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The liquid phase was proved to be a significant influencing factor among the three phases in solid-state fermentation (SSF), which determined water control was crucial. However, obvious water loss was caused by microbial utilization and moisture evaporation. Super absorbent polymer (SAP) was utilized to supply water in SSF owing to its high water-holding capacity. Adding 0.15% SAP could significantly increase the biomass of Ganoderma lucidum by 33.59% and promote filter paper activity (FPA), endocellulase activity and laccase activity by 27.11%, 29.14% and 47.39%, respectively. Water states of fermentation substrates were detected by the low-field nuclear magnetic resonance (LF-NMR). Results revealed that water present and lost was dominated by the capillary water. At the end of fermentation, the capillary water content ( C cw ) in water-supply SSF was 20.48% and 17.20% higher than that in static SSF and cold-model SSF. The relaxation time of the capillary water was reduced by 56.53% in water-supply SSF and by 53.40% in static SSF, but it just reduced by 6.82% in cold-model SSF. In addition, the C cw in SSF had a high correlation with the biomass and lignocellulose-degrading enzyme activities of G. lucidum. These results clearly demonstrated that capillary water played a very important role in improved production of G. lucidum.

The AMP-activated protein kinase (AMPK)/Sucrose-nonfermenting serine-threonine protein kinase 1 (Snf1) plays an important role in metabolic remodelling in response to energy stress. However, the role of AMPK/Snf1 in responding to other environmental stresses and metabolic remodelling in microorganisms was unclear. Heat stress (HS), which is one important environmental factor, could induce the production of reactive oxygen species and the accumulation of ganoderic acids (GAs) in Ganoderma lucidum. Here, the functions of AMPK/Snf1 were analysed under HS condition in G. lucidum. We observed that Glsnf1 was rapidly and strongly activated when G. lucidum was exposed to HS. HS significantly increased intracellular H2O2 levels (by approximately 1.6-fold) and decreased the dry weight of G. lucidum (by approximately 45.6%). The exogenous addition of N-acetyl- l -cysteine (NAC) and ascorbic acid (VC), which function as ROS scavengers, partially inhibited the HS-mediated reduction in biomass. Adding the AMPK/Snf1 inhibitor compound C (20 μM) under HS conditions increased the H2O2 content (by approximately 2.3-fold of that found in the strain without HS treatment and 1.5-fold of that found in the strain under HS treatment without compound C) and decreased the dry weight of G. lucidum (an approximately 28.5% decrease compared with that of the strain under HS conditions without compound C). Similar results were obtained by silencing the Glsnf1 gene. Further study found that Glsnf1 meditated metabolite distribution from respiration to glycolysis, which is considered a protective mechanism against oxidative stress. In addition, Glsnf1 negatively regulated the biosynthesis of GA by removing ROS. In conclusion, our results suggest that Glsnf1-mediated metabolic remodelling is involved in heat stress adaptability and the biosynthesis of secondary metabolites in G. lucidum.

Hydrogen sulfide (H2S), an emerging small-molecule signalling agent, was recently shown to play a significant role in many physiological processes, but relatively few studies have been conducted on microorganisms compared with mammals and plants. By studying the pretreatment of H2S donor sodium hydrosulfide (NaHS) and the scavenger hypotaurine (HT) and Cystathionine β-synthase silenced strains, we found that H2S could alleviate the HS-induced ganoderic acids (GAs) biosynthesis. Our transcriptome results also showed that many signaling pathways and metabolic pathways, such as the glycolysis, TCA, oxidative phosphorylation and pentose phosphate pathway, are influenced by H2S. Further experimental results indicated that H2S could affect the physiological process of Ganoderma lucidum by interacting with multiple signals, including ROS, NO, AMPK, sphingolipid, mTOR, phospholipase D and MAPK, and physiological and pharmacological analyses showed that H2S might alleviate the biosynthesis of GAs by inhibiting the intracellular calcium in G. lucidum.

