RNA-seq transcriptome and pathway analysis of the medicinal mushroom Lignosus tigris (Polyporaceae) offer insights into its bioactive compounds with anticancer and antioxidant potential.

ETHNOPHARMACOLOGICAL RELEVANCE: Medicinal mushrooms belonging to the Lignosus spp., colloquially known as Tiger Milk mushrooms (TMMs), are used as traditional medicine by communities across various regions of China and Southeast Asia to enhance immunity and to treat various diseases. At present, three Lignosus species have been identified in Malaysia: L. rhinocerus, L. tigris, and L. cameronensis. Similarities in their macroscopic morphologies and the nearly indistinguishable appearance of their sclerotia often lead to interchangeability between them. Hence, substantiation of their traditional applications via identification of their individual bioactive properties is imperative in ensuring that they are safe for consumption. L. tigris was first identified in 2013. Thus far, studies on L. tigris cultivar sclerotia (Ligno TG-K) have shown that it possesses significant antioxidant activities and has greater antiproliferative action against selected cancer cells in vitro compared to its sister species, L. rhinocerus TM02®. Our previous genomics study also revealed significant genetic dissimilarities between them. Further omics investigations on Ligno TG-K hold immense potential in facilitating the identification of its bioactive compounds and their associated bioactivities. AIM OF STUDY: The overall aim of this study was to investigate the gene expression profile of Ligno TG-K via de novo RNA-seq and pathway analysis. We also aimed to identify highly expressed genes encoding compounds that contribute to its cytotoxic and antioxidant properties, as well as perform a comparative transcriptomics analysis between Ligno TG-K and its sister species, L. rhinocerus TM02®. MATERIALS AND METHODS: Total RNA from fresh 3-month-old cultivated L. tigris sclerotia (Ligno TG-K) was extracted and analyzed via de novo RNA sequencing. Expressed genes were analyzed using InterPro and NCBI-Nr databases for domain identification and homology search. Functional categorization based on gene functions and pathways was performed using Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Clusters of Orthologous Genes (COG) databases. Selected genes were subsequently subjected to phylogenetic analysis. RESULTS: Our transcriptomics analysis of Ligno TG-K revealed that 68.06% of its genes are expressed in the sclerotium; 80.38% of these were coding transcripts. Our analysis identified highly expressed transcripts encoding proteins with prospective medicinal properties. These included serine proteases (FPKM = 7356.68), deoxyribonucleases (FPKM = 3777.98), lectins (FPKM = 3690.87), and fungal immunomodulatory proteins (FPKM = 2337.84), all of which have known associations with anticancer activities. Transcripts linked to proteins with antioxidant activities, such as superoxide dismutase (FPKM = 1161.69) and catalase (FPKM = 1905.83), were also highly expressed. Results of our sequence alignments revealed that these genes and their orthologs can be found in other mushrooms. They exhibit significant sequence similarities, suggesting possible parallels in their anticancer and antioxidant bioactivities. CONCLUSION: This study is the first to provide a reference transcriptome profile of genes expressed in the sclerotia of L. tigris. The current study also presents distinct COG profiles of highly expressed genes in Ligno TG-K and L. rhinocerus TM02®, highlighting that any distinctions uncovered may be attributed to their interspecies variations and inherent characteristics that are unique to each species. Our findings suggest that Ligno TG-K contains bioactive compounds with prospective medicinal properties that warrant further investigations. CLASSIFICATION: Systems biology and omics.

Transcriptome analysis reveals insight into the protective effect of N-acetylcysteine against cadmium toxicity in Ganoderma lucidum (Polyporales: Polyporaceae).

