Optimizing mycelial protein yield in Pleurotus djamor via ARTP mutagenesis and hybridization strategies.

With the world's population rapidly increasing, the demand for high-quality protein is on the rise. Edible fungi breeding technology stands as a crucial avenue to obtain strains with high yield, high-quality protein, and robust stress resistance. To address the protein supply gap, Atmospheric and Room Temperature Plasma (ARTP) mutagenesis, and spore hybridization techniques were employed to enhance Pleurotus djamor mycelium protein production. Beginning with the original strain Pleurotus djamor JD-1, ARTP was utilized to mutate spore suspension. The optimal treatment time for Pleurotus djamor spores, determined to achieve optimal mortality, was 240 s. Through primary and secondary screenings, 6 mutant strains out of 39 were selected, exhibiting improved protein yield and growth rates compared to the original strain. Among these mutagenic strains, 240S-4 showcased the highest performance, with a mycelial growth rate of 9.5±0.71 mm/d, a biomass of 21.45±0.54 g/L, a protein content of 28.75±0.92%, and a remarkable protein promotion rate of 128.03±7.29%. Additionally, employing spore hybridization and breeding, 7 single-nuclei strains were selected for pin-two hybridization, resulting in 21 hybrid strains. The biomass and protein content of 9 hybrid strains surpassed those of the original strains. One hybrid strain, H-5, exhibited remarkable mycelial protein production, boasting a mycelial growth rate of 26.5±0.7 mm/d, a biomass of 21.70±0.46 g/L, a protein content of 28.44±0.22%, and a protein promotion rate of 128.02±1.73%. Notably, both strains demonstrated about a 28% higher mycelial protein yield than the original strains, indicating comparable effectiveness between hybrid breeding and mutagenesis breeding. Finally, we analyzed the original and selected strains by molecular biological identification, which further proved the effectiveness of the breeding method. These findings present novel insights and serve as a reference for enhancing edible fungi breeding, offering promising avenues to meet the escalating protein demand.

Edible mycelium bioengineered for enhanced nutritional value and sensory appeal using a modular synthetic biology toolkit.

Filamentous fungi are critical in the transition to a more sustainable food system. While genetic modification of these organisms has promise for enhancing the nutritional value, sensory appeal, and scalability of fungal foods, genetic tools and demonstrated use cases for bioengineered food production by edible strains are lacking. Here, we develop a modular synthetic biology toolkit for Aspergillus oryzae, an edible fungus used in fermented foods, protein production, and meat alternatives. Our toolkit includes a CRISPR-Cas9 method for gene integration, neutral loci, and tunable promoters. We use these tools to elevate intracellular levels of the nutraceutical ergothioneine and the flavor-and color molecule heme in the edible biomass. The strain overproducing heme is red in color and is readily formulated into imitation meat patties with minimal processing. These findings highlight the promise of synthetic biology to enhance fungal foods and provide useful genetic tools for applications in food production and beyond.

Function of Transcription Factors PoMYB12, PoMYB15, and PoMYB20 in Heat Stress and Growth of Pleurotus ostreatus.

MYB transcription factors (TFs) have been extensively studied in plant abiotic stress responses and growth and development. However, the role of MYB TFs in the heat stress response and growth and development of Pleurotus ostreatus remains unclear. To investigate the function of PoMYB12, PoMYB15, and PoMYB20 TFs in P. ostreatus, mutant strains of PoMYB12, PoMYB15, and PoMYB20 were generated using RNA interference (RNAi) and overexpression (OE) techniques. The results indicated that the mycelia of OE-PoMYB12, OE-PoMYB20, and RNAi-PoMYB15 mutant strains exhibited positive effects under heat stress at 32 °C, 36 °C, and 40 °C. Compared to wild-type strains, the OE-PoMYB12, OE-PoMYB20, and RNAi-PoMYB15 mutant strains promoted the growth and development of P. ostreatus. These mutant strains also facilitated the recovery of growth and development of P. ostreatus after 24 h of 36 °C heat stress. In conclusion, the expression of PoMYB12 and PoMYB20 supports the mycelium's response to heat stress and enhances the growth and development of P. ostreatus, whereas PoMYB15 produces the opposite effect.

