[Oscillation-static cycle cultivation enhances the synthesis of triterpenes and mycelium activity in Ganoderma lucidum].

Ganoderma lucidum is a precious fungus with both edible and medicinal values and has a long history of medical use. Triterpenes as the main active components endow G. lucidum with anti-tumor, antioxidant, and other pharmacological activities. The present study endeavors to establish a proficient liquid-state fermentation technology for the enhanced production of triterpenes. In view of the limitations inherent in conventional submerged fermentation and oscillation-static two-stage cultivation, this study established an oscillation-static cycle cultivation process and optimized the cultivation conditions by building an artificial neural network model based on genetic algorithms. The cultivation conditions for the high-yield production of triterpenes were optimized as follows: 2.8 days of oscillation, 7.3 days of static cultivation, 0.2 day of oscillation, and 0.3 day of static cultivation. Under these conditions, the content of triterpenes reached 20.82 mg/g. The yield of triterpenes reached 129.09 mg/L, showing a remarkable increase of 324.78% compared with that of the Z10J0 method. Moreover, the established method shortened the cultivation cycle by 10.6 days. The mycelia cultivated under this regimen exhibited commendable anti-tumor and antioxidant activities. This study not only presents an economical liquid-state fermentation approach but also streamlines the fermentation flow, reduces fermentation duration, and effectively ameliorates drawbacks associated with conventional cultivation methods. In addition, this study gives valuable insights into the scaled application of liquid-state fermentation in the high-yield production of triterpenes, which showcases broad prospects.

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

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

Roles of α-1,3-glucosyltransferase in growth and polysaccharides biosynthesis of Ganoderma lucidum.

Ganoderma lucidum polysaccharides are valuable natural compounds possessing significant biological activity, with glycosyltransferases playing a crucial role in their biosynthesis. Although the function of ß-1,3-glucosyltransferase in polysaccharides production is well understood, the role of α-1,3-glucosyltransferase in edible fungi remains unclear. In this study, over-expression of the α-1,3-glucosyltransferase gene in G. lucidum (glagt) was found to suppress the growth, with the maximum biomass and mycelial growth rate decreasing by 21.78 % and 79.61 %, respectively, a behavior distinct from ß-1,3-glucosyltransferase. The fungal pellet diameter decreased by 38 % and the cell-wall thickness by 32.44 %, whereas intracellular and extracellular polysaccharides production increased by 27.58 % and 66.08 %, respectively. In the transcription level, overexpressing the glagt gene i) downregulated the citrate synthase and isocitrate dehydrogenase gene in the TCA cycle, disrupting energy metabolism and fungal growth; ii) upregulated key enzymes involved in UDP-glucose synthesis and glycosyltransferases (gl24465, gl24971, and gl22535); and iii) universally increased the transcriptional level of glucosidases gl21451, gl30087, and gl24581 by 22 %-397 %, contributing to cell-wall thinning to facilitate polysaccharides export. Conversely, the glagt gene downregulation promoted G. lucidum growth and decreased polysaccharides production. The results elucidate the roles of GLAGT and are expected to inspire in-depth exploration of polysaccharides biosynthesis pathways.

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.

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.

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.

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.

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.

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

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

Effect of common foods as supplements for the mycelium growth of Ganoderma lucidum and Pleurotus ostreatus on solid substrates.

The transition from a linear to a circular economy is urgently needed to mitigate environmental impacts and loss of biodiversity. Among the many potential solutions, the development of entirely natural-based materials derived from waste is promising. One such material is mycelium-bound composites obtained from the growth of fungi onto solid lignocellulosic substrates, which find applications such as insulating foams, textiles, packaging, etc. During growth, the fungus degrades and digests the substrate to create a web-like stiff network called mycelium. The development of the mycelium is influenced by several factors, including the substrate composition. As food waste accounts for nearly 44% of total municipal solid waste, incorporating food in the substrate composition could be a means to increase the nutrients absorbed by the fungus. In this paper, we study the effects of the addition of food supplements on the growth of two fungal species, Ganoderma lucidum and Pleurotus ostreatus. The substrates, the food supplements, and the mycelia are characterized using Fourier-transform infrared spectroscopy, scanning electron microscopy, and optical microscopy. Our results show that addition of barley as a supplement significantly boosts the growth of G. lucidum and P. ostreatus. Using a common food as a nutritious enrichment for the development of mycelium is a simple and straightforward strategy to create waste-based mycelium-bound biocomposites for a large range of applications, on-site, therefore promoting a circular economy.

