Basidiomycete Ganoderma Lucidum Research Articles

Transcription factor GlbHLH regulates hyphal growth, stress resistance, and polysaccharide biosynthesis in Ganoderma lucidum.

Basic helix-loop-helix (bHLH) transcription factors (TFs) participate in many physiological and cellular processes in eukaryotes. However, their functions remain unclear in the macro basidiomycete Ganoderma lucidum (G. lucidum). In this study, a gene encoding bHLH TF, GlbHLH, was identified in G. lucidum. The knockdown of GlbHLH by RNA interference reduced hyphal growth, hyphal branching, and resistant to osmotic, oxidative, and cell wall stress. The content of cell wall components β-1,3 glucan and chitin and the expression of their synthesis genes were decreased in the GlbHLH knockdown strains. The knockdown of GlbHLH led to an increase of intracellular reactive oxygen species by decreasing the enzyme activity and gene expression of antioxidant enzymes. Furthermore, the production of intracellular polysaccharides and extracellular polysaccharides was greatly decreased in the GlbHLH mutants. These results suggested that GlbHLH is involved in hyphal growth, stress response, and polysaccharide biosynthesis in G. lucidum.

Targeted Gene Insertion and Replacement in the Basidiomycete Ganoderma lucidum by Inactivation of Nonhomologous End Joining Using CRISPR/Cas9.

Targeted gene insertion or replacement is a promising genome-editing tool for molecular breeding and gene engineering. Although CRISPR/Cas9 works well for gene disruption and deletion in Ganoderma lucidum, targeted gene insertion and replacement remain a serious challenge due to the low efficiency of homologous recombination (HR) in this species. In this work, we demonstrate that the DNA double-strand breaks induced by Cas9 were mainly repaired via the nonhomologous end joining (NHEJ) pathway, at a frequency of 96.7%. To establish an efficient target gene insertion and replacement tool in Ganoderma, we first inactivated the NHEJ pathway via disruption of the Ku70 gene (ku70) using a dual single guide RNA (sgRNA)-directed gene deletion method. Disruption of the ku70 gene significantly decreased NHEJ activity in G. lucidum. Moreover, ku70 disruption strains exhibited 96.3% and 93.1% frequencies of targeted gene insertion and replacement, respectively, when target DNA with the orotidine 5'-monophosphate decarboxylase (ura3) gene and 1.5-kb homologous 5'- and 3'-flanking sequences was used as a donor template, compared to 3.3% and 0%, respectively, at these targeted sites for a control strain (Cas9 strain). Our results indicated that ku70 disruption strains were efficient recipients for targeted gene insertion and replacement. This tool will advance our understanding of functional genomics in G. lucidum. IMPORTANCE Functional genomic studies in Ganoderma have been hindered by the absence of adequate genome-engineering tools. Although CRISPR/Cas9 works well for gene disruption and deletion in G. lucidum, targeted gene insertion and replacement have remained a serious challenge due to the low efficiency of HR in these species, although such precise genome modifications, including site mutations, site-specific integrations, and allele or promoter replacements, would be incredibly valuable. In this work, we inactivated the NHEJ repair mechanism in G. lucidum by disrupting the ku70 gene using the CRISPR/Cas9 system. Moreover, we established a target gene insertion and replacement method in ku70-disrupted G. lucidum that possessed high-efficiency gene targeting. This technology will advance our understanding of the functional genomics of G. lucidum.

Prospects for using Ganoderma lucidum (Curtis) P. Karst. for biological control of phytopathogenic fungi

Antifungal, morphological and cultural properties of the Siberian and Abkhaz strains of the medicinal basidiomycete Ganoderma lucidum (Curtis) P. Karst were studied. A group of strains characterized by high growth rates in vitro on agar media (7.3-8.0 mm / day) and plant waste from timber processing (3.3-3.9 mm / day) was found. All strains of G. lucidum exhibited antifungal activity against phytopathogenic fungi; the degree of phytopathogen inhibition ranged from 10 to 58 %. Basidial rootrot fungi Heterobasidion and ascomycete fungi Bipolaris, Alternaria, Fusarium are the most sensitive to the presence of G. lucidum. The cultures most effectively limiting the phytopathogen development in the group of fast-growing Abkhaz strains (antifungal activity ranged from 21 to 58%) were identified. The high antifungal activity of strains on lignocellulosic substrates allows us to consider them as promising biocontrol agents for reducing the number, primarily, of basidial phytopathogens.

