Optimisation of hypocrellin production in Shiraia-like fungi via genetic modification involving a transcription factor gene and a putative monooxygenase gene.

Shiraia-like fungi, which are rare parasitic fungi found around bamboo, play an important role in traditional medicine. Their main active component, hypocrellin, is widely used in medicine, food, and cosmetics. By comparing strains with different hypocrellin yields, we identified a transcription factor (SbTF) in the hypocrellin biosynthesis pathway. SbTF from high-yielding zzz816 and low-yielding CNUCC C72 differed in its protein structure. Subsequently, SbTF from high-yielding zzz816 was overexpressed in several strains. This stabilised the yield in zzz816 and significantly increased the yield in low-yielding CNUCC C72. Comparing downstream non-essential genes between wild type and SbTF-overexpressing CNUCC C72 showed that SbMNF was significantly up-regulated. Therefore, it was selected for further study. SbMNF overexpression increased the hypocrellin yield in low-yielding CNUCC C72 and altered the composition of compounds in high-yielding CNUCC 1353PR and zzz816. This involved an increased elsinochrome C yield in CNUCC 1353PR and an increased hypocrellin B yield in zzz816 (by 2 and 70.3 times that in the corresponding wild type, respectively). This study is the first to alter hypocrellin synthesis to alter the levels of one bioactive agent compared to another. The results provide new insights regarding genetic modification and will help to optimise fungal fermentation.

Phylogenetic Analysis Reveals Four New Species of Otidea from China.

The emergence of molecular systematics has greatly helped researchers to identify fungal species. China has abundant Otidea species resources, and a number of new species of Otidea have been recently proposed. However, many old specimens in herbaria are mainly identified by morphology rather than molecular methods. In this study, 11 specimens deposited in Chinese herbaria and one newly collected Otidea species from northern China were identified based on morphological and phylogenetic analyses. Four gene fragments (ITS, LSU, rpb2, and tef1-α) were used to elucidate the phylogenetic relationships of species within Otidea. A total of nine phylogenetic species are recognized, of which four are described as new species, namely O. bomiensis, O. gongnaisiensis, O. hanzhongensis, and O. shennongjiana. Among the known species were O. aspera and O. sinensis.

Phylogenetic Position of Shiraia-Like Endophytes on Bamboos and the Diverse Biosynthesis of Hypocrellin and Hypocrellin Derivatives.

The main active ingredients of the fruiting bodies of Shiraia bambusicola and Rubroshiraia bambusae are Hypocrellins, belonging perylenequinones with potential photodynamic activity against cancer and microbial diseases. However, the strains of S. bambusicola and R. bambusae do not produce hypocrellins in culture, so resource exploitation of natural products was seriously restricted. In this study, a series of novel Shiraia-like fungal endophyte strains, with varying sporulation ability and synthesizing diverse secondary metabolites, was isolated from different bamboos. Based on phylogenetic analyses and morphological characteristics of the endophytes, Pseudoshiraia conidialis gen. et sp. nov. is proposed. The secondary metabolites of different fruiting bodies and strains have been comprehensively analyzed for the first time, finding that the endophytic strains are shown not only to produce hypocrellins, but also other perylenequinonoid compounds. It was noteworthy that the highest yield of total perylenequinone production and hypocrellin A appeared in P. conidialis CNUCC 1353PR (1410.13 mg/L), which was significantly higher than any other wild type P. conidialis strains in published reports. In view of these results, the identification of Shiraia-like endophytes not only confirm the phylogenetic status of similar strains, but will further assist in developing the production of valuable natural products.

Global identification of alternative splicing in Shiraia bambusicola and analysis of its regulation in hypocrellin biosynthesis.

Hypocrellins, as natural pigments from Shiraia bambusicola, have extensive applications in the agricultural, cosmetic, food, and feed industries, and play a vital role in photodynamic therapy for anticancer and antiviral treatments. However, environmental stresses are always the bottlenecks for increasing hypocrellin yield during the process of fermentation. Pre-mRNA alternative splicing (AS) is an essential mechanism in the defense of abiotic stresses in the animal and plant kingdom, but is seldom involved in fungi. In this study, AS from genome-wide sequencing and RNA-seq data for S. bambusicola was analyzed for the first time. Interestingly, the proportion of AS in S. bambusicola was 38.44% (most of them participated in metabolic processes, covering pigmentation and response to stimulus), a much higher ratio than seen in that of other fungal species (1.3-18%). Here, we identified the relationship of AS and secondary metabolic (SM) biosynthesis under a series of abiotic stresses. Suitable fungicides suppressed hypocrellin production significantly, and AS occurred in key functional genes (sbFLO, sbMFS, sbPKS) of hypocrellin biosynthesis. In contrast, H2O2 improved the yield of hypocrellins, but AS were not found in the corresponding gene cluster. A further study showed that overexpressing an isoform of sbPKS (sbPKSa) in Shiraia bambusicola could dramatically down-regulate the expression of the original gene sbPKS and nearly inhibit the production of hypocrellins. Altogether, our study strongly supported the hypothesis that AS had a vital role in the regulation of hypocrellin biosynthesis under stresses, and initially explored whether SM functional genes were relevant for fungi.