Ganoderma lucidum is widely cultivated and used as traditional medicine in China and other Asian countries. As a member of macrofungi, Ganoderma lucidum is also prone to bioaccumulation of cadmium and other heavy metals in a polluted environment, which affects the growth and production of Ganoderma lucidum, as well as human health. N-Acetyl-L-cysteine (NAC) is considered a general antioxidant and free radical scavenger that is involved in the regulation of various stress responses in plants and animals. However, whether NAC could regulate cadmium stress responses in macrofungi, particularly edible fungi, is still unknown. In this work, we found that the exogenous NAC could alleviate Cd-induced growth inhibition and reduce the cadmium accumulation in Ganoderma lucidum. The application of the NAC cloud also inhibit cadmium-induced H2O2 production in the mycelia. By using transcriptome analysis, 2920 and 1046 differentially expressed unigenes were identified in "Cd100 vs CK" and "NAC_Cd100 vs Cd100," respectively. These differential unigenes were classified into a set of functional categories and pathways, which indicated that various biological pathways may play critical roles in the protective effect of NAC against Cd­induced toxicity in Ganoderma lucidum. Furthermore, it suggested that the ATP-binding cassette transporter, ZIP transporter, heat shock protein, glutathione transferases, and Cytochrome P450 genes contributed to the increased tolerance to cadmium stress after NAC application in Ganoderma lucidum. These results provide new insight into the physiological and molecular response of Ganoderma lucidum to cadmium stress and the protective role of NAC against cadmium toxicity.

Isothermal titration calorimetric assessment of lignin conversion by laccases.

Lignin valorization may offer a sustainable approach to achieve a chemical industry that is not completely dependent on fossil resources for the production of aromatics. However, lignin is a recalcitrant, heterogeneous, and complex polymeric compound for which only very few catalysts can act in a predictable and reproducible manner. Laccase is one of those catalysts and has often been referred to as an ideal "green" catalyst, as it is able to oxidize various linkages within lignin to release aromatic products, with the use of molecular oxygen and formation of water as the only side product. The extent and rate of laccase-catalyzed lignin conversion were measured using the label-free analytical technique isothermal titration calorimetry (ITC). IITC provides the molar enthalpy of the reaction, which reflects the extent of conversion and the time-dependent power trace, which reflects the rate of the reaction. Calorimetric assessment of the lignin conversion brought about by various fungal and bacterial laccases in the absence of mediators showed marked differences in the extent and rate of conversion for the different enzymes. Kraft lignin conversion by Trametes versicolor laccase followed Michaelis-Menten kinetics and was characterized by the following thermodynamic and kinetic parameters ΔHITC = -(2.06 ± 0.06)·103 kJ mol-1 , KM = 6.6 ± 1.2 µM and Vmax = 0.30 ± 0.02 U/mg at 25°C and pH 6.5. We envision calorimetric techniques as important tools for the development of enzymatic lignin valorization strategies.

Application of CRISPR/Cas9 Tools for Genome Editing in the White-Rot Fungus Dichomitus squalens.

Dichomitus squalens is an emerging reference species that can be used to investigate white-rot fungal plant biomass degradation, as it has flexible physiology to utilize different types of biomass as sources of carbon and energy. Recent comparative (post-) genomic studies on D. squalens resulted in an increasingly detailed knowledge of the genes and enzymes involved in the lignocellulose breakdown in this fungus and showed a complex transcriptional response in the presence of lignocellulose-derived compounds. To fully utilize this increasing amount of data, efficient and reliable genetic manipulation tools are needed, e.g., to characterize the function of certain proteins in vivo and facilitate the construction of strains with enhanced lignocellulolytic capabilities. However, precise genome alterations are often very difficult in wild-type basidiomycetes partially due to extremely low frequencies of homology directed recombination (HDR) and limited availability of selectable markers. To overcome these obstacles, we assessed various Cas9-single guide RNA (sgRNA) ribonucleoprotein (RNP) -based strategies for selectable homology and non-homologous end joining (NHEJ) -based gene editing in D. squalens. We also showed an induction of HDR-based genetic modifications by using single-stranded oligodeoxynucleotides (ssODNs) in a basidiomycete fungus for the first time. This paper provides directions for the application of targeted CRISPR/Cas9-based genome editing in D. squalens and other wild-type (basidiomycete) fungi.

Taxonomy of the major rhizomorphic species of the "Melanopus group" within Polyporaceae in Yasuní National Park, Ecuador.

