Enhancing the production of a heterologous Trametes laccase (LacA) by replacement of the major cellulase CBH1 in Trichoderma reesei.

The laccases from white-rot fungi exhibit high redox potential in treating phenolic compounds. However, their application in commercial purposes has been limited because of the relatively low productivity of the native hosts. Here, the laccase A-encoding gene lacA of Trametes sp. AH28-2 was overexpressed under the control of the strong promoter of cbh1 (Pcbh1), the gene encoding the endogenous cellobiohydrolase 1 (CBH1), in the industrial workhorse fungus Trichoderma reesei. Firstly, the lacA expression cassette was randomly integrated into the T. reesei chromosome by genetic transformation. The lacA gene was successfully transcribed, but the laccase couldn't be detected in the liquid fermentation condition. Meanwhile, it was found that the endoplasmic reticulum-associated degradation (ERAD) was strongly activated, indicating that the expression of LacA probably triggered intense endoplasmic reticulum (ER) stress. Subsequently, the lacA expression cassette was added with the downstream region of cbh1 (Tcbh1) to construct the new expression cassette lacA::Δcbh1, which could replace the cbh1 locus in the genome via homologous recombination. After genetic transformation, the lacA gene was integrated into the cbh1 locus and transcribed. And the unfolded protein response (UPR) and ERAD were only slightly induced, for which the loss of endogenous cellulase CBH1 released the pressure of secretion. Finally, the maximum laccase activity of 168.3 U/l was obtained in the fermentation broth. These results demonstrated that the reduction of secretion pressure by deletion of endogenous protein-encoding genes would be an efficient strategy for the secretion of heterologous target proteins in industrial fungi. ONE-SENTENCE SUMMARY: The reduction of the secretion pressure by deletion of the endogenous cbh1 gene can contribute to heterologous expression of the laccase (LacA) from Trametes sp. AH28-2 in Trichoderma reesei.

Metabolomic comparison between natural Huaier and artificial cultured Huaier.

Vanderbylia robiniophila (Murrill) B.K. (Huaier) is a kind of higher fungal fruiting body that is parasitic on the trunk of Sophora japonica and Robinia pseudoacacia L.. As a traditional Chinese medicine with a history of more than 1,600 years, Huaier has attracted wide attention for its excellent anticancer activity. A systematic study on the metabolome differences between natural Huaier and artificial cultured Huaier was conducted using liquid chromatography-mass spectrometry in this study. Principal component analysis and orthogonal projection on latent structure-discriminant analysis results showed that cultured Huaier evidently separated and individually separated from natural Huaier, indicating metabolome differences between natural and cultured Huaier. Hierarchical clustering analysis was further performed to cluster the differential metabolites and samples based on their metabolic similarity. The higher contents of amino acids, alkaloids and terpenoids in natural Huaier make it an excellent choice as a traditional Chinese medicine for anticancer or nutritional supplementation. The results of the Bel-7,402 and A549 cell cytotoxicity tests showed that the anticancer activity of natural Huaier was better than that of cultured Huaier. This may be due to the difference in chemical composition, which makes the anticancer activities of natural and cultured Huaier different.

Medicinal Fungi with Antiviral Effect.

Pandemics from various viruses make natural organisms face challenges over and over again. Therefore, new antiviral drugs urgently need to be found to solve this problem. However, drug research and development is a very difficult task, and finding new antiviral compounds is desirable. A range of medicinal fungi such as Ganoderma lucidum and Cordyceps sinensis are widely used all over the world, and they can enhance human immunity and direct anti-virus activities and other aspects to play an antiviral role. Medicinal fungi are used as foods or as food supplements. In this review, the species of medicinal fungi with antiviral activity in recent decades and the mechanism of antiviral components were reviewed from the perspectives of human, animal, and plant viruses to provide a comprehensive theory based on better clinical utilization of medicinal fungi as antiviral agents.

Application of Metabolomics in Fungal Research.

Metabolomics is an essential method to study the dynamic changes of metabolic networks and products using modern analytical techniques, as well as reveal the life phenomena and their inherent laws. Currently, more and more attention has been paid to the development of metabolic histochemistry in the fungus field. This paper reviews the application of metabolomics in fungal research from five aspects: identification, response to stress, metabolite discovery, metabolism engineering, and fungal interactions with plants.

