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.

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.

PRMT5 regulates the polysaccharide content by controlling the splicing of thaumatin-like protein in Ganoderma lucidum.

IMPORTANCE: PRMT5 contributes to secondary metabolite biosynthesis in Ganoderma lucidum. However, the mechanism through which PRMT5 regulates the biosynthesis of secondary metabolites remains unclear. In the current study, PRMT5 silencing led to a significant decrease in the biosynthesis of polysaccharides from G. lucidum through the action of the alternative splicing of TLP. A shorter TLP2 isoform can directly bind to PGI and regulated polysaccharide biosynthesis. These results suggest that PRMT5 enhances PGI activity by regulating TLP binding to PGI. The results of the current study reveal a novel target gene for PRMT5-mediated alternative splicing and provide a reference for the identification of PRMT5 regulatory target genes.

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.

Exciton Chirality Transfer Empowers Self-Triggered Spin-Polarized Amplified Spontaneous Emission from 1D-Anchoring-3D Perovskites.

Spin-polarized lasers, arising from stimulated emission of imbalanced spin populations, play a vital role in spin-optoelectronics. It is usually tackled by external spin injection, inevitably suffering from additional losses across the barriers from injection sources to gain materials. Herein, spin-polarized coherent light emission is self-triggered from the 1D-anchoring-3D perovskites, where the imbalanced populations in achiral 3D perovskites are endowed with the spin selectivity of exciton chirality (EC) underpinned by chiral 1D perovskites. Efficient transfer of EC is enabled by rapid energy transfer, thereby creating an imbalance of the spin population of excited states. Stimulated emission of such populations brings self-triggered spin-polarized amplified spontaneous emission in the composite perovskites, yielding a higher degree of polarization (DOP) than that based on optical spin injection into bare achiral 3D perovskites. Chemical diversity of composite perovskites not only enables to adjust band gap for broadband output of spin-polarized light signals but also promises to manipulate radiative decay and spin relaxation toward remarkably increased DOP. These results highlight the importance of EC transfer mechanism for spin-polarized lasing and represent a crucial step toward the development of chiral-spintronics.

Continuous-Wave Raman Lasing from Metal-Linked Organic Dimer Microcrystals.

Stimulated Raman scattering offers an alternative strategy to explore continuous-wave (c.w.) organic lasers, which, however, still suffers from the limitation of inadequate Raman gain in organic material systems. Here we propose a metal-linking approach to enhance the Raman gain of organic molecules. Self-assembled microcrystals of the metal linked organic dimers exhibit large Raman gain, therefore allowing for c.w. Raman lasing. Furthermore, broadband tunable Raman lasing is achieved in the organic dimer microcrystals by adjusting excitation wavelengths. This work advances the understanding of Raman gain in organic molecules, paving a way for the design of c.w. organic lasers.

The AMP-Activated Protein Kinase (AMPK) Positively Regulates Lysine Biosynthesis Induced by Citric Acid in Flammulina filiformis.

Flammulina filiformis, the most produced edible mushroom species in China, is rich in lysine. Further enhancing its lysine biosynthesis is vital for improving its quality in industrialized cultivation. Citric acid induction significantly increases both the biomass and growth rate of F. filiformis hyphae, as well as the lysine content. The genes encoding enzymes in the lysine biosynthesis pathway were detected under the optimal induction, revealing that the expression levels of hcs, hac, and hah were 2.67, 1.97, and 1.90 times greater, respectively, relative to the control, whereas no significant difference was seen for hdh, aat, sr, and shd, and the expression of aar decreased. Furthermore, the transcriptional levels of Ampk, GCN2, GCN4, and TOR were found significantly upregulated, with the most upregulated, Ampk, reaching a level 42.68 times greater than that of the control, while the phosphorylation of AMPK rose by nearly 54%. In AMPK-silencing strains under the optimal induction, however, the phosphorylation increment dropped to about 16% and the lysine content remained at the same level as in the WT. Thus, AMPK is presented as the critical intermediary in citric acid's regulation of lysine biosynthesis in F. filiformis.