Yasuní National Park in Ecuador is one of the most biodiverse places on earth. The fungi in this tropical rainforest are also diverse but have received little research attention. This research paper focuses on an important group of fungi in the family Polyporaceae and examines the genera Polyporus, Atroporus, and Neodictyopus that form aerial melanized cord-like structures called rhizomorphs. Phylogenetic analyses, macro and micromorphological descriptions of basidiomata and rhizomorphs, as well as cultural characterization were completed to better understand these ecologically important fungi. Here we describe four new species: Atroporus yasuniensis, Atroporus tagaeri, Neodictyopus sylvaticus, and Polyporus taromenane, and a new variety Polyporus leprieurii var. yasuniensis. The information presented in this study adds important new knowledge about the unusual rhizomorph producing fungi found in Yasuní National Park, Ecuador and other tropical rainforests.

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.

Haploid Genome Analysis Reveals a Tandem Cluster of Four HSP20 Genes Involved in the High-Temperature Adaptation of Coriolopsis trogii.

Coriolopsis trogii is a typical thermotolerant basidiomycete fungus, but its thermotolerance mechanisms are currently unknown. In this study, two monokaryons of C. trogii strain Ct001 were assembled: Ct001_29 had a genome assembly size of 38.85 Mb and encoded 13,113 genes, while Ct001_31 was 40.19 Mb in length and encoded 13,309 genes. Comparative intra- and interstrain genomic analysis revealed the rich genetic diversity of C. trogii, which included more than 315,194 single-nucleotide polymorphisms (SNPs), 30,387 insertion/deletions (indels), and 1,460 structural variations. Gene family analysis showed that the expanded families of C. trogii were functionally enriched in lignocellulose degradation activities. Furthermore, a total of 14 allelic pairs of heat shock protein 20 (HSP20) genes were identified in the C. trogii genome. The expression profile obtained from RNA sequencing (RNA-Seq) showed that four tandem-duplicated allelic pairs, HSP20.5 to HSP20.8, had more than 5-fold higher expression at 35°C than at 25°C. In particular, HSP20.5 and HSP20.8 were the most highly expressed HSP20 genes. Allelic expression bias was found for HSP20.5 and HSP20.8; the expression of Ct29HSP20.8 was at least 1.34-fold higher than that of Ct31HSP20.8, and that of Ct31HSP20.5 was at least 1.5-fold higher than that of Ct29HSP20.5. The unique structural and expression profiles of the HSP20 genes revealed by these haplotype-resolved genomes provide insight into the molecular mechanisms of high-temperature adaptation in C. trogii. IMPORTANCE Heat stress is one of the most frequently encountered environmental stresses for most mushroom-forming fungi. Currently available fungal genomes are mostly haploid because high heterozygosity hinders diploid genome assembly. Here, two haplotype genomes of C. trogii, a thermotolerant basidiomycete, were assembled separately. A conserved tandem cluster of four HSP20 genes showing allele-specific expression was found to be closely related to high-temperature adaptation in C. trogii. The obtained haploid genomes and their comparison offer a more thorough understanding of the genetic background of C. trogii. In addition, the responses of HSP20 genes at 35°C, which may contribute to the growth and survival of C. trogii at high temperatures, could inform the selection and breeding of elite strains in the future.

Recombinant Expression of Trametes versicolor Aflatoxin B1-Degrading Enzyme (TV-AFB1D) in Engineering Pichia pastoris GS115 and Application in AFB1 Degradation in AFB1-Contaminated Peanuts.

Aflatoxins seriously threaten the health of humans and animals due to their potential carcinogenic properties. Enzymatic degradation approach is an effective and environmentally friendly alternative that involves changing the structure of aflatoxins. In this study, Trametes versicolor aflatoxin B1-degrading enzyme gene (TV-AFB1D) was integrated into the genome of Pichia pastoris GS115 by homologous recombination approach. The recombinant TV-AFB1D was expressed in engineering P. pastoris with a size of approximately 77 kDa under the induction of methanol. The maximum activity of TV-AFB1D reached 17.5 U/mL after the induction of 0.8% ethanol (v/v) for 84 h at 28 °C. The AFB1 proportion of 75.9% was degraded using AFB1 standard sample after catalysis for 12 h. In addition, the AFB1 proportion was 48.5% using AFB1-contaminated peanuts after the catalysis for 18 h at 34 °C. The recombinant TV-AFB1D would have good practical application value in AFB1 degradation in food crops. This study provides an alternative degrading enzyme for the degradation of AFB1 in aflatoxin-contaminated grain and feed via enzymatic degradation approach.

