Valorization of Ginkgo biloba Leaf Powder as a Substrate in King Oyster Mushroom (Pleurotus eryngii) Cultivation.

Ginkgo biloba is widely planted as a colorful foliage tree, and its leaf can be used as a biomass energy source, but it has been underutilized for a long time. The aim of this study was to investigate the potential of garden waste as a substrate component in the cultivation process of the king oyster mushroom (Pleurotus eryngii), with the goal of enhancing both the yield of P. eryngii and the efficiency of energy use. The percentages of G. biloba leaf powder in the substrate were 10.5% and 21% to replace sawdust or sugarcane bagasse in a typical substrate. A substrate formulation that could completely replace sawdust and sugarcane bagasse was selected by analyzing mycelial growth rate, days of production, fruiting body length, biological efficiency, yield, stipe thickness, pileus diameter and laccase activity. The results showed that Y1 (treatment with 21% G. biloba leaf powder and sugarcane bagasse) had the highest yield (303.1 ± 31.9 g), which was higher than that of CK (control) (259.3 ± 37.4 g). The crude fiber content of the samples grown on substrate Y1 (as 7.43%) was higher than CK (7.37%). In addition, P. eryngii grown on substrate Y1 had the highest laccase activity for the complete colonization of the mycelium. Thus, these findings suggest that G. biloba leaf powder represents a viable and economical supplement for enhancing both the yield and quality of P. eryngii.

Label-free comparative proteomic analysis of Pleurotus eryngii grown on sawdust, bagasse, and peanut shell substrates.

The white rot fungi Pleurotus eryngii are environmental microorganisms that can effectively break down lignocellulosic biomass. However, understanding of the mechanisms by which P. eryngii is effective in degrading lignocellulose is still limited. This work aimed to examine the extracellular secretory proteins implicated in the breakdown of lignocellulose in P. eryngii and identify degradation tactics across various cultivation substrates. Thus, a comparative analysis of the secretory proteins based on Nanoliquid chromatography combined with tandem mass spectrometry was conducted among P. eryngii cultivated on sawdusts, bagasse, peanut shells, and glucose. In total, 647, 616, 604, and 511 proteins were identified from the four samples, respectively. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis of protein expression differences identified pathways (hydrolytic enzymes, catalytic activity, metabolic processes, cellular processes, and response to stimuli) significantly enriched in proteins associated with lignocellulose degradation in P. eryngii. An integrated analysis of proteome data revealed specifically or differentially expressed genes secreted by P. eryngii in different cultivation substrates. The most prevalent carbohydrate-active enzymes involved in lignocellulose degradation in the secretome of the four samples were laccase (Lac), manganese peroxidase (MnP), aryl alcohol oxidase (AaO), and copper radical oxidase (CRO). Among them, Lac 2 mainly involved in the lignin degradation of sawdust peanut shells, and bagasse by P. eryngii, and Mnp 3 was mainly involved in the degradation of peanut shells. AaO and Lac 4 were mainly involved in glucose substrate defense and oxidative stress. It was found that exogenous addition of sawdust and peanut shells significantly increased lignolytic enzyme abundance. These findings provide insight and guidance for improving agricultural waste resource recovery. In this study, the secretomes of P. eryngii grown on four different carbon sources were compared. The findings revealed the extracellular enzymes implicated in the degradation of lignocellulose, offering avenues for further investigation into the biotransformation mechanisms of P. eryngii biomass and the potential utilization of agricultural wastes. SIGNIFICANCE: The cost of the substrate for mushroom cultivation has increased as the production of edible fungus has risen year after year. Therefore, the use of these locally available lignocellulosic wastes as substrates offers a cost-cutting option. Further, the overuse of wood for the cultivation of edible mushrooms is also detrimental to the conservation of forest resources or the ecological environment. Consequently, the use of other agricultural wastes as an alternative to sawdust or other woody substrates is a viable approach for cultivating P. eryngii. The distribution of extracellular lignocellulosic degrading enzymes, inferred in the present study could help improve the cultivation efficiency of P. eryngii vis-à-vis managing agricultural waste.

Identification of altered metabolic functional components using metabolomics to analyze the different ages of fruiting bodies of Sanghuangporus vaninii cultivated on cut log substrates.

