The CBS/H2S signalling pathway regulated by the carbon repressor CreA promotes cellulose utilization in Ganoderma lucidum.

Cellulose is an important abundant renewable resource on Earth, and the microbial cellulose utilization mechanism has attracted extensive attention. Recently, some signalling molecules have been found to regulate cellulose utilization and the discovery of underlying signals has recently attracted extensive attention. In this paper, we found that the hydrogen sulfide (H2S) concentration under cellulose culture condition increased to approximately 2.3-fold compared with that under glucose culture condition in Ganoderma lucidum. Further evidence shown that cellulase activities of G. lucidum were improved by 18.2-27.6% through increasing H2S concentration. Then, we observed that the carbon repressor CreA inhibited H2S biosynthesis in G. lucidum by binding to the promoter of cbs, a key gene for H2S biosynthesis, at "CTGGGG". In our study, we reported for the first time that H2S increased the cellulose utilization in G. lucidum, and analyzed the mechanism of H2S biosynthesis induced by cellulose. This study not only enriches the understanding of the microbial cellulose utilization mechanism but also provides a reference for the analysis of the physiological function of H2S signals.

The transcription factor GCN4 contributes to maintaining intracellular amino acid contents under nitrogen-limiting conditions in the mushroom Ganoderma lucidum.

BACKGROUND: Edible mushrooms are delicious in flavour and rich in high-quality protein and amino acids required by humans. A transcription factor, general control nonderepressible 4 (GCN4), can regulate the expression of genes involved in amino acid metabolism in yeast and mammals. A previous study revealed that GCN4 plays a pivotal role in nitrogen utilization and growth in Ganoderma lucidum. However, its regulation is nearly unknown in mushrooms. RESULTS: In this study, we found that the amino acid contents reached 120.51 mg per gram of mycelia in the WT strain under 60 mM asparagine (Asn) conditions, but decreased by 62.96% under 3 mM Asn conditions. Second, silencing of gcn4 resulted in a 54.2% decrease in amino acid contents under 60 mM Asn, especially for the essential and monosodium glutamate-like flavour amino acids. However, these effects were more pronounced under 3 mM Asn. Third, silencing of gcn4 markedly inhibited the expression of amino acid biosynthesis and transport genes. In addition, GCN4 enhanced the tricarboxylic acid cycle (TCA) and glycolytic pathway and inhibited the activity of target of rapamycin complex 1 (TORC1), thus being beneficial for maintaining amino acid homeostasis. CONCLUSION: This study confirmed that GCN4 contributes to maintaining the amino acid contents in mushrooms under low concentrations of nitrogen. In conclusion, our study provides a research basis for GCN4 to regulate amino acid synthesis and improve the nutrient contents of edible mushrooms.

Function of a hydrophobin in growth and development, nitrogen regulation, and abiotic stress resistance of Ganoderma lucidum.

Fungal hydrophobins have many important physiological functions, such as maintaining hydrophobicity and affecting virulence, growth, and development. In Ganoderma lucidum, the molecular regulation mechanisms of hydrophobins in mushroom are unclear. In this study, we investigated a hydrophobin protein 1 (Hyd1) in G. lucidum, which belongs to the fungal Class I hydrophobins. The hyd1 gene was highly expressed during the formation of primordia, and expression was the lowest in fruiting bodies. Through the construction of hyd1 silenced strains, we found that primordia formation was not initiated in these strains. This finding indicated that Hyd1 played an important role in the development of G. lucidum. Second, AreA, a key transcription factor in nitrogen metabolism, negatively regulated the expression of hyd1. In an areA-silenced strain, the expression of hyd1 increased by ∼14-fold compared with that of the wild-type (WT) strain. Electrophoretic mobility shift assays (EMSA) indicated binding of AreA to the promoter of hyd1. Additionally, expression of hyd1 was determined in the presence of different nitrogen sources. Compared with that in the ammonia nitrogen source, the expression of hyd1 in nitrate nitrogen source significantly increased. Finally, we found that hyd1 plays important roles not only in nitrogen regulation but also in the resistance to other abiotic stresses. After silencing of hyd1, the resistance to heat, cell wall, and salt stresses decreased. Our findings reveal the important roles of Hyd1 in the development and resistance to abiotic stresses in G. lucidum and provide insights into the nitrogen regulation mechanism of hydrophobins in higher basidiomycetes.

A Gene from Ganoderma lucidum with Similarity to nmrA of Filamentous Ascomycetes Contributes to Regulating AreA.

