Mitochondrial pyruvate carrier influences ganoderic acid biosynthesis in Ganoderma lucidum.

Mitochondrial pyruvate carriers (MPCs), located in the inner membrane of mitochondria, are essential carriers for pyruvate to enter mitochondria. MPCs regulate a wide range of intracellular metabolic processes, such as glycolysis, the tricarboxylic acid cycle (TCA cycle), fatty acid metabolism, and amino acid metabolism. However, the metabolic regulation of MPCs in macrofungi is poorly studied. We studied the role of MPCs in Ganoderma lucidum (GlMPC) on ganoderic acid (GA) biosynthesis regulation in G. lucidum. In this study, we found that the mitochondrial/cytoplasmic ratio of pyruvate was downregulated about 75% in GlMPC1- and GlMPC2-silenced transformants compared with wild type (WT). In addition, the GA content was 17.72 mg/g and increased by approximately 50% in GlMPC1- and GlMPC2-silenced transformants compared with WT. By assaying the expression levels of three key enzymes and the enzyme activities of isocitrate dehydrogenase (IDH) and α-ketoglutarate dehydrogenase (α-KGDH) of the TCA cycle in GlMPC1- and GlMPC2-silenced transformants, it was found that the decrease in GlMPCs activity did not significantly downregulate the TCA cycle rate, and the enzyme activity of IDH increased by 44% compared with WT. We then verified that fatty acid ß-oxidation (FAO) supplements the TCA cycle by detecting the expression levels of key enzymes involved in FAO. The results showed that compared with WT, the GA content was 1.14 mg/g and reduced by approximately 40% in co-silenced transformants. KEY POINTS: • GlMPCs affects the distribution of pyruvate between mitochondria and the cytoplasm. • Acetyl-CoA produced by FAO maintains the TCA cycle. • Acetyl-CoA produced by FAO promotes the accumulation of GA.

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.

Exogenous Salicylic Acid (SA) Promotes the Accumulation of Biomass and Flavonoid Content in Phellinus igniarius (Agaricomycetes).

Phellinus igniarius is a very important, high-value medicinal fungus; flavonoids are one of its most important components. Methods to improve the content of flavonoids in Ph. igniarius deserve attention. Moreover, because of the low production of Ph. igniarius, it is also important to increase biomass. This study demonstrated that exogenous salicylic acid (SA) treatment can significantly promote the accumulation of total flavonoids and biomass in Ph. igniarius. After treatment with different concentrations of SA, the flavonoid content and biomass of Ph. igniarius increased by 20.54-93.15% and 31.90-43.92%, respectively, compared with the control, with flavonoid content and biomass reaching maximum values of 300 µM and 200 µM SA, respectively. Additionally, we found that SA induced the biosynthesis of flavonoids not only in a concentration-dependent manner but also in a time-dependent manner. The total flavonoid content reached a maximum at 24 h after SA treatment, which was 118.29% higher than the control. Furthermore, the biomass reached maximum values at 24 h and 36 h, which were 26.11% and 23.76% higher than the control, respectively. The total flavonoid content could be increased up to 15.02 µg rutin equivalents per mg mushroom biomass with the optimum conditions determined by response surface methodology. In this treatment, the biomass was also increased by 26.48%. This approach provides an efficient strategy for improving flavonoid accumulation in Ph. igniarius, with potential value in other applications.

Effects of Exogenous Salicylic Acid on Ganoderic Acid Biosynthesis and the Expression of Key Genes in the Ganoderic Acid Biosynthesis Pathway in the Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (Agaricomycetes).

We demonstrate herein that salicylic acid (SA) can enhance ganoderic acid (GA) accumulation in the lingzhi or reishi medicinal mushroom Ganoderma lucidum. Following treatment with different concentrations of SA, the GA content was increased 22.72% to 43.04% compared with the control group. When the fungi were treated with 200 µmol/L SA at different times, the GA content was improved 10.21% to 35.24% compared with the control group. By choosing the optimum point based on response surface methodology, the GA content could be increased up to 229.03 µg/100 mg, which was improved 66.38% compared with the control group. When the fungi were treated with 200 µmol/L SA, the transcription levels of key genes in the GA biosynthesis pathway-squalene (SQ) synthase (sqs), lanosterol (Lano; osc), and hydroxy-3-methylglutaryl-coenzyme A reductase (hmgr)-were improved 119.6-, 3.2-, and 4.2-fold, respectively. In addition, following treatment with 100 µmol/L SA, the levels of Lano and SQ, which are intermediate metabolites of GA biosynthesis, were increased 2.8- and 1.4-fold, respectively. These results indicate that SA can regulate the expression of genes related to GA biosynthesis and increases the metabolic levels of Lano and SQ, thereby resulting in the accumulation of GA.

Identification of Reference Genes and Analysis of Heat Shock Protein Gene Expression in Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum, after Exposure to Heat Stress.