Antioxidant and antimicrobial activities of various extracts from Engleromyces sinensis fruiting body.

Engleromyces sinensis, as rare macro-ascomycetes and traditional ethnomedicine in the southeast part of China, have been applied in anti-infection, anti-inflammatory and anti-tumor for a long time. In this study, the antioxidant activities of ethyl acetate crude extract (EACE), acetone crude extract (ACE), 95% ethanol crude extract (ECE), methanol crude extract (MCE) and water crude extract (WCE) from E. sinensis fruiting body were investigated using conventional antioxidant assays in vitro for the first time. As results, it was noteworthy that WCE showed the greatest 2,2-diphenyl-1-picrylhydrazil (DPPH) radicals-scavenging activity and reducing power, with EC50 values of 3.56 and 19.28mg/mL. MCE and EACE exhibited higher hydroxyl radicals-scavenging activity and ferrous ion-chelating activity significantly, with EC50 values of 2.16 and 0.47mg/mL. The total phenolics and total polysaccharides content results revealed that WCE had the highest phenolics and polysaccharides contents with 1.19 mg GAEs/g extracts and 40.07 mg D-glucose/g extracts. The antimicrobial activity of the WCE, ECE, ACE, EACE was assessed in final and two of them, ACE and EACE showed a strong ability to inhibit the microbial growth. The research work demonstrated that E. sinensis fruiting body can present a promising source of antioxidant and antimicrobial agents.

Diversity and antimicrobial activity of culturable fungi from fishscale bamboo (Phyllostachys heteroclada) in China.

An important and useful bamboo species, fishscale bamboo (Phyllostachys heteroclada Oliver), is broadly distributed in Southeast China and has multiple purposes, including uses in cuisine, weaving, Chinese medicine and ecological protection. However, no previous studies have focused on the endophytes of this plant. In our article, a total of 127 fungal strains were first isolated from the healthy branches and leaves of common P. heteroclada. These endophytic fungi could be directly categorized into 50 morphotypes according to their culture characteristics, and their internal transcribed spacer (ITS) regions were analyzed for molecular identification. Using the BLAST search tool of the NCBI database and phylogenetic tree analysis, these isolates were divided into two phyla, Ascomycota (95.28%) and Basidiomycota (4.72%), including at least six orders (Xylariales, Capnodiales, Pleosporales, Hypocreales, Chaetothyriales and Polyporales) and fourteen genera (Arthrinium, Pestalotiopsis, Epicoccum, Cladosporium, Nigrospora, Setophoma, Didymella, Calcarisporium, Preussia, Nemania, Creosphaeria, Ophiobolus, Phialophora and Perenniporia). It is fascinating that four genera, Calcarisporium, Preussia, Creosphaeria and Phialophora were isolated from bamboos for the first time. The inhibitory effects against clinical pathogens were also preliminarily screened, and four isolates FB43 (Calcarisporium arbuscula), FB06 (Preussia minima), FB16 (Setophoma sp.) and FB21 (Perenniporia medulla-pains) among the candidate strains displayed broad-spectrum activities according to the agar diffusion method and the disk diffusion assay. Strain FB16 (Setophoma sp.) especially indicated high bioactivity against both clinical bacteria and yeast. This study is the first report on the diversity and antimicrobial activity of the endophytic fungi associated with P. heteroclada, which could be regarded as a potential source of drug precursors and could be used in biocontrol development.

Selection of reference genes from Shiraia bambusicola for RT-qPCR analysis under different culturing conditions.