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.

Research Progress on Edible Fungi Genetic System.

In order to obtain strains with targeted changes in genetic characteristics, molecular biology and genetic engineering techniques are used to integrate target gene fragments into the vector and transform them into recipient cells. Due to the different target genes and functional elements on the transformation plasmids, gene silencing, gene knockout, and gene overexpression can be carried out, which provides a new way to study the gene function of edible fungi. At present, the cloning vectors used in the transformation of edible fungi are modified by bacterial plasmids, among which pCAMBIA-1300 plasmid and pAN7 plasmid are the two most commonly used basic vectors. On this basis, some basic elements such as promoters, selective marker genes, and reporter genes were added to construct silencing vectors, knockout vectors, and overexpression vectors. At the same time, different expression vector systems are needed for different transformation methods. In this chapter, the main elements of the genetic system (promoters, screening markers), the current main genetic transformation methods (Agrobacterium-mediated transformation, liposome transformation, electroporation method), and the specific application of transformation were systematically summarized, which provides a reference for the study of the genetic system of edible fungi.

GCN4 Enhances the Transcriptional Regulation of AreA by Interacting with SKO1 To Mediate Nitrogen Utilization in Ganoderma lucidum.

Fungi utilize a wide range of nitrogen to adapt their metabolism. The transcription factor GCN4 has a pivotal role in nitrogen metabolism. However, the mechanism by which GCN4 regulates nitrogen utilization in Ganoderma lucidum is not well understood. In this study, we found that GCN4 physically interacts with SKO1, a bZIP (basic leucine zipper) transcription factor. GCN4 cooperated with SKO1 to positively regulate nitrogen utilization, especially for the expression of areA. Electrophoretic mobility shift assays (EMSA) indicate that GCN4 directly binds to the areA promoter region. Further affinity analysis through biolayer interferometry (BLI) experiments and surface plasmon resonance (SPR) confirmed that GCN4 specifically binds to the promoter region of areA with a strong binding affinity to activate the transcription of areA. In contrast, SKO1 showed no specified binding effect on the areA promoter. However, SKO1 activates the expression of the areA by forming a complex with GCN4, which exhibits a 14.2-fold-higher affinity than GCN4 alone. Furthermore, the presence of SKO1 promotes the stability of GCN4 protein. Accordingly, our study found that the transcription factor SKO1 enhances the transcriptional activity of GCN4 on its target gene areA by interacting with GCN4. Our study illustrates a specific regulatory mechanism for the involvement of GCN4 and SKO1 in nitrogen utilization, which provides innovative insight into the regulation of nitrogen utilization in fungi. IMPORTANCE Nitrogen is an essential nutrient for cell growth and proliferation. Limitations of nitrogen availability in organisms elicit a series of rapid transcriptional reprogramming mechanisms, which involve the participation of many transcription factors. However, the specific mechanism of coordination between different transcription factors regulating nitrogen metabolism has not been explored. Our study revealed that GCN4 interacts with SKO1 and that they are both involved in regulating nitrogen utilization by affecting the transcription level of areA. We also found that GCN4 activates transcription by directly binding to the promoter recognition region of areA. SKO1 facilitates the transcription of areA by GCN4 by forming a more stable complex with GCN4. Our study deepens our understanding of the regulatory network of nitrogen metabolism and demonstrates a further level of regulation for transcription factors.

Cross Talk between GlAQP and NOX Modulates the Effects of ROS Balance on Ganoderic Acid Biosynthesis of Ganoderma lucidum under Water Stress.