Ganoderma lucidum has been considered an emerging model species for studying how environmental factors regulate the growth, development, and secondary metabolism of Basidiomycetes. Heat stress, which is one of the most important environmental abiotic stresses, seriously affects the growth, development, and yield of microorganisms. Understanding the response to heat stress has gradually become a hotspot in microorganism research. But suitable reference genes for expression analysis under heat stress have not been reported in G. lucidum. In this study, we systematically identified 11 candidate reference genes that were measured using reverse transcriptase quantitative polymerase chain reaction, and the gene expression stability was analyzed under heat stress conditions using geNorm and NormFinder. The results show that 5 reference genes-CYP and TIF, followed by UCE2, ACTIN, and UBQ1-are the most stable genes under our experimental conditions. Moreover, the relative expression levels of 3 heat stress response genes (hsp17.4, hsp70, and hsp90) were analyzed under heat stress conditions with different normalization strategies. The results show that use of a gene with unstable expression (SAND) as the reference gene leads to biased data and misinterpretations of the target gene expression level under heat stress.

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

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

Bioinformatic Identification of Potential MicroRNAs and Their Targets in the Lingzhi or Reishi Medicinal Mushroom Ganoderma lucidum (Higher Basidiomycetes).

MicroRNAs (miRNAs) are a class of small, endogenous, noncoding RNA molecules that negatively regulate gene expression at the transcriptional or the post-transcriptional level. Although a large number of miRNAs have been identified in many species, especially model plants and animals, miRNAs in fungi remain largely unknown. In this study, based on a database of expressed sequence tags in Ganoderma lucidum, 89 potential miRNAs were identified using computational methods. Real-time polymerase chain reaction analysis of miRNA-like samples prepared from G. lucidum at different development stages revealed that miRNA-like RNAs were differentially expressed in different stages. Furthermore, a total of 28 potential targets were found based on near-perfect or perfect complementarity between the randomly selected 9 miRNA-like RNAs and the target sequences, and potential targets for G. lucidum miRNA-like RNAs were predicted. Finally, we studied the expression pattern of 4 target genes in 3 different development stages of G. lucidum to further understand the mechanism of interaction between miRNA-like RNAs and their target genes. Our analysis paves the way toward identifying fungal miRNA-like RNAs that might be involved in various physiological and cellular differentiation processes.

Functional analysis of the role of glutathione peroxidase (GPx) in the ROS signaling pathway, hyphal branching and the regulation of ganoderic acid biosynthesis in Ganoderma lucidum.

Ganoderma lucidum, a hallmark of traditional Chinese medicine, has been widely used as a pharmacologically active compound. Although numerous research studies have focused on the pharmacological mechanism, fewer studies have explored the basic biological features of this species, restricting the further development and application of this important mushroom. Because of the ability of this mushroom to reduce and detoxify the compounds produced by various metabolic pathways, glutathione peroxidase (GPx) is one of the most important antioxidant enzymes with respect to ROS. Although studies in both animals and plants have suggested many important physiological functions of GPx, there are few systematic research studies concerning the role of this enzyme in fungi, particularly in large basidiomycetes. In the present study, we cloned the GPx gene and created GPx-silenced strains by the down-regulation of GPx gene expression using RNA interference. The results indicated an essential role for GPx in controlling the intracellular H2O2 content, hyphal branching, antioxidant stress tolerance, cytosolic Ca(2+) content and ganoderic acid biosynthesis. Further mechanistic investigation revealed that GPx is regulated by intracellular H2O2 levels and suggested that crosstalk occurs between GPx and intracellular H2O2. Moreover, evidence was obtained indicating that GPx regulation of hyphal branching via ROS might occur independently of the cytosolic Ca(2+) content. Further mechanistic investigation also revealed that the effects of GPx on ganoderic acid synthesis via ROS are regulated by the cytosolic Ca(2+) content. Taken together, these findings indicate that ROS have a complex influence on growth, development and secondary metabolism in fungi and that GPx serves an important function. The present study provides an excellent framework to identify GPx functions and highlights a role for this enzyme in ROS regulation.

The cloning, characterization, and functional analysis of a gene encoding an isopentenyl diphosphate isomerase involved in triterpene biosynthesis in the Lingzhi or reishi medicinal mushroom Ganoderma lucidum (higher Basidiomycetes).

An isopentenyl diphosphate isomerase (IDI) gene, GlIDI, was isolated from Ganoderma lucidum, which produces triterpenes through the mevalonate pathway. The open reading frame of GlIDI encodes a 252 amino acid polypeptide with a theoretical molecular mass of 28.71 kDa and a theoretical isoelectric point of 5.36. GlIDI is highly homologous to other fungal IDIs and contains conserved active residues and nudix motifs shared by the IDI protein family. The color complementation assay indicated that GlIDI can accelerate the accumulation of ß-carotene and confirmed that the cloned complementary DNA encoded a functional GlIDI protein. Gene expression analysis showed that the GlIDI transcription level was relatively low in the mycelia and reached a relatively high level in the mushroom primordia. In addition, its expression level could be up-regulated by 254 µM methyl jasmonate. Our results suggest that this enzyme may play an important role in triterpene biosynthesis.

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

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

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.