Genomic and Transcriptome Analysis Reveals the Biosynthesis Network of Cordycepin in Cordyceps militaris.

Cordycepin is the primary active compound of Cordyceps militaris. However, the definitive genetic mechanism governing cordycepin synthesis in fruiting body growth and development remains elusive, necessitating further investigation. This study consists of 64 C. militaris strains collected from northeast China. The high-yielding cordycepin strain CMS19 was selected for the analysis of cordycepin production and the genetic basis of cordycepin anabolism. First, the whole-genome sequencing of CMS19 yielded a final size of 30.96 Mb with 8 contigs and 9781 protein-coding genes. The genome component revealed the presence of four additional secondary metabolite gene clusters compared with other published genomes, suggesting the potential for the production of new natural products. The analyses of evolutionary and genetic differentiation revealed a close relationship between C. militaris and Beauveria bassiana. The population of strains distributed in northeast China exhibited the significant genetic variation. Finally, functional genes associated with cordycepin synthesis were identified using a combination of genomic and transcriptomic analyses. A large number of functional genes associated with energy and purine metabolism were significantly enriched, facilitating the reconstruction of a hypothetical cordycepin metabolic pathway. Therefore, our speculation of the cordycepin metabolism pathway involved 24 genes initiating from the glycolysis and pentose phosphate pathways, progressing through purine metabolism, and culminating in the core region of cordycepin synthesis. These findings could offer fundamental support for scientific utilizations of C. militaris germplasm resources and standardized cultivation for cordycepin production.

Construction of a New Agrobacterium tumefaciens-Mediated Transformation System based on a Dual Auxotrophic Approach in Cordyceps militaris.

Cordyceps militaris is a significant edible fungus that produces a variety of bioactive compounds. We have previously established a uridine/uracil auxotrophic mutant and a corresponding Agrobacterium tumefaciens-mediated transformation (ATMT) system for genetic characterization in C. militaris using pyrG as a screening marker. In this study, we constructed an ATMT system based on a dual pyrG and hisB auxotrophic mutant of C. militaris. Using the uridine/uracil auxotrophic mutant as the background and pyrG as a selection marker, the hisB gene encoding imidazole glycerophosphate dehydratase, required for histidine biosynthesis, was knocked out by homologous recombination to construct a histidine auxotrophic C. militaris mutant. Then, pyrG in the histidine auxotrophic mutant was deleted to construct a ΔpyrG ΔhisB dual auxotrophic mutant. Further, we established an ATMT transformation system based on the dual auxotrophic C. militaris by using GFP and DsRed as reporter genes. Finally, to demonstrate the application of this dual transformation system for studies of gene function, knock out and complementation of the photoreceptor gene CmWC-1 in the dual auxotrophic C. militaris were performed. The newly constructed ATMT system with histidine and uridine/uracil auxotrophic markers provides a promising tool for genetic modifications in the medicinal fungus C. militaris.

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.

Rapid and concise quantification of mycelial growth by microscopic image intensity model and application to mass cultivation of fungi.

The microbial food fermentation industry requires real-time monitoring and accurate quantification of cells. However, filamentous fungi are difficult to quantify as they have complex cell types such as pellet, spores, and dispersed hyphae. In this study, numerous data of microscopic image intensity (MII) were used to develop a simple and accurate quantification method of Cordyceps mycelium. The dry cell weight (DCW) of the sample collected during the fermentation was measured. In addition, the intensity values were obtained through the ImageJ program after converting the microscopic images. The prediction model obtained by analyzing the correlation between MII and DCW was evaluated through a simple linear regression method and found to be statistically significant (R2 = 0.941, p < 0.001). In addition, validation with randomly selected samples showed significant accuracy, thus, this model is expected to be used as a valuable tool for predicting and quantifying fungal growth in various industries.

The transcriptome analysis on urea response mechanism in the process of ergosterol synthesis by Cordyceps cicadae.

