Bidirectional Solid-State Fermentation of Highland Barley by Edible Fungi to Improve Its Functional Components, Antioxidant Activity and Texture Characteristics.

In food industry, the characteristics of food substrate could be improved through its bidirectional solid-state fermentation (BSF) by fungi, because the functional components were produced during BSF. Six edible fungi were selected for BSF to study their effects on highland barley properties, such as functional components, antioxidant activity, and texture characteristics. After BSF, the triterpenes content in Ganoderma lucidum and Ganoderma leucocontextum samples increased by 76.57 and 205.98%, respectively, and the flavonoids content increased by 62.40% (Phellinus igniarius). Protein content in all tests increased significantly, with a maximal increase of 406.11% (P. igniarius). Proportion of indispensable amino acids increased significantly, with the maximum increase of 28.22%. Lysine content increased largest by 437.34% to 3.310 mg/g (Flammulina velutipes). For antioxidant activity, ABTS radical scavenging activity showed the maximal improvement, with an increase of 1268.95%. Low-field NMR results indicated a changed water status of highland barley after fermentation, which could result in changes in texture characteristics of highland barley. Texture analysis showed that the hardness and chewiness of the fermented product decreased markedly especially in Ganoderma lucidum sample with a decrease of 77.96% and 58.60%, respectively. The decrease indicated a significant improvement in the taste of highland barley. The results showed that BSF is an effective technology to increase the quality of highland barley and provide a new direction for the production of functional foods.

Heme Oxygenase/Carbon Monoxide Participates in the Regulation of Ganoderma lucidum Heat-Stress Response, Ganoderic Acid Biosynthesis, and Cell-Wall Integrity.

Carbon monoxide (CO), a product of organic oxidation processes, arises in vivo principally from the enzymatic reaction of heme oxygenase (HO, transcription gene named HMX1). HO/CO has been found to exert many salutary effects in multiple biological processes, including the stress response. However, whether HO/CO is involved in the regulation of the heat-stress (HS) response of Ganoderma lucidum (G. lucidum) is still poorly understood. In this paper, we reported that under heat stress, the HMX1 transcription level, HO enzyme activity, and CO content increased by 5.2-fold, 6.5-fold and 2-fold, respectively. HMX1 silenced strains showed a 12% increase in ganoderic acid (GA) content under HS as analyzed by HPLC. Furthermore, according to Western blot analysis of the protein phosphorylation levels, HMX1 attenuated the increase in phosphorylation levels of slt2, but the phosphorylation levels were prolonged over a 3 h HS time period. The chitin and glucan content in HMX1 silenced strains increased by 108% and 75%, respectively. In summary, these findings showed that the HO/CO system responds to heat stress and then regulates the HS-induced GA biosynthesis and the cell-wall integrity mediated by the Slt-MAPK phosphorylation level in G. lucidum.

Shedding light on the mechanisms underlying the environmental regulation of secondary metabolite ganoderic acid in Ganoderma lucidum using physiological and genetic methods.

The secondary metabolites of fungi are often produced at very low concentrations, and until recently the regulatory mechanisms of secondary metabolite biosynthesis have been unclear. Ganoderma lucidum is a macrofungus that is widely used as a traditional Chinese medicine or medicinal mushroom: ganoderic acid (GA) is one of the main active ingredients. Here, we review research from the last decade on which and how environmental factors regulate GA biosynthesis. These environmental factors are mainly three components: a single chemical/biological or biochemical signal, physical triggers, and nutritional conditions. Because G. lucidum is a non-model Basidiomycete, a combination of physiological and genetic research is needed to determine how those environmental factors regulate GA biosynthesis. The regulation of GA biosynthesis includes ROS, Ca2+, cAMP and phospholipid signaling, and cross-talk between different signaling pathways. The regulatory mechanisms for the synthesis of this secondary metabolite, from the perspective of physiology and genetics, in G. lucidum will provide ideas for studying the regulation of fungal secondary metabolism in other non-model species, especially those fungi with limitations in genetic manipulation.

Integrated Proteomics and Metabolomics Analysis Provides Insights into Ganoderic Acid Biosynthesis in Response to Methyl Jasmonate in Ganoderma Lucidum.

