Identification of squalene epoxidase in triterpenes biosynthesis in Poria cocos by molecular docking and CRISPR-Cas9 gene editing.

BACKGROUND: Squalene epoxidase is one of the rate-limiting enzymes in the biosynthetic pathway of membrane sterols and triterpenoids. The enzyme catalyzes the formation of oxidized squalene, which is a common precursor of sterols and triterpenoids. RESULT: In this study, the squalene epoxidase gene (PcSE) was evaluated in Poria cocos. Molecular docking between PcSE and squalene was performed and the active amino acids were identified. The sgRNA were designed based on the active site residues. The effect on triterpene synthesis in P. cocos was consistent with the results from ultra-high-performance liquid chromatography-quadruplex time-of-flight-double mass spectrometry (UHPLC-QTOF-MS/MS) analysis. The results showed that deletion of PcSE inhibited triterpene synthesis. In vivo verification of PcSE function was performed using a PEG-mediated protoplast transformation approach. CONCLUSION: The findings from this study provide a foundation for further studies on heterologous biosynthesis of P. cocos secondary metabolites.

First report on the regulation and function of carbon metabolism during large sclerotia formation in medicinal fungus Wolfiporia cocos.

The medicinal fungus Wolfiporia cocos colonizes and then grows on the wood of Pinus species, and utilizes a variety of Carbohydrate Active Enzymes (CAZymes) to degrades wood for the development of large sclerotia that is mostly built up of beta-glucans. Some differentially expressed CAZymes were revealed by comparisons between the mycelia cultured on potato dextrose agar (PDA) and sclerotia formed on pine logs in previous studies. Here, different profile of expressed CAZymes were revealed by comparisons between the mycelia colonization on pine logs (Myc.) and sclerotia (Scl.b). To further explore the regulation and function of carbon metabolism in the conversion of carbohydrates from Pine species by W. cocos, the transcript profile of core carbon metabolism was firstly analyzed, and it was characterized by the up-regulated expression of genes in the glycolysis pathway (EMP) and pentose phosphate pathway (PPP) in Scl.b, as well as high expression of genes in the tricarboxylic acid cycle (TCA) in both Myc. and Scl.b stages. The conversion between glucose and glycogen and between glucose and ß-glucan was firstly identified as the main carbon flow in the differentiation process of W. cocos sclerotia, with a gradual increase in the content of ß-glucan, trehalose and polysaccharide during this process. Additionally, gene functional analysis revealed that the two key genes (PGM and UGP1) may mediate the formation and development of W. cocos sclerotia possibly by regulating ß-glucan synthesis and hyphal branching. This study has shed light on the regulation and function of carbon metabolism during large W. cocos sclerotium formation and may facilitate its commercial production.

Poria cocos Polysaccharide Ameliorated Antibiotic-Associated Diarrhea in Mice via Regulating the Homeostasis of the Gut Microbiota and Intestinal Mucosal Barrier.