Nitrogen source is required for the growth of Cordyceps cicadae and involved in the regulation of metabolite synthesis. In order to further investigate the regulatory effects of nitrogen sources on the ergosterol synthesis by C. cicadae. We first confirmed that urea could significantly increase the ergosterol synthesis. The transcriptome analysis showed that compared with biomass cultured in the control fermentation medium (CFM), 1340 differentially expressed genes (DEGs) were obtained by Gene Ontology (GO) annotation, and 312 DEGs were obtained by Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation from the biomass cultured in CFM + CO(NH2)2. Urea up-regulated D-3-phosphoglycerate dehydrogenase gene transcription level and down-regulated enolase and L-serine/L-threonine ammonialyase gene transcription level, increased serine synthesis, allosterically activate pyruvate kinase, to promote the synthesis of pyruvate and CH3CO ~ SCOA, the primer of ergosterol; Urea increase the genes transcription related with ergosterol synthesis by up-regulating the steroid regulatory element binding protein gene transcription levels. The transcriptome results were provided by those of qRT-PCR. Collectively, our finding provided valuable insights into the regulatory effect of nitrogen source on the ergosterol synthesis by C. cicadae.

Biochemical Profiling and Cultivation of Medicinal Fungus Isaria cicadae (Ascomycetes) from India.

Isaria cicadae is an entomopathogenic fungus possessing several therapeutic properties and has a potential role in traditional Chinese medicine. The present study was designed to describe the taxonomic details of a new isolate of I. cicadae collected from the Northern Himalayas of India and to study its vegetative and reproductive growth responses under in vitro conditions. Proximate composition, biochemical profiling, and radical scavenging activities were studied to establish the bioactivity of the isolate. Micromorphological characteristics of conidia and conidiophore formation were studied using scanning electron microscopy. The optimum temperature and pH for mycelial growth was 25°C and 7.0, respectively. Pinhead initiation was observed at day 10 after inoculation, but the fully developed, branched, and coral to club-shaped fruiting bodies could be observed after 30 days of inoculation. Proximate analysis indicated that carbohydrates are the major constituents (50.2%) of the fruit bodies, along with a lower quantity of protein (4.46%), crude fat (6.4%), and crude fiber (1.55%). Vitamin D content of I. cicadae was 3,605.84 IU/g. Radical scavenging activity based on the DPPT (1,1-diphenyl-2-picrylhydrazyl) assay was 21.2%. ABTS [2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid] and potassium ferricyanide reducing activity were quite high, at around 93% and 99.3%, respectively. The findings of this study provide insight into the biochemical constituents of I. cicadae and its cultivation practices for further exploitation of this mushroom at a larger scale.

Review on Research Progress and Prospects of Cicada Flower, Isaria cicadae (Ascomycetes).

Cicada flower, Isaria cicadae Miq., has been a traditional Chinese medicine for approximately 1600 years. Many works on its identification, bioactivities, and clinical use against some disorders have been published, but some inaccuracies and inconsistencies need to be further clarified. In combination with our > 20 years of research and application of cicada flower and examination of the literature and patents published in recent years, this article summarizes and reviews the life cycle and taxonomy, genome size and mating type, molecular systematic classification and cultivation, active ingredients, and pharmacological functions of I. cicadae.

Cysteine-Rich Hydrophobin Gene Family: Genome Wide Analysis, Phylogeny and Transcript Profiling in Cordyceps militaris.

Hydrophobins are a family of small secreted proteins found exclusively in fungi, and they play various roles in the life cycle. In the present study, genome wide analysis and transcript profiling of the hydrophobin family in Cordyceps militaris, a well-known edible and medicinal mushroom, were studied. The distribution of hydrophobins in ascomycetes with different lifestyles showed that pathogenic fungi had significantly more hydrophobins than saprotrophic fungi, and class II members accounted for the majority. Phylogenetic analysis of hydrophobin proteins from the species of Cordyceps s.l. indicated that there was more variability among the class II members than class I. Only a few hydrophobin-encoding genes evolved by duplication in Cordyceps s.l., which was inconsistent with the important role of gene duplication in basidiomycetes. Different transcript patterns of four hydrophobin-encoding genes during the life cycle indicated the possible different functions for each. The transcripts of Cmhyd2, 3 and 4 can respond to light and were related with the photoreceptors. CmQHYD, with four hydrophobin II domains, was first found in C. militaris, and multi-domain hydrophobins were only distributed in the species of Cordycipitaceae and Clavicipitaceae. These results could be helpful for further function research of hydrophobins and could provide valuable information for the evolution of hydrophobins.

