Transcriptional Dynamics of Genes Purportedly Involved in the Control of Meiosis, Carbohydrate, and Secondary Metabolism during Sporulation in Ganoderma lucidum.

Ganoderma lucidum spores (GLS), the mature germ cells ejected from the abaxial side of the pileus, have diverse pharmacological effects. However, the genetic regulation of sporulation in this fungus remains unknown. Here, samples corresponding to the abaxial side of the pileus were collected from strain YW-1 at three sequential developmental stages and were then subjected to a transcriptome assay. We identified 1598 differentially expressed genes (DEGs) and found that the genes related to carbohydrate metabolism were strongly expressed during spore morphogenesis. In particular, genes involved in trehalose and malate synthesis were upregulated, implying the accumulation of specific carbohydrates in mature G. lucidum spores. Furthermore, the expression of genes involved in triterpenoid and ergosterol biosynthesis was high in the young fruiting body but gradually decreased with sporulation. Finally, spore development-related regulatory pathways were explored by analyzing the DNA binding motifs of 24 transcription factors that are considered to participate in the control of sporulation. Our results provide a dataset of dynamic gene expression during sporulation in G. lucidum. They also shed light on genes potentially involved in transcriptional regulation of the meiotic process, metabolism pathways in energy provision, and ganoderic acids and ergosterol biosynthesis.

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.

Functions of reactive oxygen species in apoptosis and ganoderic acid biosynthesis in Ganoderma lucidum.

Ganoderma lucidum is a medicinal fungus that is widely used in traditional medicine. Fungal PacC is recognized as an important transcription factor that functions during adaptation to environmental pH, fungal development and secondary metabolism. Previous studies have revealed that GlPacC plays important roles in mycelial growth, fruiting body development and ganoderic acid (GA) biosynthesis. In this study, using a terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assay, we found that the apoptosis level was increased when PacC was silenced. The transcript and activity levels of caspase-like proteins were significantly increased in the PacC-silenced (PacCi) strains compared with the control strains. Silencing PacC also resulted in an increased reactive oxygen species (ROS) levels (∼2-fold) and decreased activity levels of enzymes involved in the antioxidant system. Further, we found that the intracellular ROS levels contributed to apoptosis and GA biosynthesis. Adding N-acetyl-cysteine and vitamin C decreased intracellular ROS and resulted in the inhibition of apoptosis in the PacCi strains. Additionally, the GA biosynthesis was different between the control strains and the PacCi strains after intracellular ROS was eliminated. Taken together, the findings showed that silencing PacC can result in an intracellular ROS burst, which increases cell apoptosis and GA biosynthesis levels. Our study provides novel insight into the functions of PacC in filamentous fungi.

Roles of the Skn7 response regulator in stress resistance, cell wall integrity and GA biosynthesis in Ganoderma lucidum.

The transcription factor Skn7 is a highly conserved fungal protein that participates in a variety of processes, including oxidative stress adaptation, fungicide sensitivity, cell wall biosynthesis, cell cycle, and sporulation. In this study, a homologous gene of Saccharomyces cerevisiae Skn7 was cloned from Ganoderma lucidum. RNA interference (RNAi) was used to study the functions of Skn7, and the two knockdown strains Skn7i-5 and Skn7i-7 were obtained in G. lucidum. The knockdown of GlSkn7 resulted in hypersensitivity to oxidative and cell wall stresses. The concentrations of chitin and ß-1,3-glucan distinctly decreased in the GlSkn7 knockdown strains compared with those of the wild type (WT). In addition, the expression of cell wall biosynthesis related genes was also significantly down-regulated and the thickness of the cell wall also significantly reduced in the GlSkn7 knockdown strains. The intracellular reactive oxygen species (ROS) content and ganoderic acids biosynthesis increased significantly in the GlSkn7 knockdown strains. Interestingly, the level of intracellular ROS and the content of ganoderic acids decreased after N-acetyl-L-cysteine (NAC), an ROS scavenger, was added, indicating that GlSkn7 might regulate ganoderic acids biosynthesis via the intracellular ROS level. The transcript level of GlSkn7 were up-regulated in osmotic stress, heat stress and fungicide condition. At the same time, the content of ganoderic acids in the GlSkn7 knockdown strains also changed distinctly in these conditions. Overall, GlSkn7 is involved in stress resistance, cell wall integrity and ganoderic acid biosynthesis in G. lucidum.

