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.

Hydrogen sulfide maintains mitochondrial homeostasis and regulates ganoderic acids biosynthesis by SQR under heat stress in Ganoderma lucidum.

Hydrogen sulfide (H2S) has recently been recognized as an important gaseous transmitter with multiple physiological effects in various species. Previous studies have shown that H2S alleviated heat-induced ganoderic acids (GAs) biosynthesis, an important quality index of Ganoderma lucidum. However, a comprehensive understanding of the physiological effects and molecular mechanisms of H2S in G. lucidum remains unexplored. In this study, we found that heat treatment reduced the mitochondrial membrane potential (MMP) and mitochondrial DNA copy number (mtDNAcn) in G. lucidum. Increasing the intracellular H2S concentration through pharmacological and genetic means increased the MMP level, mtDNAcn, oxygen consumption rate level and ATP content under heat treatment, suggesting a role for H2S in mitigating heat-caused mitochondrial damage in G. lucidum. Further results indicated that H2S activates sulfide-quinone oxidoreductase (SQR) and complex III (Com III), thereby maintaining mitochondrial homeostasis under heat stress in G. lucidum. Moreover, SQR also mediated the negative regulation of H2S to GAs biosynthesis under heat stress. Furthermore, SQR might be persulfidated under heat stress in G. lucidum. Thus, our study reveals a novel physiological function and molecular mechanism of H2S signalling under heat stress in G. lucidum with broad implications for research on the environmental response of microorganisms.

Strategies to Increase the Production of Triterpene Acids in Ligzhi or Reishi Medicinal Mushroom (Ganoderma lucidum, Agaricomycetes): A Review.

Ganoderic acids (GAs) are the main active ingredient of Ganoderma lucidum, which has been widely accepted as a medicinal mushroom. Due to the low yield of GAs produced by liquid cultured Ganoderma mycelium and solid cultured fruiting bodies, the commercial production and clinical application of GAs are limited. Therefore, it is important to increase the yield of GA in G. lucidum. A comprehensive literature search was performed with no set data range using the following keywords such as "triterpene," "ganoderic acids," "Ganoderma lucidum," and "Lingzhi" within the main databases including Web of Science, PubMed, and China National Knowledge Infrastructure (CNKI). The data were screened using titles and abstracts and those relevant to the topic were included in the paper and was not limited to studies published in English. Present review focuses on the four aspects: fermentation conditions and substrate, extrinsic elicitor, genetic engineering, and mutagenesis, which play significant roles in increasing triterpene acids production, thus providing an available reference for further research on G. lucidum fermentation.

The impact of continuous cultivation of Ganoderma lucidum on soil nutrients, enzyme activity, and fruiting body metabolites.

To explore the impacts of continuous Ganoderma lucidum cultivation on soil physicochemical factors, soil enzyme activity, and the metabolome of Ganoderma lucidum fruiting bodies, this study conducted two consecutive years of cultivation on the same plot of land. Soil physicochemical factors and enzyme activity were assessed, alongside non-targeted metabolomic analysis of the Ganoderma lucidum fruiting bodies under continuous cultivation. The findings unveiled that in the surface soil layer (0-15 cm), there was a declining trend in organic matter, ammonium nitrogen, available phosphorus, available potassium, pH, polyphenol oxidase, peroxidase, alkaline phosphatase, and sucrase, whereas nitrate nitrogen, electrical conductivity (EC), and salt content exhibited an upward trend. Conversely, in the deeper soil layer (15-30 cm), organic matter, ammonium nitrogen, available potassium, alkaline phosphatase, and sucrase demonstrated a decreasing trend, while nitrate nitrogen, available phosphorus, pH, EC, salt content, polyphenol oxidase, and soil peroxidase showed an increasing trend. Metabolomic analysis of Ganoderma lucidum fruiting bodies distinguished 64 significantly different metabolites between the GCK and GT groups, with 39 components having markedly higher relative contents in GCK and 25 components having significantly lower relative contents in GCK compared to GT. Moreover, among these metabolites, there were more types with higher contents in the fruiting bodies harvested in the first year (GCK) compared to those harvested in the second year (GT), with pronounced differences. KEGG pathway analysis revealed that GCK exhibited more complex metabolic pathways compared to GT. The metabolites of Ganoderma lucidum fruiting bodies were predominantly influenced by soil physicochemical factors and soil enzyme activity. In the surface soil layer (0-15 cm), the metabolome was significantly affected by soil pH, soil organic matter, available phosphorus, and soil alkaline phosphatase, while in the deeper soil layer (15-30 cm), differences in the Ganoderma lucidum metabolome were more influenced by soil alkaline phosphatase, soil catalase, pH, nitrate nitrogen, and soil sucrase.