Identification of microRNA-like RNAs in Cordyceps guangdongensis and their expression profile under differential developmental stages.

Cordyceps guangdongensis is a well-known fungus with high nutritional and medicinal value. The metabolite profile of C. guangdongensis is similar to that of Ophiocordyceps sinensis. In plants and animals, microRNAs play important roles in regulating gene expression at the post-transcriptional level. MicroRNA-like RNAs (milRNAs) have been documented in several macro-fungi. To comprehensively investigate the milRNAs in C. guangdongensis, three small RNA libraries from the differentially developmental stages were constructed. Twenty-six conserved milRNAs were identified, and 19 novel milRNA candidates were predicted. Among them, 20 milRNAs were differentially expressed across the developmental processes, and 12 milRNAs were verified using stem-loop quantitative real-time reverse transcription polymerase chain reaction. In addition, the potential target genes of milRNA were predicted to be involved in the development of fruiting bodies and metabolite biosynthesis. This study is the first to report the milRNAs of C. guangdongensis, and provides important insights into studies of milRNA regulation pathways in ascomycete fungi.

Development of a fluid-bed coating process for soil-granule-based formulations of Metarhizium brunneum, Cordyceps fumosorosea or Beauveria bassiana.

AIM: Granule-based products of solid state fermented micro-organisms are available for biocontrol. Because liquid fermentation has several advantages, we investigated fluid-bed coating with liquid fermented biomass. METHODS AND RESULTS: Biomass containing mycelium or mycelium and submerged spores of the entomopathogenic fungi Metarhizium brunneum, Cordyceps fumosorosea and Beauveria bassiana were produced in liquid culture, separated and different biomass concentrations were adjusted. Based on the examined thermo-tolerance, we defined fluid-bed coating adjustments and investigated granule colonization and sporulation on granules. Granule colonization depended on the biomass concentration and strain. For C. fumosorosea and B. bassiana, concentrations of 0·003%dry weight resulted in nearly 100% granule colonization, for M. brunneum with concentrations of 0·7%dry weight in only 50%. The conidiation on granules in sterile soil was highly influenced by the moisture content. Because the granule colonization of M. brunneum was unsatisfactory, we pre-coated nutrients followed by coating with biomass, submerged spores or conidia. Malt extract had a positive effect on the granule colonization for biomass and submerged spores. Furthermore, aerial conidia can also be coated. CONCLUSIONS: Fluid-bed coating of fungal biomass is suitable for the development of granules. SIGNIFICANCE AND IMPACT OF THIS STUDY: With this technology, cost-efficient biocontrol products can be developed.

De novo transcriptome sequencing of Paecilomyces tenuipes revealed genes involved in adenosine biosynthesis.

The use of Paecilomyces tenuipes (P. tenuipes), a Chinese medicinal fungus in scientific research, is limited due to its low adenosine content. To improve adenosine production, the present study investigated the gene network of adenosine biosynthesis in P. tenuipes via transcriptome analysis. Mycelia of P. tenuipes cultured for 24 h (PT24), 102 h (PT102) and 196 h (PT192) were subjected to RNA sequencing. In total, 13,353 unigenes were obtained. Based on sequence similarity, 8,099 unigenes were annotated with known proteins. Of these 8,099 unigenes, 5,123 had functions assigned based on Gene Ontology terms while 4,158 were annotated based on the Eukaryotic Orthologous Groups database. Moreover, 1,272 unigenes were mapped to 281 Kyoto Encyclopedia of Genes and Genomes pathways. In addition, the differential gene expression of the three libraries was also performed. A total of 601, 1,658 and 628 differentially expressed genes (DEGs) were detected in PT24 vs. PT102, PT24 vs. PT192 and PT102 vs. PT192 groups, respectively. Reverse transcription­quantitative PCR was performed to analyze the expression levels of 14 DEGs putatively associated with adenosine biosynthesis in P. tenuipes. The results showed that two DEGs were closely associated with adenosine accumulation of P. tenuipes. The present study not only provides an improved understanding of the genetic information of P. tenuipes but also the findings can be used to aid research into P. tenuipes.

Transcriptomic analysis of the orchestrated molecular mechanisms underlying fruiting body initiation in Chinese cordyceps.

