Edible Mushroom Grifola Frondosa Research Articles

Identification and Characterization of a Predominant Hydrophobin in the Edible Mushroom Grifola frondosa.

Hydrophobins (HFBs) are a group of small, secreted amphipathic proteins of fungi with multiple physiological functions and potential commercial applications. In this study, HFB genes of the edible mushroom, Grifola frondosa, were systematically identified and characterized, and their transcriptional profiles during fungal development were determined. In total, 19 typical class I HFB genes were discovered and bioinformatically analyzed. Gene expression profile examination showed that Gf.hyd9954 was particularly highly upregulated during primordia formation, suggesting its major role as the predominant HFB in the lifecycle of G. frondosa. The wettability alteration profile and the surface modification ability of recombinant rGf.hyd9954 were greater than for the Grifola HFB HGFII-his. rGf.hyd9954 was also demonstrated to form the typical class I HFB characteristic-rodlet bundles. In addition, rGf.hyd9954 was shown to possess nanoparticle characteristics and emulsification activities. This research sheds light on the regulation of fungal development and its association with the expression of HFB genes.

TMT-based proteomic and transcriptomic analysis reveal new insights into heat stress responsive mechanism in edible mushroom Grifola frondosa

High temperature can lead to severe retardation in the growth and development of mushrooms, which influence the quality of fruiting bodies. Grifola frondosa is a popular and wide-cultivated mushroom in Asia; however, little information about the heat response mechanism in this commercially cultivated mushroom has been reported. The growth test was performed to determine the heat tolerance of different strains. Then, the TMT-based proteome and transcriptome analysis of major G. frondosa cultivar Qinghui-151 under different temperatures were performed. High temperature seriously affected the growth and recovery growth of mycelia. Thirty-four heat shock proteins (HSPs) were identified in the multiomics data and the expressions of most HSPs were induced by high temperature. The accumulation of HSPs under the control of HSFs is assumed to play a central role in the heat stress response (HSR) in G. frondosa. mTOR and Ca2+ signaling pathways were found to be activated under high temperature and might involved in heat stress signaling transduction. Polyamines synthesis enzymes were also up-regulated, suggesting the accumulation of stress protector polyamines under heat stress. In addition, expression of an L-phenylalanine ammonia-lyase (GfPAL) gene was significantly up-regulated under high temperature, which might be related to secondary metabolites synthesis. Taken together, these findings improve our understanding of the molecular mechanisms underlying the response to heat stress in G. frondosa, which could promote the breeding of new heat-tolerant G. frondosa cultivars.

Genetic and Molecular Evidence of a Tetrapolar Mating System in the Edible Mushroom Grifola frondosa.

Grifola frondosa is a valuable edible fungus with high nutritional and medicinal values. The mating systems of fungi not only offer practical strategies for breeding, but also have far-reaching effects on genetic variability. Grifola frondosa has been considered as a sexual species with a tetrapolar mating system based on little experimental data. In the present study, one group of test crosses and six groups of three-round mating experiments from two parental strains were conducted to determine the mating system in G. frondosa. A chi-squared test of the results of the test-cross mating experiments indicated that they satisfied Mendelian segregation, while a series of three-round mating experiments showed that Mendelian segregation was not satisfied, implying a segregation distortion phenomenon in G. frondosa. A genomic map of the G. frondosa strain, y59, grown from an LMCZ basidiospore, with 40.54 Mb and 12 chromosomes, was generated using genome, transcriptome and Hi-C sequencing technology. Based on the genomic annotation of G. frondosa, the mating-type loci A and B were located on chromosomes 1 and 11, respectively. The mating-type locus A coded for the β-fg protein, HD1, HD2 and MIP, in that order. The mating-type locus B consisted of six pheromone receptors (PRs) and five pheromone precursors (PPs) in a crossed order. Moreover, both HD and PR loci may have only one sublocus that determines the mating type in G. frondosa. The nonsynonymous SNP and indel mutations between the A1B1 and A2B2 mating-type strains and the reference genome of y59 only occurred on genes HD2 and PR1/2, preliminarily confirming that the mating type of the y59 strain was A1B2 and not A1B1. Based on the genetic evidence and the more reliable molecular evidence, the results reveal that the mating system of G. frondosa is tetrapolar. This study has important implications for the genetics and hybrid breeding of G. frondosa.