Removal of pharmaceutical compounds and toxicology study in wastewater using Malaysian fungal Ganoderma lucidum.

Elevated usage of pharmaceutical products leads to the accumulation of emerging contaminants in sewage. In the current work, Ganoderma lucidum (GL) was used to remove pharmaceutical compounds (PCs), proposed as a tertiary method in sewage treatment plants (STPs). The PCs consisted of a group of painkillers (ketoprofen, diclofenac, and dexamethasone), psychiatrists (carbamazepine, venlafaxine, and citalopram), beta-blockers (atenolol, metoprolol, and propranolol), and anti-hypertensives (losartan and valsartan). The performance of 800 mL of synthetic water, effluent STP, and hospital wastewater (HWW) was evaluated. Parameters, including treatment time, inoculum volume, and mechanical agitation speed, have been tested. The toxicity of the GL after treatment is being studied based on exposure levels to zebrafish embryos (ZFET) and the morphology of the GL has been observed via Field Emission Scanning Electron Microscopy (FESEM). The findings conclude that GL can reduce PCs from <10% to >90%. Diclofenac and valsartan are the highest (>90%) in the synthetic model, while citalopram and propranolol (>80%) are in the real wastewater. GL effectively removed pollutants in 48 h, 1% of the inoculum volume, and 50 rpm. The ZFET showed GL is non-toxic (LC50 is 209.95 mg/mL). In the morphology observation, pellets GL do not show major differences after treatment, showing potential to be used for a longer treatment time and to be re-useable in the system. GL offers advantages to removing PCs in water due to their non-specific extracellular enzymes that allow for the biodegradation of PCs and indicates a good potential in real-world applications as a favourable alternative treatment.

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.

Time-dependent change in Reishi (Ganoderma lingzhi) triterpenoids in culture with rumen fluid.

The purpose of this study was to investigate the time-dependent change in Reishi (Ganoderma lingzhi) triterpenoids in rumen fluid. G. lingzhi fruiting bodies were milled and incubated in a tube with rumen fluid for 0, 4, 8, 12, 24, and 48 h at 39°C. After incubation, all the tubes were freeze-dried and extracted by ethanol. The contents of 18 triterpenoids in the ethanol extract were quantitated by liquid chromatography-mass spectrometry (LC-MS/MS). Based on the results, triterpenoids were categorized into three groups: (1) rapid decrease, indicating reductions of more than 50% within 8 h; (2) mild decrease, with reductions of more than 50% within 48 h; and (3) minimal change, even after 48 h, there was not much change. Ganoderic acid C6, DM, H, K, and TR as well as Ganoderenic acid D were classified in (1); Ganoderic acid LM2 and T-Q as well as Ganoderiol F in (2); and Ganoderic acid A, B, C1, C2, I, and TN; Gnoderenic acid C; and Ganodermanontriol in (3). In addition, a relationship between chemical structure and metabolic speed was observed in some cases. The results of this study revealed that G. lingzhi triterpenoids are digested and metabolized at different speeds in ruminant fluid.

Temporal dynamics of lignin degradation in Quercus acutissima sawdust during Ganoderma lucidum cultivation.

Despite a fair amount of lignin conversion during mycelial growth, previous structural analyses have not yet revealed how lignin changes continuously and what the relationship is between lignin and ligninolytic enzymes. To clarify these aspects, Quercus acutissima sawdust attaching Ganoderma lucidum mycelium collected from different growth stage was subjected to analysis of lignin structure and ligninolytic enzyme activity. Two key periods of lignin degradation are found during the cultivation of G. lucidum: hypha rapid growth period and primordium formation period. In the first stage, laccase activity is associated with the opening of structures such as methoxyls, ß-O-4' substructures and guaiacyl units in lignin, as well as the shortening of lignin chains. Manganese peroxidases and lignin peroxidases are more suitable for degrading short chain lignin. The structure of phenylcoumarans and syringyl changes greatly in the second stage. The results from sawdust attaching mycelium provide new insights to help improve the cultivation substrate formulation of G. lucidum and understand biomass valorization better.

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.

Ganoderma lucidum: Insights on host range, diagnosis, and management strategies.