Comparative mitogenomics of Agaricomycetes: Diversity, abundance, impact and coding potential of putative open-reading frames.

The mitochondrion is an organelle found in eukaryote organisms, and it is vital for different cellular pathways. The mitochondrion has its own DNA molecule and, because its genetic content is relatively conserved, despite the variation of size and structure, mitogenome sequences have been widely used as a promising molecular biomarker for taxonomy and evolution in fungi. In this study, the mitogenomes of two fungal species of Agaricomycetes class, Phellinotus piptadeniae and Trametes villosa, were assembled and annotated for the first time. We used these newly sequenced mitogenomes for comparative analyses with other 55 mitogenomes of Agaricomycetes available in public databases. Mitochondrial DNA (mtDNA) size and content are highly variable and non-coding and intronic regions, homing endonucleases (HEGs), and unidentified ORFs (uORFs) significantly contribute to the total size of the mitogenome. Furthermore, accessory genes (most of them as HEGs) are shared between distantly related species, most likely as a consequence of horizontal gene transfer events. Conversely, uORFs are only shared between taxonomically related species, most probably as a result of vertical evolutionary inheritance. Additionally, codon usage varies among mitogenomes and the GC content of mitochondrial features may be used to distinguish coding from non-coding sequences. Our results also indicated that transposition events of mitochondrial genes to the nuclear genome are not common. Despite the variation of size and content of the mitogenomes, mitochondrial genes seemed to be reliable molecular markers in our time-divergence analysis, even though the nucleotide substitution rates of mitochondrial and nuclear genomes of fungi are quite different. We also showed that many events of mitochondrial gene shuffling probably happened amongst the Agaricomycetes during evolution, which created differences in the gene order among species, even those of the same genus. Altogether, our study revealed new information regarding evolutionary dynamics in Agaricomycetes.

Genome sequencing and annotation and phylogenomic analysis of the medicinal mushroom Amauroderma rugosum, a traditional medicinal species in the family Ganodermataceae.

Amauroderma rugosum is one of the traditional Chinese medicinal mushrooms and is used to reduce inflammation, treat diuretic and upset stomach, and prevent cancer. Here, we present a genomic resource of Amauroderma rugosum (ACCC 51706) for further understanding its biology and exploration of the synthesis pathway of bioactive compounds. Genomic DNA was extracted and then subjected to Illumina HiSeq X Ten and PacBio Sequel I sequencing. The final genome is 40.66 Mb in size, with an N50 scaffold size of 36.6 Mb, and encodes 10 181 putative predicted genes. Among them, 6931 genes were functionally annotated. Phylogenomic analysis suggested that A. rugosum and Ganoderma sinense were not clustered together into a group and the latter was grouped with the Polyporaceae. Further, we also identified 377 carbohydrate-active enzymes (CAZymes) and 15 secondary metabolite biosynthetic gene clusters. This is the first genome-scale assembly and annotation for an Amauroderma species. The identification of novel secondary metabolite biosynthetic gene clusters would promote pharmacological research and development of novel bioactive compounds in the future.

Plant Nitrilase Homologues in Fungi: Phylogenetic and Functional Analysis with Focus on Nitrilases in Trametes versicolor and Agaricus bisporus.

Fungi contain many plant-nitrilase (NLase) homologues according to database searches. In this study, enzymes NitTv1 from Trametes versicolor and NitAb from Agaricus bisporus were purified and characterized as the representatives of this type of fungal NLase. Both enzymes were slightly more similar to NIT4 type than to NIT1/NIT2/NIT3 type of plant NLases in terms of their amino acid sequences. Expression of the synthetic genes in Escherichia coli Origami B (DE3) was induced with 0.02 mM isopropyl ß-D-1-thiogalactopyranoside at 20 °C. Purification of NitTv1 and NitAb by cobalt affinity chromatography gave ca. 6.6 mg and 9.6 mg of protein per 100 mL of culture medium, respectively. Their activities were determined with 25 mM of nitriles in 50 mM Tris/HCl buffer, pH 8.0, at 30 °C. NitTv1 and NitAb transformed ß-cyano-L-alanine (ß-CA) with the highest specific activities (ca. 132 and 40 U mg-1, respectively) similar to plant NLase NIT4. ß-CA was transformed into Asn and Asp as in NIT4 but at lower Asn:Asp ratios. The fungal NLases also exhibited significant activities for (aryl)aliphatic nitriles such as 3-phenylpropionitrile, cinnamonitrile and fumaronitrile (substrates of NLase NIT1). NitTv1 was more stable than NitAb (at pH 5-9 vs. pH 5-7). These NLases may participate in plant-fungus interactions by detoxifying plant nitriles and/or producing plant hormones. Their homology models elucidated the molecular interactions with various nitriles in their active sites.