A uridine diphosphate-glycosyltransferase GFUGT88A1 derived from edible mushroom Grifola frondosa extends oligosaccharide chains

Fungal glucans play a key role to provide energy, support cell structure, and present biological functions. However, the biosynthetic machineries of fungal glucan chains remain to be elucidated. Hence, we aimed to elucidate the biochemical and catalytic characteristics of a novel glucan synthesis-associated enzyme uridine diphosphate (UDP)-glycosyltransferase GFUGT88A1 derived from Grifola frondosa and predict its potential catalytic mechanism. The purified recombinant GFUGT88A1 had a molecular weight of 51.7 kDa with an optimum temperature of 37 °C and pH of 7.0. GFUGT88A1 showed preference for oligosaccharides with relatively higher polymerization degrees (≥6) as acceptors to extend its chain when using UDP-glucose as a donor. Molecular modeling and docking of GFUGT88A1 with donor UDP-glucose, and acceptors laminaribiose, laminarihexaose, and laminarinonaose indicated that the predicted amino acid residues at the active sites may bind the donor/acceptor protein primarily via hydrogen bonds with various binding energy values. The present study revealed that GFUGT88A1 extended glucan chains, which provides a reference to understand the biosynthesis pathway of mushroom glucans.

The role of Rho1 gene in the cell wall integrity and polysaccharides biosynthesis of the edible mushroom Grifola frondosa

Grifola frondosa polysaccharides, especially β-glucans, showed the significant antitumor, hypoglycemic, and immune-stimulating activities. In the present study, a predominant regulatory subunit gfRho1p of β-1,3-glucan synthase in G. frondosa was identified with a molecular weight of 20.79 kDa and coded by a putative 648-bp small GTPase gene gfRho1. By constructing mutants of RNA interference and over-expression gfRho1, the roles of gfRho1 in the growth, cell wall integrity and polysaccharide biosynthesis were well investigated. The results revealed that defects of gfRho1 slowed mycelial growth rate by 22% to 33%, reduced mycelial polysaccharide and exo-polysaccharide yields by 4% to 7%, increased sensitivity to cell wall stress, and down-regulated gene transcriptions related to PKC-MAPK signaling pathway in cell wall integrity. Over-expression of gfRho1 improved mycelial growth rate and polysaccharide production of G. frondosa. Our study supports that gfRho1 is an essential regulator for polysaccharide biosynthesis, cell growth, cell wall integrity and stress response in G. frondosa.

The caspases-dependent apoptosis of hepatoma cells induced by an acid-soluble polysaccharide from Grifola frondosa

The edible mushroom Grifola frondosa has been used as a functional food for diseases prevention and therapy in Asian countries. In the present work, an acid-soluble polysaccharide (GFAP) was prepared from Grifola frondosa under room temperature and hydrochloric acid solution treatment, and its inhibitory effects on H22 and HepG2 cells were investigated. Results of MTT indicated that GFAP could effectively suppress proliferations of HCC cells, dose-dependently. Microscopic observation results demonstrated that GFAP-treated HCC cells showed apoptotic characteristics like membrane blebbing, chromatin condensed, nucleus pycnosis and fragmentation. Annexin V-FITC/PI staining and cell cycle distribution results showed that GFAP could induce the apoptosis of H22 and HepG2 cells via arresting them in G1 and S phases respectively. Rh123, JC-1 staining and western blotting results suggested that GFAP could significantly increase the permeability of mitochondrial membrane of HCC cells, and upregulated the expressions of Bax, cytochrome c, cleaved-caspase-3 and cleaved-caspase-9, which indicated that GFAP could trigger apoptosis of HCC cells through mitochondria apoptotic pathway in a caspases-dependent pattern. Our data demonstrated that GFAP holds great application prospect as a safe and effective antitumor drug for hepatocellular carcinoma therapy.