Poria cocos polysaccharides (PCP) have been validated for several biological activities, including antitumor, anti-inflammatory, antioxidant, immunomodulatory, hepatoprotective and modulation on gut microbiota. In this research, we aim to demonstrate the potential prebiotic effects and the therapeutic efficacies of PCP in the treatment of antibiotic-associated diarrhea (AAD), and confirm the beneficial effects of PCP on gut dysbiosis. Antibiotic-associated diarrhea mice models were established by treating them with broad-spectrum antibiotics in drinking water for seven days. Mice in two groups treated with probiotics and polysaccharide were given Bifico capsules (4.2 g/kg/d) and PCP (250 mg/kg/d) for seven days using intragastric gavage, respectively. To observe the regulatory effects of PCP on gut microbiota and intestinal mucosal barrier, we conducted the following experiments: intestinal flora analysis (16S rDNA sequencing), histology (H&E staining) and tight junction proteins (immunofluorescence staining). The levels of mRNA expression of receptors associated with inflammation and gut metabolism were assessed by real-time reverse transcription-polymerase chain reaction (RT-PCR). The study revealed that PCP can comprehensively improve the clinical symptoms of AAD mice, including fecal traits, mental state, hair quality, etc., similar to the effect of probiotics. Based on histology observation, PCP significantly improved the substantial structure of the intestine of AAD mice by increasing the expression levels of colonic tight junction protein zonula-occludens 1 (ZO-1) and its mRNA. Moreover, PCP not only increased the abundance of gut microbiota, but also increased the diversity of gut microbiota in AAD mice, including alpha diversity and beta diversity. Further analysis found that PCP can modulate seven characteristic species of intestinal flora in AAD mice, including Parabacteroides_distasonis, Akkermansia_muciniphila, Clostridium_saccharolyticum, Ruminoc-occus_gnavus, Lactobacillus_salivarius, Salmonella_enterica and Mucispirillum_schaedleri. Finally, enrichment analysis predicted that PCP may affect intestinal mucosal barrier function, host immune response and metabolic function by regulating the microbiota. RT-PCR experiments showed that PCP can participate in immunomodulatory and modulation on metabolic by regulating the mRNA expression of forkhead-box protein 3 (FOXP3) and G protein-coupled receptor 41 (GPR41). These results indicated that Poria cocos polysaccharide may ameliorate antibiotic-associated diarrhea in mice by regulating the homeostasis of the gut microbiota and intestinal mucosal barrier. In addition, polysaccharide-derived changes in intestinal microbiota were involved in the immunomodulatory activities and modulation of the metabolism.

Transcriptome analysis reveals immune and metabolic regulation effects of Poria cocos polysaccharides on Bombyx mori larvae.

Poria cocos polysaccharides (PS) have been used as Chinese traditional medicine with various pharmacological effects, including antiviral, anti-oxidative, and immunomodulatory activities. Herein Bombyx mori silkworm was used as a model animal to evaluate the immunomodulatory effects of PS via detecting the changes of innate immune parameters and explore the underlying molecular mechanism of the immunoregulatory effect of PS using Illumina HiSeq Xten platform. The results presented here demonstrated that a hemocoel injection of PS significantly enhanced the cellular immunity of silkworm, including hemocyte phagocytosis, microaggregation, and spreading ability. A total of 335 differentially expressed genes (DEGs) were screened, including 214 upregulated genes and 121 downregulated genes by differential expression analysis. Gene annotation and enrichment analyses showed that many DEGs related to immune signal recognition, detoxification, proPO activation, carbohydrate metabolism, and lipid metabolism were significantly upregulated in the treatment group. The Kyoto Encyclopedia of Genes and Genomes-based Gene Set Enrichment Analysis also revealed that the more highly expressed gene sets in the PS treatment silkworm were mainly related to immune signal transduction pathways and energy metabolism. In addition, the activity of four enzymes related to immunity and energy metabolism-including phenoloxidase, glucose-6-phosphate dehydrogenase, hexokinase, and fatty acid synthetase-were all significantly increased in the larvae injected with PS. We performed qRT-PCR to examine the expression profile of immune and metabolic-related genes, which further verified the reliability of our transcriptome data and suggested that PS can regulate the immunity of silkworm by enhancing the cellular immunity and modulating the expression levels of genes related to immune responses and physiological metabolism. These findings will lay a scientific foundation for the use of PS as an immunomodulator in disease prevention in human beings or animals.

Genome Resequencing and Transcriptome Analysis Reveal the Genetic Diversity of Wolfiporia cocos Germplasm and Genes Related to High Yield.

Wolfiporia cocos is a saprophytic fungus belonging to the phylum Basidiomycota. The dried sclerotium of this organism has been widely used in traditional Chinese medicine for several thousand years and it is prescribed in many formulations. The W. cocos germplasm resources are complex and diverse, and the molecular mechanisms underlying the growth and development of its sclerotia are unclear. In this study, we used genome resequencing and transcriptome analysis to evaluate the genetic diversity of W. cocos germplasm resources in China and the mechanism of sclerotium growth and development. Phylogenetic and population structure analyses revealed that all the 39 tested strains were divided into three major groups. Most of the strains were clustered into one group, and the remaining strains were clustered into the other two groups. There may be a shared origin of cultivated W. cocos in the main production areas. Transcriptome analysis and quantitative reverse transcription-polymerase chain reaction confirmed that candidate genes related to the yield of W. cocos were mainly enriched in oxidation-reduction and carbohydrate metabolism and highly expressed in the ShenChuan strain, which had the highest comprehensive cultivation score. The findings will be helpful for further understanding the evolution and population structure of W. cocos and determining the functional genes that contribute to the high yield of sclerotia.

