Comparative Transcriptome Analysis of Candidate Genes Associated with Mycelia Growth from a He-Ne Laser with Pulsed Light Mutant of Phellinus igniarius (Agaricomycetes).

A mutant Phellinus igniarius JQ9 with higher mycelial production was screened out by He-Ne laser with pulsed light irradiation, the mechanism underlying the higher mycelial production is still unknown. This study aims to obtain a comprehensive transcriptome assembly during the Ph. igniarius liquid fermentation and characterize the key genes associated with the mycelial growth and metabolism in Ph. igniarius JQ9. Our transcriptome data of Ph. iniarius JQ9 and the wild strain were obtained with the Illumina platform comparative transcriptome sequencing technology. The results showed that among all the 346 differentially expressed genes (DEGs), 245 were upregulated and 101 were downregulated. Candidate genes encoding endoglucanase, beta-glucosidase, cellulose 1,4-beta-cellobiosidase, glycoside hydrolase family 61 protein, were proposed to participate in the carbohydrate utilization from KEGG enrichment of the starch and sucrose metabolism pathways were upregulated in Ph. igniarius JQ9. In addition, three candidate genes encoding the laccase and another two candidate genes related with the cell growth were higher expressed in Ph. igniarius JQ9 than in the wild type of strain (CK). Analysis of these data revealed that increased these related carbohydrate metabolism candidate genes underlying one crucial way may cause the higher mycelia production.

Ultrasound Intensify the Flavonoid Production of the Willow Bracket Mushroom, Phellinus igniarius (Agaricomycetes), Fermentation Mycelia.

This research aimed to use a novel and effective ultrasound (US) approach for obtaining high bio-compound production, hence proposing strategies for boosting active ingredient biosynthesis. Furthermore, the US promotes several physiological effects on the relevant organelles in the cell, morphological effects on the structure of Phellinus igniarius mycelium, and increases the transfer of nutrients and metabolites. One suitable US condition for flavonoid fermentation was determined as once per day for 7-9 days at a frequency 22 + 40 kHz, power density 120 W/L, treated 10 min, treatment off time 7 s. The flavonoid content and production increased about 47.51% and 101.81%, respectively, compared with the untreated fermentation (P < 0.05). SEM showed that sonication changes the morphology and structure of Ph. igniarius mycelium; TEM reveals the ultrasonic treatment causes organelle aggregation. The ultrasound could affect the metabolism of the biosynthesis of the active ingredients.

Polysaccharides Derived from Mushrooms in Immune and Antitumor Activity: A Review.

Mushrooms are full of nutrition and have beneficial properties for human health. Polysaccharides are the main component of edible and medicinal mushrooms, especially ß-glucans, which have attracted much more attention for their complex structure and diverse biological activities. Among all the diverse medicinal activities of mushroom polysaccharides, antitumor and immune-enhancing activities are two excellent bioactivities that have much more potential and deserve application. Their bioactivities are highly dependent on their structural features, including molecular weight, monosaccharide composition, degree of branching, type and configuration of glycosidic bonds, substituent pattern, and chain conformation. This review summarizes the current method for obtaining polysaccharides from mushrooms, chemical characterizations of the structures and their roles in immune and antitumor activities. In addition, the methods for preparation of the polysaccharide derivatives and the potential medicinal clinical application are also discussed in this review, which may provide new guidance for mushroom polysaccharide development.

Pulsed Light Mutagenesis of the Willow Bracket Mushroom, Phellinus igniarius (Agaricomycetes), for Enhanced Production of Flavonoids, Laccase, and Fermentation Biomass.

Phellinus igniarius is a medicinal fungus possessing potent therapeutic activity due to the polysaccharides, polyphenols, flavonoids, and other secondary metabolites they contain. Laccases are crucial enzymes involved in lignin degradation in Ph. igniarius and offer great potential to accomplish several bioprocesses. To generate Ph. igniarius strains with high biomass, flavonoid, and laccase activity, we used pulsed light (PL) technology for mutagenesis of Ph. igniarius protoplasts and screened for mutants with high biomass, flavonoid, and laccase activity. At the irradiation power of 100 J, treated distance 8.5 cm, irradiation frequency was 0.5 s/time, three times treatments, after five generations of selection, three mutants were obtained with higher biomass production. Compared with control, the mycelium biomass and the flavonoid production of the screened mutant strain QB72 were increased 20.87% and 53.51%, respectively. The total amount of the accumulated extracellular laccase of the QB72 in the first 6 and 8 days increased 23.38% and 22.37% respectively, and over the total 16 days it increased 9.62%. In addition, RAPD analysis results indicated that the genetic materials of the mutant QB72 were altered. PL mutagenesis method has great potential for developing strains, especially Phellinus.

De Novo Transcriptome Analysis of the Willow Bracket Medicinal Mushroom Phellinus igniarius (Agaricomycetes) by RNA Sequencing.

Phellinus igniarius has been widely used as traditional herbal medicine in China, Korea, Japan, and other Asian countries for centuries. To gain a molecular understanding of this fungus, Illumina high-throughput technology was used to sequence and analyze the transcriptome of Ph. igniarius, and 11,847 unigenes were obtained. Additionally, based on similarity search with known proteins, unigenes were annotated with gene descriptions, gene ontology (GO), and clusters of orthologous groups (COG). Based on the GO annotation, a total of 6983 unigenes could be assigned to one or more terms, which were categorized into 39 functional groups under three main divisions (biological process, cellular component and molecular function). A total of 2315 Ph. igniarius unigenes were associated with 124 unique Kyoto Encyclopedia of Genes and Genomes pathways. A total of 832 simple sequence repeats were identified in these unigenes. In addition, nine candidate genes involved in laccases were discovered, 17 candidate genes involved in the biosynthesis of triterpenoid were identified and were worthy for further investigation. This is the first study to conduct transcriptome analyses on Ph. igniarius, which lays a basis for further functional genomics studies of Ph. igniarius.