Functions of a Glucan Synthase Gene GFGLS in Mycelial Growth and Polysaccharide Production of Grifola frondosa.

Glucan synthase (GLS) gene is known to be involved in the fungal biosynthesis of cell wall, differentiation, and growth. In the present study, a glucan synthase gene (GFGLS) in the edible mushroom Grifola frondosa with a full sequence of 5927 bp encoding a total of 1781 amino acids was cloned and characterized for the first time. GFGLSp is a membrane protein containing two large transmembrane domains connected with a hydrophilic cytoplasmic domain. With a constructed dual promoter RNA silencing vector pAN7-gfgls-dual, a GFGLS-silencing transformant iGFGLS-3 had the lowest GFGLS transcriptional expression level (26.1%) with a shorter length and thinner appearance of the mycelia, as well as decreased mycelial biomass and exo-polysaccharide production of 5.02 and 0.38 g/L, respectively. Further analysis indicated that GFGLS silence influenced slightly the monosaccharide compositions and ratios of mycelial and exo-polysaccharide. These findings suggest that GFGLS could affect mycelial growth and polysaccharide production by downregulating the glucan synthesis.

Chemical structure of a partially 3-O-methylated mannofucogalactan from edible mushroom Grifola frondosa

An unusual heteropolysaccharide was isolated from the fruiting bodies of the medicinal mushroom Grifola frondosa, via successive cold aqueous extraction, followed by fractionation through freeze-thawing, precipitation with Fehling solution and dialysis using a membrane with a size exclusion cut-off of 500 kDa. Its chemical structure was determined based on total acid hydrolysis, methylation analysis and NMR studies. The mannofucogalactan had a molar mass of 15.9 × 103 g mol−1, which was determinate by HPSEC-MALLS. This heteropolymer showed to have a main chain of (1 → 6)-linked α-d-Galp partially substituted at O-2 by 3-O-α-d-mannopyranosyl-α-l-fucopyranosyl groups and in a minor proportion with α-l-Fucp single-unit side chains. Moreover, the presence of 3-O-Me-Galp units could also be observed in the main chain of the G. frondosa mannofucogalactan.

Heterologous expression of two minor laccase isozyme cDNAs from the edible mushroom Grifola frondosa.

Two cDNAs encoding the minor laccase isozymes (Lac2 and Lac3) of Grifola frondosa were cloned, characterized, and expressed in Pichia pastoris. The recombinant Lac2 (rLac2) was stable at pH 6.0, whereas the recombinant Lac3 (rLac3) was stable in a broad pH range (pH 4.0-8.0). In addition, rLac2 and rLac3 showed the highest catalytic efficiency (kcat/Km) for 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid).

Extraction, identification and antimicrobial activity of a new furanone, grifolaone A, from Grifola frondosa

A furanone (1), (S)-methyl 2-(2-hydroxy-3,4-dimethyl-5-oxo-2,5-dihydrofuran-2-yl)acetate, was isolated from the edible mushroom Grifola frondosa. Mass spectrometry and NMR analyses were used to elucidate the structure of this compound, and its absolute configuration was determined using circular dichroism spectroscopy. Compound 1 exhibited specific antifungal activity against the plant pathogens, Fusarium oxysporum, Gibberella zeae and Piricularia oryzae and the opportunistic human pathogen, Pseudallescheria boydii, resulting in minimum inhibitory concentration values of 2.5, 2.5, 1.25 and 0.15 μg/mL, respectively. In contrast, the furanone showed only weak activity towards Aspergillus spp., Candida albicans and several other fungal strains tested as well as no appreciable antibacterial activity.