Mitochondrial pyruvate carrier regulates the lignocellulosic decomposition rate through metabolism in Ganoderma lucidum.

The activity of mitochondrial pyruvate carrier (MPC) can be modulated to regulate intracellular metabolism under different culture conditions. In Ganoderma lucidum, the role of MPC in regulating carbon sources remains unknown. By knocking down MPC genes (MPC1 and MPC2), this research found that the loss of MPC increased the growth rate of G. lucidum by ~30% in a medium with wood chips as a carbon source. Then cellulase and laccase activities were tested. Endoglucanase and laccase activity increased by ~50% and ~35%, respectively, in MPC knockdown mutants compared with that in the wild type strain. Finally, the expression levels of genes related to glycolysis were assayed, and the transcription levels of these enzymes were found to be increased by ~250% compared with the wild type strain. In conclusion, the regulation of intracellular metabolism by MPC provides a new way to improve the use of nondominant carbon sources such as lignocellulose.

Advanced mycelium materials as potential self-growing biomedical scaffolds.

Mycelia, the vegetative part of fungi, are emerging as the avant-garde generation of natural, sustainable, and biodegradable materials for a wide range of applications. They are constituted of a self-growing and interconnected fibrous network of elongated cells, and their chemical and physical properties can be adjusted depending on the conditions of growth and the substrate they are fed upon. So far, only extracts and derivatives from mycelia have been evaluated and tested for biomedical applications. In this study, the entire fibrous structures of mycelia of the edible fungi Pleurotus ostreatus and Ganoderma lucidum are presented as self-growing bio-composites that mimic the extracellular matrix of human body tissues, ideal as tissue engineering bio-scaffolds. To this purpose, the two mycelial strains are inactivated by autoclaving after growth, and their morphology, cell wall chemical composition, and hydrodynamical and mechanical features are studied. Finally, their biocompatibility and direct interaction with primary human dermal fibroblasts are investigated. The findings demonstrate the potentiality of mycelia as all-natural and low-cost bio-scaffolds, alternative to the tissue engineering systems currently in place.

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

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

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

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

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

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

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

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

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

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

Enhancement of exopolysaccharide production from Ganoderma lucidum using a novel submerged volatile co-culture system.

Based on the impact of volatile organic compounds (VOCs) on secondary metabolite pathways, a novel submerged volatile co-culture system was constructed, and the effects of thirteen fungal and bacterial VOCs were investigated on Ganoderma lucidum exopolysaccharides production. The results demonstrated at least a 2.2-fold increase in exopolysaccharide (EPS) specific production yield in 6 days submerged volatile co-culture of G. lucidum with Pleurotus ostreatus. Therefore, P. ostreatus was selected as a variable culture, and the effects of agitation speed, inoculum size, initial pH, and co-culture volume on EPSs production were investigated using a Taguchi L9 orthogonal array. Finally, the highest concentration of EPSs (3.35 ± 0.22 g L-1) was obtained under optimized conditions; initial pH 5.0, inoculum size 10%, 150 rpm, and 3:1 volume ratio of variable culture to main culture.

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

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

Comparative Studies on Bioactive Compounds, Ganoderic Acid Biosynthesis, and Antioxidant Activity of Pileus and Stipes of Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (Agaricomycetes) Fruiting Body at Different Growth Stages.

Total phenolics, flavonoids, and polysaccharides, and individual ganoderic acid (GA) contents, antioxidant capacity, and transcription levels of key enzyme genes involved in GA biosynthesis in pileus and stipes of Ganoderma lucidum fruiting body at different growth stages were investigated in this study. Results showed that the highest total phenolics and total flavonoids contents were determined in stipes at spore maturity stage, resulting in high antioxidant activity, while the highest total polysaccharide content was found in pileus at the same stage. The pileus contained more GA than the stipes, and higher contents of ganoderic acid A and D were found at fruiting body mature stage while that of ganoderic acid B, C2, and G were found at bud elongation stage. Results from quantitative real-time PCR indicated that higher gene transcription levels of hydroxyl methylglutaryl-CoA reductase (hmgr), farnesyl pyrophosphate synthase (fps), squalene synthase (sqs), and oxidosqualene cyclase (osc) were found in pileus at bud elongation stage. Our findings will be helpful for understanding the biosynthesis of bioactive components and determining the harvest time for the desired G. lucidum fruiting bodies.