Sanghuangporus vaninii is a profitable traditional and medicinal edible fungus with uncommon therapeutic properties and medicinal value. The accumulation of active ingredients in this fungus that is used in traditional Chinese medicine is affected by its years of growth, and their pharmacological activities are also affected. However, the effects of age on the medicinal value of fruiting bodies of S. vaninii cultivated on cut log substrate remain unclear. In this study, an untargeted liquid chromatography mass spectrometry (LC-MS)-based metabolomics approach was performed to characterize the profiles of metabolites from 1-, 2- and 3-year-old fruiting bodies of S. vaninii. A total of, 156 differentially accumulated metabolites (DAMs) were screened based on the criterion of a variable importance projection greater than 1.0 and p < 0.01, including 75% up regulated and 25% down regulated. The results of enrichment of metabolic pathways showed that the metabolites involved the biosynthesis of plant secondary metabolites, biosynthesis of amino acids, central carbon metabolism in cancer, steroid hormone biosynthesis, linoleic acid metabolism, prolactin signaling pathway, and arginine biosynthesis, and so on. The biosynthesis of plant secondary metabolites pathway was significantly activated. Five metabolites were significantly elevated within the growth of fruiting bodies, including 15-keto-prostaglandin F2a, (4S, 5R)-4,5,6-trihydroxy-2-iminohexanoate, adenylsuccinic acid, piplartine, and chenodeoxycholic acid. 15-keto-prostaglandin F2a is related to the pathway of arachidonic acid metabolism and was significantly increased up to 1,320- and 535-fold in the 2- and 3-year-old fruiting bodies, respectively, compared with those in the 1-year-old group. The presence of these bioactive natural products in S. vaninii is consistent with the traditional use of Sanghuang, which prompted an exploration of its use as a source of natural prostaglandin in the form of foods and nutraceuticals. These findings may provide insight into the functional components of S. vaninii to develop therapeutic strategies.

Increasing the value of Phragmites australis straw in a sustainable integrated agriculture model (SIAM) comprising edible mushroom cultivation and spent mushroom substrate compost.

Because of the regular annual harvest, Phragmites australis used in wetland protection produces an abundance of straw, resulting in a large amount of straw waste. As a result, the extra straw should be used in a convenient and efficient manner. A comprehensive analysis of P. australis straw use in Pleurotus cultivation and spent mushroom substrate compost was performed in this study to increase its value. The lignocellulose content in the straw was shown to meet the nutritional requirements of the Pleurotus mushroom. Immediately thereafter, the replacement of bagasse with P. australis proved to be reasonable for Pleurotus eryngii substrate and could generate a profit of ¥17,400 per 10,000 cultivation bags. Substituting P. australis for approximately 20 % to 40 % of bagasse is recommended for Pleurotus ostreatus cultivation and can yield a profit of approximately ¥16,000. Spent mushroom substrate compost was confirmed to increase the organic matter content, and post-compost use of this substrate as a fertilizer could increase economic income by approximately ¥1000 for every 10,000 bags. Overall, this recycling pathway for P. australis resources presents positive ecological and social benefits, and the model is a sustainable and eco-friendly solution for agricultural waste worthy of promotion and further application.

Long-read assembly of major histocompatibility complex and killer cell immunoglobulin-like receptor genome regions in cynomolgus macaque.

BACKGROUND: The major histocompatibility complex (MHC) and the killer cell immunoglobulin-like receptors (KIR) are key regulators of immune responses. The cynomolgus macaque, an Old World monkey species, can be applied as an important preclinical model for studying human diseases, including coronavirus disease 2019 (COVID-19). Several MHC-KIR combinations have been associated with either a poor or good prognosis. Therefore, macaques with a well-characterized immunogenetic profile may improve drug evaluation and speed up vaccine development. At present, a complete overview of the MHC and KIR haplotype organizations in cynomolgus macaques is lacking, and characterization by conventional techniques is hampered by the extensive expansion of the macaque MHC-B region that complicates the discrimination between genes and alleles. METHODS: We assembled complete MHC and KIR genomic regions of cynomolgus macaque using third-generation long-read sequencing approach. We identified functional Mafa-B loci at the transcriptome level using locus-specific amplification in a cohort of 33 Vietnamese cynomolgus macaques. RESULTS: This is the first physical mapping of complete MHC and KIR gene regions in a Vietnamese cynomolgus macaque. Furthermore, we identified four functional Mafa-B loci (B2, B3, B5, and B6) and showed that alleles of the Mafa-I*01, -B*056, -B*034, and -B*001 functional lineages, respectively, are highly frequent in the Vietnamese cynomolgus macaque population. CONCLUSION: The insights into the MHC and KIR haplotype organizations and the level of diversity may refine the selection of animals with specific genetic markers for future medical research.

Transcriptomics Analysis of Primordium Formation in Pleurotus eryngii.