The fungal cell wall is very important for cell growth and survival during stress, and the target of rapamycin (TOR) pathway plays a major role in regulating cell growth in response to environmental cues. Ganoderma lucidum is an important edible and medicinal fungus, and the function of TOR in this organism remains unclear. As shown in the present study, the TOR pathway regulates cell wall integrity (CWI) in G. lucidum. Inhibition of TOR signaling by RNA interference (RNAi) or rapamycin treatment reduced the growth of G. lucidum mycelia, increased contents of the cell wall components chitin and β-1,3-glucan, and increased cell wall thickness. Furthermore, inhibition of TOR signaling enhanced the relative level of phosphorylated Slt2, a member of the MAPK cascade involved in CWI signaling. Moreover, when treated with rapamycin, significantly lower chitin and β-1,3-glucan contents were observed in Slt2-silenced strains than in WT strains, indicating that TOR regulates the synthesis of these cell wall components through the Slt2-MAPK pathway. These results indicate a potential relationship between TOR signaling and CWI signaling. Additionally, participation of Slt2-MAPK in TOR-mediated regulation of cell wall component production has not previously been reported in a microorganism.

Ganoderma lucidum has high commercial value because it produces many active compounds, such as ganoderic acids (GAs). Salicylic acid (SA) was previously reported to induce the biosynthesis of GA in G. lucidum. In this study, we found that SA induces GA biosynthesis by increasing ROS production, and further research found that NADPH oxidase-silenced strains exhibited a partial reduction in the response to SA, resulting in the induction of increased ROS production. Furthermore, the localization of ROS shows that mitochondria are sources of ROS production in response to SA treatment. An additional analysis focused on the relationship between SA-induced ROS production and mitochondrial functions, and the results showed that inhibitors of mitochondrial complexes I and II exert approximately 40–50% superimposed inhibitory effects on the respiration rate and H2O2 content when co-administered with SA. However, no obvious superimposed inhibition effects were observed in the sample co-treated with mitochondrial complex III inhibitor and SA, implying that the inhibitor of mitochondrial complex III and SA might act on the same site in mitochondria. Additional experiments revealed that complex III activity was decreased 51%, 62% and 75% after treatment with 100, 200, and 400 µM SA, respectively. Our results highlight the finding that SA inhibits mitochondrial complex III activity to increase ROS generation. In addition, inhibition of mitochondrial complex III caused ROS accumulation, which plays an essential role in SA-mediated GA biosynthesis in G. lucidum. This conclusion was also demonstrated in complex III-silenced strains. To the best of our knowledge, this study provides the first demonstration that SA inhibits complex III activity to increase the ROS levels and thereby regulate secondary metabolite biosynthesis., Highlights • Mitochondria as a source of salicylic acid (SA) induced reactive oxygen species (ROS) production in Ganoderma lucidum. • SA induces the accumulation of ganoderic acids in Ganoderma lucidum by mitochondria ROS overproduction. • SA inhibits mitochondrial complex III activity to increase ROS and thereby induces ganoderic acids biosynthesis.

Heat stress (HS) is an important environmental factor that affects the growth and metabolism of edible fungi, but the molecular mechanism of the heat stress response (HSR) remains unclear. We previously reported that HS treatment increased the length between two hyphal branches and induced the accumulation of ganoderic acid biosynthesis and the gene expression of heat shock proteins (HSPs) in Ganoderma lucidum. In this study, we found that HS induced a significant increase in the cytosolic ROS concentration, and exogenously added ROS scavengers NAC, VC and NADPH oxidase (Nox) inhibitor DPI reduce the cytosolic ROS accumulation in G. lucidum. In addition, the phenomena of the increased gene expression and increased length between the two hyphal branches and the accumulation of GA biosynthesis induced by HS were mitigated. Furthermore, we investigated the effects of HS on Nox-silenced strains (NoxABi-10, NoxABi-11 and NoxRi-4, NoxRi-7) and found that the level of ROS concentration was lower than that in wild-type (WT) strains treated with HS. Additionally, Nox silenced strains reduced the HS-induced increase in HSP expression, the length between two hyphal branches and GA biosynthesis compared with the WT strain. These data indicate that HS-induced ROS participate in the regulation of HSP expression, hyphal branching and ganoderic acid biosynthesis in G. lucidum. In addition, these findings identified potential pathways linking ROS networks to HSR, physiological and metabolic processes in fungi and provide a valuable reference for studying the role of ROS in HSR, mycelium growth and secondary metabolites.

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.

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 β-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.