Dynamic content changes of cordycepin and adenosine and transcriptome in Cordyceps kyushuensis Kob at different fermentation stages.

20% (w/w) Astragali radix was added to the rice medium to cultivate C. kyushuensis Kob. The fermentation product was collected at mycelium stage, coloring stage, stromata-forming initial stage and fruiting body stage of C. kyushuensis Kob. The dynamic content changes of cordycepin and adenosine were detected at different fermentation stages. In the rice medium with Astragalus radix, both cordycepin and adenosine reached the highest content value on the 30th day of fermentation, 17.31 mg/g and 0.94 mg/g, respectively, which were 8.6 times and 2.0 times of that in rice medium at the same stage. At the same time, transcriptomics technology was used to analyze C. kyushuensis Kob during these four periods.

Systematic identification of arsenic-binding proteins reveals that hexokinase-2 is inhibited by arsenic.

Arsenic is highly effective for treating acute promyelocytic leukemia (APL) and has shown significant promise against many other tumors. However, although its mechanistic effects in APL are established, its broader anticancer mode of action is not understood. In this study, using a human proteome microarray, we identified 360 proteins that specifically bind arsenic. Among the most highly enriched proteins in this set are those in the glycolysis pathway, including the rate-limiting enzyme in glycolysis, hexokinase-1. Detailed biochemical and metabolomics analyses of the highly homologous hexokinase-2 (HK2), which is overexpressed in many cancers, revealed significant inhibition by arsenic. Furthermore, overexpression of HK2 rescued cells from arsenic-induced apoptosis. Our results thus strongly implicate glycolysis, and HK2 in particular, as a key target of arsenic. Moreover, the arsenic-binding proteins identified in this work are expected to serve as a valuable resource for the development of synergistic antitumor therapeutic strategies.

New flavonoid and their anti-A549 cell activity from the bi-directional solid fermentation products of Astragalus membranaceus and Cordyceps kyushuensis.

Astragalus membranaceus and Cordyceps kyushuensis were used to obtain Astragalus membranaceus-Cordyceps kyushuensis bi-directional solid fermentation products using the bi-directional solid fermentation technique. The fermentation products were isolated and purified to obtain 20 individual compounds, of which compound 1 was a novel isoflavane, and compounds 2, 3, and 4 were novel isoflavones, along with 16 known compounds. In vitro experiments demonstrated that compounds 4, 5, 8, 10, and 20 exhibited significant inhibitory activity against A549 lung cancer cells. Specifically, the IC50 value of the novel compound 4 was 53.4 µM, while the IC50 value of cordycepin was 9.0 µM.

Amelioration of hyperuricemia by cordycepin and Cordyceps militaris aqueous extract in mice via modulating gut microbiota and restoring metabolic profile.

In this study, we first screened and evaluated the inhibitory effects of seven medicinal fungi on diseases such as hyperuricemia (HUA). Then, using metabolomics and gut microbiome methods, the focus was on analyzing and evaluating the effects of the aqueous extract of Cordyceps. militaris (CME) and cordycepin on potassium oxyzinate induced HUA mice. It was found that CME exhibits good uric acid lowering activity in both in vivo and in vitro experiments. It can relieve hyperuricemia by inhibiting xanthine oxidase enzyme activity, reducing the production of xanthine precursors, and inhibiting insulin resistance. The uric acid-lowering efficacy of cordycepin in vivo is comparable to that of CME. The species abundance of Oscillibacter, Alistipes, Prevotellaaceae_NK3B31, Lachnospiraceae_NK4A136 were decreased after treatment with CME and cordycepin. The metabolomics analysis of cecal contents and fecal samples elucidated the mechanism of intervention of CME on hyperuricemia from different perspectives. This suggests that we should consider carefully when selecting samples. This current research provides the scientific foundation for the medicinal research of C. militaris and the maintenance of human health.

Neuroprotective Effects of Phenolic Antioxidant Tert-butylhydroquinone (tBHQ) in Brain Diseases.