Primordium formation is an important stage preceding the growth and development of the Pleurotus eryngii fruiting body. However, the molecular mechanisms underlying primordium formation remain unclear. In the present study, comparative transcriptomics was performed between mature mycelia and primordium to analyze the transcriptional properties during primordium formation in P. eryngii. A total of 19,655 differentially expressed genes (10,718 upregulated genes and 8937 downregulated genes) were identified. These differentially expressed genes were involved in cell wall degradation, carbohydrate hydrolysis, light perception, and cAMP signal transduction. These results aid further understanding of the transcriptional changes and the molecular processes underlying primordium formation and differentiation, which may lay the foundation for improving the cultivation and quality control of P. eryngii.

Transcriptome Analysis Reveals Candidate Genes Involved in Light-Induced Primordium Differentiation in Pleurotus eryngii.

Pleurotus eryngii, a highly valued edible fungus, is one of the major commercially cultivated mushrooms in China. The development of P. eryngii, especially during the stage of primordium differentiation, is easily affected by light. However, the molecular mechanism underlying the response of primordium differentiation to light remains unknown. In the present study, primordium expression profiles under blue-light stimulation, red-light stimulation, and exposure to darkness were compared using high-throughput sequencing. A total of 16,321 differentially expressed genes (DEGs) were identified from three comparisons. GO enrichment analysis showed that a large number of DEGs were related to light stimulation and amino acid biosynthesis. KEGG analyses demonstrated that the MAPK signaling pathway, oxidative phosphorylation pathway, and RNA transport were most active during primordium differentiation. Furthermore, it was predicted that the blue-light photoreceptor WC-1 and Deoxyribodipyrimidine photolyase PHR play important roles in the primordium differentiation of P. eryngii. Taken together, the results of this study provide a speculative mechanism that light induces primordium differentiation and a foundation for further research on fruiting body development in P. eryngii.

Integration of Pleurotus tuoliensis cultivation and biogas production for utilization of lignocellulosic biomass as well as its benefit evaluation.

The present study was to assess the economic benefit of integrated P. tuoliensis cultivation and biogas production based on the utilization of lignocellulosic biomass. Among the five evaluated cultivation substrates, that consisting of 55% cottonseed hull, 25% corncob, 10% wheat bran, 5% corn flour, 4% lime, and 1% gypsum was demonstrated to be optimal for the simultaneous production of P. tuoliensis mushrooms and biogas fuel. Preliminary estimation shows that, for the consumption of dry substrate per unit mass (calculated in per kg), a total of 561 g fresh mushroom product was harvested and 189.88 L biogas was generated. Accordingly, the production costs were abolished and an economic benefit of approximately $0.592 was obtained, with the high-value mushroom product being the main contributor to profit. Moreover, this integrated process also exhibited positive ecological and social benefits and as such, is worthy of promotion and further application.

Comparative transcriptomic analysis reveals molecular processes involved in pileus morphogenesis in Pleurotus eryngii under different light conditions.

Light plays an important role in pileus differentiation in Pleurotus eryngii cultivation, and pileus morphology is influenced by light quality. To understand the effects of light quality on pileus morphology at the transcriptional level, we performed a comparative transcriptomic analysis of pilei grown under blue and red light irradiation. We identified 3959 differentially expressed genes (DEGs) between the blue and red light-treated pilei, which included 1664 up-regulated and 2295 down-regulated genes. These DEGs were significantly associated with light sensing, signal transduction, cell wall degradation and melanogenesis, suggesting that these processes are involved in pileus morphogenesis. Multiple DEGs related to respiratory functions were differentially expressed, suggesting that respiratory activity increased during pileus development regardless of light quality. These results provide a valuable view of the transcriptional changes and molecular processes involved in pileus morphogenesis under different light conditions and provide a foundation for yield improvement and quality control of P. eryngii.

A Comparative Transcriptome Analysis Reveals Physiological Maturation Properties of Mycelia in Pleurotus tuoliensis.

Pleurotus tuoliensis is a precious edible fungus with extremely high nutritive and medicinal value. The cultivation period of P. tuoliensis is longer than those of other Pleurotus species, which is mainly due to a longer mycelium physiological maturation period (30-60 days). Currently, the molecular processes underlying physiological maturation of the mycelium remain unclear. We performed a comparative transcriptomic analysis of immature and mature mycelia using RNA-seq. De novo transcriptome assembly resulted in identification of 17,030 unigenes. 451 differentially expressed genes-including those encoding nucleoside diphosphate kinase (NDPK), glycoside hydrolase family proteins, exopolygalacturonase, and versatile peroxidases-were identified. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that nucleotide synthesis and energy metabolism are highly active during the physiological maturation of mycelia, and genes related to these pathways were significantly upregulated in mature mycelia. NDPK is predicted to be essential for mycelia maturation. Our findings contribute to a comprehensive understanding of mycelia maturation in a commercially important fungal species. Future efforts will focus on the function of NDPK and the mechanism by which it regulates mycelia maturation.