Ganoderma lucidum is widely recognized as a medicinal basidiomycete. It was previously reported that the plant hormone methyl jasmonate (MeJA) could induce the biosynthesis of ganoderic acids (GAs), which are the main active ingredients of G. lucidum. However, the regulatory mechanism is still unclear. In this study, integrated proteomics and metabolomics were employed on G. lucidum to globally identify differences in proteins and metabolites under MeJA treatment for 15 min (M15) and 24 h (M24). Our study successfully identified 209 differential abundance proteins (DAPs) in M15 and 202 DAPs in M24. We also identified 154 metabolites by GC-MS and 70 metabolites by LC-MS in M24 that are involved in several metabolic pathways. With an in-depth analysis, we found some DAPs and metabolites that are involved in the oxidoreduction process, secondary metabolism, energy metabolism, transcriptional and translational regulation, and protein synthesis. In particular, our results reveal that MeJA treatment leads to metabolic rearrangement that inhibited the normal glucose metabolism, energy supply, and protein synthesis of cells but promoted secondary metabolites, including GAs. In conclusion, our proteomics and metabolomics data further confirm the promoting effect of MeJA on the biosynthesis of GAs in G. lucidum and will provide a valuable resource for further investigation of the molecular mechanisms of MeJA signal response and GA biosynthesis in G. lucidum and other related species.

14-3-3 Proteins: a window for a deeper understanding of fungal metabolism and development.

Isoforms of 14-3-3 proteins, similar to their highly conserved homologs in mammals and plants, are both transcriptionally and functionally affected by their extracellular and intracellular environments. These proteins bind to phosphorylated client proteins to modulate their functions in fungi. Since phosphorylation regulates a plethora of different physiological responses in organisms, 14-3-3 proteins play roles in multiple physiological functions, including those controlling metabolisms, cell division, and responses to environmental stimulation. These proteins could also modulate signaling pathways that transduce inputs from the environment and downstream proteins that elicit physiological responses. Increasing evidence supports a prominent role for 14-3-3 proteins in regulating development and metabolism at various levels. In this review, we first provide a brief summary of the molecular structure of 14-3-3 proteins. Second, we discuss the potential roles of 14-3-3 proteins in the regulation of development and metabolism. Third, we review the roles of 14-3-3 proteins in the regulation of their binding partners, including receptors, protein kinases, and some protein kinase substrates. Finally, this review examines recent advances that further elucidate the role of 14-3-3 proteins in signaling transduction in response to environmental stress.

Conversion of phosphatidylinositol (PI) to PI4-phosphate (PI4P) and then to PI(4,5)P2 is essential for the cytosolic Ca2+ concentration under heat stress in Ganoderma lucidum.

How cells drive the phospholipid signal response to heat stress (HS) to maintain cellular homeostasis is a fundamental issue in biology, but the regulatory mechanism of this fundamental process is unclear. Previous quantitative analyses of lipids showed that phosphatidylinositol (PI) accumulates after HS in Ganoderma lucidum, implying the inositol phospholipid signal may be associated with HS signal transduction. Here, we found that the PI-4-kinase and PI-4-phosphate-5-kinase activities are activated and that their lipid products PI-4-phosphate and PI-4,5-bisphosphate are increased under HS. Further experimental results showed that the cytosolic Ca2+ ([Ca2+ ]c ) and ganoderic acid (GA) contents induced by HS were decreased when cells were pretreated with Li+ , an inhibitor of inositol monophosphatase, and this decrease could be rescued by PI and PI-4-phosphate. Furthermore, inhibition of PI-4-kinases resulted in a decrease in the Ca2+ and GA contents under HS that could be rescued by PI-4-phosphate but not PI. However, the decrease in the Ca2+ and GA contents by silencing of PI-4-phosphate-5-kinase could not be rescued by PI-4-phosphate. Taken together, our study reveals the essential role of the step converting PI to PI-4-phosphate and then to PI-4,5-bisphosphate in [Ca2+ ]c signalling and GA biosynthesis under HS.

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.

Hydrogen-rich water regulates effects of ROS balance on morphology, growth and secondary metabolism via glutathione peroxidase in Ganoderma lucidum.