Features of disruption mutants of genes encoding for hydrophobin Vmh2 and Vmh3 in mycelial formation and resistance to environmental stress in Pleurotus ostreatus.

Hydrophobins, which are small-secreted proteins with both hydrophobic and hydrophilic parts, can self-assemble into an amphiphilic film at the air-water interface, helping the fungus to form aerial hyphae. In the agaricomycete Pleurotus ostreatus, more than 20 putative hydrophobin genes have been predicted. Of these, two hydrophobin genes, vmh2 and vmh3, are predominantly expressed in the vegetative mycelium. In this study, we focused on the functions of Vmh2 and Vmh3 in vegetative mycelia. Based on the observation of the mycelial cross-section by transmission electron microscopy and the disappearance time of water droplets on the mycelial surface, Vmh2 and Vmh3 were considered essential for the maintenance of the surface hydrophobicity of the mycelium. The Δvmh3 and Δvmh2Δvmh3 strains exhibited relatively slower aerial mycelia formation on a liquid medium, and no significant alteration was observed in Δvmh2 strains. Only the Δvmh3 and Δvmh2Δvmh3 strains grew slower than the wild-type strain under stress conditions involving SDS and H2O2 on agar plates. This study revealed possible distinct roles for these hydrophobins in stress resistance. These results suggest that Agaricomycetes, including P. ostreatus, have evolved to possess multiple different hydrophobins as a means of adapting to various environments.

Uncovering Gene Expression Profiles of Three Morphologic Mycelium Forms of the Chinese Caterpillar Mushroom Ophiocordyceps sinensis (Ascomycota) Using High-Throughput Sequencing.

The asexual form of Ophiocordyceps sinensis has been controversial, but various morphologic mycelium appeared when O. sinensis was cultured under experimental conditions. To explore the generation mechanism of morphologic mycelium, developmental transcriptomes were analyzed from three kinds of mycelium (aerial mycelium, hyphae knot, and substrate mycelium). The results showed that diameter and morphology of these three kinds of mycelium were obviously different. KEGG functional enrichment analysis showed that the differential expressed genes (DEGs) of substrate mycelium were enriched in ribosomes and peroxisomes, indicating that prophase culture was rich in nutrients and the metabolism of substrate mycelium cells was vigorous in the stage of nutrient absorption. The up-DEGs of hyphae knot were mainly enriched in the oxidative phosphorylation pathway, indicating that oxidative phosphorylation was the main energy source for mycelium formation in the stage of nutrient accumulation and reproductive transformation. The up-DEGs of aerial mycelium were mainly enriched in the synthesis and degradation pathways of valine, leucine, and isoleucine, suggesting that the occurrence of aerial mycelium was related to amino acid metabolism at the later stage of culture, and nutritional stress accelerated the reproduction of asexual spores. In addition, the important roles of mycelium formation related genes were verified by combined analysis of qRT-PCR and transcriptome sequencing. Collectively, this study will provide theoretical guidance for inhibiting the occurrence of aerogenous mycelium and promoting the development of mycelium into pinhead primordia in the culture of O. sinensis in the future.

Assessment of fungal spores and spore-like diversity in environmental samples by targeted lysis.

At particular stages during their life cycles, fungi use multiple strategies to form specialized structures to survive unfavorable environmental conditions. These strategies encompass sporulation, as well as cell-wall melanization, multicellular tissue formation or even dimorphism. The resulting structures are not only used to disperse to other environments, but also to survive long periods of time awaiting favorable growth conditions. As a result, these specialized fungal structures are part of the microbial seed bank, which is known to influence the microbial community composition and contribute to the maintenance of diversity. Despite the importance of the microbial seed bank in the environment, methods to study the diversity of fungal structures with improved resistance only target spores dispersing in the air, omitting the high diversity of these structures in terms of morphology and environmental distribution. In this study, we applied a separation method based on cell lysis to enrich lysis-resistant fungal structures (for instance, spores, sclerotia, melanized yeast) to obtain a proxy of the composition of the fungal seed bank. This approach was first evaluated in-vitro in selected species. The results obtained showed that DNA from fungal spores and from yeast was only obtained after the application of the enrichment method, while mycelium was always lysed. After validation, we compared the diversity of the total and lysis-resistant fractions in the polyextreme environment of the Salar de Huasco, a high-altitude athalassohaline wetland in the Chilean Altiplano. Environmental samples were collected from the salt flat and from microbial mats in small surrounding ponds. Both the lake sediments and microbial mats were dominated by Ascomycota and Basidiomycota, however, the diversity and composition of each environment differed at lower taxonomic ranks. Members of the phylum Chytridiomycota were enriched in the lysis-resistant fraction, while members of the phylum Rozellomycota were never detected in this fraction. Moreover, we show that the community composition of the lysis-resistant fraction reflects the diversity of life cycles and survival strategies developed by fungi in the environment. To the best of our knowledge this is the first time that the fungal diversity is explored in the Salar de Huasco. In addition, the method presented here provides a simple and culture independent approach to assess the diversity of fungal lysis-resistant cells in the environment.