The structural characterization of a polysaccharide exhibiting antitumor effect from Pholiota adiposa mycelia.

PAP80-2a, purified from Pholiota adiposa mycelia, is a polysaccharide exhibiting prominent antitumor effects. However, the yield of PAP80-2a was low and its structure has not been characterized, impeding the exploration of its structure-function relationship, thus influencing the development of oral drugs for antitumor therapy and immunomodulation. In order to improve the yield of PAP80-2a, response surface methodology along with Box-Behnken design was applied to optimize the ultrasonic-assisted extraction conditions for polysaccharides. Then, the structure of PAP80-2a exhibiting antitumor activity was determined from different angles. The results showed that the extraction yield of P. adiposa polysaccharides increased by 11.5% under optimized ultrasonic extraction conditions. Structural analysis showed that PAP80-2a was mainly composed of glucose, rhamnose, xylose, and galactose in a ratio of 10.00: 2.09: 4.09: 1.13. The total amino acid content in the sugar chain was 69.92 µg/mL. The sugar chain structure was [α-Rha (1 → 3)-]n, and rhamnose was located at the non-reducing end of the sugar chain, while glucose was located at the non-reducing end or in the sugar chain in 1,2,6- and 1,3,6-linked forms. Our study clearly illuminates the primary structure of PAP80-2a, but 3D structure of PAP80-2a and its structure-function relationship is a future challenge.

Methane production and characteristics of the microbial community in the co-digestion of spent mushroom substrate with dairy manure.

Spent mushroom substrate (SMS) is a potential biomass material generated during mushroom cultivation. In this study, the methane yield and microbial community resulting from co-digestion of SMS and dairy manure (DM) at different mixing ratios (0:4, 1:1, 3:1, and 1:3), were evaluated. Co-digestion analysis showed that the methane yield from the mixtures was 6%-61% higher than the yield from SMS or DM alone, indicating a synergistic effect of co-digestion of SMS with DM. For the SMS of F.velutipes (SFv) and P.erygii var. tuoliensis (SPt), co-digestion of DM/SMS at a ratio of 1:1 was optimal, but for the SMS of P. eryngi (SPe), co-digestion of DM/SMS at a ratio of 3:1 was ideal. The pH at all co-digestion ratios was in the range of 6.8-8.0, indicating that adding DM could increase the systemic buffering capacity. Methanosaetaceae was shown to be the predominant methanogens present during the co-digestion of DM/SMS.

Metabolic Profiling of Pleurotus tuoliensis During Mycelium Physiological Maturation and Exploration on a Potential Indicator of Mycelial Maturation.

Pleurotus tuoliensis is a valuable and rare edible fungus with extremely high nutritional and medicinal value. However, the relative immaturity of P. tuoliensis cultivation technology leads to fluctuating yields and quality. The main difficulty in P. tuoliensis cultivation is estimate of mycelial maturity. There is currently no measurable indicator that clearly characterizes the physiological maturation of mycelia. The aim of this study was to identify potential indicators of physiological maturation for P. tuoliensis mycelia by using metabolomics approach. A metabolite profiling strategy involving gas chromatography-mass spectrometry (GC/MS) was used to analyze changes to extracellular metabolites in mycelia collected at mycelium physiological maturation period (MPMP) day 0, MPMP day 35 at 17°C and MPMP day 35 at 29°C. 72 differential metabolites (37.8% up-regulated and 62.2% down-regulated) were identified based on the selected criteria [variable important in projection (VIP) greater than 1.0 and p < 0.01]. Metabolic pathways enrichment analysis showed that these metabolites are involved in glycolysis, organic acid metabolism, amino acid metabolism, tricarboxylic acid cycle (TCA), sugar metabolism, nicotinate and nicotinamide metabolism, and oxidative phosphorylation. In addition, the pyrimidine synthesis pathway was significantly activated during mycelium physiological maturation of P. tuoliensis. The abundance of N-carbamoyl-L-aspartate (CA-asp), a component of this pathway, was significantly increased at MPMP day 35, which motivated us to explore its potential as an indicator for physiological maturation of mycelia. The content of CA-asp in mycelia changed in a consistent manner during physiological maturation. The feasibility of using CA-asp as an indicator for mycelial maturation requires further investigation.