When fungi are subjected to abiotic stresses, the polyamines (PAs) level alter significantly. Here, we reveal that the polyamine putrescine (Put) could play an important role in alleviating heat stress(HS)-induced accumulation of nitric oxide (NO). Ornithine decarboxylase (ODC)-silenced mutants that were defective in Put biosynthesis exhibited significantly lower NO levels than the wild type (WT) when subjected to HS. With addition of 5 mM exogenous Put, the ODC-silenced mutant endogenous Put obviously increased under HS. At the same time, the contents of NO in the ODC-silenced mutants recovered to approximately WT levels after the administration of exogenous Put. However, the elevated NO content in the ODC-silenced mutants disappeared when exogenous Put and carboxy-PTIO (PTIO is a specific scavenger of NO) were added. Intriguingly, the content of glutamine (Gln) was significantly increased in the ODC-silenced strains. When exogenous Put was added to the WT, the Gln content was significantly decreased. The appearance of a high level of Gln was accompanied by nitrate reductase (NR) activity reduction. Further studies showed that Put influenced ganoderic acids (GAs) biosynthesis by regulating NO content, possibly through NR, under HS. Our work reported that Put regulates HS-induced NO accumulation by changing the cellular Gln level in filamentous fungi. IMPORTANCE: In our present work, it was HS as an ubiquitous environmental stress that affects the important pharmacological secondary metabolite (GAs) content in G. lucidum. Afterwards, we began to explore the network formed between multiple substances to jointly reduce the massive accumulation of GAs content caused by HS. We firstly focused on Put, a substance that enhances resistance to multiple stresses. Further, we discovered an influence on Put could changing the NO content, which has been shown to decrease the accumulation of GAs via HS. Then, we also found the change of NO content may be due to Put level that would affect intracellular Gln content. It has never been reported. And ultimately, it is Put related network that could reduce HS-inducing secondary metabolite mess in fungi.

A novel composite of polyhedral MnO ( p -MnO) nanocrystals and reduced graphene oxide (RGO) nanosheets as a high performance electrode material for lithium ion batteries (LIBs) is prepared by homogeneous precipitation, subsequent reduction with hydrazine under microwave irradiation and final annealing procedure. The characterizations of structure and morphology reveal that p -MnO nanocrystals are homogeneously anchored onto the RGO nanosheets. The synergistic effect of the p -MnO nanocrystals with high crystallinity and RGO nanosheets makes the as-prepared nanocomposite possesses high contact area between electrolyte and electrode, uniform delivery of electrons and ions, short Li + insertion/extraction pathways, superior protection against volume change of electroactive materials and excellent electrical conductivity of the overall electrode during electrochemical processes. As an anode material for LIBs, the p -MnO/RGO nanocomposite exhibits superior electrochemical performances including large reversible capacity, excellent cyclic performance and high rate capability. For example, the p -MnO/RGO electrode shows a reversible capacity of 988.6 mA h g −1 after 120 cycles at a current density of 100 mA g −1 and delivers 591.1 mA h g −1 at a rate of 1600 mA g −1 . Our results highlight the importance of anchoring nanocrystals with special morphology on RGO nanosheets for maximum utilization of electroactive materials to enhance energy storage in high-performance LIBs.

The Hypsizygus marmoreus laccase gene (lcc1) sequence was cloned and analyzed. The genomic DNA of lcc1 is 2336 bp, comprising 13 introns and 14 exons. The 1626-bp full-length cDNA encodes a mature laccase protein containing 542 amino acids, with a 21-amino acid signal peptide. Phylogenetic analysis showed that the lcc1 amino acid sequence is homologous to basidiomycete laccases and shares the highest similarity with Flammulina velutipes laccase. A 2021-bp promoter sequence containing a TATA box, CAAT box, and several putative cis-acting elements was also identified. To study the function of lcc1, we first overexpressed lcc1 in H. marmoreus and found that the transgenic fungus producing recombinant laccase displayed faster mycelial growth than the wild-type (wt) strain. Additionally, primordium initiation was induced 3-5 days earlier in the transgenic fungus, and fruiting body maturation was also promoted approximately five days earlier than in the wt strain. Furthermore, we detected that lcc1 was sustainably overexpressed and that laccase activity was also higher in the transgenic strains compared with the wt strain during development in H. marmoreus. These results indicate that the H. marmoreus lcc1 gene is involved in mycelial growth and fruiting body initiation by increasing laccase activity.

