The Changes of Microbial Diversity and Isolation of Microorganism in Soil for Alleviating the Production Decreasing After Continuous Cultivation of Ganoderma lucidum.

Ganoderma lucidum is a medicinal mushroom usually cultivated in logs and covered with soil. Its production decreases after continuous cultivation. Changes of microbial diversity in soil are suggested to be one of the reasons. This study aims to investigate the changes of microbial diversity and abundance in soil during cultivation, and isolate potential microbial strains that affect the yield of G. lucidum. Soil samples were collected at two different ranges from logs during one complete growth cycle of G. lucidum. The changes in fungi and bacteria were investigated by using high-throughput sequencing and real-time PCR. Results indicated that the relative abundance of Firmicutes in the bacterial community decreased at the short-range site. In the fungal community, the relative abundance of Ganoderma increased to 70% at the long-range site at the end of the cultivation. The abundance of bacteria and fungi decreased significantly at the end of the growth cycle. Recovery of microbial changes in soil should be proceeded separately based on different ranges to logs. The microbial strains in these soil samples were also isolated and identified. Potential strains were assessed in the form of bio-fertilizer. The yield of G. lucidum in the field using bio-fertilizer with isolated bacterial strains from the Firmicutes phylum was about 13% higher than that without using bio-fertilizer, suggesting the possibility of alleviating the production decrease of G. lucidum by this method.

Changes of Active Substances in Ganoderma lucidum during Different Growth Periods and Analysis of Their Molecular Mechanism.

Ganoderma lucidum, renowned as an essential edible and medicinal mushroom in China, remains shrouded in limited understanding concerning the intrinsic mechanisms governing the accumulation of active components and potential protein expression across its diverse developmental stages. Accordingly, this study employed a meticulous integration of metabolomics and proteomics techniques to scrutinize the dynamic alterations in metabolite accumulation and protein expression in G. lucidum throughout its growth phases. The metabolomics analysis unveiled elevated levels of triterpenoids, steroids, and polyphenolic compounds during the budding stage (BS) of mushroom growth, with prominent compounds including Diplazium and Ganoderenic acids E, H, and I, alongside key steroids such as cholesterol and 4,4-dimethyl-5alpha-cholesta-8,14,24-trien-3beta-ol. Additionally, nutrients such as polysaccharides, flavonoids, and purines exhibited heightened presence during the maturation stage (FS) of ascospores. Proteomic scrutiny demonstrated the modulation of triterpenoid synthesis by the CYP450, HMGR, HMGS, and ERG protein families, all exhibiting a decline as G. lucidum progressed, except for the ARE family, which displayed an upward trajectory. Therefore, BS is recommended as the best harvesting period for G. lucidum. This investigation contributes novel insights into the holistic exploitation of G. lucidum.

Effects of different wall-breaking methods on the nutrient release of Ganoderma lucidum spore powder during in vitro digestion.

BACKGROUND: The hard double-walled structure of Ganoderma lucidum spore powder (GLSP) is difficult for the human body to digest, so it is very important to break the wall of GLSP. In this study, the wall of GLSP was broken by mechanical milling at room temperature (MM-R) and ultra-fine grinding at low temperature (UFG-L), respectively. RESULTS: Compared with MM-R, UFG-L could better retain the sporangium powder's morphological and structural integrity. During in vitro digestion, compared with unbroken GLSP, the released amounts of polysaccharides and triterpenes from broken GLSP were significantly increased, and they increased with the increase of specific surface area. The bioaccessibility of polysaccharide and triterpene from unbroken GLSP after the intestinal stage were 29.52% and 5.37%, respectively. The bioaccessibility of polysaccharides and triterpene from broken GLSP by MM-R after the intestinal phase were 39.73-72.45% and 16.44-24.97%, while those by UFG-L were 44.53-104.18% and 12.96-32.90%, respectively. CONCLUSION: The active ingredients of broken GLSP showed better digestion and absorption abilities than unbroken GLSP. Moreover, the specific surface area of GLSP by UFG-L was lower than that by MM-R, and the bioaccessibility of GLSP by UFG-L was higher than that by MM-R. © 2024 Society of Chemical Industry.

Enhancing high-efficiency breeding and microbial microdroplet cultivation techniques for Ganoderma lucidum.

