Spin Effect to Regulate the Electronic Structure of Ir─Fe Aerogels for Efficient Acidic Water Oxidation.

"Spin" has been recently reported as an important degree of electronic freedom to promote catalysis, yet how it influences electronic structure remains unexplored. This work reports the spin-induced orbital hybridization in Ir─Fe bimetallic aerogels, where the electronic structure of Ir sites is effectively regulated by tuning the spin property of Fe atoms. The spin-optimized electronic structure boosts oxygen evolution reaction (OER) electrocatalysis in acidic media, resulting in a largely improved catalytic performance with an overpotential of as low as 236 mV at 10 mA cm-2. Furthermore, the gelation kinetics for the aerogel synthesis is improved by an order of magnitude based on the introduction of a magnetic field. Density functional theory calculation reveals that the increased magnetic moment of Fe (3d orbital) changes the d-band structure (i.e., the d-band center and bandwidth) of Ir (5d orbital) via orbital hybridization, resulting in optimized binding of reaction intermediates. This strategy builds the bridge between the electron spin theory with the d-band theory and provides a new way for the design of high-performance electrocatalysts by using spin-induced orbital interaction.

Two-dimensional phosphorus carbides (ß-PC) as highly efficient metal-free electrocatalysts for lithium-sulfur batteries: a first-principles study.

Li-S batteries are considered as the next-generation batteries due to their exceptional theoretical capacity. However, their practical application is hampered by the shuttling effects of lithium polysulfides (LiPSs) and the sluggish Li2S decomposition, particularly the slow conversion from Li2S2 to Li2S. Addressing these challenges, the quest for effective catalysts that can accelerate the conversion of LiPSs and enhance the performance of Li-S batteries is crucial. In this study, we explored the electrocatalytic activity of two-dimensional phosphorus carbides (ß0-PC and ß1-PC) in Li-S batteries based on first-principles calculations. Our findings reveal that these materials demonstrate optimal binding strengths (ranging from 1.09 to 1.83 eV) with long-chain LiPSs, effectively preventing them from dissolving into the electrolyte. Additionally, they show remarkable catalytic activity during the sulfur redox reaction (SRR), with ΔG being only 0.37 eV for ß0-PC and 0.13 eV for ß1-PC. The low energy barrier induced by ß-PC enhances ion migration barrier and significantly expedites the charge/discharge cycles of Li-S batteries. Furthermore, we investigated the conversion dynamics of Li2S2 to Li2S, employing the computational lithium electrode (CLE) model. The excellent performance in these aspects underscores the potential of these materials as electrocatalysts for Li-S batteries, paving the way for advanced high-efficiency energy storage solutions.

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.

A numerical model for water hydration on nanosurfaces: from friction to hydrophilicity and hydrophobicity.

Fluidic transport down to the nanometer scale is of great importance for a wide range of applications such as energy harvesting, seawater desalination, and water treatment and may help to understand many biological processes. In this work, we studied the interfacial friction of liquid water on a series of nanostructures through molecular dynamics (MD) simulations. Our results reveal that the friction coefficient of the water-solid interface cannot be described using a previously reported simple function of the free energy corrugation. Considering that the water-solid friction is firmly correlated with the microscopic water motion, we proposed a probability parameter P(d, t) to classify water motion modes on a surface. We demonstrate that this parameter can be used to accurately predict the water-solid friction by simply monitoring the water binding time on a nanosurface. More importantly, according to the relationship between P(d, t) and friction, we found that the friction coefficient can be used as an indicative criterion for quantitatively assessing hydrophobic or hydrophilic materials, where the borderline is roughly 2 × 105 N s m-3. That is if the water-solid friction is less than 2 × 105 N s m-3, the surface is considered hydrophobic. But if the friction is larger than this value, the surface is hydrophilic. The present findings could help to better understand fluidic transport at the nanoscale and guide the future design of functional materials, such as super-hydrophobic and super-hydrophilic surfaces by structure engineering.