The MADS-box transcription factor AoRlmA is involved in the regulation of mycelium development, conidiation, cell-wall integrity, stress response, and trap formation of Arthrobotrys oligospora.

The maintenance of cell-wall integrity (CWI) is important for mycelial growth, development, and pathogenicity in fungi. Arthrobotrys oligospora is a typical nematode-trapping (NT) fungus which can capture nematodes by producing adhesive networks. In this study, we characterized an orthologous MADS-box transcription factor RlmA (AoRlmA) downstream of the CWI regulatory pathway in A. oligospora. The deletion of AorlmA caused a reduction in mycelial growth, the number of nuclei, conidiation, and trap formation, as well as increased sensitivity to cell-wall synthesis-disrupting agents, osmotic agents, and oxidants; accordingly, the transcript levels of genes associated with sporulation, cell-wall biosynthesis, and DNA damage response were downregulated in the ΔAorlmA mutant. Furthermore, the absence of AorlmA resulted in a reduction in autophagy and endocytosis. Transcriptome analysis showed that differentially expressed genes in the absence of AorlmA were involved in membrane components, the oxidation-reduction process, transmembrane transport, metabolic processes, cellular components, organelles, cellular response to stress, and DNA damage response. In addition, metabolomic analysis showed that AoRlmA was involved in the regulation of secondary metabolites of A. oligospora. To summarize, our results highlighted the important roles of transcription factor RlmA in mycelial growth, conidiation, CWI, trap formation, stress response, autophagy, endocytosis, and secondary metabolism regulation in A. oligospora, providing a basis for elucidating the regulatory mechanism of the mycelial growth and development, pathogenicity, and stress response of NT fungi.

A novel gene, Le-Dd10, is involved in fruiting body formation of Lentinula edodes.

The cDNA library prepared from Lentinula edodes, Hokken 600 (H600), primordia was screened using cDNA expressed specifically in Dictyostelium discoideum prestalk as a probe. Twenty-one clones, Le-Dd1 ~ 21, were isolated from the L. edodes primordia cDNA library. Functional analysis of each gene was carried out by transformation into protoplast cells from L. edodes Mori 252 (M252) mycelia with the overexpression vector pLG-RasF1 of each gene because M252 protoplast cells were transformed with an 11-fold higher efficiency than H600 cells. Transformants with the overexpression vector of Le-Dd10 formed a fruiting body at almost the same time as H600, a positive control, although M252, a negative control, did not form a fruiting body under culture conditions. This suggested that Le-Dd10 is involved in the formation of fruiting bodies. Single-strand conformation polymorphism analysis revealed that Le-Dd10 is located on No. 4 linkage group of L. edodes. The properties of Le-Dd10 products were investigated by Western blotting analysis using polyclonal antibodies against GST:Le-Dd10 fusion proteins. As a result, 56-kDa, 27-kDa, and 14-kDa protein bands appeared in primordial and fruiting body stages, although the expected molecular weight of the Le-Dd10 product was 50 kDa.

Transcriptome Sequencing to Identify Candidate Genes for Mycelium Period Metabolism of Chinese Caterpillar Mushroom, Ophiocordyceps sinensis (Ascomycetes), Exposed to Light Treatment.