Water stress affects both the growth and development of filamentous fungi; however, the mechanisms underlying their response to water stress remain unclear. In this study, water stress was found to increase intracellular reactive oxygen species (ROS) level, ganoderic acid (GA) content, and NADPH oxidase (NOX) activity of Ganoderma lucidum by 148.45%, 75.32%, and 161.61%, respectively. Water stress induced the expression of the G. lucidum aquaporin (GlAQP) gene, which facilitated water transfer for microbial growth. Compared to wild type (WT), exposure to water stress increased growth inhibition rate, ROS level, and GA content of GlAQP-silenced strains by 37 to 41%, 36 to 38%, and 25%, respectively. Furthermore, at the early stage of fermentation in GlAQP-silenced strains, water stress resulted in 16 to 17% and 9 to 10% lower ROS level and GA content compared to WT, respectively. However, in GlAQP-overexpressing strains, ROS level and GA content were 22 to 24% and 12 to 13% higher than in WT, respectively. In GlAQP-silenced strains, water stress at the late stage resulted in 35 to 37% and 29 to 30% higher ROS level and GA content, respectively, while in GlAQP-overexpressing strains, levels were 16 to 17% and 9% lower than WT, respectively. Cross talk between GlAQP and NOX positively regulated the GA biosynthesis of G. lucidum via ROS under water stress at the early stage but this regulation became negative at the late stage. This study deepens the understanding of fungal signaling transduction under water stress and provides a reference for analyzing environmental factors that influence the regulation of the fungal secondary metabolism. IMPORTANCE Ganoderma lucidum is an advanced basidiomycete that produces medicinally active secondary metabolites (especially ganoderic acid [GA]) with high commercial value. Water stress imposes an important environmental challenge to G. lucidum. The mechanism of GA biosynthesis under water stress and the role of G. lucidum aquaporin (GlAQP) during its biosynthesis remain unclear. Moreover, the effect of the relationship between GlAQP and NADPH oxidase (NOX) on the level of reactive oxygen species and GA production under water stress is unknown. This study provides information on the biological response mechanism of G. lucidum to water stress. A new theory on the cell signaling cascade of G. lucidum tolerance to water stress is provided that also incorporates the biosynthesis of secondary metabolites involved in NOX and GlAQP.

Heme Oxygenase/Carbon Monoxide Participates in the Regulation of Ganoderma lucidum Heat-Stress Response, Ganoderic Acid Biosynthesis, and Cell-Wall Integrity.

Carbon monoxide (CO), a product of organic oxidation processes, arises in vivo principally from the enzymatic reaction of heme oxygenase (HO, transcription gene named HMX1). HO/CO has been found to exert many salutary effects in multiple biological processes, including the stress response. However, whether HO/CO is involved in the regulation of the heat-stress (HS) response of Ganoderma lucidum (G. lucidum) is still poorly understood. In this paper, we reported that under heat stress, the HMX1 transcription level, HO enzyme activity, and CO content increased by 5.2-fold, 6.5-fold and 2-fold, respectively. HMX1 silenced strains showed a 12% increase in ganoderic acid (GA) content under HS as analyzed by HPLC. Furthermore, according to Western blot analysis of the protein phosphorylation levels, HMX1 attenuated the increase in phosphorylation levels of slt2, but the phosphorylation levels were prolonged over a 3 h HS time period. The chitin and glucan content in HMX1 silenced strains increased by 108% and 75%, respectively. In summary, these findings showed that the HO/CO system responds to heat stress and then regulates the HS-induced GA biosynthesis and the cell-wall integrity mediated by the Slt-MAPK phosphorylation level in G. lucidum.

Phospholipase D and phosphatidic acid mediate regulation in the biosynthesis of spermidine and ganoderic acids by activating GlMyb in Ganoderma lucidum under heat stress.