Bipolar system of sexual incompatibility and heterothallic life cycle in the basidiomycetes Pachyma hoelen Fr. (Fuling).

The sclerotia of Pachyma hoelen are one of the traditional Chinese medicines and foods that are widely used in East Asian countries. The strains used for cultivation showed bad performance in recent years, and breeding of superior strains has become increasingly important for this fungus. Nevertheless, the mating system and life cycle of P. hoelen were still ambiguous. In this study, the methods for distinguishing between homokaryotic offspring with different mating types were established, as well as confirmation of strain hybridization based on allelic polymorphism at a locus of the rpb2 gene. The bipolar mating system was confirmed according to the mating results of homokaryotic SSIs. The fact that heterokaryotic parents produce homokaryotic meiospores proves that the life cycle is heterothallic. Combining scanning electron microscope observation and DAPI (4',6-diamidino-2-phenylindole) fluorescent staining of hymenium and basidiospores in situ and ex situ, nuclear migration pattern from basidia to spores was revealed. The heterothallic life cycle was verified, revised, and supplemented step by step. This is the first report of systematic research on the mating system, life cycle, and outcrossing of homokaryotic offspring in P. hoelen. It will be helpful for the biological research, strain improvement, and development of the P. hoelen industry.

Analysis of weighted gene co-expression network of triterpenoid-related transcriptome characteristics from different strains of Wolfiporia cocos.

The fungus Wolfiporia cocos has wide-ranging and important medicinal value, and its dried sclerotia are used as a traditional Chinese medicine. Modern studies have shown that triterpenoid, the active ingredient of W. cocos, have a variety of pharmacological effects. The aim of our research was to determine the key genes related to triterpenoid biosynthesis, which may be useful for the genetic modification of cell-engineered bacteria for triterpenoid biosynthesis. In this study, two monospore strains, DZAC-WP-H-29 (high-yielding) and DZAC-WP-L-123 (low-yielding), were selected from the sexually propagated offspring of strain 5.78 of W. cocos, and the mycelia were cultured for 17, 34, and 51 days, respectively. Weighted gene co-expression network analysis (WGCNA) method was used to analyze transcriptional expressions. The results show that eight core genes (ACAT1-b, hgsA, mvd1, SQLE, erg6, TAT, erg26, and erg11) are associated with the triterpenoid synthesis pathway, and Pm20d2 and norA outside the pathway may be important genes that influence the biosynthesis and accumulation of W. cocos triterpenoid. The biosynthesis of W. cocos triterpenoid is closely related to the expression of sterol metabolic pathway genes. The role of these genes in triterpenoid synthesis complements our knowledge on the biosynthesis and accumulation of W. cocos triterpenoid, and also provides a reference for the target gene modification of engineered bacteria for the fermentation production of triterpenoid.

Functional Analysis of Sterol O-Acyltransferase Involved in the Biosynthetic Pathway of Pachymic Acid in Wolfiporia cocos.

Pachymic acid from Wolfiporia cocos possesses important medicinal values including anti-bacterial, anti-inflammatory, anti-viral, invigorating, anti-rejection, anti-tumor, and antioxidant activities. However, little is known about the biosynthetic pathway from lanostane to pachymic acid. In particular, the associated genes in the biosynthetic pathway have not been characterized, which limits the high-efficiency obtaining and application of pachymic acid. To characterize the synthetic pathway and genes involved in pachymic acid synthesis, in this study, we identified 11 triterpenoids in W. cocos using liquid chromatography tandem mass spectrometry (LC-MS/MS), and inferred the putative biosynthetic pathway from lanostane to pachymic acid based on analyzing the chemical structure of triterpenoids and the transcriptome data. In addition, we identified a key gene in the biosynthetic pathway encoding W. cocos sterol O-acyltransferase (WcSOAT), which catalyzes tumolusic acid to pachymic acid. The results show that silence of WcSOAT gene in W. cocos strain led to reduction of pachymic acid production, whereas overexpression of this gene increased pachymic acid production, indicating that WcSOAT is involved in pachymic acid synthesis in W. cocos and the biosynthesis of W. cocos pachymic acid is closely dependent on the expression of WcSOAT gene. In summary, the biosynthetic pathway of pachymic acid and the associated genes complement our knowledge on the biosynthesis of W. cocos pachymic acid and other triterpenoids, and also provides a reference for target genes modification for exploring high-efficiency obtaining of active components.

