Combining the Elicitor Up-Regulated Production of Unusual Linear Diterpene-Derived Variants for an In-Depth Assessment of the Application Value and Risk of the Medicinal and Edible Basidiomycete Schizophyllum commune.

To better assess the practical value and avoid potential risks of the traditionally medicinal and edible basidiomycete Schizophyllum commune, which may arise from undescribed metabolites, a combination of elicitors was introduced for the first time to discover products from cryptic and low-expressed gene clusters under laboratory cultivation. Treating S. commune NJFU21 with the combination of five elicitors led to the upregulated production of a class of unusual linear diterpene-derived variants, including eleven new ones (1-11), along with three known ones (12-14). The structures and stereochemistry were determined by 1D and 2D NMR, HRESIMS, ECD, OR and VCD calculations. Notably, the elongation terminus of all the diterpenes was decorated by an unusual butenedioic acid moiety. Compound 1 was a rare monocyclic diterpene, while 2-6 possessed a tetrahydrofuran moiety. The truncated metabolites 4, 5 and 13 belong to the trinorditerpenes. All the diterpenes displayed approximately 70% scavenging of hydroxyl radicals at 50 µM and null cytotoxic activity at 10 µM. In addition, compound 1 exhibited potent antifungal activity against the plant pathogenic fungi Colletotrichum camelliae, with MIC values of 8 µg/mL. Our findings indicated that this class of diterpenes could provide valuable protectants for cosmetic ingredients and the lead compounds for agricultural fungicide development.

A new Schizophyllum commune strain as a potential biocontrol agent against blueberry root rot.

In recent years, blueberry root rot has been caused mainly by Fusarium commune, and there is an urgent need for a green and efficient method to control this disease. To date, research on Schizophyllum commune has focused on antioxidant mechanisms, reactive dye degradation, etc., but the mechanism underlying the inhibition of pathogenic microorganisms is still unclear. Here, the control effects of S. commune on F. commune and blueberry root rot were studied using adversarial culture, tissue culture, and greenhouse pot experiments. The results showed that S. commune can dissolve insoluble phosphorus and secrete various extracellular hydrolases. The results of hyphal confrontation and fermentation broth antagonism experiments showed that S. commune had a significant inhibitory effect on F. commune, with inhibition rates of 70.30% and 22.86%, respectively. Microscopy results showed distortion of F. commune hyphae, indicating that S. commune is strongly parasitic. S. commune had a significant growth-promoting effect on blueberry tissue-cultured seedlings. After inoculation with S. commune, inoculation with the pathogenic fungus, or inoculation at a later time, the strain significantly reduced the root rot disease index in the potted blueberry seedlings, with relative control effects of 79.14% and 62.57%, respectively. In addition, S. commune G18 significantly increased the antioxidant enzyme contents in the aboveground and underground parts of potted blueberry seedlings. We can conclude that S. commune is a potential biocontrol agent that can be used to effectively control blueberry root rot caused by F. commune in the field.

The regulatory network of the White Collar complex during early mushroom development in Schizophyllum commune.

Blue light is an important signal for fungal development. In the mushroom-forming basidiomycete Schizophyllum commune, blue light is detected by the White Collar complex, which consists of WC-1 and WC-2. Most of our knowledge on this complex is derived from the ascomycete Neurospora crassa, where both WC-1 and WC-2 contain GATA zinc-finger transcription factor domains. In basidiomycetes, WC-1 is truncated and does not contain a transcription factor domain, but both WC-1 and WC-2 are still important for development. We show that dimerization of WC-1 and WC-2 happens independent of light in S. commune, but that induction by light is required for promoter binding by the White Collar complex. Furthermore, the White Collar complex is a promoter of transcription, but binding of the complex alone is not always sufficient to initiate transcription. For its function, the White Collar complex associates directly with the promoters of structural genes involved in mushroom development, like hydrophobins, but also promotes the expression of other transcription factors that play a role in mushroom development.

Anti-Inflammatory Sesquiterpenes from Fruiting Bodies of Schizophyllum commune.