Plantaricin A reverses resistance to ciprofloxacin of multidrug-resistant Staphylococcus aureus by inhibiting efflux pumps.

Overexpression of Staphylococcus aureus efflux pumps is commonly associated with antibiotic resistance, causing conventional antibiotics to be unsuccessful in combating multidrug-resistant bacterial infections. Reducing the activity of the efflux pump is an urgently required to tackle this problem. Here, we found that plantaricin A (PlnA), an antimicrobial peptide derived from Lactobacillus plantarum, had a synergistic effect with ciprofloxacin (CIP), reducing the IC90 of CIP by eight times. Subsequently, changes in membrane permeability, membrane potential, and reactive oxygen species (ROS) were determined; changes that did not explain the synergistic effect were previously observed. Ethidium bromide intake and efflux experiments showed that PlnA inhibited the function of the efflux pump by binding it and altering the structure of MepA, NorA, and LmrS. Then, a series of PlnA mutants were designed to explore the underlying mechanism; they showed that the charge and foaming of PlnA were the predominant factors affecting the structure of NorA. In a skin wound infection model, PlnA significantly reduced the dose of CIP, relieved inflammation, and promoted wound healing, indicating that PlnA and CIP synergy persisted in vivo. Overall, PlnA reduced the use of CIP for combination therapy, and allowing the continued used of CIP to kill MDR S. aureus. Multidrug-resistant Staphylococcus aureus threatens our life as a tenacious pathogen, which causes infections in hospitals, communities and animal husbandry. Various studies have showed that efflux pump inhibitors (EPIs) have been considered potential therapeutic agents for rejuvenating the activity of antibiotics. Unfortunately, small molecule EPIs exhibit several side effects that limit their use for clinical application. The present study showed a new EPI (plantaricin A) produced by Lactobacillus plantarum, which has low cytotoxicity and haemolysis and powerful inhibitory activity on efflux pumps. Therefore, it helps the design of new EPIs and controls the infection of MDR S. aureus.

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.

Plasmonic Nanozymes: Leveraging Localized Surface Plasmon Resonance to Boost the Enzyme-Mimicking Activity of Nanomaterials.

Nanozymes, a type of nanomaterials that function similarly to natural enzymes, receive extensive attention in biomedical fields. However, the widespread applications of nanozymes are greatly plagued by their unsatisfactory enzyme-mimicking activity. Localized surface plasmon resonance (LSPR), a nanoscale physical phenomenon described as the collective oscillation of surface free electrons in plasmonic nanoparticles under light irradiation, offers a robust universal paradigm to boost the catalytic performance of nanozymes. Plasmonic nanozymes (PNzymes) with elevated enzyme-mimicking activity by leveraging LSPR, emerge and provide unprecedented opportunities for biocatalysis. In this review, the physical mechanisms behind PNzymes are thoroughly revealed including near-field enhancement, hot carriers, and the photothermal effect. The rational design and applications of PNzymes in biosensing, cancer therapy, and bacterial infections elimination are systematically introduced. Current challenges and further perspectives of PNzymes are also summarized and discussed to stimulate their clinical translation. It is hoped that this review can attract more researchers to further advance the promising field of PNzymes and open up a new avenue for optimizing the enzyme-mimicking activity of nanozymes to create superior nanocatalysts for biomedical applications.

Fine Mapping and Functional Analysis of the Gene PcTYR, Involved in Control of Cap Color of Pleurotus cornucopiae.