Ganoderma lucidum is one of the most important medicinal fungi, but the lack of basic study on the fungus has hindered the further development of its value. To investigate the roles of the redox system in G. lucidum, acetic acid (HAc) was applied as a reactive oxygen species (ROS) stress inducer, and hydrogen-rich water (HRW) was used to relieve the ROS stress in this study. Our results demonstrate that the treatment of 5% HRW significantly decreased the ROS content, maintained biomass and polar growth morphology of mycelium, and decreased secondary metabolism under HAc-induced oxidative stress. Furthermore, the roles of HRW were largely dependent on restoring the glutathione system under HAc stress in G. lucidum. To provide further evidence, we used two glutathione peroxidase (GPX)-defective strains, the gpxi strain, the mercaptosuccinic acid (MS, a GPX inhibitor)-treated wide-type (WT) strain, and gpx overexpression strains for further research. The results show that HRW was unable to relieve the HAc-induced ROS overproduction, decreased biomass, mycelium morphology change and increased secondary metabolism biosynthesis in the absence of GPX function. The gpx overexpression strains exhibited resistance to HAc-induced oxidative stress. Thus, we propose that HRW regulates morphology, growth and secondary metabolism via glutathione peroxidase under HAc stress in the fungus G. lucidum. Furthermore, our research also provides a method to study the ROS system in other fungi.

Membrane fluidity is involved in the regulation of heat stress induced secondary metabolism in Ganoderma lucidum.

Ganoderma lucidum has become a potential model system for evaluating how environmental factors regulate the secondary metabolism of basidiomycetes. Heat stress (HS) is one of the most important environmental factors. It was previously reported that HS could induce the biosynthesis of ganoderic acids (GA). In this study, we found that HS increased GA biosynthesis and also significantly increased cell membrane fluidity. Furthermore, our results showed that addition of the membrane rigidifier dimethylsulfoxide (DMSO) could revert the increased GA biosynthesis elicited by HS. These results indicate that an increase in membrane fluidity is associated with HS-induced GA biosynthesis. Further evidence showed that the GA content was decreased in D9des-silenced strains and could be reverted to WT levels by addition of the membrane fluidizer benzyl alcohol (BA). In contrast, GA content was increased in D9des-overexpression strains and could be reverted to WT levels by the addition of DMSO. Furthermore, both membrane fluidity and GA biosynthesis induced by HS could be reverted by DMSO in WT and D9des-silenced strains. To the best of our knowledge, this is the first report demonstrating that membrane fluidity is involved in the regulation of heat stress induced secondary metabolism in filamentous fungi.

Phospholipase D and phosphatidic acid mediate heat stress induced secondary metabolism in Ganoderma lucidum.

Phospholipid-mediated signal transduction plays a key role in responses to environmental changes, but little is known about the role of phospholipid signalling in microorganisms. Heat stress (HS) is one of the most important environmental factors. Our previous study found that HS could induce the biosynthesis of the secondary metabolites, ganoderic acids (GA). Here, we performed a comprehensive mass spectrometry-based analysis to investigate HS-induced lipid remodelling in Ganoderma lucidum. In particular, we observed a significant accumulation of phosphatidic acid (PA) on HS. Further genetic tests in which pld-silencing strains were constructed demonstrated that the accumulation of PA is dependent on HS-activated phospholipase D (PLD) hydrolysing phosphatidylethanolamine. Furthermore, we determined the role of PLD and PA in HS-induced secondary metabolism in G. lucidum. Exogenous 1-butanol, which decreased PLD-mediated formation of PA, reverses the increased GA biosynthesis that was elicited by HS. The pld-silenced strains partly blocked HS-induced GA biosynthesis, and this block can be reversed by adding PA. Taken together, our results suggest that PLD and PA are involved in the regulation of HS-induced secondary metabolism in G. lucidum. Our findings provide key insights into how microorganisms respond to heat stress and then consequently accumulate secondary metabolites by phospholipid remodelling.

Heat Stress Modulates Mycelium Growth, Heat Shock Protein Expression, Ganoderic Acid Biosynthesis, and Hyphal Branching of Ganoderma lucidum via Cytosolic Ca2.