Schizophyllum commune, a fleshy fungus, is an important medicinal and food-homologous mushroom in China. In this work, eight undescribed sesquiterpenes schizomycins A-H (1-8) and one new meroterpenoid schizomycin I (9) together with three known analogues (10-12) were isolated from fruiting bodies of S. commune. Their planar structures were established by extensive spectroscopic and mass spectrometric data. The absolute configurations of compounds 1, 2, and 4 were determined by single crystal X-ray diffraction, and compounds 3 and 5-9 were confirmed by electronic circular dichroism calculations. Anti-inflammatory activities of all isolated compounds were evaluated for their inhibitory effects on IL-6 and IL-1ß production in RAW 264.7 cells. Among them, compound 7 exhibited significant IL-6 inhibitory activity with an IC50 value of 3.6 µM. The results of molecular docking showed that compound 7 interacts with amino acid residues (Gly117, Lys118, Asp120, Thr166, and Try168) of the IL-6 receptor protein through hydrogen bonding.

Emulsion for stabilizing ß-carotene and curcumin prepared directly using a continuous phase of polysaccharide-rich Schizophyllum commune fermentation broth.

In this study, we examined the effect of Schizophyllum commune fermentation broth (SCFB) rich in polysaccharides (SCFP) on the stability and bioaccessibility of ß-carotene and curcumin. An SCFB-stabilized oil-in-water (o/w) emulsion (SCFBe) was prepared using SCFB as the continuous phase, and then evaluated for storage stability using an SCFP-based emulsion (SCFPe) as the control. The findings revealed that SCFBe is more stable at 60 °C than SCFPe, and stratification or droplet size varied at differing pH levels (3-9) and concentrations of Na+ (0.1-0.5 M) and Ca2+ (0.01-0.05 M). Since the absolute value of the zeta potential of SCFBe is much lower at 60 °C than that at 4 °C and 25 °C, a higher temperature (60 °C) may enhance the reactivity of polysaccharides and proteins in SCFB to improve the stability of SCFBe. Both the protective impact of SCFB on functional food molecules and their capacity to block lipid oxidation increased as polysaccharide content improved. The bioaccessibility of ß-carotene after in vitro simulated gastrointestinal digestion is 11.18 %-12.28 %, whereas that of curcumin is 31.64 %-33.00 %. By fermenting edible and medicinal fungi in liquid, we created a unique and environmentally friendly approach for getting food-grade emulsifiers without extraction.

Schizophyllan from Schizophyllum commune BRM 060008: Potential application as an inhibitor of lipase.

ß-D-glucan has significant implications in regulating lipid metabolism and preventing diseases associated with lipid accumulation. Schizophyllan (SPG) from Schizophyllum commune fungus is a commercially important ß-glucan with applications in the health food industry, pharmacy, and cosmetics. However, SPG was obtained by submerged culture of the wood-rotting and filamentous fungus S. commune BRM 060008, which may have been isolated from the Cerrado Biome of Brazil. In this study, to confirm that the polysaccharide produced by BRM 060008 strain fermentation was indeed (1→3)(1→6)-ß-D-glucan, it was purified and characterized using Fourier transform infrared spectroscopy, thermogravimetric analysis, high-performance size exclusion chromatography, nuclear magnetic resonance, and methylation analysis. The polysaccharide produced was identified as the ß-D-glucan expected with a high molecular weight (1.093 × 106 g/mol) and the thermogravimetric analysis indicated a maximum degradation temperature of ~324 °C and a 60 % residual weight, lower than commercial SPG. The molecular structure and thermal properties of the ß-D-glucan were similar to the commercial sample. Additionally, the in vitro pancreatic lipase inhibitory activity was evaluated, investigating anti-obesity and anti-lipidemic properties. The results showed unprecedented lipase inhibition activity to SPG prepared using the S. commune strain BRM 060008, making it promising for food and pharmaceutical applications.

Anaerobic production and biosynthesis mechanism of exopolysaccharides in Schizophyllum commune 20R-7-F01.

Exopolysaccharides (EPS) produced by microorganisms play a vital role in physiological and ecological processes. However, the mechanisms of EPS synthesis and release in anaerobic environments remain poorly understood. Here, we provide the first evidence of anaerobic EPS synthesis by the fungus Schizophyllum commune 20R-7-F01, isolated from coal-bearing sediments ~2.0 km below the seafloor. Under anaerobic conditions, the fungus exhibited significantly higher specific EPS production (1.57 times) than under aerobic conditions. Transcriptomic analysis revealed 2057 differentially expressed genes (DEGs) in the strain cultured anaerobically for 7 days compared to aerobically. Among these genes, 642 were significantly upregulated, while 1415 were significantly downregulated, mainly associated with carbon metabolism pathways. Genes involved in glycolysis and EPS synthesis, including hexokinase (HK), phosphoglucomutase (PGM), and (1 â†’ 3)-ß-glucan synthase (GLS), were significantly upregulated, while those related to the TCA cycle, respiratory chain, and pentose phosphate pathway were downregulated under anaerobic conditions. These findings highlight the oxygen-dependent regulation of EPS synthesis and suggest that EPS may serve as a key mechanism for fungal adaptation to anaerobic environments.