Forest ecosystems play an important role in upholding life on our planet. However, the onslaught of fungal pathogens like Ganoderma lucidum, poses a threat by decimating numerous tree species. G. lucidum identified as a root pathogen, causing root rot in numerous tree species of horticulture and forestry importance. The fungus initiates infection through basidiospores, which germinate and penetrate within roots and start to degrade lignocellulosic components of plant cells. Early-stage detection of G. lucidum, is challenging, while in advance stages, the wood undergoes softening and a loss of tensile strength, rendering the disease incurable. Hence, effective management of G. lucidum necessitates a pivotal role of disease diagnostic techniques, which are currently underutilized or inadequately accessible. Subsequent implementation of suitable control measures becomes imperative to thwart disease occurrence and mitigate its impact in early stages, thus preserving the vitality of forest ecosystems. This study provides comprehensive overview of G. lucidum, covering taxonomy, pathogenicity, disease cycle, diagnosis and effective control measures, which will be helpful in formulating effective diagnostic techniques for early management of root rot disease.

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.

Glsirt1-mediated deacetylation of GlCAT regulates intracellular ROS levels, affecting ganoderic acid biosynthesis in Ganoderma lucidum.

Lysine acetylation is a reversible, dynamic protein modification regulated by lysine acetyltransferases and deacetylases. However, in Basidiomycetes, the extent of lysine acetylation of nonhistone proteins remains largely unknown. Recently, we identified the deacetylase Glsirt1 as a key regulator of the biosynthesis of ganoderic acid (GA), a key secondary metabolite of Ganoderma lucidum. To gain insight into the characteristics, extent, and biological function of Glsirt1-mediated lysine acetylation in G. lucidum, we aimed to identify additional Glsirt1 substrates via comparison of acetylomes between wild-type (WT) and Glsirt1-silenced mutants. A large amount of Glsirt1-dependent lysine acetylation occurs in G. lucidum according to the results of this omics analysis, involving energy metabolism, protein synthesis, the stress response and other pathways. Our results suggest that GlCAT is a direct target of Glsirt1 and that the deacetylation of GlCAT by Glsirt1 reduces catalase activity, thereby leading to the accumulation of intracellular reactive oxygen species (ROS) and positively regulating the biosynthesis of GA. Our findings provide evidence for the involvement of nonhistone lysine acetylation in the biological processes of G. lucidum and help elucidate the involvement of important ROS signaling molecules in regulating physiological and biochemical processes in this organism. In conclusion, this proteomic analysis reveals a striking breadth of cellular processes affected by lysine acetylation and provides new nodes of intervention in the biosynthesis of secondary metabolites in G. lucidum.

GlPRMT5 inhibits GlPP2C1 via symmetric dimethylation and regulates the biosynthesis of secondary metabolites in Ganoderma lucidum.

PRMT5, a type II arginine methyltransferase, is involved in transcriptional regulation, RNA processing and other biological processes and signal transduction. Secondary metabolites are vital pharmacological compounds in Ganoderma lucidum, and their content is an important indicator for evaluating the quality of G. lucidum. Here, we found that GlPRMT5 negatively regulates the biosynthesis of secondary metabolites. In further in-depth research, GlPP2C1 (a type 2C protein phosphatase) was identified out as an interacting protein of GlPRMT5 by immunoprecipitation-mass spectrometry (IP-MS). Further mass spectrometry detection revealed that GlPRMT5 symmetrically dimethylates the arginine 99 (R99) and arginine 493 (R493) residues of GlPP2C1 to weaken its activity. The symmetrical dimethylation modification of the R99 residue is the key to affecting GlPP2C1 activity. Symmetrical demethylation-modified GlPP2C1 does not affect the interaction with GlPRMT5. In addition, silencing GlPP2C1 clearly reduced GA content, indicating that GlPP2C1 positively regulates the biosynthesis of secondary metabolites in G. lucidum. In summary, this study reveals the molecular mechanism by which GlPRMT5 regulates secondary metabolites, and these studies provide further insights into the target proteins of GlPRMT5 and symmetric dimethylation sites. Furthermore, these studies provide a basis for the mutual regulation between different epigenetic modifications.

4-Coumarate-CoA ligase (4-CL) enhances flavonoid accumulation, lignin synthesis, and fruiting body formation in Ganoderma lucidum.