Oyster mushrooms have a high biological efficiency and are easy to cultivate, which is why they are produced all over the world. Cap color is an important commercial trait for oyster mushrooms. Little is known about the genetic mechanism of the cap color trait in oyster mushrooms, which limits molecular breeding for the improvement of cap color-type cultivars. In this study, a 0.8-Mb major quantitative trait locus (QTL) region controlling cap color in the oyster mushroom Pleurotus cornucopiae was mapped on chromosome 7 through bulked-segregant analysis sequencing (BSA-seq) and extreme-phenotype genome-wide association studies (XP-GWAS). Candidate genes were further selected by comparative transcriptome analysis, and a tyrosinase gene (PcTYR) was identified as the highest-confidence candidate gene. Overexpression of PcTYR resulted in a significantly darker cap color, while the cap color of RNA interference (RNAi) strains for this gene was significantly lighter than that of the wild-type (WT) strains, suggesting that PcTYR plays an essential role in cap color formation. This is the first report about fine mapping and functional verification of a gene controlling cap color in oyster mushrooms. This will enhance our understanding of the genetic basis for cap color formation in oyster mushrooms and will facilitate molecular breeding for cap color. IMPORTANCE Oyster mushrooms are widely cultivated and consumed over the world for their easy cultivation and high biological efficiency (mushroom fresh weight/substrate dry weight × 100%). Fruiting bodies with dark caps are more and more popular according to consumer preferences, but dark varieties are rarely seen on the market. Little is known about the genetic mechanism of the cap color trait in oyster mushrooms, which limits molecular breeding for the improvement of cap color-type cultivars. A major QTL of cap color in oyster mushroom P. cornucopiae was fine mapped by using bulked-segregant analysis (BSA) and extreme-phenotype genome-wide association study (XP-GWAS) analysis. A candidate gene PcTYR coding tyrosinase was further identified with the help of comparative transcriptome analysis. qPCR analysis and genetic transformation tests proved that PcTYR played an essential role in cap color formation. This study will contribute to revealing the genetic mechanism of cap color formation in mushrooms, thereby facilitating molecular breeding for cap color trait.

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.

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.

Plantaricin A, Derived from Lactiplantibacillus plantarum, Reduces the Intrinsic Resistance of Gram-Negative Bacteria to Hydrophobic Antibiotics.

The outer membrane of Gram-negative bacteria is one of the major factors contributing to the development of antibiotic resistance, resulting in a lack of effectiveness of several hydrophobic antibiotics. Plantaricin A (PlnA) intensifies the potency of antibiotics by increasing the permeability of the bacterial outer membrane. Moreover, it has been proven to bind to the lipopolysaccharide of Escherichia coli via electrostatic and hydrophobic interactions and to interfere with the integrity of the bacterial outer membrane. Based on this mechanism, we designed a series of PlnA1 analogs by changing the structure, hydrophobicity, and charge to enhance their membrane-permeabilizing ability. Subsequent analyses revealed that among the PlnA1 analogs, OP4 demonstrated the highest penetrating ability, weaker cytotoxicity, and a higher therapeutic index. In addition, it decelerated the development of antibiotic resistance when the E. coli cells were continuously exposed to sublethal concentrations of erythromycin and ciprofloxacin for 30 generations. Further in vivo studies in mice with sepsis showed that OP4 heightens the potency of erythromycin against E. coli and relieves inflammation. In summary, our results showed that the PlnA1 analogs investigated in the present study, especially OP4, reduce the intrinsic antibiotic resistance of Gram-negative pathogens and expand the antibiotic sensitivity spectrum of hydrophobic antibiotics in Gram-negative bacteria. IMPORTANCE Antibiotic resistance is a global health concern due to indiscriminate use of antibiotics, resistance transfer, and intrinsic resistance of certain Gram-negative bacteria. The asymmetric bacterial outer membrane prevents the entry of hydrophobic antibiotics and renders them ineffective. Consequently, these antibiotics could be employed to treat infections caused by Gram-negative bacteria, after increasing their outer membrane permeability. As PlnA reportedly penetrates outer membranes, we designed a series of PlnA1 analogs and proved that OP4, one of these antimicrobial peptides, effectively augmented the permeability of the bacterial outer membrane. Furthermore, OP4 effectively improved the potency of erythromycin and alleviated inflammatory responses caused by Escherichia coli infection. Likewise, OP4 curtailed antibiotic resistance development in E. coli, thereby prolonging exposure to sublethal antibiotic concentrations. Thus, the combined use of hydrophobic antibiotics and OP4 could be used to treat infections caused by Gram-negative bacteria by decreasing their intrinsic antibiotic resistance.