Chinese cordyceps, the fruiting body of the Chinese caterpillar fungus (Ophiocordyceps sinensis, syn. Cordyceps sinensis), is among the most valuable traditional Chinese medicine fungi. Transcriptomic analysis of O. sinensis has revealed several aspects of its life cycle and ecological importance. However, the molecular mechanisms involved in fruiting body initiation remain unclear. The developmental transcriptomes were analyzed from three tissues at the fruiting body initiation stage, namely, the mycelium, sclerotium and primordium. Principal component analysis showed that in the three tissues, the gene expression patterns differed from each other. The functional analysis of differentially expressed genes showed that DNA synthesis and cell division were active in the primordium. In addition, the function of the mycelium was to absorb certain substances from the environment and the sclerotium was the metabolism center of O. sinensis. Genes participating in the mitogen-activated protein kinase (MAPK) signal pathway were involved in fruiting body initiation. Two environmental sensing genes, including a pheromone receptor gene (OSIN6252) and an amino acid sensing gene (OSIN6398), were highly expressed in the primordium, suggesting their important roles in initiation. These results provided insights into the orchestrated functions and gene profiles of different O. sinensis tissues at the key stage. These findings will aid in revealing the underlying mechanisms of fruiting body initiation, which will further benefit artificial cultivation.

Biotransformation of Methoxyflavones by Selected Entomopathogenic Filamentous Fungi.

The synthesis and biotransformation of five flavones containing methoxy substituents in the B ring: 2'-, 3'-, 4'-methoxyflavones, 2',5'-dimethoxyflavone and 3',4',5'-trimethoxyflavone are described. Strains of entomopathogenic filamentous fungi were used as biocatalysts. Five strains of the species Beauveria bassiana (KCh J1.5, J2.1, J3.2, J1, BBT), two of the species Beauveria caledonica (KCh J3.3, J3.4), one of Isaria fumosorosea (KCh J2) and one of Isaria farinosa (KCh KW 1.1) were investigated. Both the number and the place of attachment of the methoxy groups in the flavonoid structure influenced the biotransformation rate and the amount of nascent products. Based on the structures of products and semi-products, it can be concluded that their formation is the result of a cascading process. As a result of enzymes produced in the cells of the tested strains, the test compounds undergo progressive demethylation and/or hydroxylation and 4-O-methylglucosylation. Thirteen novel flavonoid 4-O-methylglucosides and five hydroxy flavones were isolated and identified.

Bioactive Metabolites and Potential Mycotoxins Produced by Cordyceps Fungi: A Review of Safety.

Ascomycete Cordyceps fungi such as C.militaris, C. cicadae, and C.guangdongensis have been mass produced on artificial media either as food supplements or health additives while the byproducts of culture substrates are largely used as animal feed. The safety concerns associated with the daily consumption of Cordyceps fungi or related products are still being debated. On the one hand, the known compounds from these fungi such as adenosine analogs cordycepin and pentostatin have demonstrated different beneficial or pharmaceutical activities but also dose-dependent cytotoxicities, neurological toxicities and or toxicological effects in humans and animals. On the other hand, the possibility of mycotoxin production by Cordyceps fungi has not been completely ruled out. In contrast to a few metabolites identified, an array of biosynthetic gene clusters (BGCs) are encoded in each genome of these fungi with the potential to produce a plethora of as yet unknown secondary metabolites. Conservation analysis of BGCs suggests that mycotoxin analogs of PR-toxin and trichothecenes might be produced by Cordyceps fungi. Future elucidation of the compounds produced by these functionally unknown BGCs, and in-depth assessments of metabolite bioactivity and chemical safety, will not only facilitate the safe use of Cordyceps fungi as human food or alternative medicine, but will also benefit the use of mass production byproducts as animal feed. To corroborate the long record of use as a traditional medicine, future efforts will also benefit the exploration of Cordyceps fungi for pharmaceutical purposes.

Effects of Substrates on the Production of Fruiting Bodies and the Bioactive Components by Different Cordyceps militaris Strains (Ascomycetes).