Insights into the mechanism of cyanobacteria removal by the algicidal fungi Bjerkandera adusta and Trametes versicolor.

Fungal mycelia can eliminate almost all cocultured cyanobacterial cells within a short time. However, molecular mechanisms of algicidal fungi are poorly understood. In this study, a time-course transcriptomic analysis of algicidal fungus Bjerkandera adusta T1 was applied to investigate gene expression and regulation. A total of 132, 300, 422, and 823 differentially expressed genes (DEGs) were identified at 6, 12, 24, and 48 hr, respectively. Most DEGs exhibited high endopeptidase activity, cellulose catabolic process, and transmembrane transporter activity by using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. Many decomposition genes encoding endopeptidases were induced a little later in B. adusta T1 when compared with previously investigated algicidal fungus Trametes versicolor F21a. Besides, the accumulated expression of Polysaccharide lyases8 (PL8) gene with peptidoglycan and alginate decomposition abilities was greatly delayed in B. adusta T1 relative to T. versicolor F21a. It was implied that endopeptidases and enzymes of PL8 might be responsible for the strong algicidal ability of B. adusta T1 as well as T. versicolor F21a.

GC-MS Analysis and Cytotoxicity Detection of Volatile Oil from the Fragrant Bracket Mushroom, Trametes suaveolens (Agaricomycetes).

Trametes suaveolens is a medicinal mushroom known as Baizhi in traditional Chinese medicine. Our previous research has found that it has some pharmacological activity in vivo. The aim of the study was to investigate the chemical compounds and cytotoxic effects of volatile oil from T. suaveolens. In this study, internal transcribed spacer (ITS) sequence analysis was used to determine wild T. suaveolens collected. To fully analyze the composition of volatile oil extracted from T. suaveolens, hydrodistillation (HD) and solid phase microextraction (SPME) were adopted simultaneously. In both cases, the analysis was carried out using gas chromatography-mass spectrometry (GC-MS) and the cytotoxic effects of T. suaveolens volatile oil on human NCI-H460 lung non-small cell carcinoma cells and MCF-7 breast adenocarcinoma cells were investigated. The results indicated that all these wild samples were identified as T. suaveolens. Thirty-one components in HD and 62 components in SPME were identified, respectively. Furthermore, the volatile compounds obtained from T. suaveolens by HD indicated that esters compounds were a major class (68.47%), followed by acids (25.06%), aldehydes (4.20%), and alcohols (1.48%). SPME found that the largest content were aldehydes (45.47%), followed by alcohols (31.42%), ketones (6.89%), and esters (6.72%). In the cytotoxic assays, the volatile oil was found to have toxic effect on NCI-H460 and MCF-7 tumor cells but not BEAS-2B and MCF-10A normal cells, and the IC50 values of NCI-H460 and MCF-7 tumor cells were 24.1 and 19.2 µg/ml, respectively. The present study shows that the composition of essential oil from T. suaveolens has potential value for the prevention and treatment of lung cancer.

Genome and secretome analysis of jute endophyte Grammothele lineata strain SDL-CO-2015-1: Insights into its lignocellulolytic structure and secondary metabolite profile.