Polyamines are essential for all kinds of organisms and take part in the regulation of multiple biological processes. Our previous study revealed that heat stress promoted the conversion of putrescine to spermidine and subsequently promoted the accumulation of ganoderic acids (GAs). However, how heat stress affects polyamine homeostasis remains unclear. To explore the underlying mechanism by which heat stress promoted spermidine biosynthesis, we assessed the effects of signalling molecules that respond to heat stress on spermidine biosynthesis. Our data suggested that heat stress-induced spermidine biosynthesis and GAs accumulation via a phospholipase D (PLD)-mediated phosphatidic acid (PA) signal. Further research revealed that the transcription factor GlMyb promoted spermidine biosynthesis via regulating spermidine synthase genes (spds1 and spds2) expression by directly bonding to their promoters and it responded to heat stress and PA signal. In summary, heat stress activated GlMyb by PLD-mediated PA signalling and subsequently induced the expression of spds1 and spds2 to promote the biosynthesis of spermidine and the accumulation of GAs. Our findings firstly reveal a detailed mechanism by which heat signalling regulates polyamine homeostasis by PLD-mediated PA signal in fungi and provide a greater understanding of how organisms alter polyamine levels in response to environmental changes.

Target of Rapamycin Mediated Ornithine Decarboxylase Antizyme Modulate Intracellular Putrescine and Ganoderic Acid Content in Ganoderma lucidum.

Putrescine (Put) has been shown to play an important regulatory role in cell growth in organisms. As the primary center regulating the homeostasis of polyamine (PA) content, ornithine decarboxylase antizyme (AZ) can regulate PA content through feedback. Nevertheless, the regulatory mechanism of Put is poorly understood in fungi. Here, our analysis showed that GlAZ had a modulate effect on intracellular Put content by interacting with ornithine decarboxylase (ODC) proteins and reducing its intracellular protein levels. In addition, GlAZ upregulated the metabolic pathway of ganoderic acid (GA) biosynthesis in Ganoderma lucidum by modulating the intracellular Put content. However, a target of rapamycin (TOR) was found to promote the accumulation of intracellular Put after the GlTOR inhibitor Rap was added exogenously, and unbiased analyses demonstrated that GlTOR may promote Put production through its inhibitory effect on the level of GlAZ protein in GlTOR-GlAZ-cosilenced strains. The effect of TOR on fungal secondary metabolism was further explored, and the content of GA in the GlTOR-silenced strain after the exogenous addition of the inhibitor Rap was significantly increased compared with that in the untreated wild-type (WT) strain. Silencing of TOR in the GlTOR-silenced strains caused an increase in GA content, which returned to the WT state after replenishing Put. Moreover, the content of GA in GlTOR-GlAZ-cosilenced strains was also not different from that in the WT strain. Consequently, these results strongly indicate that GlTOR affects G. lucidum GA biosynthesis via GlAZ. IMPORTANCE Research on antizyme (AZ) in fungi has focused on the mechanism by which AZ inhibits ornithine decarboxylase (ODC). Moreover, there are existing reports on the regulation of AZ protein translation by TOR. However, little is known about the mechanisms that influence AZ in fungal secondary metabolism. Here, both intracellular Put content and GA biosynthesis in G. lucidum were shown to be regulated through protein interactions between GlAZ and GlODC. Furthermore, exploration of upstream regulators of GlAZ suggested that GlAZ was regulated by the upstream protein GlTOR, which affected intracellular Put levels and ganoderic acid (GA) biosynthesis. The results of our work contribute to the understanding of the upstream regulation of Put and provide new insights into PA regulatory systems and secondary metabolism in fungi.

Chitosan Increases Lysine Content through Amino Acid Transporters in Flammulina filiformis.