A novel aspartic protease with HIV-1 reverse transcriptase inhibitory activity from fresh fruiting bodies of the wild mushroom Xylaria hypoxylon.

A novel aspartic protease with HIV-1 RT inhibitory activity was isolated and characterized from fruiting bodies of the wild mushroom Xylaria hypoxylon. The purification protocol comprised distilled water homogenization and extraction step, three ion exchange chromatographic steps (on DEAE-cellulose, Q-Sepharose, and CM-cellulose in succession), and final purification was by FPLC on Superdex 75. The protease was adsorbed on all the three ion exchangers. It was a monomeric protein with a molecular mass of 43 kDa as estimated by SDS-PAGE and FPLC. Its N-terminal amino acid sequence was HYTELLSQVV, which exhibited no sequence homology to other proteases reported. The activity of the protease was adversely affected by Pepstatin A, indicating that it is an aspartic protease. The protease activity was maximal or nearly so in the pH range 6-8 and in the temperature range 35-60°C. The purified enzyme exhibited HIV-1 RT inhibitory activity with an IC50 value of 8.3 µM, but was devoid of antifungal, ribonuclease, and hemagglutinating activities.

Isolation and purification of polysaccharides with anti-tumor activity from Pholiota adiposa (Batsch) P. Kumm. (higher Basidiomycetes).

Pholiota adiposa is a mushroom with excellent medicinal and nutritional properties. After culture in fermentation medium, Ph. Adiposa mycelia were filtered, lyophilized, and powdered. A crude polysaccharide (PAP) of Ph. Adiposa was prepared from the mycelial powder with hot water, centrifuged, and the resulting supernatant lyophilized. PAP was fractionated by 30%, 60%, and 80% ethanol precipitation steps to yield PAP30, PAP60, and PAP80. Subsequently PAP30-1 and PAP30-2, PAP60-1 and PAP60-2, and PAP80-1 and PAP80-2 were isolated from PAP30, PAP60, and PAP80, respectively, by ion-exchange chromatography on a DEAE-Sepharose column. Polysaccharide content increased from 43.8% in PAP to 50.54%~73.19% in PAP30-1~PAP80-2. The protein content was 4.92% at minimum in these polysaccharide products. In order to identify the chemical composition, the six polysaccharides (PAP30-1, PAP30-2, PAP60-1, PAP60-2, PAP80-1, and PAP80-2) were further purified by gel filtration on Sephacryl S-100-500. Finally, three water-soluble polysaccharides (PAP30-2a, PAP60-2b, and PAP80-2a) were obtained. HPLC analysis revealed that PAP30-2a, PAP60-2b, and PAP80-2a exhibited a molecular weight of 6.6 × 105 Da, 8.4 × 103 Da, and 3.5 × 103 Da, respectively. The glucose content in PAP80-2a, PAP60-2b, and PAP30-2a was 57.8%, 72.7%, and 68.9%, respectively. PAP30-2a, PAP60-2b, and PAP80-2a demonstrated significant differences in anti-tumor activity in mice. PAP80-2a is the optimal bioactive constituent with anti-tumor and T-lymphocyte proliferation stimulating effects.

Effect of inoculation with Penicillium expansum on the microbial community and maturity of compost.

Compost prepared from wheat straw and cattle/chicken mature was inoculated with the lignocellulolytic fungus, Penicillium expansum. Compared to uninoculated compost, the inoculated compost exhibited a 150% higher germination index, more than 1.2 g kg(-1)-dw of changes in NH(4)(+)-N concentrations, a ca. 12.0% higher humus content and a lignocellulose degradation that proceeded 57.5% faster. Culture-based determinations of microbial populations demonstrated that aerobic heterotrophic bacteria and fungi were about 1-2 orders of magnitude higher in inoculated than in uninoculated compost. The number of ammonifying, ammonium-oxidizing, nitrite-oxidizing, denitrifying bacteria and cellulose-decomposing bacteria was 6.1-9.0 log(10) CFU g(-1)-dw, 1.2-4.3 log(10) MPN g(-1)-dw, 3.5-6.8 log(10) MPN g(-1)-dw, 3.58-4.34 log(10) MPN g(-1)-dw, 1.4-3.8 log(10)MPN g(-1)-dw, and 4.2-8.8 log(10) CFU g(-1)-dw higher in the compost inoculated with P. expansum.