Human life and health are gravely threatened by brain diseases. The onset and progression of the illnesses are influenced by a variety of factors, including pathogenic causes, environmental factors, mental issues, etc. According to scientific studies, neuroinflammation and oxidative stress play a significant role in the development and incidence of brain diseases by producing pro-inflammatory cytokines and oxidative tissue damage to induce inflammation and apoptosis. Neuroinflammation, oxidative stress, and oxidative stress-related changes are inseparable factors in the etiology of several brain diseases. Numerous neurodegenerative diseases have undergone substantial research into the therapeutic alternatives that target oxidative stress, the function of oxidative stress, and the possible therapeutic use of antioxidants. Formerly, tBHQ is a synthetic phenolic antioxidant, which has been widely used as a food additive. According to recent researches, tBHQ can suppress the processes that lead to neuroinflammation and oxidative stress, which offers a fresh approach to treating brain diseases. In order to achieve the goal of decreasing inflammation and apoptosis, tBHQ is a specialized nuclear factor erythroid 2-related factor (Nrf2) activator that decreases oxidative stress and enhances antioxidant status by upregulating the Nrf2 gene and reducing nuclear factor kappa-B (NF-κB) activity. This article reviews the effects of tBHQ on neuroinflammation and oxidative stress in recent years and looks into how tBHQ inhibits neuroinflammation and oxidative stress through human, animal, and cell experiments to play a neuroprotective role in Alzheimer's disease (AD), stroke, depression, and Parkinson's disease (PD). It is anticipated that this article will be useful as a reference for upcoming research and the creation of drugs to treat brain diseases.

A novel promising neuroprotective agent: Ganoderma lucidum polysaccharide.

Nervous system diseases (NSDs) are characterized by a wide range of symptoms, a complex pathophysiology, an unclear etiology, a great deal of variation in treatment response, and lengthy therapy cycles, all of which pose considerable hurdles to clinical treatment. A traditional valuable medicine known as Ganoderma lucidum (GL) has a significant role to play in preserving health and treating diseases. Ganoderma lucidum polysaccharides (GLP) is one of the cardinal effective active ingredients of GL, which has a number of pharmacological actions, including liver protection, immune regulation, antioxidant activity, anticancer activity, antibacterial activity, and antiviral activity. Recently, studies on the structural characterization and biological functions of GLP were presented in this article to review the progress of researches about GLP on NSDs and summarize the potential mechanisms of action. These studies were anticipated to provide new research ideas for GLP as a novel promising neuroprotective agent and provide a reference for better development and utilization of GLP.

Cordyceps militaris extracts and cordycepin ameliorate type 2 diabetes mellitus by modulating the gut microbiota and metabolites.

Introduction: Cordyceps militaris, which has many potential medicinal properties, has rarely been reported to alleviate type 2 diabetes mellitus (T2DM). Methods: The effects of C. militaris extracts (CE) and cordycepin (CCS) on high-fat diet and streptozotocin (STZ) induced T2DM mice were analysed by gut microbiome and metabolomics methods in this study. Results: The results demonstrated that glucose and lipid metabolism parameters, oxidative stress biomarkers and inflammation cytokines were down-regulated in the CCS and CE groups. A comparative analysis of the fecal samples from mice in the model and experimental groups showed that experimental groups resulted in a higher abundance of Firmicutes/Bacteroidetes. Conclusion: This study provides evidence that C. militaris can be used as a food supplement to relieve T2DM, which provides a promising prospect for new functional food in it.

[Analysis and evaluation of different radial parts of Cordyceps kyushuensis by Fourier transform infrared spectroscopy].

Fourier-transform infrared spectroscopy (FTIR) and second derivative spectra were used to analyze and evaluate the different parts of Cordyceps kyushuensis Kob in the present work. The results showed that C. kyushuensis contained proteins, polysaccharides, nucleosides, lipids and other active ingredients, the single-dimensional IR spectra of the various parts were highly similar, the similarity coefficient between the cultured stroma and medium reached up to 0.992 7, and the natural stroma was more different from the parasites, with similarity coefficient of 0.949 9. Second derivative spectrum further enriched and confirmed the feature of corresponding spectrum peaks, proved the existence of active substances such as cordycepin and adenosine, and prompted the presence of alpha- and beta-glycosidic bonds. The diversity and complexity of chemical constituents in different parts of C. kyushuensis were synthetically described by IR spectra, which provided a fast, comprehensive and objective approach to the analysis and evaluation of the imperceptible differences, and the quality control of Cordyceps. This work supplies a theoretical basis for development and utilization of genetic resources C. kyushuensis.