It is now understood that 4-Coumarate-CoA ligases (4-CL) are pivotal in bridging the phenylpropanoid metabolic pathway and the lignin biosynthesis pathway in plants. However, limited information on 4-CL genes and their functions in fungi is available. In this study, we cloned the 4-CL gene (Gl21040) from Ganoderma lucidum, which spans 2178 bp and consists of 10 exons and 9 introns. We also developed RNA interference and overexpression vectors for Gl21040 to investigate its roles in G. lucidum. Our findings indicated that in the Gl21040 interference transformants, 4-CL enzyme activities decreased by 31 %-57 %, flavonoids contents decreased by 10 %-22 %, lignin contents decreased by 20 %-36 % compared to the wild-type (WT) strain. Conversely, in the Gl21040 overexpression transformants, 4-CL enzyme activity increased by 108 %-143 %, flavonoids contents increased by 8 %-37 %, lignin contents improved by 15 %-17 % compared to the WT strain. Furthermore, primordia formation was delayed by approximately 10 days in the Gl21040-interferenced transformants but occurred 3 days earlier in the Gl21040-overexpressed transformants compared to the WT strain. These results underscored the involvement of the Gl21040 gene in flavonoid synthesis, lignin synthesis, and fruiting body formation in G. lucidum.

Determination of selenium-containing species, including nanoparticles, in selenium-enriched Lingzhi mushrooms.

Mushrooms are considered a valuable food source due to their high protein and fibre and low fat content, among the other health benefits of their consumption. Selenium is an essential nutrient and is renowned for its chemo-preventative properties. In this study, batches of selenium-enriched Lingzhi mushrooms were prepared by growing mycelium and fruit in substrates containing various concentrations of sodium selenite. The mushroom fruit accumulated low levels of selenium with selenomethionine being the most abundant form in all enriched samples. Conversely, the mycelium showed significant selenium accumulation but relatively low proportions of selenomethionine. The red colour of the selenium-enriched mycelia indicated the probable presence of selenium nanoparticles, which was confirmed by single-particle inductively coupled plasma-mass spectrometry. Mean particle diameters of 90-120 nm were observed, with size distributions of 60-250 nm. Additional analysis with transmission electron microscopy confirmed this size distribution and showed that the biogenic selenium nanoparticles were roughly spherical in shape and contained elemental selenium.

Developing Ganoderma lucidum as a next-generation cell factory for food and nutraceuticals.

Ganoderma lucidum holds a colossal reservoir of hydrolytic enzymes and therapeutic compounds and can be a sustainable source of proteins and bioactive compounds. Its metabolic versatility, propelled by its rich genome content, provides excellent biosynthetic machinery for innovation-driven pathway engineering. However, robust regulatory networks and low frequency of homologous recombination are critical bottlenecks that limit the development of molecular tools and precise genetic markers for biomanufacturing innovations in this organism. Modern synthetic biology provides tools that could help to accelerate precise multiple gene targeting and editing and untangling the biosynthetic machinery of G. lucidum. This review provides insight into molecular strategies to unwind the regulatory bottlenecks and transform G. lucidum into efficient cell factories for food and nutraceuticals.

Ganoderma lucidum ethanol extracts ameliorate hepatic fibrosis and promote the communication between metabolites and gut microbiota g_Ruminococcus through the NF-κB and TGF-ß1/Smads pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Ganoderma lucidum, a traditional edible medicinal mushroom, has been widely reported to improve liver diseases as a dietary intervention for people. Ganoderma lucidum extracts, primarily total triterpenoids (GLTTs), are one of the bioactive ingredients that have excellent beneficial effects on hepatic fibrosis. Therefore, its prevention and reversal are particularly critical due to the increasing number of patients with chronic liver diseases worldwide. AIM OF THE STUDY: The study aimed to evaluate whether GLTTs had a hepatoprotective effect against hepatic fibrosis through metabolic perturbations and gut microbiota changes and its underlying mechanisms. MATERIALS AND METHODS: The compound compositions of GLTTs were quantified, and carbon tetrachloride (CCl4)-induced hepatic fibrosis rats were used to investigate the cause of the improvement in various physiological states with GLTTs treatment, and to determine whether its consequent effect was associated with endogenous metabolites and gut microbiota using UPLC-Q-TOF-MSE metabolomics and 16S rRNA gene sequencing technology. RESULTS: GLTTs alleviated physical status, reduced liver pathological indicators, proinflammatory cytokines, and deposition of hepatic collagen fibers via regulating the NF-κB and TGF-ß1/Smads pathways. The untargeted metabolomics analysis identified 16 potential metabolites that may be the most relevant metabolites for gut microbiota dysbiosis and the therapeutic effects of GLTTs in hepatic fibrosis. Besides, although GLTTs did not significantly affect the α-diversity indexes, significant changes were observed in the composition of microflora structure. In addition, Spearman analysis revealed strong correlations between endogenous metabolites and gut microbiota g_Ruminococcus with hepatic fibrosis. CONCLUSION: GLTTs could provide a potential target for the practical design and application of novel functional food ingredients or drugs in the therapy of hepatic fibrosis.