Development of a transformation system for the edible mushroom Grifola frondosa: Demonstrating heterologous gene expression and RNAi-mediated gene silencing

Grifola frondosa is a well-known edible mushroom that is industrially produced year-round under artificial cultivation. To improve the productivity and quality of cultivated mushrooms, transcriptomic and genomic data for G. frondosa have been used to investigate genes involved in fruiting body differentiation. However, further functional analysis of genes has been limited by the lack of specific genetic engineering tools for this species. Hence, the primary goal of this study is to establish an efficient transformation method and vector system for G. frondosa. To express transgenes efficiently, a transformation vector was constructed using homologous regulatory sequences from constitutively expressed genes identified from comprehensive gene expression profile of G. frondosa. Using this vector system, we have successfully expressed enhanced green fluorescent protein (EGFP) and firefly luciferase (Luc) in G. frondosa. Furthermore, we have tested RNA interference (RNAi)-mediated gene silencing using an RNAi vector, and showed that expression of the hairpin RNA (hpRNA) encoded by this vector can trigger downregulation of expression of Gf.HydA1, a model target hydrophobin gene from G. frondosa. Thus, the transformation system established in this study should be a useful tool for further functional analysis of other genes in G. frondosa.

Growth, fruiting and lignocellulolytic enzyme production by the edible mushroom Grifola frondosa (maitake)

Cultivation of specialty mushrooms on lignocellulosic wastes represents one of the most economical organic recycling processes. Compared with other cultivated mushrooms, very little is known about the nature of the lignocellulolytic enzymes produced by the edible and medicinal fungus Grifola frondosa, the parameters affecting their production, and enzyme activity profiles during different stages of the developmental cycle. In this work we investigated the enzymes that enable G. frondosa, to colonize and deconstruct two formulations based on industrial lignocellulosic by-products. G. frondosa degraded both substrates (oak-sawdust plus corn bran, and oak/corn bran supplemented with coffee spent-ground) decreasing 67 and 50% of their lignin content, along with 44 and 37% of the polysaccharides (hemicellulose and cellulose) respectively. 35.3% biological efficiency was obtained when using oak sawdust plus corn bran as substrate. Coffee spent-ground addition inhibited mushroom production, decreased growth, xylanase and cellulase activities. However, taking into account that G. frondosa successfully colonized this residue; this substrate formula might be considered for its growth and medicinal polysaccharide production. Although G. frondosa tested positive for Azure B plate degradation, a qualitative assay for lignin-peroxidase, attempts to detect this activity during solid state fermentation were unsuccessful. Enzyme activities peaked during colonization but declined drastically during fruiting body formation. Highest activities achieved were: endoglucanase 12.3, exoglucanase 16.2, β-glucosidase 2.3, endoxylanase 20.3, amylase 0.26, laccase 14.8 and Mn-peroxidase 7.4 U/g dry substrate.

Prokaryotic expression, purification, and polyclonal antibody production of a hydrophobin from <italic>Grifola frondosa</italic>

Hydrophobins are small fungal proteins that self-assemble spontaneously at hydrophilic-hydrophobic interfaces and change the polar nature of the surfaces to which they attach. A new hydrophobin gene hgfI was identified recently from the edible mushroom Grifola frondosa. In this paper, the cloning, expression, purification, and polyclonal antibody preparation of the HGFI were described. The hgfI gene was cloned into pET-28a expression plasmid at the EcoRI and NdeI restriction sites and then transformed into Escherichia coli BL21 strain. SDSPAGE analysis showed that recombinant HGFI protein was satisfactorily expressed by optimizing the concentration and induction time of IPTG. The expressed recombinant HGFI protein was purified by electroelution because its inclusion body was insoluble in traditional processing method. After a desalting procedure with Sephadex G-25, the recombinant HGFI protein was used to immunize adult rabbits following standard protocol. ELISA and western blot analysis indicated that the produced antiserum could detect both HGFI protein expressed in the prokaryotic (E. coli) and in the eukaryotic cells (G. frondosa). Furthermore, the antiserum was used to determine the localization of HGFI protein in G. frondosa cells using an immunofluorescence technique. The results demonstrated that HGFI protein was localized in the cell wall, especially at the budding position of hypha. The polyclonal antibody against HGFI will facilitate further production and functional study of HGFI protein.