The Chinese caterpillar mushroom, Ophiocordyceps sinensis, is rare in traditional Chinese herbal medicine. It was reported that the development of ascospores in sexual stage of Chinese Cordyceps requires certain UV irradiation, but whether light has effect on the asexual stage of O. sinensis is unclear. It is important and necessary to identify the candidate genes involved in asexual stages (mycelium period) metabolism of O. sinensis exposed to light treatment. In this study, the isolated and purified monoascospore strains from O. sinensis were treated with 10 days light (L10) and dark as control. Transcriptome sequencing (RNA-seq) was conducted to investigate the effect of light treatment on O. sinensis at the gene level and the changes in various metabolic pathways. The results showed that the colony surface was covered with villous aerial hyphae with a yellow circular mycelium ribbon in the center of the colony, and the content of polysaccharides, urea, adenosine, and cordycepin were significantly enhanced by L10 treatment. Kyoto Encyclopedia of Genes and Genomes (KEGG) functional annotation analysis showed that cell structure and catalytic metabolism were the significant items classified, and crucial genes affecting the anabolic pathways of polysaccharides, urea, adenosine, and cordycepin were also identified. In addition, the important roles of photoreceptor genes CRYD, WC-1, and FRQ were verified by combined analysis of qRT-PCR and transcriptome sequencing. Collectively, this study would be helpful to better understand the influence of light on the asexual stage of Chinese Cordyceps and provide a preliminary light treatment reference for Cordyceps artificial cultivation.

Mnsod1 promotes the development of Pleurotus ostreatus and enhances the tolerance of mycelia to heat stress.

BACKGROUND: Superoxide dismutases (SODs, EC 1.15.1.1) are defense proteins that can be used as sweepers to clear reactive oxygen species (ROS). They have been widely studied in the plant. Intensive research demonstrates that SOD plays an essential role in plants. However, in Pleurotus ostreatus, the function and regulatory pathway of SOD in the growth and development and the abiotic stress response have not been clear. RESULTS: In this study, three MnSOD-encoding genes of the P. ostreatus CCMSSC00389 strain were cloned and identified. Mnsod1, Mnsod2, and Mnsod3 were interrupted by 3, 7, and 2 introns, and encoded proteins of 204, 220, and 344 amino acids, respectively. By comparing the relative expression of three MnSOD-encoding genes in mycelia, the results showed that the gene with the highest primary expression was Mnsod1. Subsequently, the function of P. ostreatus Mnsod1 was explored by overexpression (OE) and RNA interference (RNAi). The results showed that during the growth and development of P. ostreatus, MnSOD1 protein increased gradually from mycelia to the fruiting body, but decreased in spores. The change of Mnsod1 transcription level was not consistent with the changing trend of MnSOD1 protein. Further studies showed that during primordia formation, the expression of Mnsod1 gradually increased, reaching a peak at 48 h, and the transcription level was 2.05-folds compared to control. H2O2 content progressively accumulated during the formation of primordia, and its change trend was similar to that of Mnsod1 transcription. OE-Mnsod1-1 and OE-Mnsod1-21 strains accelerated the formation of primordia. The results suggested that Mnsod1 may participate in the formation rate of P. ostreatus primordium by regulating the signal molecule H2O2. In addition, OE-Mnsod1-1 and OE-Mnsod1-21 strains shortened the mycelial recovery time after heat stress and improved the tolerance of the strains to 2.5 mM and 5 mM H2O2, which showed that Mnsod1 was involved in the response of P. ostreatus mycelium to heat stress. CONCLUSIONS: This study indicates that Mnsod1 plays an active role in the formation of P. ostreatus primordia and the response to abiotic stress.

Autophagy-Related Gene PlATG6a Is Involved in Mycelial Growth, Asexual Reproduction and Tolerance to Salt and Oxidative Stresses in Peronophythora litchii.