Highly-anisotropic plasmons in two-dimensional hyperbolic copper borides.

Hyperbolic materials have wide application prospects, such as all-angle negative refraction, sub-diffraction imaging and nano-sensing, owning to the unusual electromagnetic response characteristics. Compared with artificial hyperbolic metamaterials, natural hyperbolic materials have many advantages. Anisotropic two-dimensional (2D) materials show great potential in the field of optoelectronics due to the intrinsic in-plane anisotropy. Here, the electronic and optical properties of two hyperbolic 2D materials, monolayer CuB6 and CuB3, are investigated using first-principles calculations. They are predicted to have multiple broadband hyperbolic windows with low loss and highly-anisotropic plasmon excitation from infrared to ultraviolet regions. Remarkably, plasmon propagation along the x-direction is almost forbidden in CuB3 monolayer. The hyperbolic windows and plasmonic properties of these 2D copper borides can be effectively regulated by electron (or hole) doping, which offers a promising strategy for tuning the optical properties of the materials.

Structural and energetic features of the dimerization of the main proteinase of SARS-CoV-2 using molecular dynamic simulations.

The COVID-19 pandemic caused by SARS-CoV-2 has been declared a global health crisis. The development of anti-SARS-CoV-2 drugs heavily depends on the systematic study of the critical biological processes of key proteins of coronavirus among which the main proteinase (Mpro) dimerization is a key step for virus maturation. Because inhibiting the Mpro dimerization can efficiently suppress virus maturation, the key residues that mediate dimerization can be treated as targets of drug and antibody developments. In this work, the structure and energy features of the Mpro dimer of SARS-CoV-2 and SARS-CoV were studied using molecular dynamics (MD) simulations. The free energy calculations using the Generalized Born (GB) model showed that the dimerization free energy of the SARS-CoV-2 Mpro dimer (-107.5 ± 10.89 kcal mol-1) is larger than that of the SARS-CoV Mpro dimer (-92.83 ± 9.81 kcal mol-1), indicating a more stable and possibly a quicker formation of the Mpro dimer of SARS-CoV-2. In addition, the energy decomposition of each residue revealed 11 key attractive residues. Furthermore, Thr285Ala weakens the steric hindrance between the two protomers of SARS-CoV-2 that can form more intimate interactions. It is interesting to find 11 repulsive residues which effectively inhibit the dimerization process. At the interface of the Mpro dimer, we detected three regions that are rich in interfacial water which stabilize the SARS-CoV-2 Mpro dimer by forming hydrogen bonds with two protomers. The key residues and rich water regions provide important targets for the future design of anti-SARS-CoV-2 drugs through inhibiting Mpro dimerization.

pH-switchable nanozyme cascade catalysis: a strategy for spatial-temporal modulation of pathological wound microenvironment to rescue stalled healing in diabetic ulcer.