Investigation of the Influence of Sensor Films Made of the Mycelium of Basidiomycetes on the Characteristics of a Me1/AlN/Me2/Diamond Ultrahigh-Frequency Resonator

Organic films based on extracts of the biomass of basidiomycete strain Ganoderma lucidum are proposed to use as sensor coatings for acoustic gas sensors based on a layered Me1/AlN/Me2/diamond microwave acoustic resonator. A biomass of basidiomycetes is obtained by submerged cultivation in a selected liquid nutrient medium after which biomass extracts are obtained using two-component solvent systems (water/ethanol) with different volumetric ratios. A base technique has been developed and organic films have been created based on the biomass of basidiomycete Ganoderma lucidum on the resonator surface. The morphological properties of the created films are studied using scanning electron microscopy. The Q factor and resonance frequency of the developed layered Me1/AlN/Me2/diamond microwave acoustic resonator are measured. The effect of the created organic films on the parameters of the microwave acoustic resonator is analyzed. A conclusion is drawn that these films can be used as sensor coatings for a corresponding acoustic gas sensor.

In Ganoderma lucidum, Glsnf1 regulates cellulose degradation by inhibiting GlCreA during the utilization of cellulose.

Cellulose is a by-product of agricultural production and an abundant waste. As a carbon source, cellulose can be degraded and utilized by fungi. Carbon sources, which act as nutrients, not only provide energy but also serve as regulators of gene expression, metabolism and growth, through various signalling networks that enable cells to sense and adapt to varying environmental conditions. Nutrient-sensing pathways prioritize the use of preferred carbon sources and regulate the production of cellulose-degrading enzymes when necessary. Understanding the regulation of the fungal cellulolytic response will become increasingly important because we strive to increase the efficiency of the utilization of these renewable energy sources. Here, we show that Glsnf1, a sucrose-nonfermenting serine-threonine-protein kinase 1 (Snf1)/AMP-activated protein kinase homologue in medicinal macro basidiomycete Ganoderma lucidum, actively responds to carbon alterations and positively regulates cellulase activity and cellulase-related gene transcription. The carbon catabolite repressor CreA, a zinc binuclear cluster transcription factor that mediates the sensing of nutrients and suppression of the transcription of a number of genes necessary for the consumption of a less preferred carbon source, participates in the Glsnf1-mediated regulation of cellulases. Glsnf1 not only negatively regulates the transcription level of the CreA gene but also hinders its localization in the nucleus. Overall, our findings reveal a key nutrient-sensing mechanism that is critical for the modulation of carbon source adaptation in G. lucidum.

Conditions Affecting Lingzhi or Reishi Medicinal Mushroom Ganoderma lucidum (Agaricomycetes) Basidiome Quality, Morphogenesis, and Biodegradation of Wood By-products in Argentina.

Solid-state fermentation (SSF) with the medicinal higher Basidiomycete Ganoderma lucidum was studied as a strategy to use pine (Pinus radiata D. Don) and poplar (Populus nigra L.) wood chips and sawdust. Fruiting bodies were produced and the value of the biotransformed substrate was assessed. The highest mushroom yield (63 g dry weight per kilogram of dry substrate) was obtained with poplar sawdust and wood chips. Immersion of the bioreactors was a simple watering method that obtained suitable yields. Two morphological types were induced using 2 different incandescent light intensities. High light irradiation induced the highest valued mushroom morphology (as a whole product). Time course study of substrate biodegradation and mycelial growth dynamics indicated that the trophophase lasted 20 days and presented laccase activity of 0.01-0.03 units · g-1. The activity at idiophase was 10 times higher. Aqueous and alkali extracts, as well as carbohydrase enzyme profile activity, revealed differences in the properties of the residual substrate; some related to the substrate source are considered to be of concern for further use of this pretreated biomass. In view of the results obtained, we propose use of SSF of pine and poplar with G. lucidum to profitably recycle softwood by-products from the timber industry.