Ganoderma lucidum is known for its bioactive compounds, such as polysaccharides and triterpenoids, which are crucial in food and medicine. However, liquid fermentation encounters challenges in terms of strain differentiation and stability. In this research, we employed atmospheric room temperature plasma mutation and a microbial microdroplet culture system to identify strains with enhanced biomass and triterpenoid production. The three mutant strains, YB05, YB09, and YB18, exhibited accelerated growth rates and antagonized the initial strain G0023 more effectively than the controls. Notably, YB18 displayed the fastest growth, with a 17.25% increase in colony radius. Shake flask cultivation demonstrated that, compared with the initial strain, YB05 and YB18 had 26.33% and 17.85% greater biomass, respectively. Moreover, the triterpenoid production of YB05 and YB18 surpassed that of the control by 32.10% and 15.72%, respectively, as confirmed by colorimetric detection. Importantly, these mutant strains remained stable for five generations. This study revealed a comprehensive screening system utilizing atmospheric pressure, room temperature plasma mutation technology and microbial droplet cultivation. This innovative approach offers a promising pathway for obtaining advantageous Ganoderma strains for liquid fermentation. The methodology of atmospheric room temperature plasma mutation and microbial microdroplet culture systems is detailed for better comprehension.

Integrative Analysis of Selected Metabolites and the Fungal Transcriptome during the Developmental Cycle of Ganoderma lucidum Strain G0119 Correlates Lignocellulose Degradation with Carbohydrate and Triterpenoid Metabolism.

To systemically understand the biosynthetic pathways of bioactive substances, including triterpenoids and polysaccharides, in Ganoderma lucidum, the correlation between substrate degradation and carbohydrate and triterpenoid metabolism during growth was analyzed by combining changes in metabolite content and changes in related enzyme expression in G. lucidum over 5 growth phases. Changes in low-polarity triterpenoid content were correlated with changes in glucose and mannitol contents in fruiting bodies. Additionally, changes in medium-polarity triterpenoid content were correlated with changes in the lignocellulose content of the substrate and with the glucose, trehalose, and mannitol contents of fruiting bodies. Weighted gene coexpression network analysis (WGCNA) indicated that changes in trehalose and polyol contents were related to carbohydrate catabolism and polysaccharide synthesis. Changes in triterpenoid content were related to expression of the carbohydrate catabolic enzymes laccase, cellulase, hemicellulase, and polysaccharide synthase and to the expression of several cytochrome P450 monooxygenases (CYPs). It was concluded that the products of cellulose and hemicellulose degradation participate in polyol, trehalose, and polysaccharide synthesis during initial fruiting body formation. These carbohydrates accumulate in the early phase of fruiting body formation and are utilized when the fruiting bodies mature and a large number of spores are ejected. An increase in carbohydrate metabolism provides additional precursors for the synthesis of triterpenoids. IMPORTANCE Most studies of G. lucidum have focused on its medicinal function and on the mechanism of its activity, whereas the physiological metabolism and synthesis of bioactive substances during the growth of this species have been less studied. Therefore, theoretical guidance for cultivation methods to increase the production of bioactive compounds remains lacking. This study integrated changes in the lignocellulose, carbohydrate, and triterpenoid contents of G. lucidum with enzyme expression from transcriptomics data using WGCNA. The findings helped us better understand the connections between substrate utilization and the synthesis of polysaccharides and triterpenoids during the cultivation cycle of G. lucidum. The results of WGCNA suggest that the synthesis of triterpenoids can be enhanced not only through regulating the expression of enzymes in the triterpenoid pathway, but also through regulating carbohydrate metabolism and substrate degradation. This study provides a potential approach and identifies enzymes that can be targeted to regulate lignocellulose degradation and accelerate the accumulation of bioactive substances by regulating substrate degradation in G. lucidum.

GCN4 Regulates Secondary Metabolism through Activation of Antioxidant Gene Expression under Nitrogen Limitation Conditions in Ganoderma lucidum.