Stable reference genes are necessary to analyse quantitative real-time reverse transcription PCR (qRT-PCR) data and determine the reliability of the final results. For further studies of the valuable fungus Shiraia bambusicola, the identification of suitable reference genes has become increasingly urgent. In this study, three conventional reference genes and nine novel candidates were evaluated under different light conditions (all-dark, all-light and 12-h light/dark) and in different media (rice medium, PD medium, and Czapek-Dox medium). Three popular software programs (geNorm, NormFinder and BestKeeper) were used to analyse these genes, and the final ranking was determined using RefFinder. SbLAlv9, SbJsn1, SbSAS1 and SbVAC55 displayed the best stability among the genes, while SbFYVE and SbPKI showed the worst. These emerging genes exhibited significantly better properties than the three existing genes under almost all conditions. Furthermore, the most reliable reference genes were identified separately under different nutrient and light conditions, which would help accessible to make the most of the existing data. In summary, a group of novel housekeeping genes from S. bambusicola with more stable properties than before was explored, and these results could also provide a practical approach for other filamentous fungi.

High-efficiency biosynthesis of hypocrellin A in Shiraia sp. using gamma-ray mutagenesis.

Hypocrellin A (HA), well known as one of the best natural pigments and bioactive agent to treat skin diseases, is further anticipated to play a vital role in photodynamic therapy (PDT) in anticancer and antiviral treatments. In this study, an HA-producing strain ZZZ816 (Shiraia sp.) was isolated from the moso bamboo (Phyllostachys edulis) seeds, and gamma irradiation was used to mutagenize spores of the original strain. After treatment with cobalt-60 gamma ((60)Coγ) with different doses (20, 50, 80, 100, 150, 180, 300, and 500 Gy), the 100 Gy was selected as the optimal condition, which led to 77.2 % lethality of spores and 35 % positive mutant frequency. The extracted compound of the most excellent HA-producing strain (H-4-2) was precisely analyzed by a combination of seven detection methods, and the maximum HA content was shown to reach 2018.3 mg/L. HA production in H-4-2 increased by 414.9 % compared to that of original strain ZZZ816 (392 mg/L) and was significantly higher than all the other industrial HA-producing strains in published reports.

Identification and Evaluation of Reliable Reference Genes in the Medicinal Fungus Shiraia bambusicola.

The stability of reference genes plays a vital role in real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis, which is generally regarded as a convenient and sensitive tool for the analysis of gene expression. A well-known medicinal fungus, Shiraia bambusicola, has great potential in the pharmaceutical, agricultural and food industries, but its suitable reference genes have not yet been determined. In the present study, 11 candidate reference genes in S. bambusicola were first evaluated and validated comprehensively. To identify the suitable reference genes for qRT-PCR analysis, three software-based algorithms, geNorm, NormFinder and Best Keeper, were applied to rank the tested genes. RNA samples were collected from seven fermentation stages using different media (potato dextrose or Czapek medium) and under different light conditions (12-h light/12-h dark and all-dark). The three most appropriate reference genes, ubi, tfc and ags, were able to normalize the qRT-PCR results under the culturing conditions of 12-h light/12-h dark, whereas the other three genes, vac, gke and acyl, performed better in the culturing conditions of all-dark growth. Therefore, under different light conditions, at least two reference genes (ubi and vac) could be employed to assure the reliability of qRT-PCR results. For both the natural culture medium (the most appropriate genes of this group: ubi, tfc and ags) and the chemically defined synthetic medium (the most stable genes of this group: tfc, vac and ef), the tfc gene remained the best gene used for normalizing the gene expression found with qRT-PCR. It is anticipated that these results would improve the selection of suitable reference genes for qRT-PCR assays and lay the foundation for an accurate analysis of gene expression in S. bambusicola.

Characterization and phylogenetic analysis of the mitochondrial genome of Shiraia bambusicola reveals special features in the order of pleosporales.

Shiraia bambusicola P. Henn. is a pathogenic fungus of bamboo, and its fruiting bodies are regarded as folk medicine. We determined and analyzed its complete mitochondrial DNA sequence (circular DNA molecule of 39,030 bp, G + C content of 25.19%). It contains the typical genes encoding proteins involved in electron transport and coupled oxidative phosphorylation (nad1-6 and nad4L, cob and cox1-3), one ATP synthase subunit (atp6), 4 hypothetical proteins, and two genes for large and small rRNAs (rnl and rns). There is a set of 32 tRNA genes comprising all 20 amino acids, and these genes are evenly distributed on the two strands. Phylogenetic analyses based on concatenated mitochondrial proteins indicated that S. bambusicola clustered with members of the order Pleosporales, which is in agreement with previous results. The gene arrangements of Dothideomycetes species contained three regions of gene orders partitioned in their mitochondrial genomes, including block 1 (nad6-atp6), block 2 (nad1-cox3) and block 3 (genes around rns). S. bambusicola displayed unique special features that differed from the other Pleosporales species, especially in the coding regions around rns (trnR-trnY). Moreover, a comparison of gene orders in mitochondrial genomes from Pezizomycotina revealed that although all encoded regions are located on the same strand in most Pezizomycotina mtDNAs, genes from Dothideomycetes species had different orientations, as well as diverse positions and colocalization of genes (such as cox3, cox1-cox2 and nad2-nad3); these distinctions were regarded as class-specific features. Interestingly, two incomplete copies of the atp6 gene were found on different strands of the mitogenomic DNA, a finding that has not been observed in the other analyzed fungal species. In our study, mitochondrial genomes from Dothideomycetes species were comprehensively analyzed for the first time, including many species that have not appeared in previous reports.