The Wolfiporia cocos Genome and Transcriptome Shed Light on the Formation of Its Edible and Medicinal Sclerotium.

Wolfiporia cocos (F. A. Wolf) has been praised as a food delicacy and medicine for centuries in China. Here, we present the genome and transcriptome of the Chinese strain CGMCC5.78 of W. cocos. High-confidence functional prediction was made for 9277 genes among the 10,908 total predicted gene models in the W. cocos genome. Up to 2838 differentially expressed genes (DEGs) were identified to be related to sclerotial development by comparing the transcriptomes of mycelial and sclerotial tissues. These DEGs are involved in mating processes, differentiation of fruiting body tissues, and metabolic pathways. A number of genes encoding enzymes and regulatory factors related to polysaccharide and triterpenoid production were strikingly regulated. A potential triterpenoid gene cluster including the signature lanosterol synthase (LSS) gene and its modified components were annotated. In addition, five nonribosomal peptide synthase (NRPS)-like gene clusters, eight polyketide synthase (PKS) gene clusters, and 15 terpene gene clusters were discovered in the genome. The differential expression of the velevt family proteins, transcription factors, carbohydrate-active enzymes, and signaling components indicated their essential roles in the regulation of fungal development and secondary metabolism in W. cocos. These genomic and transcriptomic resources will be valuable for further investigations of the molecular mechanisms controlling sclerotial formation and for its improved medicinal applications.

The development of an efficient RNAi system based on Agrobacterium-mediated transformation approach for studying functional genomics in medical fungus Wolfiporia cocos.

Genetic transformation methods reported for Wolfiporia cocos are limited. In this study, we describe an efficient RNA interference (RNAi) system based on Agrobacterium-mediated transformation approach in W. cocos for the first time. Actively growing mycelial plugs were used as recipients for transformation using endogenous orotidine-5'-phosphate decarboxylase gene (URA3) as both a selective marker and a silencing gene, under the control of the dual promoters of Legpd and Leactin from Lentinula edodes and the single promoter of Wcgpd from W. cocos, respectively. The results showed that both the two kinds of promoters effectively drive the expression of URA3 gene, and the URA3-silenced transformants could be selected on CYM medium containing 5'-fluoroorotic acid. In addition, silencing URA3 gene has no effect on the growth of W. cocos hyphae. The incomplete silencing of the URA3 locus was also observed in this study. This study will promote further study on the mechanism of substrate degradation, sclerotial formation, and biosynthesis network of pharmacological compounds in W. cocos.

Comparative Analysis of the Characteristics of Triterpenoid Transcriptome from Different Strains of Wolfiporia cocos.

The dried sclerotia of Wolfiporia cocos (Schwein.) Ryvarden & Gilb., a traditional Chinese medicine, has triterpenoid as its main active component. Breeding high-yield triterpenoid in W. cocos is an important research topic at present. We screened out two monosporal strains from the same W. cocos 5.78, high-yielding DZAC-Wp-H-29 (H) and low-yielding DZAC-Wp-L-123 (L), and cultured mycelia for 17 days, 34 days, and 51 days, respectively. Transcriptome analysis results showed that triterpenoid synthesis is closely related to gene expression in triterpenoid synthesis pathways (hydroxymethyl glutaryl-CoA reductase (HMGCR), farnesyl diphosphate synthase (FDPS), 4-hydroxybenzoate polyprenyltransferase (COQ2), C-8 sterol isomerase (ERG2), sterol O-acyltransferase (ACAT), tyrosine aminotransferase (TAT), torulene dioxygenase (CAO2), and sterol-4alpha-carboxylate 3-dehydrogenase (erg26)), and is limited by the expression of enzyme M20 combined with domain protein peptide (Pm20d2), aryl-alcohol dehydrogenase (norA), ISWI chromatin-remodeling complex ATPase ISW2, GroES-like protein (adh), cytochrome P450 (ftmP450-1), and unknown proteins unigene0001029 and unigene0011374. In addition, maintaining high triterpenoid accumulation in W. cocos may require a stable membrane structure, so the accumulation ability may be related to the high synthesis ability of sterols. The low accumulation of triterpenoid in W. cocos may be due to the products of key enzymes increasing flow to other pathways.