Unlocking the benefits of split gill mushroom: Chemical analysis and prebiotic properties of schizophyllan extract.

The edible split gill mushroom is considered both a nutritive and therapeutic superfood, as well as rich in schizophyllan and protein. Prebiotic properties and other biological effects distinguish the schizophyllan (ß-glucan). Thus, this research investigates the identity of the mushroom strain, the nutritional composition of this mushroom, and the schizophyllan extract for further analysis, including its prebiotic activity and so on. The experimental results revealed that this mushroom was identified as Schizophyllum commune, comprising more excellent carbohydrates, protein, crude fiber, lower fat, and no heavy metal detection. Moreover, this extract consisted of pharmaceutical hydrophobin (14.0-18.5 kDa), lectin protein (21-35 kDa), bioactive purpurin or red pigment, including the prebiotic ß-glucan stimulating the proliferation of probiotic bacteria isolated from yogurt. Therefore, both S. commune and the schizophyllan extract can be used as a prebiotic candidate, functional food, and nutraceutical product.

Production, purification, and characterization of p-diphenol oxidase (PDO) enzyme from lignolytic fungal isolate Schizophyllum commune MF-O5.

Fungi are producers of lignolytic extracellular enzymes which are used in industries like textile, detergents, biorefineries, and paper pulping. This study assessed for the production, purification, and characterization of novel p-diphenol oxidase (PDO; laccase) enzyme from lignolytic white-rot fungal isolate. Fungi samples collected from different areas of Pakistan were initially screened using guaiacol plate method. The maximum PDO producing fungal isolate was identified on the basis of ITS (internal transcribed spacer sequence of DNA of ribosomal RNA) sequencing. To get optimum enzyme yield, various growth and fermentation conditions were optimized. Later PDO was purified using ammonium sulfate precipitation, size exclusion, and anion exchange chromatography and characterized. It was observed that the maximum PDO producing fungal isolate was Schizophyllum commune (MF-O5). Characterization results showed that the purified PDO was a monomeric protein with a molecular mass of 68 kDa and showed stability at lower temperature (30 °C) for 1 h. The Km and Vmax values of the purified PDO recorded were 2.48 mM and 6.20 U/min. Thermal stability results showed that at 30 °C PDO had 119.17 kJ/K/mol Ea value and 33.64 min half-life. The PDO activity was stimulated by Cu2+ ion at 1.0 mM showing enhanced activity up to 111.04%. Strong inhibition effect was noted for Fe2+ ions at 1 mM showing 12.04% activity. The enzyme showed stability against 10 mM concentration oxidizing reducing agents like DMSO, EDTA, H2O2, NaOCl, and urea and retained more than 75% of relative activity. The characterization of purified PDO enzyme confirmed its tolerance against salt, metal ions, organic solvents, and surfactants indicating its ability to be used in the versatile commercial applications.

An endophytic fungus Schizophyllum commune isolated from Panax ginseng enhances hairy roots growth and ginsenoside biosynthesis.

Using endophytic fungal elicitors to increase the accumulation of valuable secondary metabolites in plant tissue culture is an effective biotechnology strategy. In this study, a collection of 56 strains of endophytic fungi were isolated from different organs of cultivated Panax ginseng, of which seven strains can be symbiotically co-cultured with the hairy roots of P. ginseng. Further experiments observed that strain 3R-2, identified as endophytic fungus Schizophyllum commune, can not only infect hairy roots but also promote the accumulation of specific ginsenosides. This was further verified because S. commune colonization significantly affected the overall metabolic profile of ginseng hairy roots. By comparing the effects of S. commune mycelia and its mycelia extract (EM) on ginsenoside production in P. ginseng hairy roots, the EM was confirmed to be a relatively better stimulus elicitor. Additionally, the introduction of EM elicitor can significantly enhance the expressions of key enzyme genes of pgHMGR, pgSS, pgSE, and pgSD involved in the biosynthetic pathway of ginsenosides, which was deemed the most relevant factor for promoting ginsenosides production during the elicitation period. In conclusion, this study is the first to show that the EM of endophytic fungus S. commune can be considered as an effective endophytic fungal elicitor for increasing the biosynthesis of ginsenosides in hairy root cultures of P. ginseng.