Cordyceps militaris is a type of food and medicinal species and is widely cultured in Asia. Substrate and strain are important factors for the production of fruiting bodies and bioactive components contents in fruiting bodies of C. militaris. This study aimed to select the excellent strains and suitable substrates by six strains of C. militaris cultivated on rice, wheat, and tussah (Antheraea pernyi) pupae. The results showed that the rice and wheat were suitable for fruiting body formation of strain CM3, with yields of 23.19 and 19.07 g per bottle, and biological efficiency of strain CM3 were 62.26% and 54.48%, respectively, which were significantly higher than other strains. Tussah pupae is suitable for fruiting body formation of strain CM9, with fruiting body length, yield, and biological efficiency of 5.57 cm, 6.80 g per each, and 291.70%, respectively, which were significantly higher than other strains. The content of adenosine in fruiting bodies of strain CM9 cultivated on tussah pupae was 2.62 mg g-1, followed by that of strain CM3 on rice of 2.51 mg g-1. The content of cordycepin in fruiting bodies of strain CM4 cultivated on wheat was 5.68 mg g-1, followed by that of strain CM9 on wheat of 5.41 mg g-1. To improve the product quality and the contents of bioactive components, C. militaris strains and substrates should both be considered, that is, different strains should be appropriate for different substrates.

Cmfhp Gene Mediates Fruiting Body Development and Carotenoid Production in Cordyceps militaris.

Cordyceps militaris fruiting bodies contain a variety of bioactive components that are beneficial to the human body. However, the low yield of fruiting bodies and the low carotenoid content in C. militaris have seriously hindered the development of the C. militaris industry. To elucidate the developmental mechanism of the fruiting bodies of C. militaris and the biosynthesis mechanism of carotenoids, the function of the flavohemoprotein-like Cmfhp gene of C. militaris was identified for the first time. The Cmfhp gene was knocked out by the split-marker method, and the targeted gene deletion mutant ΔCmfhp was obtained. An increased nitric oxide (NO) content, no fruiting body production, decreased carotenoid content, and reduced conidial production were found in the mutant ΔCmfhp. These characteristics were restored when the Cmfhp gene expression cassette was complemented into the ΔCmfhp strain by the Agrobacterium tumefaciens-mediated transformation method. Nonetheless, the Cmfhp gene had no significant effect on the mycelial growth rate of C. militaris. These results indicated that the Cmfhp gene regulated the biosynthesis of NO and carotenoids, the development of fruiting bodies, and the formation of conidia. These findings potentially pave the way to reveal the developmental mechanism of fruiting bodies and the biosynthesis mechanism of carotenoids in C. militaris.

Expression profiling of Cordyceps DnaJ protein family in Tolypocladium guangdongense during developmental and temperature stress processes.

DnaJ is an important molecular chaperone, with significant roles in growth, development, and stress resistance. Studies on the DnaJ gene family in macro-fungi such as Cordyceps spp. s.l. is scare. In this study, 22, 20, and 24 putative DnaJ genes were identified in Tolypocladium guangdongense, Ophiocordyceps sinensis, and C. militaris, respectively. They were classified into four groups based on the presence of the J, zinc finger, and C-terminal domains. We mainly studied the T. guangdongense DnaJ genes being located in the endoplasmic reticulum, cytoplasm, mitochondrion, and nucleus. Phylogenetic analysis revealed gene duplications during the evolutionary process. Multiple cis-elements and transcription factor binding sites were observed in the promoter, suggesting their involvement in the response to multiple stresses. qRT-PCR analysis showed that 63.63% and 45.45% of T. guangdongense DnaJ genes were differentially expressed under cold and heat stress, respectively, indicating their involvement in the response to temperature stress. Many T. guangdongense DnaJ genes in the primordium and fruiting body exhibited differential expression, in comparison to those in the mycelium, suggesting a regulatory role in its growth and development process. These findings will facilitate further functional analysis, and provide information on the classification and conservative functions of DnaJ proteins in macro-fungi.

Selection and validation of reliable reference genes for Tolypocladium guangdongense gene expression analysis under differentially developmental stages and temperature stresses.

