High Throughput Identification of the Potential Antioxidant Peptides in Ophiocordyceps sinensis

Ophiocordyceps sinensis, an ascomycete caterpillar fungus, has been used as a Traditional Chinese Medicine owing to its bioactive properties. However, until now the bio-active peptides have not been identified in this fungus. Here, the raw RNA sequences of three crucial growth stages of the artificially cultivated O. sinensis and the wild-grown mature fruit-body were aligned to the genome of O. sinensis. Both homology-based prediction and de novo-based prediction methods were used to identify 8541 putative antioxidant peptides (pAOPs). The expression profiles of the cultivated mature fruiting body were similar to those found in the wild specimens. The differential expression of 1008 pAOPs matched genes had the highest difference between ST and MF, suggesting that the pAOPs were primarily induced and play important roles in the process of the fruit-body maturation. Gene ontology analysis showed that most of pAOPs matched genes were enriched in terms of ‘cell redox homeostasis’, ‘response to oxidative stresses’, ‘catalase activity’, and ‘ integral component of cell membrane’. A total of 1655 pAOPs was identified in our protein-seqs, and some crucial pAOPs were selected, including catalase, peroxiredoxin, and SOD [Cu–Zn]. Our findings offer the first identification of the active peptide ingredients in O. sinensis, facilitating the discovery of anti-infectious bio-activity and the understanding of the roles of AOPs in fungal pathogenicity and the high-altitude adaptation in this medicinal fungus.

New Insights Into the Biosynthesis of Typical Bioactive Components in the Traditional Chinese Medicinal Fungus Cordyceps militaris

Cordyceps militaris, a traditional medicinal ingredient with a long history of application in China, is regarded as a high-value fungus due to its production of various bioactive ingredients with a wide range of pharmacological effects in clinical treatment. Several typical bioactive ingredients, such as cordycepin, D-mannitol, cordyceps polysaccharides, and N6-(2-hydroxyethyl)-adenosine (HEA), have received increasing attention due to their antitumor, antioxidant, antidiabetic, radioprotective, antiviral and immunomodulatory activities. Here, we systematically sorted out the latest research progress on the chemical characteristics, biosynthetic gene clusters and pathways of these four typical bioactive ingredients. This summary will lay a foundation for obtaining low-cost and high-quality bioactive ingredients in large amounts using microbial cell factories in the future.

Characterization and Heterologous Expression of UDP-Glucose 4-Epimerase From a Hericium erinaceus Mutant with High Polysaccharide Production

Hericium erinaceus is an important medicinal fungus in traditional Chinese medicine because of its polysaccharides and other natural products. Compared terpenoids and polyketides, the analysis of synthetic pathway of polysaccharides is more difficult because of the many genes involved in central metabolism. In previous studies, A6180, encoding a putative UDP-glucose 4-epimerase (UGE) in an H. erinaceus mutant with high production of active polysaccharides, was significantly upregulated. Since there is no reliable genetic manipulation technology for H. erinaceus, we employed Escherichia coli and Saccharomyces cerevisiae to study the function and activity of A6180. The recombinant overexpression vector pET22b-A6180 was constructed for heterologous expression in E. coli. The enzymatic properties of the recombinant protein were investigated. It showed that the recombinant A6180 could strongly convert UDP-α-D-glucose into UDP-α-D-galactose under optimal conditions (pH 6.0, 30°C). In addition, when A6180 was introduced into S. cerevisiae BY4742, xylose was detected in the polysaccharide composition of the yeast transformant. This suggested that the protein coded by A6180 might be a multifunctional enzyme. The generated polysaccharides with a new composition of sugars showed enhanced macrophage activity in vitro. These results indicate that A6180 plays an important role in the structure and activity of polysaccharides. It is a promising strategy for producing polysaccharides with higher activity by introducing A6180 into polysaccharide-producing mushrooms.