Enhanced Mycelium Production of Phellinus igniarius (Agaricomycetes) Using a He-Ne Laser with Pulsed Light.

This study used a He-Ne laser with pulsed light irradiation to produce mutant strains of Phellinus igniarius strain JQ9 with enhanced characteristics for fermentation (17.685 ± 3.092 g/L) compared with the parent strain (12.062 ± 1.119 g/L). The combined treatment conditions were as follows: He-Ne laser irradiation for 30 min using a spot diameter of 10 mm, pulsed light treatment power set at 100 J, a treatment distance of 14.5 cm, and a flash frequency of 0.5 s. The production of bioactive polysaccharides and small biocompounds such as polyphenols, flavonoids, and triterpenes increased together with mycelium production. The results showed that polyphenol content was significantly correlated with L*, a*, and b* values (R = -0.594, P < 0.01; R = 0.571, P < 0.01; and R = 0.500, P < 0.05; respectively). Antagonistic and random amplified polymorphic DNA analyses indicated that the genetic material of the screened mutants was altered. The mutant screening using a He-Ne laser with pulsed light irradiation could be an effective method for the development of Phellinus strains and could thus improve mycelium production.

Review of Advances in Bioactive Low-Molecular-Weight Compounds, Extracts, and Biology of Phellinus sensu lato Mushrooms (Agaricomycetes) from 2011 to 2017.

Various diseases such as cancer, hyperglycemia, and obesity negatively influence people's health and daily life. Mushroom species are miniature pharmaceutical factories producing hundreds of novel constituents with a long history of use in Oriental medicine. As a new therapy, they have attracted much attention owing to their potent therapeutic activity. Phellinus sensu lato (s.l.) is a well-known medicinal mushroom genus that has long been used in preventing ailments, including gastroenteric dysfunction, diarrhea, hemorrhage, and cancers, in Oriental countries, particularly in China, Japan, and Korea. Isolated compounds or complex extracts from Phellinus demonstrate specific bioactivity. Low-molecular-weight components, especially terpenoids, polyphenols, and other secondary metabolites, still exhibit biological activity. This review highlights the bioactive compounds from Phellinus s. l. mushrooms as being potent and having unlimited applications, especially the bioactive low-molecular-weight compounds, such as terpenoids, polyphenols, and their derivatives; also, pharmacological properties of the extracts and fractions from the fruiting bodies and the mycelia of Phellinus s. l., and the mechanism of the pharmacological activity have been discussed. This review summarized the two parts as mentioned above over a period of 7 years from 2011 to 2017.

Employment of ARTP to Generate Phellinus baumii (Agaricomycetes) Strain with High Flavonoids Production and Validation by Liquid Fermentation.

To obtain Phellinus baumii strain with high flavonoids yield, ARTP was employed to generate mutants of a Ph. baumii strain, which were screened for higher flavonoids content. After five rounds of screening, four mutants were identified to produce more flavonoids than the wild type strain under optimal conditions, of which A67 was the mutant with the highest flavonoids productive capacity. When cultured in shake flasks, the maximum intracellular total flavonoids production of A67 reached 0.56 g/L, 86.67% higher than the total flavonoids in CK. Antagonistic testing, RAPD, and HPLC analysis suggested that ARTP caused changes of the genetic material and metabolites in Ph. baumii. In addition, the superiority of A67 to CK was proved by liquid fermentation using unstructured kinetic models, which was performed in a 50-L fermentor. The maximum intracellular total flavonoids production and dry mycelium weight of A67 reached 0.64 g/L and 15.24 g/L, which was an increase of 88.24% and 18.23% compared with CK, respectively. This work could provide an efficient and practical strategy to obtain high flavonoids production strains and the superiority of A67 could also provide a reference to further increase flavonoids production of Ph. baumii in large-scale production mode by submerged fermentation process.

Proteomic analysis of hepatocellular carcinoma HepG2 cells treated with platycodin D.

Platycodin D (PD), a triterpenoid saponin isolated from Platycodonis Radix, is a famous Chinese herbal medicine that has been shown to have anti-proliferative effects in several cancer cell lines. The aim of this study was to determine the changes in cellular proteins after the treatment of hepatocellular carcinoma HepG2 cells with PD using proteomics approaches. The cell viability was determined using the MTT assay. The proteome was analyzed by two-dimensional difference gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Western blot analysis was used to confirm the expression of changed proteins. Our results showed that PD inhibited the proliferation of HepG2 cells in concentration- and time-dependent manners. Sixteen proteins were identified to be up-regulated in PD-treated HepG2 cells, including ATP5H, OXCT1, KRT9, CCDC40, ERP29, RCN1, ZNF175, HNRNPH1, HSP27, PA2G4, PHB, BANF1, TPM3, ECH1, LGALS1, and MYL6. Three proteins (i.e., RPS12, EMG1, and KRT1) decreased in HepG2 cells after treatment with PD. The changes in HSP27 and PHB were further confirmed by Western blotting. In conclusion, our results shed new lights on the mechanisms of action for the anti-cancer activity of PD.