Diversity and antimicrobial activity of culturable endophytic fungi isolated from moso bamboo seeds.

Bamboos, regarded as therapeutic agents in ethnomedicine, have been used to inhibit inflammation and enhance natural immunity for a long time in Asia, and there are many bamboo associated fungi with medical and edible value. In the present study, a total of 350 fungal strains were isolated from the uncommon moso bamboo (Phyllostachys edulis) seeds for the first time. The molecular diversity of these endophytic fungi was investigated and bioactive compound producers were screened for the first time. All the fungal endophytes were categorized into 69 morphotypes according to culturable characteristics and their internal transcriber spacer (ITS) regions were analyzed by BLAST search with the NCBI database. The fungal isolates showed high diversity and were divided in Ascomycota (98.0%) and Basidiomycota (2.0%), including at least 19 genera in nine orders. Four particular genera were considered to be newly recorded bambusicolous fungi, including Leptosphaerulina, Simplicillium, Sebacina and an unknown genus in Basidiomycetes. Furthermore, inhibitory effects against clinical pathogens and phytopathogens were screened preliminarily and strains B09 (Cladosporium sp.), B34 (Curvularia sp.), B35 (undefined genus 1), B38 (Penicillium sp.) and zzz816 (Shiraia sp.) displayed broad-spectrum activity against clinical bacteria and yeasts by the agar diffusion method. The crude extracts of isolates B09, B34, B35, B38 and zzz816 under submerged fermentation, also demonstrated various levels of bioactivities against bambusicolous pathogenic fungi. This study is the first report on the antimicrobial activity of endophytic fungi associated with moso bamboo seeds, and the results show that they could be exploited as a potential source of bioactive compounds and plant defense activators. In addition, it is the first time that strains of Shiraia sp. have been isolated and cultured from moso bamboo seeds, and one of them (zzz816) could produce hypocrellin A at high yield, which is significantly different from the other strains published.

[Use of high-risk human papillomavirus DNA testing to deal with atypical squamous cells undetermined significance and low squamous intraepithelial lesion].

OBJECTIVE: To explore the role of high-risk human papillomavirus (HPV) DNA testing in improvement of recognition of cervical intraepithelial lesions (CIN) 2, 3 or cervical cancer confirmed by biopsy in women with abnormal cervical cytology including atypical squamous cells undetermined significance (ASC-US) and low squamous intraepithelial lesion (LSIL). METHODS: Total 940 patients with abnormal cervical liquid-based cytology including ASC-US and LSIL were included in this study. Hybrid capture II assay was applied in the high-risk HPV detection. The cervical pathologic diagnoses were obtained under colposcopy guided biopsy. RESULTS: Of the 940 cases, 497 were ASC-US, and 443 LSIL. With pathological diagnosis of cervical biopsy, 417 cases were chronic inflammation, 315 condyloma, 124 CIN 1, 65 CIN 2, 19 CIN 3 or cervical cancer. The positive rate of high-risk HPV DNA in groups of ASC-US and LSIL were 59.6% and 84.9%, respectively. In each group of abnormal cytology, detection rates of CIN 2, 3 or cervical cancer in patients with positive HPV DNA were 8.8% and 14.1%, respectively, which were higher than those with negative HPV DNA (0.2% and 6.0%, P < 0.05). In group of ASC-US, high-risk HPV DNA test had a negative predictive value of 99.5%; detection rates of CIN 2, 3 or cervical cancer in patients with HPV DNA virus load 1-9.99 and > or = 10 was 1.6% and 10.7%, respectively. CONCLUSION: High-risk HPV DNA test were helpful to improve the detection rates of CIN 2, 3 or cervical cancer in patients with ASC-US.