UNLABELLED: Heat stress (HS) influences the growth and development of organisms. Thus, a comprehensive understanding of how organisms sense HS and respond to it is required. Ganoderma lucidum, a higher basidiomycete with bioactive secondary metabolites, has become a potential model system due to the complete sequencing of its genome, transgenic systems, and reliable reverse genetic tools. In this study, we found that HS inhibited mycelium growth, reduced hyphal branching, and induced the accumulation of ganoderic acid biosynthesis and heat shock proteins (HSPs) in G. lucidum Our data showed that HS induced a significant increase in cytosolic Ca(2+) concentration. Further evidence showed that Ca(2+) might be a factor in the HS-mediated regulation of hyphal branching, ganoderic acid (GA) biosynthesis, and the accumulation of HSPs. Our results further showed that the calcium-permeable channel gene (cch)-silenced and phosphoinositide-specific phospholipase gene (plc)-silenced strains reduced the HS-induced increase in HSP expression compared with that observed for the wild type (WT). This study demonstrates that cytosolic Ca(2+) participates in heat shock signal transduction and regulates downstream events in filamentous fungi. IMPORTANCE: Ganoderma lucidum, a higher basidiomycete with bioactive secondary metabolites, has become a potential model system for evaluating how environmental factors regulate the development and secondary metabolism of basidiomycetes. Heat stress (HS) is an important environmental challenge. In this study, we found that HS inhibited mycelium growth, reduced hyphal branching, and induced HSP expression and ganoderic acid biosynthesis in G. lucidum Further evidence showed that Ca(2+) might be a factor in the HS-mediated regulation of hyphal branching, GA biosynthesis, and the accumulation of HSPs. This study demonstrates that cytosolic Ca(2+) participates in heat shock signal transduction and regulates downstream events in filamentous fungi. Our research offers a new way to understand the mechanism underlying the physiological and metabolic responses to other environmental factors in G. lucidum This research may also provide the basis for heat shock signal transduction studies of other fungi.

The pH-responsive transcription factor PacC regulates mycelial growth, fruiting body development, and ganoderic acid biosynthesis in Ganoderma lucidum.

Ganoderma lucidum is a medicinal macrofungus that is widely used in traditional Chinese medicine. Nonetheless, the scarcity of basic biological studies of this organism has hindered the further development of its commercial value. The pH-responsive transcription factor PacC/Rim101 governs the adaptation to environmental pH, the development and the secondary metabolism of many fungi. In this study, a homologue of PacC/Rim101 that encodes GlPacC was identified in the higher basidiomycete G. lucidum. GlPacC is composed of 807 amino acids and contains three typical C2H2 zinc-finger domains, two potential PEST domains, a putative PKA phosphorylation site, and a putative nuclear localization signal (NLS). GlPacC was transcribed at a high level when the fungus was under neutral and alkaline conditions, and silencing of GlPacC impaired the fungal response to ambient pH. The distance between the hyphal branches (of vegetative hyphae and aerial hyphae) was significantly increased in the GlPacC-silenced strains. The GlPacC-silenced strains grew abnormally or became sickly on solid culture medium and were unable to form primordia and fruiting bodies. The ganoderic acid content, levels of the sqs and ls transcripts, and contents of the metabolic intermediates squalene and lanosterol were all up-regulated in the GlPacC-silenced strains. Our results indicate that GlPacC is functional and plays complex roles in mycelial growth, fruiting body development and ganoderic acid biosynthesis in G. lucidum.

UDP-glucose pyrophosphorylase influences polysaccharide synthesis, cell wall components, and hyphal branching in Ganoderma lucidum via regulation of the balance between glucose-1-phosphate and UDP-glucose.

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.

Molecular characterization and expression analysis of GlHMGS, a gene encoding hydroxymethylglutaryl-CoA synthase from Ganoderma lucidum (Ling-zhi) in ganoderic acid biosynthesis pathway.

A hydroxymethylglutaryl-CoA synthase gene, designated as GlHMGS (GenBank accession No. JN391469) involved in ganoderic acid (GA) biosynthesis pathway was cloned from Ganoderma lucidum. The full-length cDNA of GlHMGS (GenBank accession No. JN391468) was found to contain an open reading frame of 1,413 bp encoding a polypeptide of 471 amino acid residues. The deduced amino acid sequence of GlHMGS shared high homology with other known hydroxymethylglutaryl-CoA synthase (HMGS) enzymes. In addition, functional complementation of GlHMGS in a mutant yeast strain YSC1021 lacking HMGS activity demonstrated that the cloned cDNA encodes a functional HMGS. A 1,561 bp promoter sequence was isolated and its putative regulatory elements and potential specific transcription factor binding sites were analyzed. GlHMGS expression profile analysis revealed that salicylic acid, abscisic acid and methyl jasmonate up-regulated GlHMGS transcript levels over the control. Further expression analysis revealed that the developmental stage and carbon source had significant effects on GlHMGS transcript levels. GlHMGS expression peaked on day 16 before decreasing with prolonged culture time. The highest mRNA level was observed when the carbon source was maltose. Overexpression of GlHMGS enhanced GA content in G. lucidum. This study provides useful information for further studying this gene and on its function in the ganoderic acid biosynthetic pathway in G. lucidum.