New Metabolites, Antifeedant, Insecticidal Activities, and Reciprocal Relationship Between Insect and Fungus from Endophyte Schizophyllum commune.

Five new bisabolane sesquiterpenes, a new polyketide, along with seven known compounds, were isolated from endophyte Schizophyllum commune associated with a famous medicinal and edible plant, Gastrodia elata. Most compounds 1-12, and extract indicated antifeedant activities against silkworm with feeding deterrence index (FDI) of 21-85 %, at concentrations of 20 µg/cm2 , 40 µg/cm2 , respectively. Compound 6 indicated obvious insecticidal activity with fatality rate of 60 %, at the concentration of 20 µg/cm2 . Five bisabolane sesquiterpenes, two ergosterols, and a glyceride showed insecticidal synergism by combining with abamectin. Interesting, ergosterol peroxide (13) distributed widely in mushrooms and fungi, was found to have feeding attractant activities on insects and antifungal activity against entomopathogen Beauveria bassiana. The reciprocal relationship should be occurred between S. commune and pests for the fungus produced ergosterol peroxide to attract the pests propagating spore, and its anti-entomopathogen activity was also benefit for the health of insects.

The Transcription Factor Roc1 Is a Key Regulator of Cellulose Degradation in the Wood-Decaying Mushroom Schizophyllum commune.

Wood-decaying fungi of the class Agaricomycetes (phylum Basidiomycota) are saprotrophs that break down lignocellulose and play an important role in nutrient recycling. They secrete a wide range of extracellular plant cell wall degrading enzymes that break down cellulose, hemicellulose, and lignin, the main building blocks of plant biomass. Although the production of these enzymes is regulated mainly at the transcriptional level, no activating regulators have been identified in any wood-decaying fungus in the class Agaricomycetes. We studied the regulation of cellulase expression in the wood-decaying fungus Schizophyllum commune. Comparative genomics and transcriptomics on two wild isolates revealed a Zn2Cys6-type transcription factor gene (roc1) that was highly upregulated during growth on cellulose, compared to glucose. It is only conserved in the class Agaricomycetes. A roc1 knockout strain showed an inability to grow on medium with cellulose as sole carbon source, and growth on cellobiose and xylan (other components of wood) was inhibited. Growth on non-wood-related carbon sources was not inhibited. Cellulase gene expression and enzyme activity were reduced in the Δroc1 strain. ChIP-Seq identified 1474 binding sites of the Roc1 transcription factor. Promoters of genes involved in lignocellulose degradation were enriched with these binding sites, especially those of LPMO (lytic polysaccharide monooxygenase) CAZymes, indicating that Roc1 directly regulates these genes. A conserved motif was identified as the binding site of Roc1, which was confirmed by a functional promoter analysis. Together, Roc1 is a key regulator of cellulose degradation and the first identified in wood-decaying fungi in the phylum Basidiomycota. IMPORTANCE Wood-degrading fungi in the phylum Basidiomycota play a crucial role in nutrient recycling by breaking down all components of wood. Fungi have evolved transcriptional networks that regulate expression of wood-degrading enzymes, allowing them to prioritize one nutrient source over another. However, to date all these transcription factors have been identified in the phylum Ascomycota, which is only distantly related to the phylum Basidiomycota. Here, we identified the transcription factor Roc1 as a key regulator of cellulose degradation in the mushroom-forming and wood-degrading fungus Schizophyllum commune. Roc1 is highly conserved in the phylum Basidiomycota. Using comparative genomics, transcriptomics, ChIP-Seq and promoter analysis we have identified direct targets of Roc1, as well as other aspects of the transcriptional response to cellulose.

A new metabolite, mannogeranylnerol, specifically produced at body temperature by Schizophyllum commune, a causative fungus of human mycosis.

Schizophyllum commune is a causative fungus of human mycosis. Its metabolites produced at 27 °C were compared with those produced at 37 °C, to obtain a candidate low-molecular-weight virulence factor related to the pathogenicity of this fungus. We found that S. commune specifically produces two acyclic terpene mannosides at 37 °C. They were identified as nerolidol ß-D-mannoside (1) and geranylnerol ß-D-mannoside (2) by NMR, MS, and CD analyses. Compound 2, a new compound named mannogeranylnerol, showed weak antibiotic activity that was slightly stronger than that of compound 1.