Fungal AreA is a key nitrogen metabolism transcription factor in nitrogen metabolism repression (NMR). Studies have shown that there are different ways to regulate AreA activity in yeast and filamentous ascomycetes, but in Basidiomycota, how AreA is regulated is unknown. Here, a gene from Ganoderma lucidum with similarity to nmrA of filamentous ascomycetes was identified. The NmrA interacted with the C-terminal of AreA according to yeast two-hybrid assay. In order to determine the effect of NmrA on the AreA, 2 nmrA silenced strains of G. lucidum, with silencing efficiencies of 76% and 78%, were constructed using an RNA interference method. Silencing nmrA resulted in a decreased content of AreA. The content of AreA in nmrAi-3 and nmrAi-48 decreased by approximately 68% and 60%, respectively, compared with that in the WT in the ammonium condition. Under the nitrate culture condition, silencing nmrA resulted in a 40% decrease compared with the WT. Silencing nmrA also reduced the stability of the AreA protein. When the mycelia were treated with cycloheximide for 6 h, the AreA protein was almost undetectable in the nmrA silenced strains, while there was still approximately 80% of the AreA protein in the WT strains. In addition, under the nitrate culture, the content of AreA protein in the nuclei of the WT strains was significantly increased compared with that under the ammonium condition. However, when nmrA was silenced, the content of the AreA protein in the nuclei did not change compared with the WT. Compared with the WT, the expression of the glutamine synthetase gene in nmrAi-3 and nmrAi-48 strains increased by approximately 94% and 88%, respectively, under the ammonium condition, while the expression level of the nitrate reductase gene in nmrAi-3 and nmrAi-48 strains increased by approximately 100% and 93%, respectively, under the nitrate condition. Finally, silencing nmrA inhibited mycelial growth and increased ganoderic acid biosynthesis. Our findings are the first to reveal that a gene from G. lucidum with similarity to the nmrA of filamentous ascomycetes contributes to regulating AreA, which provides new insight into how AreA is regulated in Basidiomycota.

Effects of glutamate oxaloacetate transaminase on reactive oxygen species in Ganoderma lucidum.

Nitrogen metabolism can regulate mycelial growth and secondary metabolism in Ganoderma lucidum. As an important enzyme in intracellular amino acid metabolism, glutamate oxaloacetate transaminase (GOT) has many physiological functions in animals and plants, but its function in fungi has been less studied. In the present study, two GOT isoenzymes were found in G. lucidum; one is located in the mitochondria (GOT1), and the other is located in the cytoplasm (GOT2). The reactive oxygen species (ROS) level was increased in got1 silenced strains and was approximately 1.5-fold higher than that in the wild-type (WT) strain, while silencing got2 did not affect the ROS level. To explore how GOT affects ROS in G. lucidum, experiments related to the generation and elimination of intracellular ROS were conducted. First, compared with that in the WT strain, the glutamate content, one of the substrates of GOT, decreased when got1 or got2 was knocked down, and the glutathione (l-γ-glutamyl-l-cysteinylglycine) (GSH) content decreased by approximately 38.6%, 19.3%, and 40.1% in got1 silenced strains, got2 silenced strains, and got1/2 co-silenced strains respectively. Second, GOT also affects glucose metabolism. The pyruvate (PA), acetyl-CoA and α-ketoglutarate (α-KG) contents decreased in got1 and got2 silenced strains, and the transcription levels of most genes involved in the glycolytic pathway and the tricarboxylic acid cycle increased. The NADH content was increased in got1 silenced strains and got2 silenced strains, and the NAD+/NADH ratio was decreased, which might result in mitochondrial ROS production. Compared with the WT strain, the mitochondrial ROS level was approximately 1.5-fold higher in the got1 silenced strains. In addition, silencing of got1 or got2 resulted in a decrease in antioxidant enzymes, including superoxide dismutase, catalase, glutathione reductase, and ascorbate peroxidase. Finally, ganoderic acid (GA) was increased by approximately 40% in got1 silenced strains compared with the WT strain, while silencing of got2 resulted in a 10% increase in GA biosynthesis. These findings provide new insights into the effect of GOT on ROS and secondary metabolism in fungi. KEY POINTS: • GOT plays important roles in ROS level in Ganoderma lucidum. • Silencing of got1 resulted in decrease in GSH content and antioxidant enzymes activities, but an increase in mitochondrial ROS level in G. lucidum. • Silencing of got1 and got2 resulted in an increase in ganoderic acid biosynthesis in G. lucidum.

Citric Acid Induces the Increase in Lenthionine Content in Shiitake Mushroom, Lentinula edodes.

Shiitake mushroom, Lentinula edodes, is the second largest edible fungus in the world, with a characteristic aroma. 1,2,3,5,6-pentathioheterocycloheptane, commonly known as lenthionine, is the main source of this aroma. Lenthionine has high commercial value, and if we explore the possible induction mechanism of citric acid in lenthionine synthesis, we can provide a reference for the effective application of citric acid as an inducer. In this paper, the single-factor treatment of Lentinula edodes with variable citric acid concentration and treatment duration showed that the best citric acid concentration for L. edodes was 300 µM, and the best treatment duration was 15 days. Additionally, the optimal design conditions were obtained using the response surface method (RSM); the treatment concentration was 406 µM/L, the treatment duration was 15.6 days, and the lenthionine content was 130 µg/g. γ-Glutamyl transpeptidase (LEGGT) and cystine sulfoxide lyase (LECSL) are the key enzymes involved in the biosynthesis of lanthionine. The expression levels of LEGGT and LECSL genes increased significantly under citric acid treatment. Additionally, the lenthionine content of the silenced strains of LEGGT and LECSL was significantly decreased.