Evaluation of diuron tolerance and biotransformation by the white-rot fungus Ganoderma lucidum

The white rot basidiomycete Ganoderma lucidum was evaluated for its capability to tolerate and to degrade the herbicide diuron. Diuron at a subtoxic concentration was added at the start of the cultivation in glucose liquid stationary cultures. Under this condition diuron was a laccase inducer. Almost 50% of the initially present diuron was removed after 15 d of cultivation. Two diuron metabolites were found N′-(3,4-dichlorophenyl)-N-methylurea (DCPMU) and 3,4-dichlorophenylurea (DCPU). The addition of the cytochrome P450 inhibitors 1-aminobenzotriazole and piperonyl butoxide reduced significantly the capability of the fungus in degrading diuron. The activities of superoxide dismutase and catalase were significantly increased in the mycelial extracts by the presence of diuron. On the other hand, diuron did not cause any significant alteration in the levels of reactive oxygen species. Additionally, laccase could also degrade diuron in vitro and this degradation was increased by the addition of synthetic mediators, 3-ethylbenzthiazoline-6-sulphonic acid and acetylacetone. Significant reduction in the toxicity, as evaluated by the Lactuca sativa bioassay, was observed after G. lucidum treatment. In conclusion, G. lucidum is able to metabolize diuron by intra- and extracellular mechanisms, without the accumulation of toxic products.

The Influence of the Fungus, the Basidiomycete Ganoderma Lucidum for Short-Term Shaped Memory and Performance of People with Dyscirculatory Encephalopathy

The Influence of the Fungus, the Basidiomycete Ganoderma Lucidum for Short-Term Shaped Memory and Performance of People with Dyscirculatory Encephalopathy

Ethylene promotes mycelial growth and ganoderic acid biosynthesis in Ganoderma lucidum.

To investigate the effects of ethylene, in the form of ethephon (2-chloroethylphosphonic acid), on mycelial growth and ganoderic acid (GA) accumulation in the higher basidiomycete Ganoderma lucidum. Treatment with both 10 and 15mM ethephon enhanced the growth of G. lucidum on solid CYM plates and in CYM liquid medium. After optimization using response surface methodology, GA reached 33mg/g dry cell weight (DW), an increase of 90%, compared with the control. Lanosterol and squalene contents were 31 and 2.4μg/g DW, being increased by 1.2- and 0.6-fold, respectively, in response to ethephon. Additionally, the transcriptional levels of hydroxymethylglutaryl-CoA reductase, squalene synthase and oxidosqualene cyclase were up-regulated by 2.6-, 4.3- and 3.8-fold, respectively, compared with the control group. This approach provides an efficient strategy for improving GA accumulation in G. lucidum, with potential future applications.

Microbiological and physicochemical analysis of pumpkin juice fermentation by the basidiomycetous fungus Ganoderma lucidum.

A new protocol for processing of pumpkin juice was set up which included fermentation by the basidiomycete Ganoderma lucidum at 28 °C for 7 d. The growth curve of G. lucidum in pumpkin juice was successfully (R(2) =0.99) fitted by a 4-parameter logistic model and the ideal highest biomass was estimated to be 4.79 g/L. G. lucidum was found to have a significant acidification effect on pumpkin juice. The lowest pH (4.05 ± 0.05) and highest total titratable acidity (14.31± 0.16 mL 0.1 M NaOH/100 mL) were found on the 4th day during fermentation. Sugars in pumpkin juice fermented with G. lucidum showed a significant decrease, especially glucose and fructose. On the contrary, the release of exo-polysaccharides and free amino acids greatly enriched the pumpkin juice. The variation of color index and viscosity also mirrored the above behavior. Based on headspace solid phase microextraction and gas chromatography-mass spectrometry, 68 volatile compounds were identified, including 17 esters, 14 alcohols, 13 phenyl compounds, 11 aldehydes, 8 ketones, 3 acids, 1 furan, and 1 benzothiazole. The pumpkin juices fermented for different days were markedly differentiated with principal component analysis and the fermentation process was tentatively divided into 3 periods: the booming (from the 1st to 4th day), steady (from the 5th to 6th day), and decline (the 7th day) period.

Enhancement of Ganoderic Acid Accumulation by Overexpression of an N-Terminally Truncated 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Gene in the Basidiomycete Ganoderma lucidum

Ganoderic acids produced by Ganoderma lucidum, a well-known traditional Chinese medicinal mushroom, exhibit antitumor and antimetastasis activities. Genetic modification of G. lucidum is difficult but critical for the enhancement of cellular accumulation of ganoderic acids. In this study, a homologous genetic transformation system for G. lucidum was developed for the first time using mutated sdhB, encoding the iron-sulfur protein subunit of succinate dehydrogenase, as a selection marker. The truncated G. lucidum gene encoding the catalytic domain of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) was overexpressed by using the Agrobacterium tumefaciens-mediated transformation system. The results showed that the mutated sdhB successfully conferred carboxin resistance upon transformation. Most of the integrated transfer DNA (T-DNA) appeared as a single copy in the genome. Moreover, deregulated constitutive overexpression of the HMGR gene led to a 2-fold increase in ganoderic acid content. It also increased the accumulation of intermediates (squalene and lanosterol) and the upregulation of downstream genes such as those of farnesyl pyrophosphate synthase, squalene synthase, and lanosterol synthase. This study demonstrates that transgenic basidiomycete G. lucidum is a promising system to achieve metabolic engineering of the ganoderic acid pathway.