Autophagy is ubiquitously present in eukaryotes. During this process, intracellular proteins and some waste organelles are transported into lysosomes or vacuoles for degradation, which can be reused by the cell to guarantee normal cellular metabolism. However, the function of autophagy-related (ATG) proteins in oomycetes is rarely known. In this study, we identified an autophagy-related gene, PlATG6a, encoding a 514-amino-acid protein in Peronophythora litchii, which is the most destructive pathogen of litchi. The transcriptional level of PlATG6a was relatively higher in mycelium, sporangia, zoospores and cysts. We generated PlATG6a knockout mutants using CRISPR/Cas9 technology. The P. litchii Δplatg6a mutants were significantly impaired in autophagy and vegetative growth. We further found that the Δplatg6a mutants displayed decreased branches of sporangiophore, leading to impaired sporangium production. PlATG6a is also involved in resistance to oxidative and salt stresses, but not in sexual reproduction. The transcription of peroxidase-encoding genes was down-regulated in Δplatg6a mutants, which is likely responsible for hypersensitivity to oxidative stress. Compared with the wild-type strain, the Δplatg6a mutants showed reduced virulence when inoculated on the litchi leaves using mycelia plugs. Overall, these results suggest a critical role for PlATG6a in autophagy, vegetative growth, sporangium production, sporangiophore development, zoospore release, pathogenesis and tolerance to salt and oxidative stresses in P. litchii.

Genomic Sequence and Transcriptome Analysis of the Medicinal Fungus Keithomyces neogunnii.

The filamentous fungus Keithomyces neogunnii can infect the larvae of Lepidoptera (Hepialus sp.) and form an insect-fungi complex, which is utilized as an important traditional Chinese medicine. As a valuable medicinal fungus, K. neogunnii produces diverse bioactive substances (e.g., polysaccharide, vitamins, cordycepic acid, and adenosine) under cultivation conditions. Herein, we report the first high-quality genome of the K. neogunnii single-spore isolate Cg7.2a using single-molecule real-time sequencing technology in combination with Illumina sequencing. The assembled genome was 32.6 Mb in size, containing 8,641 predicted genes and having a GC content of 52.16%. RNA sequencing analysis revealed the maximum number of differentially expressed genes in the fungus during the stroma formation stage compared with those during the mycelium stage. These data are valuable to enhance our understanding of the biology, development, evolution, and physiological metabolism of K. neogunnii.

Quantitative Monitoring of Mycelial Growth of Aspergillus fumigatus in Liquid Culture by Optical Density.

Filamentous fungi form multicellular hyphae, which generally form pellets in liquid shake cultures, during the vegetative growth stage. Because of these characteristics, growth-monitoring methods commonly used in bacteria and yeast have not been applied to filamentous fungi. We have recently revealed that the cell wall polysaccharide α-1,3-glucan and extracellular polysaccharide galactosaminogalactan (GAG) contribute to hyphal aggregation in Aspergillus oryzae. Here, we tested whether Aspergillus fumigatus shows dispersed growth in liquid media that can be quantitatively monitored, similar to that of yeasts. We constructed a double disruptant mutant of both the primary α-1,3-glucan synthase gene ags1 and the putative GAG synthase gene gtb3 in A. fumigatus AfS35 and found that the hyphae of this mutant were fully dispersed. Although the mutant lost α-1,3-glucan and GAG, its growth and susceptibility to antifungal agents were not different from those of the parental strain. Mycelial weight of the mutant in shake-flask cultures was proportional to optical density for at least 18 h. We were also able to quantify the dose response of hyphal growth to antifungal agents by measuring optical density. Overall, we established a convenient strategy to monitor A. fumigatus hyphal growth. Our method can be directly used for screening for novel antifungals against Aspergillus species. IMPORTANCE Filamentous fungi generally form hyphal pellets in liquid culture. This property prevents filamentous fungi so that we may apply the methods used for unicellular organisms such as yeast and bacteria. In the present study, by using the fungal pathogen Aspergillus fumigatus strain with modified hyphal surface polysaccharides, we succeeded in monitoring the hyphal growth quantitatively by optical density. The principle of this easy measurement by optical density could lead to a novel standard of hyphal quantification such as those that have been used for yeasts and bacteria. Dose response of hyphal growth by antifungal agents could also be monitored. This method could be useful for screening for novel antifungal reagents against Aspergillus species.

The arbuscular mycorrhizal fungus Rhizophagus irregularis harmonizes nuclear dynamics in the presence of distinct abiotic factors.