Protein HGFI from the edible mushroom Grifola frondosa is a novel 8 kDa class I hydrophobin that forms rodlets in compressed monolayers

Hydrophobins are a group of low-molecular-mass, cysteine-rich proteins that have unusual biophysical properties. They are highly surface-active and can self-assemble at hydrophobic-hydrophilic interfaces, forming surface layers that are able to reverse the hydropathy of surfaces. Here we describe a novel hydrophobin from the edible mushroom Grifola frondosa, which was named HGFI and belongs to class I. The hydrophobin gene was identified during sequencing of random clones from a cDNA library, and the corresponding protein was isolated as a hot SDS-insoluble aggregate from the cell wall. The purified HGFI was found to have 83 amino acids. The protein sequence deduced from the cDNA sequence had 107 amino acids, from which a 24 aa signal sequence had been cleaved off in the mature protein. This signal sequence was 5 aa longer than had been predicted on the basis of signal peptide analysis of the cDNA. Rodlet mosaic structures were imaged using atomic force microscopy (AFM) on mica surfaces after drying-down HGFI solutions. Using Langmuir films we were also able to take images of both the hydrophobic and hydrophilic sides of films formed at the air-water interface. No distinct structure was observed in films compressed once, but in films compressed several times rodlet structures could be seen. Most rodlets were aligned in the same direction, indicating that formation of rodlets may be promoted during compression of the monolayer.

Effect of culture parameters on the production of the edible mushroom Grifola frondosa (maitake) in tropical weathers

Hitherto, little effort has been directed to improve culture conditions for commercial development of maitake (Grifola frondosa), an edible and medicinal fungus, due to the short history of cultivation, particularly in tropical weathers. The purpose of this research was analyzing the environmental factors required for successful basidiome development on synthetic substrates in Colombia. We evaluated different cereal grains (corn, barley, sorghum and rice) for spawn production; and industrial by-products (such as coffee spent-ground and oak-sawdust) as substrates for mushroom production. Exploiting these residues for G. frondosa solid culturing would primarily provide edible mushroom and simultaneously help in resolving their disposal problem. The use of corn grains as substrate for spawn production results an important factor for reducing crop cycle time. A cold shock to 10°C was requisite for basidiome formation. Coffee spent-ground was a good substrate for mycelial growth, but not for mushroom production. When using oak sawdust plus corn bran as substrate, we obtained consistent yields with combined high biological efficiency (BE) (35.3%), best quality mushrooms, and a crop cycle of 12–14 weeks. The results achieved in this investigation contribute to expand the knowledge on this fungus, and compare favorably with previous works in the northern hemisphere with respect to BE, mushroom quality and crop cycle time.

Production of Polysaccharide by the Edible Mushroom, Grifola frondosa

The production of polysaccharide according to various developmental stages (mycelium growth, primordium appearance, and fruiting-body formation) in the edible mushroom Grifola frondosa was studied. The cap of the mature mushroom showed the highest amount of polysacchride. Mycelial growth and polysaccharide synthesis were optimal at pH 5 and 20°C. Polysaccharide synthesis was maximal after 12 days of cultivation, whereas maximum mycelial growth was shown after 18 days. Mannose, cellobiose and starch increased the level of polysaccharide as well as growth in submerged culture. Glucose and sucrose appeared to be good substrates for fruiting of Grifola frondosa.

Structure of a new `aspzincin' metalloendopeptidase fromGrifola frondosa: implications for the catalytic mechanism and substrate specificity based on several different crystal forms

Crystal structures of a peptidyl-Lys metalloendopeptidase (MEP) from the edible mushroom Grifola frondosa (GfMEP) were solved in four crystal forms. This represents the first structure of the new family 'aspzincins' with a novel active-site architecture. The active site is composed of two helices and a loop region and includes the HExxH and GTxDxxYG motifs conserved among aspzincins. His117, His121 and Asp130 coordinate to the catalytic zinc ligands. An electrostatically negative region composed of Asp154 and Glu157 attracts a positively charged Lys side chain of a substrate in a specific manner. A Tyr133 side chain located on the S1' pocket had different configurations in two crystal forms and was not observed in the other crystal forms. The flexible Tyr133 plays two roles in the enzymatic function of GfMEP. The first is to provide a hydrophobic environment with Phe83 in order to accommodate the alkyl part of the Lys side chain of a substrate and the second is as a 'proton donor' to the oxyanion of the tetrahedral transition state to stabilize the reaction transition state.