Tolypocladium guangdongense, formerly known as Cordyceps guangdongensis, is a widely cultivated fungus of the Cordyceps s.l. species that has been investigated over the last 12 years. It has the potential to be used in a number of applications in the health and pharmaceutical industries for it has shown its high nutritional and medicinal values according to previous animal studies. qRT-PCR (quantitative reverse transcription polymerase chain reaction) is extensively used to analyze the expression pattern and molecular mechanisms of functional genes under differentially experimental conditions. The expression stability of reference genes used for normalization determines the reliability of qRT-PCR results, indicating the importance of selection and validation of reference genes before gene expression analysis. In the present study, three statistical algorithms, geNorm, NormFinder and BestKeeper, were used for analyzing the expression stability of nineteen candidate reference genes (CRGs) in T. guangdongense. Investigation were carried out under differentially experimental conditions, which included differentially developmential stages (mycelia, primordia, young and mature fruiting bodies), different carbon sources, cold and heat stresses. The results showed that histone H4 and tubulin beta chain 2 (ß-tub2) were the most and least stable genes, respectively, across all the experimental samples. Moreover, analysis of individual data sets exhibited different stability and expression profiles of reference genes. The vacuolar protein sorting gene VPS was the most stable gene expressed under the differentially developmental stages and temperature stresses, whereas H4 was the most stably expressed gene under different carbon sources. Therefore, it can be proposed that VPS and H4 are the preferred reference genes for normalization of gene expression under different experimental conditions. The results of our present study will enable more accurate evaluation of gene expression in T. guangdongense using the optimal reference gene for qRT-PCR analysis.

Vegetative development and host immune interaction of Ophiocordyceps sinensis within the hemocoel of the ghost moth larva, Thitarodes xiaojinensis.

Ophiocordyceps sinensis is an entomopathogenic fungus that infects ghost moth larva, forming the most valuable and rare traditional Chinese medicine, Chinese cordyceps. Our knowledge of the basic morphology and developmental biology of Chinese cordyceps is limited. In this study, morphological and ultrastructural observations of O. sinensis development in the hemocoel of Thitarodes xiaojinensis were obtained by multiple light and electron microscopy techniques, and the host immune reaction activities were determined. Our results indicated that fungal cells in the host hemocoel underwent morphotype transformations from blastospores to prehyphae to hyphae in sequence. The fusiform yeast-like blastospores were the initial cell type present in the host hemocoel and remained for 5 months or more; the encapsulation reaction and phenoloxidase activity of T. xiaojinensis hemolymph were inhibited during this period. When larvae entered the last instar, the blastospores switched to prehyphae and expanded throughout the host tissues, and then hyphae germinated from the prehyphae and mycelia formed, which finally led to host death. Considering the distinct differences between blastospores and hyphae, we identified prehyphae, which play important roles in fungal expansion, hyphae germination, and fusion formation among filaments. Notably, the elongation of prehyphae was strongly presumed to occur through fission but without separation of the two sister cells, in contrast to blastospore budding. During the morphotype transformation, the amount and composition of lipid droplets changed greatly, suggesting their important roles in these events. Overall, we provide a morphological and ultrastructural characterization of O. sinensis vegetative development within the hemocoel of T. xiaojinensis, identify and name the prehypha fungal cell type in entomopathogenic fungi for the first time, and conclude that O. sinensis infection causes sustained immunosuppression in T. xiaojinensis.

Enhanced Cordycepin Production in Caterpillar Medicinal Mushroom, Cordyceps militaris (Ascomycetes), Mutated by a Multifunctional Plasma Mutagenesis System.

A multifunctional plasma mutation system (MPMS) method was used to create high cordycepin-yielding mutations from wild Cordyceps militaris, which yielded many viable mutants, many of which produced more cordycepin compared to the wild strain. One particular mutant strain (GYS60) produced 7.883 mg/mL, which is much higher than those reported to date and is more than 20 times higher than that of the wild strain, whereas the cordycepin production of another viable mutant (GYS80) was almost zero. The extraction and purification of cordycepin, using the fermentation broth of C. militaris GYS60, was also investigated. Cordycepin was extracted by using AB-8 macroporous resin and purified by using reversed-phase column chromatography. When the sample was adsorbed onto the macroporous resin, 20% ethanol was used as the desorption solvent yielding various fractions. The fractions containing cordycepin were loaded onto a reversed-phase chromatography column packed with octadecyl bonded silica as the stationary phase and ethanol (95%)/acetic acid solution (5%) at pH 6.0 as the mobile phase. The combination of this two-step extraction-purification process yielded cordycepin at 95% purity with a total recovery rate of 90%.