in the same medium and under different physical conditions by Mucor hiemalis. Both biomolecules showed thermal stability, as well as have significant effect on the viscosity of hydrophobic compounds, indicating the excellent potential for environmental safety or industrial applications to improve the efficiency of sustainable and economic technologies. How to cite: Ferreira INS, Rodríguez DM, Campos-Takaki, GM, et al. Biosurfactant and bioemulsifier as promissing molecules produced by Mucor hiemalis isolated from Caatinga soil. Electron J Biotechnol 2020; 47. https://doi.org/10.1016/j.ejbt.2020.06.006.

Talaromyces albobiverticillius 30548, a marine-derived fungus, produces Monascus-like azaphilone red/orange pigments which have the potential for various industrial applications. The objective of this study was to scale up pigment production in a 2 L bioreactor with a working volume of 1.3 L media and to compare its biomass growth and pigment production against small volume (500 mL) shake flasks with 200 mL working volume. Additionally, fungal morphology, pigment intensity, fermentation length and duration of pigment production were also compared. Experiments were carried out at laboratory scale in 200 mL shake flasks without controlling pH and oxygen. In parallel, fermentation was performed in a 2 L bioreactor as an initial scale-up to investigate the influence of dissolved oxygen, agitation speed and controlled pH on pigment production and biomass growth of T. albobiverticillius 30548. The highest orange and red pigment production in bioreactor at 24 °C was noticed after 160 h of fermentation (70% pO2) with 25.95 AU 470 nm for orange pigments and 22.79 AU 500 nm for red pigments, at pH set point 5.0. Meanwhile, the fermentation using 200 mL shake flasks effectively produced orange pigments with 22.39 AU 470 nm and red pigments with 14.84 AU 500 nm at 192 h under the same experimental conditions (24 °C, pH 5.0, 150 rpm). Regarding fungal morphology, growth of fungus in the bioreactor was in the form of pellets, whereas in the shake flasks it grew in the form of filaments. From the observed differences in shake flasks and closed bioreactor, it is known that the bioprocess was significantly influenced by dissolved oxygen saturation and agitation speed in scale-up. Thus, oxygen transfer appears to be the rate-limiting factor, which highly influences overall growth and production of pigments in Talaromyces albobiverticillius 30548 liquid culture.

Like many hemibiotrophic plant pathogens, the root-infecting vascular wilt fungus Fusarium oxysporum induces an increase in the pH of the surrounding host tissue. How alkalinization promotes fungal infection is not fully understood, but recent studies point towards the role of cytosolic pH (pHc) and mitogen-activated protein kinase (MAPK) signaling. In fungi, pHc is mainly controlled by the essential plasma membrane H+-ATPase Pma1. Here we created mutants of F. oxysporum lacking casein kinase 1 (Ck1), a known negative regulator of Pma1. We found that the ck1Δ mutants have constitutively high Pma1 activity and exhibit reduced alkalinization of the surrounding medium as well as decreased hyphal growth and conidiation. Importantly, the ck1Δ mutants exhibit defects in hyphal chemotropism towards plant roots and in pathogenicity on tomato plants. Thus, Ck1 is a key regulator of the development and virulence of F. oxysporum.

Glucose, a key component of traditional Japanese fermented foods, is derived from rice starch via saccharification by hydrolytic enzymes produced by Aspergillus oryzae . The δ 13 C value of glucose reflects that of its rice source. However, the influence of saccharification parameters (glucose concentration, degradation temperature, and reaction time) on glucose δ 13 C values is unclear. Here, we investigated the influence of saccharification on the δ 13 C value of glucose. Our experiments showed a significant difference in the δ 1 ³C value of glucose (-27.0 ± 0.1 ‰) obtained from saccharification compared to the ingredient rice (-27.1 ± 0.1 ‰) and remaining solid residue (-27.1 ± 0.1 ‰); however, it did not differ significantly from those of rice koji (-27.0 ± 0.1 ‰) and steamed rice (-27.1 ± 0.1 ‰), despite all values being within 0.1 ‰. Notably, glucose concentration, degradation temperature, and reaction time did not significantly affect glucose δ 13 C values. These findings demonstrate the remarkable preservation of glucose δ 13 C values. The δ 13 C values remain aligned with the original δ 13 C value of the rice, even with up to 60 % degradation during A. oryzae saccharification. This persistence of the δ 13 C value throughout the process offers a potential tool for authenticating the origin of rice-fermented beverages based on the δ 13 C value of their glucose component., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

The protein O- mannosyltransferase catalyzes O- mannosylation in the endoplasmic reticulum by transferring mannose to the seryl or threonyl residues of substrate proteins. We previously reported a deletion mutant of O -mannosyltransferase C (Δ pmtC) in Aspergillus nidulans with impaired vegetative growth and sterigmatocystin (ST) production. In this study, we investigated whether osmotic conditions contribute to the developmental processes and ST biosynthesis of the Δ pmtC deletion mutant. We found that hyphal growth and ST production partially improved in the presence of NaCl, KCl or sorbitol as osmotic stabilizers. Conidiation of the Δ pmtC deletion mutant was not restored under osmotic stress conditions when the hogA gene was deleted. The hogA gene encodes a protein required for the cellular response to osmotic pressure. However, the yield of ST and the vegetative growth of the Δ hogA Δ pmtC double deletant was restored by high osmolarity in a HogA-independent manner. [ABSTRACT FROM AUTHOR]

Purpose: There are several studies on the use of RNA interference (RNAi) for gene function analysis in fungi. However, most studies on filamentous fungi are based on in vitro-transcribed or -synthesized small interfering RNA (siRNA), and only a few have reported the use of vector-based RNAi. Here we want to develop and evaluate a new vector-based RNAi method using the mouse U6 promoter to drive short hairpin RNA (shRNA) expression in the filamentous fungi. Methods: Molecular techniques were employed to develop and evaluate a new vector-based RNAi method using the mouse U6 promoter to drive short hairpin RNA (shRNA) expression in the filamentous fungus Blakeslea trispora. Results: We characterized the mouse U6 promoter and utilized it for the expression of shRNA in B. trispora. Using real-time polymerase chain reaction and western blotting analyses, we confirmed the decrease in the mRNA and protein expression of carRA, respectively, in cells transformed with the mouse U6 promoter-driven shRNA expression vector. This indicated that the shRNA was transcribed from the mouse U6 promoter and correctly processed into siRNA and that the mouse U6 promoter exhibited transcription ability in the filamentous fungi. Conclusions: The results suggest that the mouse U6 promoter that drives the expression of shRNA vectors may serve as a novel tool for RNAi induction in filamentous fungi. [ABSTRACT FROM AUTHOR]