Production of a novel Poria cocos immunomodulatory protein in Pichia pastoris: cloning, expression, purification and activities assays.

In this study, the cDNA of immunomodulatory protein from Poria cocos (PCP) was amplified by reverse transcription polymerase chain reaction and used to transform P. Pastoris cells, resulting in rPCP expression as a secreted protein to a concentration of ~ 38 mg/L following methanol induction in shake flasks. Approximately 1.6 mg of high purity rPCP was obtained from a 100-mL culture by Ni+-affinity chromatography, and sodium dodecyl sulfate polyacrylamide gel electrophoresis results indicated rPCP as a homologous dimer glycoprotein formed by different molecular-weight monomers. Peptide-N-glycosidase F-mediated deglycosylation analysis showed the presence of an N-glycosylated rPCP monomer, and bioactivity assays showed that rPCP activity upregulated tumor necrosis factor (TNF)-α and interleukin-1ß transcription and increased TNF-α secretion from mouse macrophage RAW 264.7 cells. Shortly, we demonstrated successful purification of active rPCP from P. pastoris, which promoted further study of its biological activities and medical applications.

Transcriptome analysis of three cultivars of Poria cocos reveals genes related to the biosynthesis of polysaccharides.

Poria cocos (P. cocos) polysaccharides (PCPs) are used to improve immunity and possess antitumor activities. We compared three cultivars of P. cocos (5.78, XJ 28 and JHYH) PCP contents. Then we determined that malZ, galA, SORD, gnl and bglX are key enzymes within the PCP biosynthetic pathway by using HiSeq2500 transcriptome and qRT-PCR validation. Our results provide more detailed information about the PCP biosynthesis pathway at the molecular level in P. cocos and establish the functions for the molecular breeding to produce polysaccharides in general for therapeutic use in Chinese medicinal plants.

The Phosphatome of Medicinal and Edible Fungus Wolfiporia cocos.

Wolfiporia cocos is an important medicinal and edible fungus that grows in association with pine trees, and its dried sclerotium has been used as a traditional medicine in China for centuries. However, the commercial production of W. cocos sclerotia is currently limited by shortages in pine wood resources. Since protein phosphatases (PPs) play significant roles in growth, signal transduction, development, metabolism, sexual reproduction, cell cycle, and environmental stress responses in fungi, the phosphatome of W. cocos was analyzed in this study by identifying PP genes, studying transcript profiles and assigning PPs to orthologous groups. Fifty-four putative PP genes were putatively identified in W. cocos genome based on homologous sequences searching using BLASTx program against the Saccharomyces cerevisiae, Fusarium graminearum, and Sclerotinia sclerotiorum databases. Based on known and presumed functions of orthologues of these PP genes found in other fungi, the putative roles of these W. cocos PPs in colonization, hyphal growth, sclerotial formation, secondary metabolism, and stress tolerance to environment were discussed in this study. And the level of transcripts from PP genes in the mycelium and sclerotium stages was also analyzed by qRT-PCR. Our study firstly identified and functional discussed the phosphatome in the medicinal and edible fungus W. cocos. The data from our study contribute to a better understanding of PPs potential roles in various cellar processes of W. cocos, and systematically provide comprehensive and novel insights into W. cocos economically important traits that could be extended to other fungi.

Characterisation of three fungal glucuronoyl esterases on glucuronic acid ester model compounds.