Nitrogen limitation has been widely reported to affect the growth and development of fungi, and the transcription factor GCN4 (general control nonderepressible 4) is involved in nitrogen restriction. Here, we found that nitrogen limitation highly induced the expression of GCN4 and promoted the synthesis of ganoderic acid (GA), an important secondary metabolite in Ganoderma lucidum. The activated GCN4 is involved in regulating GA biosynthesis. In addition, the accumulation of reactive oxygen species (ROS) also affects the synthesis of GA under nitrogen restrictions. The silencing of the gcn4 gene led to further accumulation of ROS and increased the content of GA. Further studies found that GCN4 activated the transcription of antioxidant enzyme biosynthesis genes gr, gst2, and cat3 (encoding glutathione reductase, glutathione S-transferase, and catalase, respectively) through direct binding to the promoter of these genes to reduce the ROS accumulation. In conclusion, our study found that GCN4 directly interacts with the ROS signaling pathway to negatively regulate GA biosynthesis under nitrogen-limiting conditions. This provides an essential insight into the understanding of GCN4 transcriptional regulation of the ROS signaling pathway and enriches the knowledge of nitrogen regulation mechanisms in fungal secondary metabolism of G. lucidum.IMPORTANCE Nitrogen has been widely reported to regulate secondary metabolism in fungi. Our study assessed the specific nitrogen regulatory mechanisms in Ganoderma lucidum. We found that GCN4 directly interacts with the ROS signaling pathway to negatively regulate GA biosynthesis under nitrogen-limiting conditions. Our research highlights a novel insight that GCN4, the nitrogen utilization regulator, participates in secondary metabolism through ROS signal regulation. In addition, this also provides a theoretical foundation for exploring the regulation of other physiological processes by GCN4 through ROS in fungi.

Temperature affects substrate-associated bacterial composition during Ganoderma lucidum hyphal growth.

The growth of the well-known fungus Ganoderma lucidum is influenced by temperature, which has an impact on the associated microbial structure in the substrate. In this study, we analyzed the bacterial diversity of the substrate at different temperatures using next-generation sequencing technology. A total of 513 733 sequences from 15 samples were assigned to 19 bacterial phyla. The samples were dominated by Proteobacteria, followed by Firmicutes; the 2 phyla exhibited opposite changes with elevated temperature. Bacterial genera showed different abundances at different temperatures, in which Sediminibacterium maintained a stable abundance below 40 °C, while Ochrobactrum and Rhodococcus were enriched with elevated temperature and both showed their highest abundances at 40 °C. Functional prediction uncovered 39 identified KEGG pathways, and bacterial genes involved in the membrane transport pathway exhibited the highest abundance subject to heat (40 °C) during the growth of G. lucidum. In general, our findings illustrated the influence of temperatures on G. lucidum mycelial morphology and the bacterial community in the substrate, and the results will facilitate cultivation of this fungus.

De novo transcriptome assembly and protein profiling of copper-induced lignocellulolytic fungus Ganoderma lucidum MDU-7 reveals genes involved in lignocellulose degradation and terpenoid biosynthetic pathways.

Ganoderma lucidum is an important medicinal fungus that possesses exceedingly high lignocellulose degrading ability. Evidently, Cu2+ has decisive roles on the mycelial growth and enzyme production. To reveal the effect of Cu2+ on G. lucidum transcriptome, predominantly associated with lignocellulolytic progression, we conducted comparative NGS based de novo transcriptome assembly using Illumina Hi SeqTM sequencing platform. We obtained 26,083,372 and 35,713,076 high-quality reads from induced and uninduced cultures. For wood degrading activity, 194 transcript coding for oxidoreductases and 402 transcripts for CAZymes were predicted. Further, secretome studies revealed high score GO terms related to oxidoreductases, glycosyl hydrolases, and chitinases from Cu-induced mycelia. The increased Cu2+ concentrations showed higher secretion of lignocellulases such as laccases, cellulases, and xylanases along with increased production of phenolics and antioxidants. Several differences in the transcriptomic and proteomic signatures for lignocellulolytic enzymes provide vital clues about Cu2+ mediated gene regulation and metabolic pathways in basidiomycetous fungi.

Enhanced exopolysaccharide production in submerged fermentation of Ganoderma lucidum by Tween 80 supplementation.

Bioactive polysaccharides extracted from Ganoderma lucidum (G. lucidum) have been widely applied in food and medicine for their multiple functions. In this study, G. lucidum exopolysaccharide (EPS) production in submerged fermentation was stimulated by Tween 80. The addition of 0.25% Tween 80 on day 3 gave a maximum production of mycelial biomass and EPS, with an increase of 19.76 and 137.50%, respectively. Analysis of fermentation kinetics showed that glucose was consumed faster after adding Tween 80, while the expression of EPS biosynthesis-related genes and ATP generation were greatly improved. Moreover, Tween 80 resulted in the significant accumulation of reactive oxygen species and increased cell membrane and cell wall permeability. The EPS from Tween 80-containing medium had higher contents of carbohydrate and uronic acid, lower molecular weight, and higher antioxidant activity against 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals than those of EPS produced in the absence of Tween 80. This study provides further evidence to clarify the stimulatory effects of Tween 80 in fermentation and provides a guide for the production of bioactive G. lucidum EPS.