Cross Talk between Nitric Oxide and Calcium-Calmodulin Regulates Ganoderic Acid Biosynthesis in Ganoderma lucidum under Heat Stress.

We previously reported that high temperature impacts ganoderic acid (GA) biosynthesis in Ganoderma lucidum via Ca2+ Therefore, to further understand the signal-regulating network of the organism's response to heat stress (HS), we examined the role of nitric oxide (NO) under HS. After HS treatment, the NO level was significantly increased by 120% compared to that under the control conditions. The application of a NO scavenger resulted in a 25% increase in GA compared with that found in the sample treated only with HS. Additionally, the application of a NO donor to increase NO resulted in a 30% lower GA content than that in the sample treated only with HS. These results show that the increase in NO levels alleviates HS-induced GA accumulation. Subsequently, we aimed to detect the effects of the interaction between NO and Ca2+ on GA biosynthesis under HS in G. lucidum Our pharmacological approaches revealed that the NO and Ca2+ signals promoted each other in response to HS. We further constructed the silenced strain of nitrate reductase (NR) and calmodulin (CaM), and the results are in good agreement with the silenced strain and pharmacological experiment. The cross-promotion between NO and Ca2+ signals is involved in the regulation of HS-induced GA biosynthesis in G. lucidum, and this finding is supported by studies with NR-silenced (NRi) and CaM-silenced (CaMi) strains. However, Ca2+ may have a more direct and significant effect on the HS-induced GA increase than NO. These data indicate that NO functions in signaling and has a close relationship with Ca2+ in HS-induced GA biosynthesis.IMPORTANCE HS is an important environmental stress affecting the growth and development of organisms. We previously reported that HS modulates GA biosynthesis in G. lucidum via Ca2+ However, the signal-regulating network of the organism's response to HS has not yet been elucidated. In this study, we found that NO relieved HS-induced GA accumulation, and NO and Ca2+ could exert promoting effects on each other in response to HS. Further research on the effect of NO and Ca2+ on the production of GAs in response to HS indicated that Ca2+ has a notably more direct and significant effect on the HS-induced GA increase than NO. Our results improve our understanding of the mechanism of HS signal transduction in fungi. A greater understanding of the regulation of secondary metabolism in response to environmental stimuli will provide clues regarding the role of these products in fungal biology.

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.

Preparation and characterization of Ganoderma lucidum spores-loaded alginate microspheres by electrospraying.

Ganoderma lucidum spores (GLSs), popular functional food in preventive medicine, are susceptible to oxidative and acidic degradation during processing, storage and oral administration, resulting in the loss of sensory and nutritional qualities. The main objective of the study was to encapsulate the GLS in order to fully preserve the bioactivity of the ingredients as well as providing controlled and targeted delivery. Electrospraying was applied to prepare GLS-Alginate (GLS/A) micro beads in the current study. The size of GLS/A beads can be tailored by varying the applied voltage and drying processes. pH responsive release profiles of GLS/A beads were revealed from in vitro study in a simulated gastrointestinal environment: no release of GLS encapsulated beads in the simulated gastric fluid (pH of 1.8) was observed; while a rapid, size dependent release was found in the simulated intestinal solution (pH of 7.5). The release from smaller beads (e.g. 600 µm) was 1.5 times faster than that of larger beads (e.g. 2000 µm). In addition, the GLS release from freeze dried beads was almost 3 times faster than those of air and vacuum dried beads in the first 90 min. The present results illustrate the potential to protect GLS by encapsulation using electrospraying to achieve the controlled release of GLS ingredients. This will pave the way to develop effective GLS products with desirable bioactive components for healthcare applications.

Increased polysaccharide production and biosynthetic gene expressions in a submerged culture of Ganoderma lucidum by the overexpression of the homologous α-phosphoglucomutase gene.