BIOLOGICALLY ACTIVE SUBSTANCES OF MEDICAL MUSHROOM GANODERMA LUCIDUM (CURT.:FR) P. KARST

The issue related the current data on the biologically active substances of medicinal basidiomycete Ganoderma lucidum and their actions. There were given the chemical formulas glucans and terpenoids – the substances that determine the main medicinal properties of the fungus. There were presented the information on the other substances involved in the forming of the therapeutic effect of Ganoderma: they were proteins and glicoproteins, nucleotides and nucleosides, sterols and steroids, fatty acids, alkaloids, glycosides, volatile essential oils and vitamins. In addition, Ganoderma as a xylotrophic-saprophyte has the set of ligninolytic enzymes which have been used in food processing, pulp and paper, and textile industries, as well as in the processes of biological treatment. Immunomodulatory, antimicrobial, antiviral and antitumor activities are confirmed by many experiments. The studies show the biological effects and mechanisms of action of G. lucidum metabolites.

Purification, crystallization and preliminary X-ray structure analysis of the laccase fromGanoderma lucidum

The ligninolytic enzymes of the basidiomycetes play a key role in the global carbon cycle. A characteristic property of these enzymes is their broad substrate specificity, which has led to their use in various biotechnologies, thus stimulating research into the three-dimensional structures of ligninolytic enzymes. This paper presents the purification, crystallization and preliminary X-ray analysis of the laccase from the ligninolytic basidiomycete Ganoderma lucidum.

Influence of NaCl and Na2SO4 on the kinetics and dye decolorization ability of crude laccase from Ganoderma lucidum

In a solid state medium using yellow passion fruit waste as substrate, the basidiomycete Ganoderma lucidum produced a laccase as the main ligninolytic enzyme. This crude enzyme presented Michaelian behavior with both substrates tested, namely 3-ethylbenzthiazoline-6-sulphonic acid (ABTS) and the anthraquinone dye remazol brilliant blue R (RBBR). The KM’s for these substrates were, respectively, 0.232×10−3 and 0.602×10−3M. The actions of NaCl and Na2SO4, two important salts usually found in textile wastewaters, were investigated. The enzyme was inhibited by NaCl, but not by Na2SO4. Inhibition by NaCl was of the mixed type with two different inhibition constants. The enzyme was able to completely decolorize RBBR in the presence of 1.0M Na2SO4 and 50% decolorization was found in the presence of 0.1M NaCl. Such properties certainly make the enzyme a good agent for textile dye effluent treatment considering the fact that wastewaters of this industry usually contain high concentrations of NaCl and Na2SO4.

Polysaccharides of basidiomycetes. Alkali-soluble polysaccharides from the mycelium of white rot fungus Ganoderma lucidum (Curt.: Fr.) P. Karst

Two polysaccharides were isolated from submergedly cultured mycelium of the basidiomycete Ganoderma lucidum by extraction with alkali followed by fractionation with Fehling reagent. The polysaccharides were shown to be a linear (1-->3)-alpha-D-glucan and a highly branched xylomannan containing a backbone built up of (1-->3)-linked alpha-D-mannopyranose residues, the majority of which are substituted at O-4 by single beta-D-xylopyranose residues or by disaccharide fragments beta-D-Manp-(1-->3)-beta-D-Xylp-(1-->. Polysaccharide structures were elucidated by NMR spectroscopy in combination with methylation analysis and periodate oxidation. An interesting feature of the xylomannan is the simultaneous presence of alpha-D-mannopyranose and beta-D-mannopyranose residues, the first forming the backbone, and the second being the non-reducing terminal units of disaccharide side chains.