Arbuscular mycorrhizal fungi (AMF) are widespread obligate root symbionts that assist plants in obtaining nutrients and protection against environmental stresses. In the model species Rhizophagus irregularis, heterokaryotic strains (AMF dikaryons) carry thousands of nuclei originating from two parental strains whose frequency varies depending on strains and host identity. Here, using digital droplet PCR, we demonstrate that surrounding abiotic factors (temperature, phosphorus, and pH) also change the nuclear dynamics of such strains in root organ cultures. Furthermore, when spatially separated portions of the AMF mycelium grow under different abiotic conditions, all the produced spores carry highly similar nuclear ratios. Overall, these findings demonstrate that abiotic stressors impact the nuclear organization of a widespread group of multinucleate plant symbionts, and reveal remarkable mechanisms of nuclear ratio harmonization across the mycelium in these prominent symbionts.

Transcriptomics Analysis of Primordium Formation in Pleurotus eryngii.

Primordium formation is an important stage preceding the growth and development of the Pleurotus eryngii fruiting body. However, the molecular mechanisms underlying primordium formation remain unclear. In the present study, comparative transcriptomics was performed between mature mycelia and primordium to analyze the transcriptional properties during primordium formation in P. eryngii. A total of 19,655 differentially expressed genes (10,718 upregulated genes and 8937 downregulated genes) were identified. These differentially expressed genes were involved in cell wall degradation, carbohydrate hydrolysis, light perception, and cAMP signal transduction. These results aid further understanding of the transcriptional changes and the molecular processes underlying primordium formation and differentiation, which may lay the foundation for improving the cultivation and quality control of P. eryngii.

Optimized methodology for obtention of high-yield and -quality RNA from the mycelium of the bioluminescent fungus Neonothopanus gardneri.

Neonothopanus gardneri, also known as coconut flower mushroom (flor-de-coco), is a Brazilian bioluminescent basidiomycete found in Palm Forest, a transitional biome between the Amazonian Forest and Caatinga (Savanna-like vegetation) in Northeast Brazil, especially in Piauí State. Recent advances toward the elucidation of fungal bioluminescence have contributed to the discovery of four genes (hisps, h3h, luz and cph) involved with the bioluminescence process, the so-called Caffeic Acid Cycle (CAC) and to develop biotechnological applications such autoluminescent tobacco plants and luciferase-based reporter genes. High-yield and -quality RNA-extraction methods are required for most of these purposes. Herein, four methods for RNA isolation from the mycelium of N. gardneri were evaluated: RNeasy® kit (QIAGEN), TRI+, TRI18G+, and TRI26G+. Highest RNA yield was observed for TRI18G+ and TRI26G+ methods, an increase of ~130% in comparison to the RNeasy® method and of ~40% to the TRI+ protocol. All the RNA samples showed good purity and integrity, except by gDNA contamination in RNA samples produced with the RNeasy® method. High quality of RNA samples was confirmed by successful cDNA synthesis and PCR amplification of the coding sequence of h3h gene, responsible for the hydroxylation of the precursor of fungal luciferin (3-hydroxyhispidin). Similarly, RT-qPCR amplification of ef-tu gene, related to the protein biosynthesis in the cell, was demonstrated from RNA samples. This is the first report of a reproducible, time-saving and low-cost optimized method for isolation of high-quality and -yield, DNA-free RNA from a bioluminescent fungus, but that can also be useful for other basidiomycetes.

Identification of a key protein set involved in Moniliophthora perniciosa necrotrophic mycelium and basidiocarp development.

Moniliophthora perniciosa is a hemibiotrophic fungus that causes witches' broom disease in cacao (Theobroma cacao L.). The biotrophic fungal phase initiates the disease and is characterized by a monokaryotic mycelium, while the necrotrophic phase is characterized by a dikaryotic mycelium and leads to necrosis of infected tissues. A study of the necrotrophic phase was conducted on bran-based solid medium, which is the only medium that enables basidiocarp and basidiospore production. Six different fungal developmental phases were observed according to the mycelium colour or the organ produced: white, yellow, pink, dark pink, primordium and basidiocarp. In this study, we identified notable proteins in each phase, particularly those accumulated prior to basidiocarp formation. Proteins were analysed by proteomics; 2-D gels showed 300-550 spots. Statistically differentially accumulated spots were sequenced by mass spectrometry and 259 proteins were identified and categorized into nine functional classes. Proteins related to energy metabolism, protein folding and morphogenesis that were potentially involved in primordium and basidiocarp formation were identified; these proteins may represent useful candidates for further analysis related to the spread and pathogenesis of this fungus. To the best of our knowledge, this report describes the first proteomic analysis of the developmental phases of Moniliophthora perniciosa.