This study aimed to improve the production of polysaccharide by engineering the biosynthetic pathway in Ganoderma lucidum through the overexpression of α-phosphoglucomutase (PGM) gene. PGM is responsible for the linkage between sugar catabolism and sugar anabolism. The effects of PGM gene overexpression on intracellular polysaccharide (IPS) content, extracellular polysaccharide (EPS) production and transcription levels of three genes encoding the enzymes involved in polysaccharide biosynthesis, including PGM, UDP-glucose pyrophosphorylase (UGP), and ß-1,3-glucan synthase (GLS), were investigated. The maximum IPS content and EPS production in G. lucidum overexpressing the PGM gene were 23.67 mg/100 mg dry weight and 1.76 g/L, respectively, which were higher by 40.5 and 44.3% than those of the wild-type strain. The transcription levels of PGM, UGP and GLS were upregulated by 4.77-, 1.51- and 1.53-fold, respectively, in the engineered strain, suggesting that increased polysaccharide biosynthesis may result from a higher expression of those genes.

Ganoderma lucidum ameliorate mitochondrial damage in isoproterenol-induced myocardial infarction in rats by enhancing the activities of TCA cycle enzymes and respiratory chain complexes.

BACKGROUND: Decreased mitochondrial function has been suggested to be one of the important pathological events in isoproterenol (ISO)-induced cardiotoxicity. In this communication, we have evaluated the protective effect of Ganoderma lucidum against ISO induced cardiac toxicity and mitochondrial dysfunction. METHODS: Cardiac toxicity was assessed by determining the activities of creatine kinase (CK) and lactate dehydrogenases (LDH) after subcutaneous injection of ISO (85 mg/kg) at an interval of 24h for 2 days. The animals were sacrificed 24h after last ISO administration. G. lucidum (100 and 250 mg/kg, p.o.) was given to the rats once daily for 15 days prior to the ISO challenge. Similarly, α-Tocopherol (100mg/kg, p.o) was kept as the standard. To assess the extent of cardiac mitochondrial damage, the activities of Krebs cycle dehydrogenases and mitochondrial complexes I, II, III, and IV as well as the level of ROS and mitochondrial membrane potential (ΔΨmt) were evaluated. RESULTS: Administration of G. lucidum and α-tocopherol significantly protected the elevated activities of CK and LDH. Further, the activities of mitochondrial enzymes and the level of ΔΨmt were significantly enhanced and the level of ROS was significantly declined in the G. lucidum and α-tocopherol treatments. CONCLUSION: The present study concluded that the cardiac mitochondrial enzymes are markedly declined by the ISO challenge and the administration G. lucidum and α-Tocopherol significantly protected mitochondria by preventing the decline of antioxidant status and ΔΨmt or by directly scavenging the free radicals.

Development of a simple and efficient transformation system for the basidiomycetous medicinal fungus Ganoderma lucidum.

In this study, we report the development of a simple and efficient system for genetic transformation of the medicinal fungus Ganoderma lucidum. Various parameters were optimized to obtain successful Agrobacterium tumefaciens-mediated transformation. Co-cultivation of bacteria and protoplast at a ratio of 1,000:1 at 25°C in medium containing 0.2 mM acetosyringone was found to be the optimum condition for high efficiency transformation. Four plasmids, each carrying a different promoter driving the expression of an antibiotic resistance marker, were tested. The construct carrying the Ganoderma lucidum glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter showed good transformation efficiency, whereas constructs with the GPD promoter from ascomycetes were ineffective. Our analysis showed that over 70% of the transformants tested remained mitotically stable even after five successive rounds of subculturing. We were able to detect the expression of EGFP and GUS reporter genes in the Ganoderma lucidum transformants by fluorescence imaging and histochemical staining assays respectively. Our results demonstrate a new transgenic approach that will facilitate Ganoderma lucidum research.

Isolation and analysis of differentially expressed genes during asexual sporulation in liquid static culture of Ganoderma lucidum by suppression subtractive hybridization.