Decolorization of direct dyes with manganese peroxidase from white-rot basidiomycete Ganoderma lucidum IBL-05

Decolorization of direct dyes with manganese peroxidase from white-rot basidiomycete Ganoderma lucidum IBL-05

Changes in some components of soymilk during fermentation with the basidiomycete Ganoderma lucidum

Soymilk was fermented with the basidiomycete Ganoderma lucidum WZ02 and the changes in the contents of polysaccharide, sugars, crude protein, B-vitamins, free amino acids and isoflavones were analyzed. Polysaccharide and crude protein were increased by the fermentation of G. lucidum while most free amino acids were reduced. The flatulence factor (e.g. stachyose and raffinose) was significantly decreased and stachyose was not detected after 72 h of fermentation. The contents of thiamin, riboflavin, and niacin were increased during the fermentation. Most of isoflavone glycosides were converted to aglycones and the contents of daidzein and genistein were increased by the fermentation of G. lucidum. The results suggested that fermentation by G. lucidum could improve the acceptability and health properties of soymilk.

Structure and bioactivities of a galactose rich extracellular polysaccharide from submergedly cultured Ganoderma lucidum

A galactose rich extracellular polysaccharide (GLP-2), which is composed of galactose, mannose, glucose, arabinose and rhamnose in the molar ratios 103:17:12:10:3, was isolated from the submerged culture broth of a basidiomycete Ganoderma lucidum. Its structure was evaluated with chemical techniques, NMR and IR spectroscopy and ESI-MS experiments and the following structure was suggested: [Display omitted] The bioactivity tests showed that GLP-2 enhanced the T and B lymphocyte proliferation at polysaccharide concentrations of 10 and 50 μg/mL and exhibited lymphocyte activity at concentrations of 100 and 200 μg/mL in vitro. The test in mice also showed that GLP-2 significantly enhanced the T and B lymphocyte proliferation and antibody production and increased the mass of spleen tissue at a dose of 25 or 75 mg/kg, but did not show the same effect at a high dose of 150 or 300 mg/kg. However, GLP-2 did not significantly influence the serum IgG and C-3 levels in mice. GLP-2 also showed a hepatoprotective activity in liver injury mice induced by Bacille Calmette-Guérin and lipopolysaccharide.

Solid-state fermentation of cornmeal with the basidiomycete Ganoderma lucidum for degrading starch and upgrading nutritional value

The objective of this research was to study the ability of the basidiomycete Ganoderma lucidum to degrade starch and upgrade nutritional value of cornmeal during solid-state fermentation (SSF). On the basal medium that consisted of cornmeal and salt solution, alpha-amylase activity of G. lucidum reached its maximum value of 267 U g(-1) of culture on day 20 after inoculation. Prolongation of fermentation time from 10 to 25 days increased significantly the degradation rate of starch and ergosterol yield (a kind of physiologically active substances of G. lucidum, also as an indicator of mycelial biomass) (P < 0.01). Supplementation of glucose, sucrose or maltose to the basal medium also caused a significant increase in either the degradation rate of starch or the ergosterol yield as compared with control (P < 0.01). Among five kinds of nitrogen sources supplemented, yeast extract, casamino acid and peptone were more effective than (NH4)2SO4 and NH4NO3, and yeast extract gave the highest degradation rate of starch and ergosterol yield, followed by peptone. Through orthogonal experiments, the theoretical optimum culture medium for SSF of this fungus was the following: 100 g cornmeal, ground to 30-mesh powder, moistened with 67 ml of nutrient salt solution supplemented with 3 g yeast extract and 7.5 g glucose per litre. Under the optimum culture condition, the degradation rate of starch reached its maximum values of 70.4%; the starch content of the fermented product decreased from 64.5 to 25.3%, while the reducing sugar content increased from 4.2 to 20.6%. SSF also produced a significant increase (P < 0.01) from 11.0 to 16.5% in protein content. After SSF by G. lucidum, the digesting and absorbing ratio of cornmeal was strikingly increased and some active substances originated from G. lucidum remained in the fermented product. This implied that cornmeal could be processed into many kinds of special functional foods by SSF of G. lucidum.

Genetic transformation and mutant isolation in Ganoderma lucidum by restriction enzyme-mediated integration

A white-rot basidiomycete Ganoderma lucidum has long been used as a medicinal mushroom in Asia, and it has an array of enzymes important for wood degrading activity. There have been many reports about the ingredients which show health aiding effects. In order to analyze gene functions and introduce foreign genes into this fungus, genetic transformation is required. We have successfully transformed G. lucidum to geneticin resistance using pJS205-1 which has the antibiotic resistance genes against geneticin and phosphinothricin. Many different mutants have been generated during the transformation by restriction enzyme mediated integration, and the transformation yield was 4–17 transformants (μg plasmid DNA) −1. The plasmid was integrated stably into the recipient chromosome, which was confirmed by PCR with the plasmid-specific primers.