Effect of the heme oxygenase gene on mycelial growth and polysaccharide synthesis in Ganoderma lucidum.

The heme oxygenase gene has antioxidant and cytoprotective effects in organisms, but no related research has been conducted in Ganoderma lucidum. For the first time, we cloned the HMX1 gene in G. lucidum. The CDS is 1092 bp in length and encodes 363 amino acids. The HMX1 protein was prokaryotically expressed and purified, and the enzyme activity of the purified protein was measured. The value of Km was 0.699 µM, and Vm was 81.9 nmol BV h-1 nmol-1 protein. By constructing the silencing vector pAN7-dual-HMX1i, the transformants HMX1i1 and HMX1i2 were obtained. Compared with the wild-type (WT), the average growth rate of HMX1i1 and HMX1i2 decreased by 31% and 23%, respectively, and the mycelium biomass decreased by 53% and 48%, respectively. Compared with the WT, the extracellular polysaccharide content of HMX1i1 and HMX1i2 increased by 59% and 51%, and the intracellular polysaccharide content increased by 24% and 22%, respectively. These results indicate that the HMX1 gene affects mycelial growth and polysaccharide synthesis in G. lucidum.

Contribution of vitamin B12 to biogas upgrading and nutrient removal by different microalgae-based technology.

The algae-based technology has a positive effect on the treatment of biogas slurry and the purification of biogas, while vitamin B12 (VB12) is one of the important regulatory substances in the algae-based cultivation system. In this study, different concentrations of VB12 were used in three microalgal treatment technologies to assess their effect on simultaneous removal of nutrients from biogas slurry and removal of CO2 from raw biogas. Results showed that Chlorella vulgaris exhibited higher growth rate, mean daily productivity, chlorophyll a content, carbonic anhydrase activity and better photosynthetic properties when co-cultivated with Ganoderma lucidum, rather than when co-cultivated with activated sludge or under mono-cultivation. Maximum mean chemical oxygen demand, total nitrogen, total phosphorus and CO2 removal efficiencies were found to be 84.29 ± 8.28%, 83.27 ± 8.14%, 85.27 ± 8.46% and 65.71 ± 6.35%, respectively when microalgae were co-cultivated with Ganoderma lucidum under 100 ng L-1 of VB12. This study shows the potential of microalgae and fungi co-cultivation supplemented with VB12 for the simultaneous upgradation of biogas production as well as for the purification of biogas slurry.

Overexpression of nicotinamide mononucleotide adenylyltransferase (nmnat) increases the growth rate, Ca2+ concentration and cellulase production in Ganoderma lucidum.

Identifying new and economical means to utilize diverse lignocellulosic biomass is an urgent task. Ganoderma lucidum is a well-known edible and medicinal basidiomycete with an excellent ability to degrade a wide range of cellulosic biomass, and its nutrient use efficiency is closely related to the activity of extracellular cellulase. Intracellular nicotinamide adenine dinucleotide (NAD+) biosynthesis is controlled in response to nutritional status, and NAD+ is an essential metabolite involved in diverse cellular processes. Nicotinamide mononucleotide adenylyltransferase (NMNAT) is a common enzyme in three NAD+ synthesis pathways. In this study, a homologous gene of nmnat was cloned from G. lucidum and two G. lucidum overexpression strains, OE::nmnat4 and OE::nmnat19, were constructed using an Agrobacterium tumefaciens-mediated transformation method. The G. lucidum nmnat overexpression strains showed obviously increased colony growth on different carbon sources, and intracellular Ca2+ concentrations in the G. lucidum OE::nmnat4 and OE::nmnat19 strains were increased by 2.04- and 2.30-fold, respectively, compared with those in the wild-type (WT) strains. In the G. lucidum OE::nmnat4 and OE::nmnat19 strains, endo-ß-glucanase (CMCase) activity increased by approximately 2.8- and 3-fold, while ß-glucosidase (pNPGase) activity increased by approximately 1.9- and 2.1-fold, respectively, compared with the activity in the WT strains. Furthermore, overexpression of NAD+ synthesis pathways was found to elicit cellulase production by increasing the intracellular Ca2+ concentration. In summary, this study is the first to demonstrate that increased intracellular NAD+ contents through overexpression of the nmnat gene of NAD+ synthesis pathways may increase cellulase production by increasing intracellular Ca2+ concentrations in G. lucidum. KEY POINTS: • The concentration of NAD+influences cellulase production in G. lucidum. • The concentration of NAD+influences the intracellular Ca2+concentration in G. lucidum. • The concentration of NAD+influences cellulase production by eliciting a change in intracellular Ca2+in G. lucidum.