Ganoderma lucidum differentiates in liquid static culture by forming aerial mycelia and asexual spores, and this differentiation process is accompanied by higher production of anti-tumor compounds ganoderic acids. To gain an insight into the molecular events during asexual sporulation of G. lucidum, comparative transcriptome analysis using suppression subtractive hybridization (SSH) technique was performed to identify preferentially expressed genes in liquid static culture vs. in traditional shaking culture. After macroarray analysis of 1920 cDNAs from SSH library, 147 unigenes which exhibited high expression in static culture were identified. Among these sequences, putative translations of 88 unigenes possessed much similarity to known proteins involved in cell organization, signal transduction, cell metabolism, protein biosynthesis and transcription regulation; 13 had significant similarity to hypothetical proteins; the remaining 46 showed little or no similarity to GenBank sequences. RT-qPCR analysis confirmed increases in transcripts of selected genes under liquid static culture condition. The results of this study present the useful application of EST analysis on G. lucidum and provide preliminary indication of gene expression putatively involved in asexual sporulation process.

Neuroprotective effect of preadministration with Ganoderma lucidum spore on rat hippocampus.

The aim of this study was to investigate if preadministration with Ganoderma lucidum spore (GLS) could (1) alleviate oxidative stress and mitochondrial dysfunction in rat hippocampus of intracerebroventricular (ICV) injection of streptozotocin (STZ), (2) protect neurons from apoptosis, and (3) improve cognitive dysfunction. Three groups of Sprague-Dawley rats were preadministrated with GLS at doses of 2.0, 4.0 and 8.0 g/kg, respectively, for 3 weeks before the ICV STZ injury. Thereafter the rats were operated with ICV STZ (1.5 mg/kg) bilaterally on days 1 and 3. The behavioral alterations, oxidative stress indexes, ATP, cytochrome oxidase (CytOx), and histopathology of hippocampal neurons were studied. The results showed that ICV STZ model rats exhibited a significant increase of malondialdehyde (MDA), a significant decrease of glutathione reductase (GR), reduced glutathione (GSH), ATP and CytOx, accompanied with marked impairments in spatial learning and memory, and severe damage of hippocampal neuron. In conclusion, preadministration with GLS at dose of 8.0 g/kg in ICV STZ rats significantly reversed these abnormalities. In conclusion, preadministration with GLS might protect hippocampus from oxidative impairment and energy metabolism disturbance of ICV STZ. This may also provide useful information for future research on the pathogenesis and prevention of Alzheimer's disease (AD).

Efecto de nutrientes sobre la producción de biomasa del hongo medicinal Ganoderma lucidum / Effect of nutrients in the biomass production of the medicinal mushroom Ganoderma lucidum

El hongo Ganoderma lucidum, en los constituyentes de su biomasa, tiene compuestos con propiedades benéficas para la salud; es por esto que el conocimiento de las condiciones nutricionales adecuadas para su crecimiento permitirá su producción industrial y a bajo costo. En este trabajo se evaluó a nivel de matraz el efecto de la relación C/N, y la presencia de diferentes fuentes de carbono, nitrógeno y micronutrientes sobre la producción de biomasa. Empleando glucosa y peptona como fuentes de carbono y nitrógeno, respectivamente, se encontró una relación C/N óptima de 16,7:1 para la cual la máxima producción de biomasa fue de 25 g/L. Manteniendo esta relación C/N, y sustituyendo la glucosa por lactosa o harina de cebada y la peptona por extracto de levadura, la producción de biomasa se incrementó a 35 g/L. En presencia de harina de cebada la adición al medio de cultivo de sales de Mg y K, y de tiamina, no generó un mayor incremento en la producción de biomasa. La producción de biomasa de G. lucidum se ve favorecida por la presencia en el medio de cultivo de relaciones C/N cercanas a las reportadas conforme a la composición típica de los hongos, así como por la presencia de sustratos complejos como la harina de cebada que le aportan además de la fuente de carbono micronutrientes necesarios para su desarrollo.