Grammothele lineata strain SDL-CO-2015-1, jute (Corchorus olitorius) endophyte has been reported to produce anti-cancer drug paclitaxel in culture condition. Here we investigated the genome using different bioinformatic tools to find its association with the production of commercially important compounds including taxol. Carbohydrate-active enzymes, proteases, and secretory proteins were annotated revealing a complex endophytic relationship with its plant host. The presences of a diverse range of CAZymes including numerous lignocellulolytic enzymes support its potentiality in biomass degradation. Genome annotation led to the identification of 28 clusters for secondary metabolite biosynthesis. Several biosynthesis gene clusters were identified for terpene biosynthesis from antiSMASH analysis but none could be specifically pinned to taxol synthesis. This study will direct us to understand the genomic organization of endophytic basidiomycetes with a potential for producing numerous commercially important enzymes and secondary metabolites taking G. lineata as a model.

Combined Effect of Light and Nutrients on the Micromorphology of the White rot Fungus Cerrena Unicolor.

Light influences developmental pathways in fungi. Recent transcriptomic and biochemical analyses have demonstrated that light influences the metabolism of a white-rot basidiomycete Cerrena unicolor. However, the expression profile of genes involved in the growth and development, or micromorphological observations of the mycelium in response to variable lighting and culturing media, have not performed. We aim to reveal the effect of light and nutrients on C. unicolor growth and a potential relationship between the culture medium and lighting conditions on fungus micromorphological structures. Confocal laser scanning microscopy and scanning electron microscopy were employed for morphological observations of C. unicolor mycelium cultivated in red, blue, green, and white light and darkness on mineral and sawdust media. A comprehensive analysis of C. unicolor differentially expressed genes (DEGs) was employed to find global changes in the expression profiles of genes putatively involved in light-dependent morphogenesis. Both light and nutrients influenced C. unicolor growth and development. Considerable differences in the micromorphology of the mycelia were found, which were partially reflected in the functional groups of DEGs observed in the fungus transcriptomes. A complex cross-interaction of nutritional and environmental signals on C. unicolor growth and morphology was suggested. The results are a promising starting point for further investigations of fungus photobiology.

Notes on Medicinal Polypore Species from the Genus Daedaleopsis (Agaricomycetes), Distributed in the Asian Part of Russia.

We present a study on the medicinal value, taxonomy, and ecology of the polypore mushrooms Daedaleopsis confragosa and D. tricolor isolated from the Asian part of Russia (the Urals, Siberia, and the Far East). The phylogenetic analysis of recombinant DNA internal transcribed spacer sequences data has shown that D. confragosa and D. tricolor do not differ taxonomically and should be considered as one species. However, because D. confragosa and D. tricolor differ significantly in their ecological characteristics, they may be considered as two morpho-ecological varieties: D. confragosa var. confragosa and D. confragosa var. tricolor (both nomen provisiorum).

Cloning, overexpression, purification, and modeling of a lectin (Rhinocelectin) with antiproliferative activity from Tiger Milk Mushroom, Lignosus rhinocerus.

A lectin gene from the Tiger Milk Mushroom Lignosus rhinocerus TM02® was successfully cloned and expressed via vector pET28a in Escherichia coli BL21(DE3). The recombinant lectin, Rhinocelectin, with a predicted molecular mass of 22.8 kDa, was overexpressed in water-soluble form without signal peptide and purified via native affinity chromatography Ni-NTA agarose. Blast protein analysis indicated the lectin to be homologous to jacalin-related plant lectin. In its native form, Rhinocelectin exists as a homo-tetramer predicted with four chains of identical proteins consisting of 11 beta-sheet structures with only one alpha-helix structure. The antiproliferative activity of the Rhinocelectin against human cancer cell lines was concentration dependent and selective. The IC50 values against triple negative breast cancer cell lines MDA-MB-231 and breast cancer MCF-7 are 36.52 ± 13.55 µg mL-1 and 53.11 ± 22.30 µg mL-1 , respectively. Rhinocelectin is only mildly cytotoxic against the corresponding human nontumorigenic breast cell line 184B5 with IC50 value at 142.19 ± 36.34 µg mL-1 . The IC50 against human lung cancer cell line A549 cells is 46.14 ± 7.42 µg mL-1 while against nontumorigenic lung cell line NL20 is 41.33 ± 7.43 µg mL-1 . The standard anticancer drug, Doxorubicin exhibited IC50 values mostly below 1 µg mL-1 for the cell lines tested. Flow cytometry analysis showed the treated breast cancer cells were arrested at G0/G1 phase and apoptosis induced. Rhinocelectin agglutinated rat and rabbit erythrocytes at a minimal concentration of 3.125 µg mL-1 and 6.250 µg mL-1 , respectively.