Production of laccase by repeated batch semi-solid fermentation using wheat straw as substrate and support for fungal growth.

Repeated batch semi-solid fermentation (sSF) process using wheat straw substrate and fungal growth of Ganoderma lucidum on solid substrate was studied for production of laccase. pH showed significant effect on laccase production. Highest laccase activity with pH controlled to 5.0 in batch sSF was 15257.2 ± 353.4 U L- 1 on 9th day. In repeated batch process at pH 5.0, insoluble biomass substrate and fungal growth were reused after liquid part of medium was replaced with glucose, ammonium phosphate (best nitrogen source) and combined glucose and ammonium phosphate solution separately. Refilled to 80% w v- 1 of initial soluble sugar of first batch resulted in highest laccase production with peak activity after 4 days from replacement. Production of enzyme increased from 15257.2 U L- 1 in first batch to cumulative 90164.4 U L- 1 in 29 days after six repeated batches, productivity increased from 1680.2 to 3110.3 U L- 1 day- 1 (∼ 1.9 times) due to reductions in inhibitory effects and time required for fungal growth. Utilization of wheat straw in repeated batch sSF was supported by composition analysis and morphological changes (scanning electron microscopy) of substrate. Economic production of laccase using agricultural residues in repeated batch sSF could be possible.

Mass Spectrometry-Based Untargeted Metabolomics and α-Glucosidase Inhibitory Activity of Lingzhi (Ganoderma lingzhi) During the Developmental Stages.

Lingzhi is a Ganoderma mushroom species which has a wide range of bioactivities. Analysis of the changes in metabolites during the developmental stages of lingzhi is important to understand the underlying mechanism of its biosynthesis, as well as its bioactivity. It may also provide valuable information for the cultivation efficiency of lingzhi. In this study, mass spectrometry based untargeted metabolomics was carried out to analyze the alteration of metabolites during developmental stages of lingzhi. Eight developmental stages were categorized on the basis of morphological changes; starting from mycelium stage to post-mature stage. GC/MS and LC/MS analyses along with multivariate analysis of lingzhi developmental stages were performed. Amino acids, organic acids, sugars, polyols, fatty acids, fatty alcohols, and some small polar metabolites were extracted as marker metabolites from GC/MS analysis, while, lanostane-type triterpenoids were observed in LC/MS analysis of lingzhi. The marker metabolites from untargeted analysis of lingzhi developmental stages were correlated with the α-glucosidase inhibitory activity. Two metabolites, compounds 34 and 35, were identified as potential contributors of the α-glucosidase inhibitory activity. The current result shows that some metabolites are involved in the developmental process and α-glucosidase inhibitory activity of lingzhi.

Submerged cultivation of medicinal mushroom in hydrolysate of ligno-cellulosic material.

Ganoderma lucidum belongs to the family Ganodermataceae and found in Japan, China and some other parts of Asia. Traditionally it is used in herbal medicine as anti-diabetic, cancer prevention agent, antitumor, an immunomodulatory, antimicrobial and antiviral agent. Due to difficulty in field cultivation, submerged fermentation was employed as a promising method for efficient and large-scale production of mycelia biomass and bioactive metabolites. Cellulose was used in the form of a lignocellulosic substrate. The Ganoderma lucidum which is medicinal and edible mushrooms were successfully grown in the form of mycelial biomass in static submerged culture in Petri plates and flasks. The present study is based on the utilization of hydrolyzates of lignocellulosic materials such as Peanut cort, Sugarcane bagasse, and Wheat Straw was used after hydrolysis. A Static Fermentation Technique was employed to investigate the mycelial growth, instead of Fruiting Body. Ganoderma lucidum was kept up on PDA (potato dextrose agar) medium in Petri dishes at 4°C and brooded at 25°C for 5 days for the development of G. lucidum and generation of Ganoderic Acid. Morphology of G. lucidum on various Hydrolysates was white and delicate like cotton unpredictable shape, Cloud-like appearance spread in general plate and multiple little sporadic white cotton-like shape with string-like projections. We got a Ganoderic Acid from the Hydrolysates of Peanut cort concentrate, Sugarcane bagasse concentrate and Wheat straw concentrate at a concentration of 0.006g/L, 0.011g/L and 0.017g/L respectively.