Ganoderma lucidum fungus has some biomass components with beneficial health properties. The knowledge about its nutritionals requirements for growing will favor its industrial production at lower cost. In this work, the effect of C/N ratio, the presence of different carbon, nitrogen and micronutrients sources, on fungal biomass production, were evaluated. Using glucose and peptone as carbon and nitrogen sources, respectively, an optimal C/N ratio of 16,7:1 was found, for which the maximal biomass production was 25 g/L. Replacing glucose by lactose or barley flour and peptone by yeast extract at the same C/N ratio, the biomass production was enhanced to 35 g/L. With barley flour in the culture medium, the presence of Mg and K salts and thiamine did not turn out into a major increase of biomass. The G. lucidum biomass production is promoted by C/N ratios in the culture medium nearly equivalent to that found in the fungus, as well as the presence of complex substrates as barley flour which, additionally, contributes with important micronutrients along with the carbon source.

Transcriptome analysis of cadmium response in Ganoderma lucidum.

Ganoderma species are white-rot fungi widespread throughout the world. In this study, a wild isolate of Ganoderma lucidum was first collected and its tolerance was tested in a medium containing 3.0 mM CdCl(2). The cDNA-amplified fragment length polymorphism method was conducted to analyze the transcription profiling of this Ganoderma species in response to Cd treatment. In total, 12 925 transcript-derived fragments (TDFs) were amplified using 256 primer combinations. Forty-nine differentially expressed TDFs were confirmed by DNA dot-blot analysis. Northern blot analysis was used to verify the transcription levels of 34 Cd-inducible TDFs. Sequence analysis indicated that genes involved in reactive oxygen species generation, synthesis of sulfur-containing metabolites, translation machinery, DNA repair, transporting system, proteolysis pathway, mitochondria function, and cell wall biosynthesis were upregulated by Cd treatment. Our results provide a genome-wide transcriptome profiling of Cd response in Ganoderma species.

[Effects of ganoderma lucidum spores on mitochondrial calcium ion and cytochrome C in epididymal cells of type 2 diabetes rats].

OBJECTIVE: To observe the effects of ganoderma lucidum spores (GLS) on mitochondrial calcium ion and cytochrome C in the epididymal cells of type 2 diabetes rats. METHODS: Fifty adolescent rats were randomly divided into a model group (n=20), a GLS group (n=20) and a control group (n=10). The animals of the former two groups were injected with 2% STZ via vena caudalis for one time to induce type 2 diabetes. Then the model group was given high-fat-sugar diet, the GLS group high-fat-sugar diet + GLS (250 mg/kg x d), and the control group normal diet + CA-citrate sodium buffer. The bilateral epididymides were obtained 10 weeks later and the contents of mitochondrial calcium and cytochrome C detected. RESULTS: Type 2 diabetes models were successfully constructed. The content of mitochondrial calcium in the epididymal cells was significantly higher in the model group ([3.279 +/- 0.502] mg/L) than in the control group ([2.606 +/- 0.048] mg/L, P < 0.01), with no significant difference between the GLS group ([2.693 +/- 0. 196] mg/L) and the control (P > 0.05). In the model group, the content of mitochondrial cytochrome C ([3.213 +/- 1.511] micromol/L) was significantly lower (P < 0.05) while that of cytoplasm cytochrome C ([2.484 +/- 0.661] micromol/L) significantly higher (P < 0.05) than in the control ([5.688 +/- 1.679] micromol/L and [1.574 +/- 0.329] micromol/L, respectively). In the GLS group, the content of mitochondrial cytochrome C ([5.258 +/- 1.560] micromol/L) was higher, with no significant difference (P > 0.05), and that of cytoplasm cytochrome C ([1.727 +/- 0.396] micromol/L) significantly lower than in the model group (P < 0.05), but the difference between the GLS and the control group was not significant (P > 0.05). CONCLUSION: With disequilibrium of calcium homeostasis and damage to mitochondria, there might be excessive apoptosis in the epididymal cells of type 2 diabetes rats. Ganoderma lucidum spores could protect epididymal cells and counteract their apoptosis in diabetic condition.

Culture pH affects exopolysaccharide production in submerged mycelial culture of Ganoderma lucidum.