The AMP-Activated Protein Kinase (AMPK) Positively Regulates Lysine Biosynthesis Induced by Citric Acid in Flammulina filiformis.

Flammulina filiformis, the most produced edible mushroom species in China, is rich in lysine. Further enhancing its lysine biosynthesis is vital for improving its quality in industrialized cultivation. Citric acid induction significantly increases both the biomass and growth rate of F. filiformis hyphae, as well as the lysine content. The genes encoding enzymes in the lysine biosynthesis pathway were detected under the optimal induction, revealing that the expression levels of hcs, hac, and hah were 2.67, 1.97, and 1.90 times greater, respectively, relative to the control, whereas no significant difference was seen for hdh, aat, sr, and shd, and the expression of aar decreased. Furthermore, the transcriptional levels of Ampk, GCN2, GCN4, and TOR were found significantly upregulated, with the most upregulated, Ampk, reaching a level 42.68 times greater than that of the control, while the phosphorylation of AMPK rose by nearly 54%. In AMPK-silencing strains under the optimal induction, however, the phosphorylation increment dropped to about 16% and the lysine content remained at the same level as in the WT. Thus, AMPK is presented as the critical intermediary in citric acid's regulation of lysine biosynthesis in F. filiformis.

Cross Talk between GlAQP and NOX Modulates the Effects of ROS Balance on Ganoderic Acid Biosynthesis of Ganoderma lucidum under Water Stress.

Water stress affects both the growth and development of filamentous fungi; however, the mechanisms underlying their response to water stress remain unclear. In this study, water stress was found to increase intracellular reactive oxygen species (ROS) level, ganoderic acid (GA) content, and NADPH oxidase (NOX) activity of Ganoderma lucidum by 148.45%, 75.32%, and 161.61%, respectively. Water stress induced the expression of the G. lucidum aquaporin (GlAQP) gene, which facilitated water transfer for microbial growth. Compared to wild type (WT), exposure to water stress increased growth inhibition rate, ROS level, and GA content of GlAQP-silenced strains by 37 to 41%, 36 to 38%, and 25%, respectively. Furthermore, at the early stage of fermentation in GlAQP-silenced strains, water stress resulted in 16 to 17% and 9 to 10% lower ROS level and GA content compared to WT, respectively. However, in GlAQP-overexpressing strains, ROS level and GA content were 22 to 24% and 12 to 13% higher than in WT, respectively. In GlAQP-silenced strains, water stress at the late stage resulted in 35 to 37% and 29 to 30% higher ROS level and GA content, respectively, while in GlAQP-overexpressing strains, levels were 16 to 17% and 9% lower than WT, respectively. Cross talk between GlAQP and NOX positively regulated the GA biosynthesis of G. lucidum via ROS under water stress at the early stage but this regulation became negative at the late stage. This study deepens the understanding of fungal signaling transduction under water stress and provides a reference for analyzing environmental factors that influence the regulation of the fungal secondary metabolism. IMPORTANCE Ganoderma lucidum is an advanced basidiomycete that produces medicinally active secondary metabolites (especially ganoderic acid [GA]) with high commercial value. Water stress imposes an important environmental challenge to G. lucidum. The mechanism of GA biosynthesis under water stress and the role of G. lucidum aquaporin (GlAQP) during its biosynthesis remain unclear. Moreover, the effect of the relationship between GlAQP and NADPH oxidase (NOX) on the level of reactive oxygen species and GA production under water stress is unknown. This study provides information on the biological response mechanism of G. lucidum to water stress. A new theory on the cell signaling cascade of G. lucidum tolerance to water stress is provided that also incorporates the biosynthesis of secondary metabolites involved in NOX and GlAQP.

Chitosan Increases Lysine Content through Amino Acid Transporters in Flammulina filiformis.