The glucuronoyl esterases (GEs) that have been identified so far belong to family 15 of the carbohydrate esterases in the CAZy classification system and are presumed to target ester bonds between lignin alcohols and (4-O-methyl-)D-glucuronic acid residues of xylan. Few GEs have been cloned, expressed and characterised to date. Characterisation has been done on a variety of synthetic substrates; however, the number of commercially available substrates is very limited. We identified novel putative GEs from a wide taxonomic range of fungi and expressed the enzymes originating from Acremonium alcalophilum and Wolfiporia cocos as well as the previously described PcGE1 from Phanerochaete chrysosporium. All three fungal GEs were active on the commercially available compounds benzyl glucuronic acid (BnGlcA), allyl glucuronic acid (allylGlcA) and to a lower degree on methyl glucuronic acid (MeGlcA). The enzymes showed pH stability over a wide pH range and tolerated 6-h incubations of up to 50 °C. Kinetic parameters were determined for BnGlcA. This study shows the suitability of the commercially available model compounds BnGlcA, MeGlcA and allylGlcA in GE activity screening and characterisation experiments. We enriched the spectrum of characterised GEs with two new members of a relatively young enzyme family. Due to its biotechnological significance, this family deserves to be more extensively studied. The presented enzymes are promising candidates as auxiliary enzymes to improve saccharification of plant biomass.

De novo assembly and transcriptome analysis of sclerotial development in Wolfiporia cocos.

Wolfiporia cocos Ryvarden et Gilbertson, a well-known medicinal fungus in the Basidiomycetes, is widely distributed in East Asia. Its dried sclerotium, which is known as Fuling in China, has been used as a traditional crude drug in Chinese traditional medicine for thousand years. However, little is known about how the sclerotium is developed at the genetic level. In this study, the de novo sequencing of sclerotia of W. cocos (S1_initial stage; S2_developmental stage and S3_mature stage) was carried out by illumina HiSeq 2000 technology. 27,438 unigenes were assembled from ~30Gbp raw data, and 12,093 unigenes were significantly annotated. The analysis of expression profiles during development returned 304 differentially expressed genes (DEGs), which were clustered into four different groups according to their expression trends. Especially for the maturation stage (S3), the sclerotium exhibited a markedly different expression profile from other stages. We further showed that peroxisome, unsaturation of fatty acids and degradation pathway were respectively prevalent in S1, S2 and S3 stages as evidenced by enrichment analysis. To our knowledge, this study represents the first report of sclerotial development transcriptomics in W. cocos. The obtained results provide novel insights into the developmental biology of the sclerotia, which is helpful for future studies about cultivation and breeding of W. cocos.

Transcriptome and Secretome Analyses of the Wood Decay Fungus Wolfiporia cocos Support Alternative Mechanisms of Lignocellulose Conversion.

UNLABELLED: Certain wood decay basidiomycetes, collectively referred to as brown rot fungi, rapidly depolymerize cellulose while leaving behind the bulk of cell wall lignin as a modified residue. The mechanism(s) employed is unclear, but considerable evidence implicates the involvement of diffusible oxidants generated via Fenton-like chemistry. Toward a better understanding of this process, we have examined the transcriptome and secretome of Wolfiporia cocos when cultivated on media containing glucose, purified crystalline cellulose, aspen (Populus grandidentata), or lodgepole pine (Pinus contorta) as the sole carbon source. Compared to the results obtained with glucose, 30, 183, and 207 genes exhibited 4-fold increases in transcript levels in cellulose, aspen, and lodgepole pine, respectively. Mass spectrometry identified peptides corresponding to 64 glycoside hydrolase (GH) proteins, and of these, 17 corresponded to transcripts upregulated on one or both woody substrates. Most of these genes were broadly categorized as hemicellulases or chitinases. Consistent with an important role for hydroxyl radical in cellulose depolymerization, high transcript levels and upregulation were observed for genes involved in iron homeostasis, iron reduction, and extracellular peroxide generation. These patterns of regulation differ markedly from those of the closely related brown rot fungus Postia placenta and expand the number of enzymes potentially involved in the oxidative depolymerization of cellulose. IMPORTANCE: The decomposition of wood is an essential component of nutrient cycling in forest ecosystems. Few microbes have the capacity to efficiently degrade woody substrates, and the mechanism(s) is poorly understood. Toward a better understanding of these processes, we show that when grown on wood as a sole carbon source the brown rot fungus W. cocos expresses a unique repertoire of genes involved in oxidative and hydrolytic conversions of cell walls.