In submerged culture of Ganoderma lucidum, the pH optimum for cell growth has been shown to be lower than that for exopolysaccharides (EPS) formation. Therefore, in the present study, a two-stage pH-control strategy was employed to maximize the productions of mycelial biomass and EPS. When compared, a batch culture without pH control had a maximum concentration of EPS and endopolysaccharides, which was much lower than those with pH control. Maximum mycelial growth (12.5 g/L) and EPS production (4.7 g/L) were achieved by shifting the controlled pH from 3.0 to 6.0 after day 4. The contrast between the controlled-pH process and uncontrolled pH was marked. By using various two-stage culture processes, it was also observed that culture pH has a significant affect on the yield of product, mycelial morphology, chemical composition, and molecular weight of EPS. A detailed observation of mycelial morphology revealed that the productive morphological form for EPS production was a dispersed pellet (controlled pH shifting from 3.0 to 6.0) rather than a compact pellet with a dense core area (controlled pH 4.5) or a feather-like pellet (controlled pH shifting from 6.0 to 3.0). Three different polysaccharides were obtained from each pH conditions, and their molecular weights and chemical compositions were significantly different.

Cellular and physiological effects of Ganoderma lucidum (Reishi).

In Asia, a variety of dietary products have been used for centuries as popular remedies to prevent or treat different diseases. A large number of herbs and extracts from medicinal mushrooms are used for the treatment of diseases. Mushrooms such as Ganoderma lucidum (Reishi), Lentinus edodes (Shiitake), Grifola frondosa (Maitake), Hericium erinaceum (Yamabushitake), and Inonotus obliquus (Chaga) have been collected and consumed in China, Korea, and Japan for centuries. Until recently, these mushrooms were largely unknown in the West and were considered 'fungi' without any nutritional value. However, most mushrooms are rich in vitamins, fiber, and amino acids and low in fat, cholesterol, and calories. These mushrooms contain a large variety of biologically active polysaccharides with immunostimulatory properties, which contribute to their anticancer effects. Furthermore, other bioactive substances, including triterpenes, proteins, lipids, cerebrosides, and phenols, have been identified and characterized in medicinal mushrooms. This review summarizes the biological effects of Ganoderma lucidum upon specific signaling molecules and pathways, which are responsible for its therapeutic effects.

Links between morphology and physiology of Ganoderma lucidum in submerged culture for the production of exopolysaccharide.

Ganoderma lucidum was grown in submerged culture in shake flasks on a medium containing peptone, yeast extract and glucose. In pre-cultures, inoculated from an agar-grown culture, morphological and metabolic events were linked: the pellets originally produced protuberances when glucose was present in the medium, although glucose was not consumed. The protuberances were then liberated into the medium as second-generation pellets, at which time glucose consumption began and the rate of exopolysaccharide (EPS) production increased. The synchrony between events was repeated in cultures fed with either glucose or peptone and yeast extract. In main cultures, inoculated from a 16-day-old pre-culture, the biomass concentration increased linearly, while glucose consumption and EPS production were initially slow but then accelerated. Protuberances were produced and liberated similarly to the pre-culture, but there was less synchrony amongst the pellets. When glucose was added to such a culture on day 10, an EPS concentration of 5.7 g L(-1) was achieved on day 13, this being the highest reliable EPS concentration yet reported for submerged culture of G. lucidum. We conclude that a greater understanding of the morphological and physiological events during the culture of G. lucidum will allow the proposal of culture strategies to improve EPS production.

Antitumor activity of the sporoderm-broken germinating spores of Ganoderma lucidum.

The inhibitory effects of the dormant spores, the germinating spores, the sporoderm-broken germinating spores (SBGS), and the lipids extracted from the germinating spores of Ganoderma lucidum on the growth of mouse hepatoma, sarcoma S-180, and reticulocyte sarcoma L-II cells were investigated, respectively. The dormant spores could be activated by germination, and thus the bioactivities of the spores might be enhanced. The sporoderm-broken spores could show much higher bioactivities than the whole spores. Both the lipids extracted from the germinating spores and the SBGS of G. lucidum had remarkable antitumor effects in a dose-dependent manner, and could significantly inhibit three tumors with an inhibition of 80-90%.