Enediyne antitumor antibiotic maduropeptin biosynthesis featuring a C-methyltransferase that acts on a CoA-tethered aromatic substrate.

The enediyne antitumor antibiotic maduropeptin (MDP) is produced by Actinomadura madurae ATCC 39144. The biosynthetic pathway for the 3,6-dimethylsalicylic acid moiety of the MDP chromophore is proposed to be comprised of four enzymes: MdpB, MdpB1, MdpB2, and MdpB3. Based on the previously characterized biosynthesis of the naphthoic acid moiety of neocarzinostatin (NCS), we expected a biosynthetic pathway featuring carboxylic acid activation by the MdpB2 CoA ligase immediately before its coupling to an enediyne core intermediate. Surprisingly, the MDP aromatic acid biosynthetic pathway employs an unusual logic in which MdpB2-catalyzed CoA activation occurs before MdpB1-catalyzed C-methylation, demonstrating that MdpB1 is apparently unique in its ability to C-methylate a CoA-tethered aromatic acid. MdpB2 is a promiscuous CoA ligase capable of activating a variety of salicylic acid analogues, a property that could be potentially exploited to engineer MDP analogues.

Immunomodulatory and Antioxidant Effects of Polysaccharides from the Parasitic Fungus Cordyceps kyushuensis.

The ascomycete Cordyceps genus has been used as valued traditional Chinese medicine. Cordyceps kyushuensis is a unique species of Cordyceps, which parasitizes on the larvae of Clanis bilineata Walker, and its major component cordycepin and aqueous extract are known to have many pharmacological effects. However, the physiological function of water-soluble polysaccharides has not been explored in detail. In this study, to resolve these doubts, we extracted and separated Cordyceps-derived polysaccharides and then evaluated the immunomodulatory and antioxidant activities. Four polysaccharide fractions were purified from Cordyceps-cultured stroma by DEAE-cellulose 23 and Sephadex G-150 column chromatography. Basic structural information was elucidated on the basis of physicochemical property and spectroscopic evidences. The antioxidant activities were evaluated by a 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical method and protective effect of DNA damage. The qualified immunologic activities were also determined in vivo and in vitro. The polysaccharides could stimulate the proliferation of mouse splenocytes whether concanavalin A (ConA) and lipopolysaccharide (LPS) existed or not, strengthen peritoneal macrophages to devour neutral red, and increase the content of interleukin-2 (IL-2) and tumor necrosis factor-alpha (TNF-α) in serum. The research provides the corresponding evidence for Cordyceps polysaccharides as a potential candidate for functional foods and therapeutic agents.

Cordycepin and pentostatin biosynthesis gene identified through transcriptome and proteomics analysis of Cordyceps kyushuensis Kob.

Cordyceps kyushuensis is the only species of cordyceps growing on the larvae of Clanis bilineata Walker, and has been demonstrated that there are lots of pharmacological components including cordycepin. Cordycepin shows lots of pharmacological action but it could be converted to 3'-deoxyinosine by adenosine deaminase in vivo, which weakens the efficiency of cordycepin. That pentostatin, which has been reported to inhibit adenosine deaminase, combining cordycepin could enhance the efficiency of cordycepin in vivo. During transcriptome and proteomics analysis of Cordyceps kyushuensis, a single gene cluster including four genes we named ck1-ck4 which can synthesis both cordycepin and pentostatin has been identified using BLAST. Meanwhile, KEGG, KOG, GO analysis and differentially expressed genes were analyzed in transcriptome and proteomics. This study first sequenced transcriptome and proteomics of C. kyushuensis, and demonstrated that there is a single gene cluster related to biosynthesis of cordycepin and pentostatin, which can be employed to improve the yield of cordycepin and find more functional proteins.

Aqueous Extracts of Cordyceps kyushuensis Kob Induce Apoptosis to Exert Anticancer Activity.