Lysine content is considered an important indicator of the quality of Flammulina filiformis. In this study, chitosan was used to improve lysine content of F. filiformis. Optimal design conditions were obtained using central combination design (CCD): treatment concentration was 14.61 µg/mL, treatment time was 52.90 h, and the theoretical value of lysine content was 30.95 mg/g. We used Basic Local Alignment Search Tool Protein (BLASTP) to search the F. filiformis genome database using known AATs in the NCBI database. There were 11 members of AAT in F. filiformis. The expression levels of AAT3 and AAT4 genes increased significantly with chitosan treatment. Subsequently, AAT3 and AAT4 silencing strains were constructed using RNAi technology. The lysine content of the wild-type (WT) strain treated with chitosan increased by 26.41%. Compared with the chitosan-induced WT strain, chitosan-induced lysine content decreased by approximately 24.87% in the AAT3 silencing strain, and chitosan-induced lysine content in the AAT4 silencing strain increased by approximately 13.55%. The results indicate that AAT3 and AAT4 are involved in the regulation of the biosynthesis of lysine induced by chitosan in F. filiformis. AAT3 may participate in the absorption of lysine, and AAT4 may be involved in the excretion of lysine with chitosan treatment.

Establishment of CRISPR/Cas9 Genome-Editing System Based on Dual sgRNAs in Flammulina filiformis.

Flammulina filiformis, previously known as Asian Flammulina velutipes, is one of the most commercially important edible fungi, with nutritional value and medicinal properties worldwide. However, precision genome editing using CRISPR/Cas9, which is a revolutionary technology and provides a powerful tool for molecular breeding, has not been established in F. filiformis. Here, plasmids harboring expression cassettes of Basidiomycete codon-optimized Cas9 and dual sgRNAs targeting pyrG under the control of the gpd promoter and FfU6 promoter, respectively, were delivered into protoplasts of F. filiformis Dan3 strain through PEG-mediated transformation. The results showed that an efficient native U6 promoter of F. filiformis was identified, and ultimately several pyrG mutants exhibiting 5-fluorooric acid (5-FOA) resistance were obtained. Additionally, diagnostic PCR followed by Sanger sequencing revealed that fragment deletion between the two sgRNA target sites or small insertions and deletions (indels) were introduced in these pyrG mutants through the nonhomologous end joining (NHEJ) pathway, resulting in heritable changes in genomic information. Taken together, this is the first report in which a successful CRISPR/Cas9 genome-editing system based on dual sgRNAs was established in F. filiformis, which broadens the application of this advanced tool in Basidiomycetes.

Target of Rapamycin Mediated Ornithine Decarboxylase Antizyme Modulate Intracellular Putrescine and Ganoderic Acid Content in Ganoderma lucidum.

Putrescine (Put) has been shown to play an important regulatory role in cell growth in organisms. As the primary center regulating the homeostasis of polyamine (PA) content, ornithine decarboxylase antizyme (AZ) can regulate PA content through feedback. Nevertheless, the regulatory mechanism of Put is poorly understood in fungi. Here, our analysis showed that GlAZ had a modulate effect on intracellular Put content by interacting with ornithine decarboxylase (ODC) proteins and reducing its intracellular protein levels. In addition, GlAZ upregulated the metabolic pathway of ganoderic acid (GA) biosynthesis in Ganoderma lucidum by modulating the intracellular Put content. However, a target of rapamycin (TOR) was found to promote the accumulation of intracellular Put after the GlTOR inhibitor Rap was added exogenously, and unbiased analyses demonstrated that GlTOR may promote Put production through its inhibitory effect on the level of GlAZ protein in GlTOR-GlAZ-cosilenced strains. The effect of TOR on fungal secondary metabolism was further explored, and the content of GA in the GlTOR-silenced strain after the exogenous addition of the inhibitor Rap was significantly increased compared with that in the untreated wild-type (WT) strain. Silencing of TOR in the GlTOR-silenced strains caused an increase in GA content, which returned to the WT state after replenishing Put. Moreover, the content of GA in GlTOR-GlAZ-cosilenced strains was also not different from that in the WT strain. Consequently, these results strongly indicate that GlTOR affects G. lucidum GA biosynthesis via GlAZ. IMPORTANCE Research on antizyme (AZ) in fungi has focused on the mechanism by which AZ inhibits ornithine decarboxylase (ODC). Moreover, there are existing reports on the regulation of AZ protein translation by TOR. However, little is known about the mechanisms that influence AZ in fungal secondary metabolism. Here, both intracellular Put content and GA biosynthesis in G. lucidum were shown to be regulated through protein interactions between GlAZ and GlODC. Furthermore, exploration of upstream regulators of GlAZ suggested that GlAZ was regulated by the upstream protein GlTOR, which affected intracellular Put levels and ganoderic acid (GA) biosynthesis. The results of our work contribute to the understanding of the upstream regulation of Put and provide new insights into PA regulatory systems and secondary metabolism in fungi.

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

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