Optimization of Solid-State Fermentation Extraction of Inonotus hispidus Fruiting Body Melanin

Melanin has good nutritional and medicinal value; however, its extraction rate is extremely low. This study explored the edible and medicinal fungus Inonotus hispidus fruiting body melanin (IHFM) extraction process and solid-state fermentation conditions. The results showed that the best way to extract IHFM is the compound enzymatic method, with complex enzyme 26.63 mg/g, liquid material ratio 5:1, enzymatic hydrolysis 80 min, pH 4.61, and enzymolysis temperature at 36.07 °C. The yield of IHFM was 23.73 ± 0.57%, which was equivalent to 1.27 times before optimization. The best solid medium formula was normal pH, rice 20 g per cultivation bottle, maltose 22 g/L, beef extract 4.4 g/L, carbon-nitrogen ratio 5:1, and liquid-to-material ratio 1.1:1, where the IHFM yield was 31.80 ± 1.34%, which was equivalent to 1.7 times that before optimization. In summary, solid-state fermentation and extraction optimization greatly improved the yield of melanin, provided a reference to produce melanin, and laid a foundation for the development and utilization of melanin.

Effect of Inonotus obliquus polysaccharide on composition of the intestinal flora in mice with acute endometritis

Inonotus obliquus Polysaccharide (IOP) is a large molecule extracted from Inonotus obliqus, a medicinal fungus, which has a wide range of biological activities and has been shown to be associated with inflammation. The purpose of this study is to investigate whether IOP can help to reduce acute endometritis by regulating intestinal flora. We observed pathological changes in mice with endometritis following treatment with IOP and evaluated changes in the levels of interleukin-6 (IL-6), interleukin-1β (IL-1β) and tumor necrosis factor α (TNF-α), and further studied the effects of IOP on the intestinal flora of endometritis mice using 16S rRNA high-throughput sequencing. The results showed that IOP improved the condition of uterine tissues and reduced the release of pro-inflammatory cytokines. Meanwhile, the 16S rRNA sequencing results showed that IOP could regulate the changes in intestinal microflora at the level of genera, possibly by changing the relative abundance of some genera.

Pan-genome Analyses of 3 Strains of Inonotus obliquus and Prediction of Polysaccharide and Terpenoid Genes

Inonotus obliquus is a rare, edible and medicinal fungus that is widely used as a remedy for various diseases. Its main bioactive substances are polysaccharides and terpenoids. In this study, we characterized and investigated the pan-genome of three strains of I. obliquus. The genome sizes of JL01, HE, and NBRC8681 were 32.04, 29.04, and 31.78 Mb, respectively. There were 6 543 core gene families and 6 197 accessory gene families among the three strains, with 14 polysaccharide-related core gene families and seven accessory gene families. For terpenoids, there were 13 core gene families and 17 accessory gene families. Pan-genome sequencing of I. obliquus has improved our understanding of biological characteristics related to the biosynthesis of polysaccharides and terpenoids at the molecular level, which in turn will enable us to increase the production of polysaccharides and terpenoids by this mushroom.

Addition of Vegetable Oil to Improve Triterpenoids Production in Liquid Fermentation of Medicinal Fungus Antrodia cinnamomea

The liquid fermentation of Antrodia cinnamomea is a promising alternative source for fungus production compared to the wildly grown fruiting body. Elicitation is a strong tool to enhance the productivity in microbial cells to obtain more compounds of interest. In this study, in order to improve the fungus growth and its terpenoids production, various vegetable oils were added in the fermentation broth of A. cinnamomea. It was found that corn oil from a group of vegetable oils exhibited the best effect on the biomass and triterpenoid content. After optimization, the initial addition of 1% (v/v) corn oil plus the inoculation of 10% (v/v) mycelia led to a maximum triterpenoid yield (532.3 mg L−1), which was increased as much as fourfold compared to the blank control. Differential transcriptome analysis demonstrated that corn oil significantly enriched several metabolic pathways including glycolysis/gluconeogenesis, propanoate metabolism and transmembrane hydrophobins. The enriched pathways interacted with deferentially expressed genes (DEGs) induced by corn oil treatment. Our research provides a potential strategy for the large production of triterpenoids by the improved fermentation of A. cinnamomea.