Lysine content is considered an important indicator of the quality of Flammulina filiformis. In this study, chitosan was used to improve lysine content of F. filiformis. Optimal design conditions were obtained using central combination design (CCD): treatment concentration was 14.61 µg/mL, treatment time was 52.90 h, and the theoretical value of lysine content was 30.95 mg/g. We used Basic Local Alignment Search Tool Protein (BLASTP) to search the F. filiformis genome database using known AATs in the NCBI database. There were 11 members of AAT in F. filiformis. The expression levels of AAT3 and AAT4 genes increased significantly with chitosan treatment. Subsequently, AAT3 and AAT4 silencing strains were constructed using RNAi technology. The lysine content of the wild-type (WT) strain treated with chitosan increased by 26.41%. Compared with the chitosan-induced WT strain, chitosan-induced lysine content decreased by approximately 24.87% in the AAT3 silencing strain, and chitosan-induced lysine content in the AAT4 silencing strain increased by approximately 13.55%. The results indicate that AAT3 and AAT4 are involved in the regulation of the biosynthesis of lysine induced by chitosan in F. filiformis. AAT3 may participate in the absorption of lysine, and AAT4 may be involved in the excretion of lysine with chitosan treatment.

Establishment of CRISPR/Cas9 Genome-Editing System Based on Dual sgRNAs in Flammulina filiformis.

Flammulina filiformis, previously known as Asian Flammulina velutipes, is one of the most commercially important edible fungi, with nutritional value and medicinal properties worldwide. However, precision genome editing using CRISPR/Cas9, which is a revolutionary technology and provides a powerful tool for molecular breeding, has not been established in F. filiformis. Here, plasmids harboring expression cassettes of Basidiomycete codon-optimized Cas9 and dual sgRNAs targeting pyrG under the control of the gpd promoter and FfU6 promoter, respectively, were delivered into protoplasts of F. filiformis Dan3 strain through PEG-mediated transformation. The results showed that an efficient native U6 promoter of F. filiformis was identified, and ultimately several pyrG mutants exhibiting 5-fluorooric acid (5-FOA) resistance were obtained. Additionally, diagnostic PCR followed by Sanger sequencing revealed that fragment deletion between the two sgRNA target sites or small insertions and deletions (indels) were introduced in these pyrG mutants through the nonhomologous end joining (NHEJ) pathway, resulting in heritable changes in genomic information. Taken together, this is the first report in which a successful CRISPR/Cas9 genome-editing system based on dual sgRNAs was established in F. filiformis, which broadens the application of this advanced tool in Basidiomycetes.

Single-Crystalline Perovskite p-n Junction Nanowire Arrays for Ultrasensitive Photodetection.

Highly sensitive photodetectors play significant roles in modern optoelectronic integrated circuits. Constructing p-n junctions has been proven to be a particularly powerful approach to realizing sensitive photodetection due to their efficient carrier separation. Recently, p-n-junction photodetectors based on organic-inorganic hybrid perovskites, which combine favorable optoelectronic performance with facile processability, hold great potential in practical applications. So far, these devices have generally been made of polycrystalline films, which exhibit poor carrier-transport efficiency, impeding the further improvement of their photoresponsivities. Here, a type of ultrasensitive photodetector based on single-crystalline perovskite p-n-junction nanowire arrays is demonstrated. The single-crystalline perovskite p-n-junction nanowire arrays not only possess high crystallinity that enables efficient carrier transport but also form a built-in electric field facilitating effective carrier separation. As a result, the devices show excellent photosensitivity over a wide spectral range from 405 to 635 nm with an outstanding responsivity of 2.65 × 102  A W-1 at 532 nm. These results will provide new insights into the design and construction of high-performance photodetectors for practical optoelectronic applications.

Trehalose-6-phosphate synthase influences polysaccharide synthesis and cell wall components in Ganoderma lucidum.

Trehalose-6-phosphate synthase (TPS) is a key enzyme that participates in trehalose metabolism, which can synthesize trehalose in a two-step pathway with trehalose phosphatase, but its role in fungi is rarely studied, especially in large basidiomycetes. In this study, the tps gene of Ganoderma lucidum was cloned and named as gltps. And gltps-silenced strains were constructed by RNA interference. In this study, it is found that the extracellular polysaccharide content increased 1.6-2-fold, but there was no significant change on intracellular polysaccharide content in gltps-silenced strains compared with the wild-type (WT) strain. Furthermore, the cell wall compositions of the gltps-silenced strains were also altered, which showed that the chitin and ß-1,3-glucan contents were significantly decreased. Compared with WT, the concentration of chitin decreased by 20%-50% and that of ß-1, 3-glucan decreased by 15%-30%. The study found that the cells of gltps-silenced strains were more sensitive to cell wall stress, which might be due to changes in the compounds and structure of the cell wall. These results showed that gltps had an important effect on carbohydrate metabolism of G. lucidum cells.