Cross Talk between Calcium and Reactive Oxygen Species Regulates Hyphal Branching and Ganoderic Acid Biosynthesis in Ganoderma lucidum under Copper Stress.

Ganoderma lucidum is among the best known medicinal basidiomycetes due to its production of many pharmacologically active compounds. To study the regulatory networks involved in its growth and development, we analyzed the relationship between reactive oxygen species (ROS) and Ca2+ signaling in the regulation of hyphal branching and ganoderic acid (GA) biosynthesis after Cu2+ treatment. Our results revealed that Cu2+ treatment decreased the distance between hyphal branches and increased the GA content and the intracellular levels of ROS and Ca2+ Further research revealed that the Cu2+-induced changes in hyphal branch distance, GA content, and cytosolic Ca2+ level were dependent on increases in cytosolic ROS. Our results also showed that increased cytosolic Ca2+ could reduce cytosolic ROS by activating antioxidases and modulating Cu2+ accumulation, resulting in feedback to adjust hyphal growth and GA biosynthesis. These results indicated that cytosolic ROS and Ca2+ levels exert important cross talk in the regulation of hyphal growth and GA biosynthesis induced by Cu2+ Taken together, our results provide a reference for analyzing the interactions among different signal transduction pathways with regard to the regulation of growth and development in other filamentous fungi.IMPORTANCEGanoderma lucidum, which is known as an important medicinal basidiomycete, is gradually becoming a model organism for studying environmental regulation and metabolism. In this study, we analyzed the relationship between reactive oxygen species (ROS) and Ca2+ signaling in the regulation of hyphal branching and ganoderic acid (GA) biosynthesis under Cu2+ stress. The results revealed that the Cu2+-induced changes in the hyphal branch distance, GA content, and cytosolic Ca2+ level were dependent on increases in cytosolic ROS. Furthermore, the results indicated that increased cytosolic Ca2+ could reduce cytosolic ROS levels by activating antioxidases and modulating Cu2+ accumulation. The results in this paper indicate that there was important cross talk between cytosolic ROS and Ca2+ levels in the regulation of hyphal growth and GA biosynthesis induced by Cu2.

14-3-3 proteins are involved in growth, hyphal branching, ganoderic acid biosynthesis, and response to abiotic stress in Ganoderma lucidum.

Ganoderma lucidum, which contains many pharmacologically active compounds, is regarded as a traditional medicinal fungus. Nevertheless, the scarcity of basic research limits the commercial value and utilization of G. lucidum. As a class of highly conserved, phosphopeptide-binding proteins present in all eukaryotes, 14-3-3 proteins play vital roles in controlling multiple physiological processes, including signal transduction, primary metabolism, and stress responses. However, knowledge of the roles of 14-3-3 proteins in Basidiomycetes is sparse. In this article, two homologs of 14-3-3 proteins, encoded by the two distinct genes GlBmh1 and GlBmh2, were distinguished in G. lucidum. We found that GlBmh1 and GlBmh2 were expressed at various developmental stages, including in vegetative mycelium cultivated on solid medium and in primordia and fruiting bodies. Moreover, we constructed GlBmh1 single-silenced strains, GlBmh2 single-silenced strains, and 14-3-3 double-silenced mutants for further study. When GlBmh1 and GlBmh2 were inhibited by RNA interference, the growth rate of mycelia was decreased, and the distance between the aerial hyphal branches was reduced; responses to various abiotic stresses such as oxidants and cell wall and osmotic stressors were also changed. Furthermore, the contents of secondary metabolite ganoderic acids (GAs) were increased after GlBmh1 and GlBmh2 were simultaneously silenced. Taken together, we provide evidence that implicates potential roles for the two 14-3-3 proteins in affecting growth and GA biosynthesis, thereby providing new insights into the basic functions of 14-3-3 proteins in G. lucidum.