Ceramide synthases (CERSs) catalyse an N-acyltransferase reaction using long-chain base (LCB) and fatty acyl-coenzyme A (CoA) as substrates to synthesize ceramide (Cer), which is the backbone of all complex sphingolipids. In the present study, three CERSs (LAG1, LAG2 and LAG3) form Ganoderma lucidum were analysed. The silencing of lag1 by RNA interference reduced ganoderic acid biosynthesis and Cer and complex sphingolipids contents, which contain long-chain-fatty-acids (LCFAs, including C16 and C18). In contrast, the silencing of lag2 or lag3 did not result in obvious phenotypic and sphingolipid homeostasis changes, although the lag2/lag3 double-silenced mutants exhibited increased ganoderic acid biosynthesis as well as reduced growth, reduced Cer and complex sphingolipids contents, which contain very-long-chain fatty acids (VLCFAs, including C22, C24 and C26). The results of the present study indicate that the three assayed CERSs have distinct physiological functions and substrate specificities in G. lucidum.

UDP-glucose pyrophosphorylase (UGP) is a key enzyme involved in carbohydrate metabolism, but there are few studies on the functions of this enzyme in fungi. The ugp gene of Ganoderma lucidum was cloned, and enzyme kinetic parameters of the UGP recombinant protein were determined in vitro, revealing that this protein was functional and catalyzed the reversible conversion between Glc-1-P and UDP-Glc. ugp silencing by RNA interference resulted in changes in the levels of the intermediate metabolites Glc-1-P and UDP-Glc. The compounds and structure of the cell wall in the silenced strains were also altered compared with those in the wild-type strains. Moreover, the number of hyphal branches was also changed in the silenced strains. To verify the role of UGP in hyphal branching, a ugp-overexpressing strain was constructed. The results showed that the number of hyphal branches was influenced by UGP. The mechanism underlying hyphal branching was further investigated by adding exogenous Glc-1-P. Our results showed that hyphal branching was regulated by a change in the cytosolic Ca(2+) concentration, which was affected by the level of the intermediate metabolite Glc-1-P, in G. lucidum. Our findings indicate the existence of an interaction between carbon metabolism and Ca(2+) signaling in this fungus.

Ganoderma lucidum is one of the best known medicinal basidiomycetes because it produces many pharmacologically active compounds, and methyl jasmonate (MeJA) was previously reported to induce the biosynthesis of ganoderic acids (GA) in G. lucidum. In this study, we found that MeJA not only increased the amount of GA but also increased the distance between hyphal branches by approximately 1.2-fold. Further analysis showed that MeJA could increase the intracellular ROS (reactive oxygen species) content by approximately 2.2-2.7-fold. Furthermore, the hyphal branching and GA biosynthesis regulated by MeJA treatment could be abolished by ROS scavengers to a level similar to or lower than that of the control group. These results indicated that the regulation of hyphal branching and GA biosynthesis by MeJA might occur via a ROS signaling pathway. Further analysis revealed that NADPH oxidase (NOX) plays an important role in MeJA-regulated ROS generation. Importantly, our results highlight that NOX functions in signaling cross-talk between ROS and MeJA. In addition, these findings provide an excellent opportunity to identify potential pathways linking ROS networks to MeJA signaling in fungi and suggest that plants and fungi share a conserved signaling-crosstalk mechanism.

Hypsizygus marmoreus is one of the major edible mushrooms in East Asia. As no efficient transformation method, the molecular and genetics studies were hindered. The glyceraldehyde-3-phosphate dehydrogenase (GPD) gene of H. marmoreus was isolated and its promoter was used to drive the hygromycin B phosphotransferase (HPH) and enhanced green fluorescent protein (EGFP) in H. marmoreus. Agrobacterium tumefaciens-mediated transformation (ATMT) was successfully applied in H. marmoreus. The transformation parameters were optimized, and it was found that co-cultivation of bacteria with protoplast at a ratio of 1000:1 at a temperature of 26 °C in medium containing 0.3 mM acetosyringone resulted in the highest transformation efficiency for Agrobacterium strain. Besides, three plasmids, each carrying a different promoter (from H. marmoreus, Ganoderma lucidum and Lentinula edodes) driving the expression of an antibiotic resistance marker, were also tested. The construct carrying the H. marmoreus gpd promoter produced more transformants than other constructs. Our analysis showed that over 85% of the transformants tested remained mitotically stable even after five successive rounds of subculturing. Putative transformants were analyzed for the presence of hph gene by PCR and Southern blot. Meanwhile, the expression of EGFP in H. marmoreus transformants was detected by fluorescence imaging. This ATMT system increases the transformation efficiency of H. marmoreus and may represent a useful tool for molecular genetic studies in this mushroom species.