NAD+-dependent Glsirt1 has a key role on secondary metabolism in Ganoderma lucidum.

Nicotinamide adenine dinucleotide (NAD+) is an important intracellular metabolite that is involved in different cel1lular processes. Glnmnat is the key enzyme that can affect intracellular NAD+ content. Here, we found that exogenous NAD+ treatment significantly increased ganoderic acid (GA) content in Ganoderma lucidum by 56.2%. Further experimental results showed that the acetylation level in Glnmnat-silenced strains significantly increased by about 35% and the transcript level of deacetylase Glsirt1 decreased by about 70%. Moreover, silencing Glnmnat led to a decrease in GA content, and this decrease could be rescued by the Glsirt1 activator. In addition, the acetylation of Glsirt1i-11 and Glsirt1i-21 was significantly increased by 28.8% and 41.0%. Furthermore, the decrease in GA content caused by silencing Glsirt1 could not be completely rescued by NAD+ treatment. Taken together, our study reveals that Glsirt1 is essential for the downstream regulation of GA biosynthesis by Glnmnat/NAD+, emphasizes the importance of acetylation modification in the mechanism of GA biosynthesis, and provides ideas for other fungi to study secondary metabolic regulatory networks in epigenetics.

Spermidine Regulates Mitochondrial Function by Enhancing eIF5A Hypusination and Contributes to Reactive Oxygen Species Production and Ganoderic Acid Biosynthesis in Ganoderma lucidum.

Spermidine, a kind of polycation and one important member of the polyamine family, is essential for survival in many kinds of organisms and participates in the regulation of cell growth and metabolism. To explore the mechanism by which spermidine regulates ganoderic acid (GA) biosynthesis in Ganoderma lucidum, the effects of spermidine on GA and reactive oxygen species (ROS) contents were examined. Our data suggested that spermidine promoted the production of mitochondrial ROS and positively regulated GA biosynthesis. Further research revealed that spermidine promoted the translation of mitochondrial complexes I and II and subsequently influenced their activity. With a reduction in eukaryotic translation initiation factor 5A (eIF5A) hypusination by over 50% in spermidine synthase gene (spds) knockdown strains, the activities of mitochondrial complexes I and II were reduced by nearly 60% and 80%, respectively, and the protein contents were reduced by over 50%, suggesting that the effect of spermidine on mitochondrial complexes I and II was mediated through its influence on eIF5A hypusination. Furthermore, after knocking down eIF5A, the deoxyhypusine synthase gene (dhs), and the deoxyhypusine hydroxylase gene (dohh), the mitochondrial ROS level was reduced by nearly 50%, and the GA content was reduced by over 40%, suggesting that eIF5A hypusination contributed to mitochondrial ROS production and GA biosynthesis. In summary, spermidine maintains mitochondrial ROS homeostasis by regulating the translation and subsequent activity of complexes I and II via eIF5A hypusination and promotes GA biosynthesis via mitochondrial ROS signaling. The present findings provide new insight into the spermidine-mediated biosynthesis of secondary metabolites. IMPORTANCE Spermidine is necessary for organism survival and is involved in the regulation of various biological processes. However, the specific mechanisms underlying the various physiological functions of spermidine are poorly understood, especially in microorganisms. In this study, we found that spermidine hypusinates eIF5A to promote the production of mitochondrial ROS and subsequently regulate secondary metabolism in microorganisms. Our study provides a better understanding of the mechanism by which spermidine regulates mitochondrial function and provides new insight into the spermidine-mediated biosynthesis of secondary metabolites.