Biosynthetic Pathway and the Potential Role of Melatonin at Different Abiotic Stressors and Developmental Stages in Tolypocladium guangdongense

Melatonin, a bioactive compound and an important signaling molecule produced in plants and animals, is involved in many biological processes. However, its function and synthetic pathways in fungi are poorly understood. Here, the samples from Tolypocladium guangdongense, a highly valued edible fungus with functional food properties, were collected under different experimental conditions to quantify the levels of melatonin and its intermediates. The results showed that the intracellular melatonin content was markedly improved by Congo red (CR), cold, and heat stresses; the levels of intracellular melatonin and its intermediates increased at the primordial (P) and fruiting body (FB) stages. However, the levels of most intermediates exhibited a notable decrease under CR stress. Several genes related to melatonin synthesis, excluding AADC (aromatic-L-amino-acid decarboxylase), were markedly upregulated at an early stage of CR stress but downregulated later. Compared to the mycelial stage, those genes were significantly upregulated at the P and FB stages. Additionally, exogenous melatonin promoted resistance to several abiotic stressors and P formation in T. guangdongense. This study is the first to report melatonin biosynthesis pathway in macro-fungi. Our results should help in studying the diversity of melatonin function and melatonin-synthesis pathways and provide a new viewpoint for melatonin applications in the edible-medicinal fungus.

The Small Molecule Fractions of Floccularia luteovirens Induce Apoptosis of NSCLC Cells through Activating Caspase-3 Activity

Floccularia luteovirens is a rare wild edible and medicinal fungus endemic to the Qinghai-Tibet Plateau. In this study, the hollow fiber membranes with molecular weights of 50 kDa, 6 kDa and 3 kDa were used to extract different fractions of F. luteovirens, which were named as #1, #2 and #3. Then the antitumor activity of these fractions on NSCLC cell lines, PC9 and NCI-H460, were investigated by using MTT assay, flow cytometry analysis and Western blot assay. The results indicated that the #2 and #3 fractions showed obviously inhibitory activities on PC9 and NCI-H460 tumor cells and proved that these small molecule fractions induced apoptosis of NSCLC cells by activating caspase-3. Finally, a total of 15 components, including six amino acids, two nucleosides, two glycosides, two terpenoids, one phenylpropanoid, one ester and one alkaloid, were identified in #2 and #3 fractions. This is the first evidence that the small molecule components of F. luteovirens were able to inhibit lung cancer by inducing apoptosis in a caspase-3 manner. The present study indicated the benefits of F. luteovirens in lung cancer treatment, which might be a potential resource of functional food and drugs.

Optimization of exopolysaccharide synthesys by medicinal fungus Trametes versicolor in submerged culture

The species Trametes versicolor refers to medicinal mushroom well known in traditional Asian medicine for over 2000 years. Due to the long time required for basidiocarp formation, attention has recently been given to the submerged cultivation method for the production of mycelial biomass and various bioactive components. Exopolysaccharides (EPS) produced by Trametes versicolor are essential components possessing numerous functionalities and exhibiting potential medicinal applications. The subject of this investigation is higher fungus Trametes versicolor isolated from Bulgaria. Four well-defined culture media were studied to select the medium that maximizes production of EPS in submerged cultivation. The M3 was shown to provide the highest yields of EPS and was further investigated to optimize EPS production conditions. The initial glucose concentration was found to be the most important factor in both EPS production and cell growth. The maximum biopolymer quantity of 1.067 g/L was obtained at 40 g/L glucose. For examination and evaluation of the correlation between the carbon source and the complex influence of the nitrogen sources over the mycelial growth and the EPS synthesis and the optimization of the media orthogonal central composition design 23 with star arm ±α=1.454671 was applied. The experimental design was based on 21 combinations. Dynamic cultivation was carried out after the optimization of the media for determination of the effect of the duration of the cultivation process over the Trametes versicolor growth and EPS gain. Maximum EPS yield was observed after 216 hours.