Expression of F-actin and ß-tubulin genes in free mycelia and robust biofilms of the filamentous fungus Aspergillus niger.

The morphology and growth of the filamentous fungi are influenced by different factors as the culture conditions and the type of fermentative process. The production and secretion of metabolites by these organisms present a direct relationship with their morphology. The organization of the microtubules and actin in the cytoskeleton is determinant for both the fungal growth and morphology. In this context, this study aimed to analyze the expression of the ß-tubulin, F-actin, and glucan synthase in the A. niger mycelia obtained from submerged fermentation and biofilm fermentation through qPCR, as well as the analysis of the nucleus distribution in the hypha. Herein, we showed that ß-tubulin and the F-actin gene were more expressed in the biofilm condition, while the glucan synthase was in the submerged condition. No significant difference was observed in the nucleus distribution between the mycelia obtained from both the fermentative processes. In conclusion, the different morphologies observed for the mycelia from submerged fermentation and biofilm fermentation might be influenced by the differential modulation of genes that codify cytoskeleton proteins, which seems to be potentially regulated by mechanosensing during fungal contact with solid supports.

Coriolopsis trogii hydrophobin genes favor a clustering distribution and are widely involved in mycelial growth and primordia formation.

Hydrophobins are small, secreted proteins with important physiological functions in mycelial growth and fungal development. Here, 1 nucleus-specific and 35 allelic hydrophobin genes were identified in the genome of a white rot fungus, Coriolopsis trogii. Among these, 22 were eight-cysteine class I hydrophobin genes and the other 14 were uncommon six-cysteine hydrophobin genes. The six-cysteine hydrophobins were speculated to have originated from a common ancestor. The hydrophobin genes favored a clustering distribution and two recent duplication pairs were identified. The genes had conserved gene structures with three exons and two introns. Cthyd18, Cthyd19, and Cthyd32 were constitutively highly expressed in all developmental stages. Cthyd20, Cthyd21, Cthyd22, Cthyd28, Cthyd30, Cthyd31, and Cthyd33 were highly expressed in mycelia, and Cthyd12 and Cthyd35 in the reproductive stages. Sixteen hydrophobin genes were regulated differently in the transition from mycelia to primordia; Cthyd35 showed maximal upregulation of 1922-fold, and Cthyd23 showed maximal downregulation of 552-fold. Most (32) hydrophobin genes showed significant differential expression between mycelia cultured in different media (potato dextrose agar or broth). Weighted gene co-expression network analysis and promoter analysis revealed that C2H2 zinc finger proteins may regulate hydrophobin genes. These results may support further research into the function and evolution of hydrophobins.

Mitochondrial pyruvate carrier regulates the lignocellulosic decomposition rate through metabolism in Ganoderma lucidum.

The activity of mitochondrial pyruvate carrier (MPC) can be modulated to regulate intracellular metabolism under different culture conditions. In Ganoderma lucidum, the role of MPC in regulating carbon sources remains unknown. By knocking down MPC genes (MPC1 and MPC2), this research found that the loss of MPC increased the growth rate of G. lucidum by ~30% in a medium with wood chips as a carbon source. Then cellulase and laccase activities were tested. Endoglucanase and laccase activity increased by ~50% and ~35%, respectively, in MPC knockdown mutants compared with that in the wild type strain. Finally, the expression levels of genes related to glycolysis were assayed, and the transcription levels of these enzymes were found to be increased by ~250% compared with the wild type strain. In conclusion, the regulation of intracellular metabolism by MPC provides a new way to improve the use of nondominant carbon sources such as lignocellulose.