The management of diabetic ulcer (DU) to rescue stalled wound healing remains a paramount clinical challenge due to the spatially and temporally coupled pathological wound microenvironment that features hyperglycemia, biofilm infection, hypoxia and excessive oxidative stress. Here we present a pH-switchable nanozyme cascade catalysis (PNCC) strategy for spatial-temporal modulation of pathological wound microenvironment to rescue stalled healing in DU. The PNCC is demonstrated by employing the nanozyme of clinically approved iron oxide nanoparticles coated with a shell of glucose oxidase (Fe3O4-GOx). The Fe3O4-GOx possesses intrinsic glucose oxidase (GOx), catalase (CAT) and peroxidase (POD)-like activities, and can catalyze pH-switchable glucose-initiated GOx/POD and GOx/CAT cascade reaction in acidic and neutral environment, respectively. Specifically, the GOx/POD cascade reaction generating consecutive fluxes of toxic hydroxyl radical spatially targets the acidic biofilm (pH ~ 5.5), and eradicates biofilm to shorten the inflammatory phase and initiate normal wound healing processes. Furthermore, the GOx/CAT cascade reaction producing consecutive fluxes of oxygen spatially targets the neutral wound tissue, and accelerates the proliferation and remodeling phases of wound healing by addressing the issues of hyperglycemia, hypoxia, and excessive oxidative stress. The shortened inflammatory phase temporally coupled with accelerated proliferation and remodeling phases significantly speed up the normal orchestrated wound-healing cascades. Remarkably, this Fe3O4-GOx-instructed spatial-temporal remodeling of DU microenvironment enables complete re-epithelialization of biofilm-infected wound in diabetic mice within 15 days while minimizing toxicity to normal tissues, exerting great transformation potential in clinical DU management. The proposed PNCC concept offers a new perspective for complex pathological microenvironment remodeling, and may provide a powerful modality for the treatment of microenvironment-associated diseases.

Cloning and characterization of phosphoglucose isomerase in Lentinula edodes.

Phosphoglucose isomerase (PGI) is a key enzyme that participates in polysaccharide synthesis, which is responsible for the interconversion of glucose-6-phosphate (G-6-P) and fructose-6-phosphate (F-6-P), but there is little research focusing on its role in fungi, especially in higher basidiomycetes. The pgi gene was cloned from Lentinula edodes and named lepgi. Then, the lepgi-silenced strains were constructed by RNA interference. In this study, we found that lepgi-silenced strains had significantly less biomass than the wild-type (WT) strain. Furthermore, the extracellular polysaccharide (EPS) and intracellular polysaccharide (IPS) levels increased 1.5- to 3-fold and 1.5-fold, respectively, in lepgi-silenced strains. Moreover, the cell wall integrity in the silenced strains was also altered, which might be due to changes in the compounds and structure of the cell wall. The results showed that compared to WT, silencing lepgi led to a significant decrease of approximately 40% in the ß-1,3-glucan content, and there was a significant increase of 2-3-fold in the chitin content. These findings provide support for studying the biological functions of lepgi in L. edodes.

Improvement of laccase activity by silencing PacC in Ganoderma lucidum.

Ganoderma lucidum is a representative white-rot fungus that has great potential to degrade lignocellulose biomass. Laccase is recognized as a class of the most important lignin-degrading enzymes in G. lucidum. However, the comprehensive regulatory mechanisms of laccase are still lacking. Based on the genome sequence of G. lucidum, 15 laccase genes were identified and their encoding proteins were analyzed in this study. All of the laccase proteins are predicted to be multicopper oxidases with conserved copper-binding domains. Most laccase proteins were secreted enzymes in addition to Lac14 in which the signal peptide could not be predicted. The activity of all laccases showed the highest level at pH 3.0 or pH 7.0, with total laccase activity of approximately 200 U/mg protein. Silencing PacC resulted in a 5.2 fold increase in laccase activity compared with WT. Five laccase genes (lac1, lac6, lac9, lac10 and lac14) showed an increased transcription levels (approximately 1.5-5.6 fold) in the PacC-silenced strains versus that in WT, while other laccase genes were downregulated or unchanged. The extracellular pH value was about 3.1, which was more acidic in the PacC-silenced strains than in the WT (pH 3.5). Moreover, maintaining the fermentation pH resulted in a downregulation of laccase activity which is induced by silencing PacC. Our findings indicate that in addition to its function in acidification of environmental pH, PacC plays an important role in regulating laccase activity in fungi.

Regulation of glutamine synthetase activity by transcriptional and posttranslational modifications negatively influences ganoderic acid biosynthesis in Ganoderma lucidum.