Effect of surfactants on the production of polysaccharides from Schizophyllum commune through submerged fermentation.

Schizophyllum commune (S. commune) polysaccharides are biomacromolecules with multiple biological activities and wide applications. In this study, polysaccharide production through submerged fermentation of S. commune using different surfactants was investigated. The addition of 1 g/L of polyoxyethylene sorbitan monooleate (Tween 80) at the beginning of the fermentation showed the best promotional effects on collective exopolysaccharide (EPS) production (which increased by 37.17%) while shortening the production cycle by 2 days. The monosaccharide composition of the EPS produced when the added Tween 80 was similar to that of the control; however, the molecular weight (Mw) was lower. Notably, the addition of Tween 80 significantly increased the ATP levels and the transcription levels of phosphoglucomutase and ß-glucan synthase genes in the polysaccharide synthesis pathway. The addition of Tween 80 reduced the pellet size of the mycelium compared to that of the control, but did not significantly change the microstructure of the mycelial cells. This study proposes an efficient strategy for the production of polysaccharides through submerged fermentation of S. commune, and elucidates the detailed mechanism of using Tween 80 as a fermentation stimulatory reagent.

Synthesis of branched ß-1,3-glucan oligosaccharide with narrow degree of polymerization by fungi co-cultivation.

The large molecular weight and poor water solubility of ß-1,3-glucan impede its potential applications. In this study, the ß-1,3-glucan producing fungi and Trichoderma harzianum capable of secreting endo-ß-1,3-glucanase were co-cultivated to produce branched ß-1,3-glucan oligosaccharides (bOßGs) by fermentation with Sclerotium rolfsii and Schizophyllum commune. The highest bOßG yields from S. rolfsii in flasks were 4.53 and 9.94 g/L in a 7 L fermenter. Structural analysis proved that bOßG from S. rolfsii had a narrow degree of polymerization of 5-12, whereas bOßG from S. commune had a degree of polymerization of 5-15. Antioxidant tests showed that both bOßGs had remarkable DPPH radical scavenging activity and hydroxyl radical scavenging activity, and the activity of bOßG from S. commune was better than that of bOßG from S. rolfsii. In addition, bOßGs could promote the secretion of NO by mouse macrophages and increase the production of TNF-α, IL-1ß, and IL-6 in RAW264.7.

A screening method for decoloration of xenobiotic dyes by fungi.

Wood degrading fungi are often screened for their ability to degrade xenobiotics such as dyes. Dye decoloration by these fungi on solid media could until now only be assessed qualitatively. We here describe a fast quantitative method to screen for dye decoloration on such media. Decoloration of crystal violet (CV), malachite green (MG), orange G (OG), rose bengal (RB) and remazol brilliant blue R (RBBR) by 124 isolates of the basidiomycete Schizophyllum commune was quantified with a flatbed scanner and the CIE-L*a*b* model. Colour and intensity changes were calculated with the Euclidean distance formula. More than 10 strains showed high MG decoloration. Isolates 136, 140 and 213 showed superior CV decoloration, while OG was most effectively decolorized by isolates 183, 216 and 227. Six strains showed high RB decoloration with isolate 216 being superior. The latter strain was also highly active on RBBR together with isolates 177 and 227. Together, dye decoloration was highly variable between the 124 isolates but strain 216 showed high activity on 3 out of 5 dyes. The fast screening method described in this paper enables identification of strains effectively decolorizing dyes.

Effect of oxygen concentrations and branched-chain amino acids on the growth and development of sub-seafloor fungus, Schizophyllum commune 20R-7-F01.