Ganoderma lucidum phosphoglucomutase is required for hyphal growth, polysaccharide production, and cell wall integrity.

Phosphoglucomutase (pgm) is an important enzyme in carbohydrate metabolism that is located at the branching point between glycolysis and the Leloir pathway. pgm catalyzes the reversible conversion reaction between glucose-6-phosphate (Glc-6-P) and glucose-1-phosphate (Glc-1-P). The glpgm gene was cloned in Escherichia coli, and the recombinant pgm protein from Ganoderma lucidum was purified in this study. The activity of native pgm was also detected to demonstrate that this predicted gene was functional in G. lucidum. Interestingly, silencing the glpgm gene in the fungus reduced hyphal growth. Moreover, glpgm silencing was associated with declining extracellular polysaccharide (EPS) production (approximately 20-40% of that in the WT strain) and increasing intracellular polysaccharide (IPS) production (approximately 1.7-fold that in the WT strain). Additionally, in our research, cell wall components were also shown to differ according to the glpgmi strain. Compared with WT, chitin significantly increased by 1.5-fold; however, the content of ß-1,3-glucan was observably reduced to 60-70% that of the WT. Further research showed that the cell wall component changes were associated with the transcription of related genes. These findings provide references for further study on the potential physiological function of pgm in G. lucidum.

Optimization of Culture Condition for Ganoderic Acid Production in Ganoderma lucidum Liquid Static Culture and Design of a Suitable Bioreactor.

Ganoderma lucidum, a famous medicinal mushroom used worldwide, is a rich source of triterpenoids which, together with polysaccharides, are believed to be the main effective constituents of G. lucidum. With the increase of market demand, the wild resource is facing serious limitations, and the quality of cultivated fruiting bodies can be seriously affected by the availability of wood resources and by cultivation management practices. In the present study, we aimed to develop an alternative way to produce useful triterpenoids from G. lucidum. We cultured the strain using a two-stage liquid culture strategy and investigated the effects of nitrogen limitation, carbon supply, static culture volume and air supply in the static culture stage on the accumulation of five triterpenoids (GA-P, GA-Q, GA-T, GA-S, GA-R). Our results showed that, under optimized condition, the total yield of the five triterpenoids reached 963 mg/L (as determined by HPLC). Among the five triterpenoids, GA-T accounted for about 75% of the total yield. Besides, a bioreactor suitable for fungal liquid static culture with a 10 L extensible plastic bag shaped culture unit was designed and in which the maximum total yield of the five GAs reached 856.8 mg/L, and the GAs content reached 5.99%. Our results demonstrate the potential of industrial application of G. lucidum culture for the production of triterpenoids, especially GA-T. Air supply significantly improved the accumulation of triterpenoids, and this will provide important clues to understand why more triterpenoids are produced in the mycelia mat under static liquid culture conditions.

The mitogen-activated protein kinase GlSlt2 regulates fungal growth, fruiting body development, cell wall integrity, oxidative stress and ganoderic acid biosynthesis in Ganoderma lucidum.

The mitogen-activated protein kinases (MAPKs) are crucial signaling instruments in eukaryotes that play key roles in regulating fungal growth, development, and secondary metabolism and in adapting to the environment. In this study, we characterized an Slt2-type MAPK in Ganoderma lucidum, GlSlt2, which was transcriptionally induced during the primordium and fruiting body stages. RNA interference was used to examine the function of GlSlt2. Knockdown of GlSlt2 caused defects in growth and increased hyphal branching as well as hypersensitivity to cell wall-disturbing substances. Consistently, the chitin and ß-1,3-d-glucan contents and the expression of cell wall biosynthesis genes were decreased and down-regulated, respectively, in GlSlt2 knockdown strains compared with those in the wild type (WT). In addition, no primordium or fruiting body could be observed in GlSlt2 knockdown strains. Furthermore, the intracellular reactive oxygen species (ROS) content and ganoderic acid biosynthesis also decreased in GlSlt2 knockdown strains. Addition of H2O2 could recover the decreased ganoderic acid content in GlSlt2 knockdown strains, indicating that GlSlt2 might regulate ganoderic acid biosynthesis via the intracellular ROS level. Overall, GlSlt2 is involved in hyphal growth, fruiting body development, cell wall integrity, oxidative stress and ganoderic acid biosynthesis in G. lucidum.