Transcriptome analysis of differential gene expression in Dichomitus squalens during interspecific mycelial interactions and the potential link with laccase induction.

Interspecific mycelial interactions between white rot fungi are always accompanied by an increased production of laccase. In this study, the potential of the white rot fungus Dichomitus squalens to enhance laccase production during interactions with two other white rot fungi, Trametes versicolor or Pleurotus ostreatus, was assessed. To probe the mechanism of laccase induction and the role that laccase plays during combative interaction, we analyzed the differential gene expression profile of the laccase induction response to stressful conditions during fungal interaction. We further confirmed the expression patterns of 16 selected genes by qRT-PCR analysis. We noted that many differentially expressed genes (DEGs) encoded proteins that were involved in xenobiotic detoxification and reactive oxygen species (ROS) generation or reduction, including aldo/keto reductase, glutathione S-transferases, cytochrome P450 enzymes, alcohol oxidases and dehydrogenase, manganese peroxidase and laccase. Furthermore, many DEG-encoded proteins were involved in antagonistic mechanisms of nutrient acquisition and antifungal properties, including glycoside hydrolase, glucanase, chitinase and terpenoid synthases. DEG analyses effectively revealed that laccase induction was likely caused by protective responses to oxidative stress and nutrient competition during interspecific fungal interactions.

Functional analysis of an APSES transcription factor (GlSwi6) involved in fungal growth, fruiting body development and ganoderic-acid biosynthesis in Ganoderma lucidum.

The APSES transcription factors have been identified as key regulators of fungal development and other biological processes in fungi. In the present study, the function of Ganoderma lucidum GlSwi6, a homolog of Saccharomyces cerevisiae Swi6, was characterized. RNAi was used to examine the function of GlSwi6 in G. lucidum. Silencing GlSwi6 resulted in multiple developmental defects, including reduced fungal growth and increased hyphal branching, and the GlSwi6-silenced strains did not exhibit primordium or fruiting body formation. In addition, the H2O2 and ganoderic-acid (GA) levels of the GlSwi6-silenced strains decreased approximately 50% and 25%, respectively, compared with those of the WT strain. Furthermore, the addition of H2O2 led to the recovery of the GA levels of GlSwi6-silenced strains, implying that GlSwi6 might regulate GA biosynthesis by regulating the intracellular ROS levels. Taken together, these results indicate that GlSwi6 is involved in fungal growth, development and GA biosynthesis in G. lucidum.

Solid state fermentation and crude cellulase based bioconversion of potential bamboo biomass to reducing sugar for bioenergy production.

BACKGROUND: Lignocellulosic biomass from bamboo is an attractive feedstock for the bioethanol industry owing to its high cellulosic content and fast growth rate. In this study, powdery biomass was first enzymatically delignified and then saccharified using crude enzymes. RESULTS: The biological pretreatment decreased the lignin content of the biomass from an initial value of 295 to 137.7 g kg-1 , with a simultaneous increase in exposed cellulose content from 379.3 to 615.9 g kg-1 . For optimization of the saccharification, response surface methodology was adopted using a three-factor/three-level Box-Behnken design with crude fungal cellulase loading (FPU g-1 substrate), substrate concentration (% w/v) and saccharification temperature (°C) as the main process parameters. A maximum saccharification yield of 47.19% was achieved under the optimized conditions (cellulase enzyme 18.4 FPU g-1 substrate, substrate concentration 1.0% w/v, temperature 39.49 °C). Biological delignification and saccharification of the biomass were further confirmed through scanning electron microscopy analysis. CONCLUSION: It is evident from the study that bamboo, as a renewable energy bioresource, can be hydrolysed to reducing sugars by using crude laccase/cellulase enzymes of fungal origin with good saccharification yield. Thus crude enzyme preparations could be utilized efficiently for eco-friendly and cost-effective bioethanol production. © 2018 Society of Chemical Industry.