Abscisic acid-mediated cytosolic Ca2+ modulates triterpenoid accumulation of Ganoderma lucidum. / è„±è½é ¸ä»‹å¯¼èƒžå† Ca2+参与调控灵芝三萜积累.

Ganoderma lucidum is a mushroom widely used for its edible and medicinal properties. Primary bioactive constituents of G. lucidum are ganoderic triterpenoids (GTs), which exhibit important pharmacological activity. Abscisic acid (ABA), a plant hormone, is associated with plant growth, development, and stress responses. ABA can also affect the growth, metabolism, and physiological activities of different fungi and participates in the regulation of the tetracyclic triterpenes of some plants. Our findings indicated that ABA treatment promoted GT accumulation by regulating the gene expression levels (squalene synthase (sqs), 3-hydroxy-3-methylglutaryl-CoA reductase (hmgr), and lanosterol synthase (ls)), and also activated cytosolic Ca2+ channels. Furthermore, under ABA mediation, exogenous Ca2+ donors and inhibitors directly affected the cytosolic Ca2+ concentration and related gene expression in Ca2+ signaling. Our study also revealed that ABA-mediated cytosolic Ca2+ played a crucial regulatory role in GT biosynthesis, accompanied by antioxidant defense modulation with increasing superoxide dismutase (SOD) activity and ascorbate peroxidase (APX) activity, and the resistance ability of O2•- and glutathione (GSH) contents.

Application of Miscanthus substrates in the cultivation of Ganoderma lingzhi.

Ganoderma lingzhi is a traditional Chinese medicine that has been used to improve health and longevity for thousands of years. It is usually cultivated on hardwood log- or sawdust-based formulations. Conversely, in this study, we used Miscanthus sacchariflorus (MSF), M. floridulus, and M. sinensis (MSS), fast-growing perennial grasses widely distributed in China, for G. lingzhi cultivation. Mycelial growth rate, activities of lignin-degrading enzymes on colonized mushroom substrates, and expression levels of CAZymes and laccase genes based on different substrates were analyzed. Total triterpenoids, sterols, and polysaccharides content of fruiting bodies obtained from different substrates were investigated. The activities of laccase and manganese peroxidase in mycelia increased in the MSF- and MSS-based formulations compared with that in the sawdust-based formulation. The results of mycelial growth- and cultivation-related experiments showed that the Miscanthus substrates could be used as the substrates for cultivating G. lingzhi. The content of active ingredients, namely triterpenoids, sterols, and polysaccharides, in fruiting bodies cultivated on the Miscanthus substrates did not decrease compared with those in substrate obtained from the sawdust-based formulation. Therefore, the present study provides alternative substrates for the cultivation of G. lingzhi, and a reference for better utilization of inexpensive substrate in future.

Improving nanochemoimmunotherapy efficacy by boosting "eat-me" signaling and downregulating "don't-eat-me" signaling with Ganoderma lucidum polysaccharide-based drug delivery.

To address the challenges posed by low immunogenicity and immune checkpoints during cancer treatment, we propose an alternative strategy that combines immunogenic cell death (ICD) effects with CD47/SIRPα blockade to reactivate phagocytosis of tumor cells by macrophages with polysaccharide-based drug delivery. In this study, the EGFR inhibitor gefitinib was identified as a novel CD47 modulator, which promoted the translocation of CD47 molecules from the cell membrane to endosomes through the EGFR-Rab5 pathway, leading to reduced cell surface CD47 levels and limiting interaction with SIRPα. Based on this finding, we developed prophagocytic mixed nanodrugs to enhance macrophage phagocytosis by encapsulating ICD inducer doxorubicin and CD47 inhibitor gefitinib with immunostimulatory polysaccharides from Ganoderma lucidum. This approach downregulated cell surface CD47 expression to attenuate "don't-eat-me" signaling, while increasing doxorubicin accumulation in tumors by inhibiting drug-resistance proteins, leading to more exposure of calreticulin and amplifying the "eat-me" signaling. In vivo experiments demonstrated that this approach significantly suppressed intraperitoneal tumor dissemination, reversed doxorubicin-induced weight loss, and effectively induced macrophage polarization, dendritic cell maturation, and CD8+ T cell activation. These findings highlighted the significant potential of our macrophage-centered therapeutic strategy using polysaccharide-based nanocarriers and provided new perspectives for chemoimmunotherapy.