Fungi have been reported to be the dominant eukaryotic group in anoxic sub-seafloor sediments, but how fungi subsist in the anoxic sub-marine sedimental environment is rarely understood. Our previous study demonstrated that the fungus, Schizophyllum commune 20R-7-F01 isolated from a ~2 km sediment below the seafloor, can grow and produce primordia in the complete absence of oxygen with enhanced production of branched-chain amino acids (BCAAs), but the primordia cannot be developed into fruit bodies without oxygen. Here, we present the individual and synergistic effects of oxygen and BCAAs on the fruit-body development of this strain. It was found that the fungus required a minimum oxygen concentration of 0.5% pO2 to generate primordia and 1% pO2 to convert primordia into mature fruit body. However, if BCAAs (20 mM) were added to the medium, the primordium could be developed into fruit body at a lower oxygen concentration up to 0.5% pO2 where genes fst4 and c2h2 playing an important role in compensating oxygen deficiency. Moreover, under hypoxic conditions, the fungus showed an increase in mitochondrial number and initiation of auto-phagocytosis. These findings suggest that the fruit-body formation of S. commune may have multiple mechanisms, including energy and amino acid metabolism in response to oxygen concentrations.

Submerged cultivation, characterization and in vitro antitumor activity of polysaccharides from Schizophyllum radiatum.

Production of polysaccharides by white-rot-fungi in submerged cultivation has several advantages due to process control. This work deals with the submerged cultivation, extraction and antitumor activity of polysaccharides from a wild strain of Schizophyllum radiatum isolated from a tropical forest of Colombia. The mushroom was cultivated in laboratory conditions, and classified by classical and molecular taxonomy. Submerged cultivation was performed in a bioreactor of 5 L using a ligninolytic residue as substrate. The fermentation conditions were 30 ± 1 °C, pH 4.5, 300 rpm and 1.5 vvm of air for 4 days. The yields were 16.8 g/L (w/v) of biomass, and after extraction, 0.6 g/L of water-soluble exopolysaccharide (SEPS) and 2.01 % (w/w) of water-soluble intrapolysaccharide (SIPS) were obtained. In each extract total carbohydrate, glucans and protein contents were determined. Also, nuclear magnetic resonance (NMR), scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy, X-ray diffractometry (XRD), high performance liquid chromatography with refraction index detection (HPLC-RI), high performance gel permeation chromatography (HPGPC) and Nuclear Magnetic Resonance (NMR) analysis were performed. Results indicated that SEPS and SIPS are heteropolysaccharides with amorphous structure and high molecular weights. Antitumor and immunostimulant activity was evaluated in different cancer cell lines. The results suggest these polysaccharides have direct and indirect antitumor activity activating immune cells such as macrophages. These findings enhance our knowledge about new sources of fungal metabolites that serve as adjuvant, cheaper and less harmful alternatives to cancer treatment.

Function of sesquiterpenes from Schizophyllum commune in interspecific interactions.

Wood is a habitat for a variety of organisms, including saprophytic fungi and bacteria, playing an important role in wood decomposition. Wood inhabiting fungi release a diversity of volatiles used as signaling compounds to attract or repel other organisms. Here, we show that volatiles of Schizophyllum commune are active against wood-decay fungi and bacteria found in its mycosphere. We identified sesquiterpenes as the biologically active compounds, that inhibit fungal growth and modify bacterial motility. The low number of cultivable wood inhabiting bacteria prompted us to analyze the microbial community in the mycosphere of S. commune using a culture-independent approach. Most bacteria belong to Actinobacteria and Proteobacteria, including Pseudomonadaceae, Sphingomonadaceae, Erwiniaceae, Yersiniaceae and Mariprofundacea as the dominating families. In the fungal community, the phyla of ascomycetes and basidiomycetes were well represented. We propose that fungal volatiles might have an important function in the wood mycosphere and could meditate interactions between microorganisms across domains and within the fungal kingdom.

The anaerobic survival mechanism of Schizophyllum commune 20R-7-F01, isolated from deep sediment 2 km below the seafloor.

Fungi dominated the eukaryotic group in the anaerobic sedimentary environment below the ocean floor where they play an essential ecological role. However, the adaptive mechanism of fungi to these anaerobic environments is still unclear. Here, we reported the anaerobic adaptive mechanism of Schizophyllum commune 20R-7-F01, isolated from deep coal-bearing sediment down to ~2 km below the seafloor, through biochemical, metabolomic and transcriptome analyses. The fungus grows well, but the morphology changes obviously and the fruit body develops incompletely under complete hypoxia. Compared with aerobic conditions, the fungus has enhanced branched-chain amino acid biosynthesis and ethanol fermentation under anaerobic conditions, and genes related to these metabolisms have been significantly up-regulated. Additionally, the fungus shows novel strategies for synthesizing ethanol by utilizing both glycolysis and ethanol fermentation pathways. These findings suggest that the subseafloor fungi may adopt multiple mechanisms to cope with lack of oxygen.