Glutamine synthetase (GS), a central nitrogen metabolic enzyme, plays important roles in the nitrogen regulation network and secondary metabolism in fungi. However, the mechanisms by which external nitrogen sources regulate fungal GS activity have not been determined. Here, we found that GS activity was inhibited under nitrate conditions in Ganoderma lucidum. By constructing gs-silenced strains and adding 1 mM GS inhibitor to inhibit GS activity, we found that a decrease in GS activity led to a decrease in ganoderic acid biosynthesis. The transcription of gs increased approximately five fold under nitrate conditions compared with that under ammonia. Electrophoretic mobility shift and yeast one-hybrid assay showed that gs was transcriptionally regulated by AreA. Although both gs expression and GS protein content increased under nitrate conditions, the GS activity still decreased. Treatment of recombinant GS with SIN-1 (protein nitration donor) resulted in a strengthened nitration accompanied by a 71% decrease in recombinant GS activity. Furthermore, intracellular GS could be nitrated from mycelia cultivated under nitrate conditions. These results indicated that GS activity could be inhibited by NO-mediated protein nitration. Our findings provide the first insight into the role of transcriptional and posttranslational regulation of GS activity in regulating secondary metabolism in fungi.

Nitric oxide regulates ganoderic acid biosynthesis by the S-nitrosylation of aconitase under heat stress in Ganoderma lucidum.

Nitric oxide (NO) is an important signalling molecule in stress response of organisms. We previously reported that NO decreases heat stress (HS)-induced ganoderic acid (GA) accumulation in Ganoderma lucidum. To explore the mechanisms by which NO modulates GA biosynthesis under HS, the effect of NO on the reactive oxygen species (ROS) content was examined. The results showed that NO decreased the production of mitochondrial ROS (mitROS) by 60% under HS. Further research revealed that NO reduced the mitROS content by inhibiting aconitase (Acon) activity. The GA content in Acon-silenced (Aconi) strains treated with NO donor did not differ significantly from that in untreated Aconi strains. To study the mechanism by which Acon activity is inhibited, the S-nitrosylation level of Acon was determined. Biotin-switch technology and mass spectrometry analysis were used to show that Acon is S-nitrosylated at the Cys-594 amino acid residue. Substitution of Cys-594 with a Ser, which cannot be S-nitrosylated, abolished the responsiveness of Acon to the NO-induced reduction in its enzymatic activity. These findings demonstrate that NO inhibits Acon activity through S-nitrosylation at Cys-594. In summary, these findings describe mechanism by which NO regulates GA biosynthesis via S-nitrosylation of Acon under HS condition in G. lucidum.

Swi6B, an alternative splicing isoform of Swi6, mediates the cell wall integrity of Ganoderma lucidum.

The cell wall integrity (CWI) signaling activates the transcription factor Swi6 through a MAPK signaling cascade in response to cell wall stresses. In this study, we observed two different mRNA variants of swi6 (GlSwi6A and GlSwi6B) existed, due to alternative splicing. Besides, the expression level of GlSwi6B was higher than that of the GlSwi6A mRNA variant. The co-silencing of GlSwi6A and GlSwi6B was more sensitive to cell wall stress compared with WT, resulting in a decrease of 78% and 76% in chitin and ß-1,3-d-glucan content respectively. However, only the overexpression of GlSwi6B decreased the sensitivity to cell wall stress and increased the content of chitin and ß-1,3-d-glucan compared with the WT strain. Furthermore, Y1H, EMSA and BLI assays revealed that the GlSwi6B could bind to the promoters of chitin and glucan synthesis genes (GL24454 and GL18134). However, the binding phenome has not been observed in the isoform GlSwi6A. Taken together, our results found two different transcripts generated from Swi6, in which the alternative splice isoform of GlSwi6B participates in regulating the CWI of G. lucidum. This study provides the first insight into the alternative splicing isoform of GlSwi6B in the regulation of CWI signaling in fungi.

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.