Cancer has become the leading cause of mortality since 2010 in China. Despite the remarkable advances in cancer therapy, a low survival rate is still a burden to the society. The antineoplastic activity of aqueous extracts of Cordyceps kyushuensis Kob (AECK) was measured in this study. Results showed that AECK can significantly inhibit the proliferation and viability of U937 and K562 when treated with different concentrations of AECK, and the IC50 values of U937 and K562 were 31.23 µg/ml and 62.5 µg/ml, respectively. Hoechst 33258 staining showed that AECK could cause cell shrinkage, chromatin, condensation, and cytoplasmic blebbing, and DNA ladder experiment revealed the evident feature of DNA fragmentation which showed that AECK could induce cell apoptosis. Moreover, AECK gave rise to intrinsic apoptosis through increasing the amount of Ca2+ and downregulating the expression of Bcl-2. Meanwhile, the level of Fas death receptor was elevated which indicated that AECK could lead to exogenous apoptosis in U937. The expressions of oncogene c-Myc and c-Fos were suppressed which manifested that AECK could negatively regulate the growth, proliferation, and tumorigenesis of U937 cells. This research presented the primary antitumor activity of AECK which would contribute to the widely use of Cordyceps kyushuensis Kob as a functional food and medicine.

Genome shuffling improved the nucleosides production in Cordyceps kyushuensis.

Genome shuffling was first applied to improve the production of nucleosides in Cordyceps kyushuensis. Six improved strains were selected for genome shuffling by UV and HNO2 mutagenesis. Ten improved genome shuffling strains with good genetic stability were obtained, among which, the production of cordycepin in R6 was 9.624 times higher than that of the ancestor. While in R18 and R19, the yield of cordycepin, adenosine, guanosine and uridine were all increased greatly compared with the ancestor. Based on the four phenotypes of the content of cordycepin, adenosine, guanosine and uridine, hierarchical clustering analysis (HCA) and principal component analysis (PCA) were applied to infer the relationships between genome shuffling strains and mutants.

Cordycepin induces autophagy-mediated c-FLIPL degradation and leads to apoptosis in human non-small cell lung cancer cells.

Cordycepin, a main active composition extracted from Cordyceps militaris, has been reported to exert anti-tumor activity in a broad spectrum of cancer types. However, the function of cordycepin on human non-small cell lung cancer cells is still obscure. Our present work showed that cordycepin inhibited cell growth by inducing apoptosis and autophagy in human NSCLC cells. Further study revealed that cordycepin triggered extrinsic apoptosis associated with down-regulation of c-FLIPL which suppresses the activity of caspase-8. And ectopic expression of c-FLIPL dramatically prevented cordycepin-caused apoptosis. Meanwhile, cordycepin stimulated autophagy through suppressing mTOR signaling pathway in lung cancer cells. When autophagy was blocked by Atg5 siRNA or PI3K inhibitor LY294002, the levels of apoptosis caused by cordycepin were obviously attenuated. In addition, suppression of autophagy could also elevate the level of c-FLIPL which indicated cordycepin-triggered autophagy promoted the degradation of c-FLIPL. Therefore, we conclude that cordycepin induces apoptosis through autophagy-mediated downregulation of c-FLIPL in human NSCLC cells. Taken together, our findings provide a novel prospect on the anti-tumor property of cordycepin, which may further prompt cordycepin to serve as a promising therapeutic approach in NSCLC treatment.

Physicochemical characteristics of epichlorohydrin, pyridine and trimethylamine functionalized cotton stalk and its adsorption/desorption properties for perchlorate.

Amine-impregnated cotton stalk (AICS) prepared by the reaction of cotton stalk with epichlorohydrin, pyridine and trimethylamine was used as the effective adsorbent for perchlorate removal. Solid-state (13)C NMR spectra, FT-IR, BET principle and element analysis provided evidence that amine groups have been successfully introduced onto the surface of AICS. The adsorption capacity of perchlorate by AICS was about 83.8 mg g(-1) at 20 °C. It was decreased to 80.6 mg g(-1) as the temperature was increased to 40 °C, which implied an exothermic nature for this adsorption process. Perchlorate adsorption capacity in fixed-bed column was optimum at neutral condition (pH: 6.0, 70.8 mg g(-1)) with bed depth of 2.7 cm and flow rate of 5 ml min(-1). In addition, chemical regeneration by HCl or NaOH (0.1 mol L(-1)) achieved more than 95% of regeneration efficiency. Biological regeneration of the saturated AICS with mixed bacteria has shown its merit with regeneration and biological perchlorate destruction simultaneously although its regeneration efficiency was only 56.8-74.8%.