Cordycepin, a nucleoside analog, is widely used in medicine and health products. However, the production of cordycepin from Cordyceps militaris faces the challenges of low productivity and high rate of greenhouse gas emissions. In this study, by optimizing the cordycepin biosynthesis pathway through promoter combination, Kozak sequence, and enzyme fusion, enhancing the methanol assimilation capacity in peroxisomes, adjusting the synthesis of NADPH and ATP, and combining the enhanced supply of adenosine and 3\'-AMP, the cordycepin high-yield strain Pp29 was constructed, which produced 1551.44 mg/L cordycepin by shake-flask fermentation. In fed-batch fermentation, Pp29 achieved the highest yield (8.11 g/L, 67.64 mg/g DCW, and 1.35 g/L/d) to date in 10 L fermenter, and the CO2-eq emissions were 1.9-17.3 times lower than C. militaris and other yeast systems. This study provide basis for Pichia pastoris to be used as chassis cell for synthesizing cordycepin and other nucleoside analogs by methanol as carbon source.

Cordyceps militaris, a medicinal fungus, has gained considerable attention owing to its potential health benefits, notably the production of bioactive compounds such as cordycepin. Cordycepin possesses significant antifungal, antibacterial, and antiviral properties. The present study focused on optimizing the fermentation conditions for C. militaris to boost the production of mycelia and cordycepin, alongside investigating its antifungal properties using in silico and in vitro approaches. The optimal conditions, yielding the highest cordycepin and mycelial biomass, were a temperature of 20°C and a pH range of 4-6, with glucose and sucrose as carbon sources and yeast extract and casein hydrolysate as nitrogen sources. Under these conditions, cordycepin production peaked at low pH (600-1000 mg/L) and with carbon and maltose (400-500 mg/L). The low temperature favored cordycepin production (400 mg/L), whereas casein hydrolysate as a nitrogen source boosted cordycepin yield (600 mg/L). The docking analysis indicated that cordycepin had the highest binding affinity for the tubulin beta chain 2 (-10.4 kcal/mol) compared to the fungicide tebuconazole (-7.9 kcal/mol for both targets). The in silico results were corroborated by in vitro studies, where the mycelial extract of C. militaris inhibited approximately 75% of fungal growth at a concentration of 6000 ppm. These findings suggest that optimizing fermentation conditions significantly enhances cordycepin production, and cordycepin shows antifungal solid activity, making it a promising agent for biocontrol in agriculture.

To explain the effect and mechanism of cordycepin (COR) in resisting acute kidney injury (AKI). Network pharmacology was employed to analyze the correlations between COR, AKI, and pyroptosis, as well as the action target of COR. A mouse model of AKI was established by ischemia reperfusion injury (IRI), and after treatment with COR, the renal function, tissue inflammatory cytokine levels, and pyroptosis-related signals were detected in mice. In in-vitro experiments, damage of renal macrophages was caused by the oxygen-glucose deprivation model, and pyroptosis indicators and inflammatory cytokine levels were assayed after COR treatment. Network pharmacological analysis revealed that nuclear factor kappa-B (NF-κB) was the primary action target of COR and that COR could inhibit kidney injury and tissue inflammation during IRI by inhibiting NF-κB-mediated gasdermin D cleavage. When NF-κB was inhibited, the effect of COR was weakened. COR in renal macrophages could inhibit pyroptosis and lower the levels of inflammatory cytokines, whose effect was associated with NF-κB. Our study finds that COR can play an anti-inflammatory role and inhibit the progression of AKI through the NF-κB-mediated pyroptosis, which represents its nephroprotective mechanism.

Cordyceps genus is entomopathogenic mushrooms that have traditionally been used in ethnomedicine in Asian countries. Nucleosides (Ns), nucleotide(Nt), Nucleobases (Nb) and their analogues play a critically physiological role and have a great potential in drug development, such as pentostatin and cordycepin (COR). Due to their significance bioactivity, several Nt/Ns were used as markers for quality evaluation for medicinal Cordyceps, including adenosine, inosine, guanosine, uridine and COR. Among them, COR is the most considerable adenosine analogue, exhibiting significant therapeutic potential and has many intracellular targets. Nt/Ns contains polar compounds and the phosphate groups of Nt deprotonate and carry negative charges with a broad range of pH values. Recent years, various advanced methods of extraction and separation, and nanomaterials have been developed to extract, isolate and determine these molecules, such as ultrasound-assisted extraction (UAE), Supercritical fluid extraction (SFE) and pressurized liquid extraction (PLE) for the extraction, the solid phase extraction (SPE) methods (microextraction SPE (SPME), magnetic SPE (MSPE), and unique SPE materials based on the boronate affinity for the separation, and chromatography methods employing ultraviolet (UV), fluorescence, MS detection and electrospray ionization (ESI), along with matrix-assisted laser desorption/ ionization (MALDI) for the determination. COR derived from adenosine and its structure is very similar to that of 2\'-deoxyadenosine (2\'-dA) and adenosine, resulting in an incorrect identification, which will influence its therapeutic effects. Therefore, this review primarily focused on the characteristics of Nt/Ns, the advanced methods, strategies, nanomaterials for extracting and determining Nt/Ns (COR in particular) in Cordyceps spp, as well as the methods for distinguishing COR from its structure analogs.

Cordyceps militaris infects insects and forms sclerotia within the insect remains, establishing insect-microbe complexes. Here, C. militaris sclerotia samples from a single location in China over a 5-year period were subjected to high-throughput DNA sequencing, and the core microbes (which were stably enriched in the sclerotia over the 5 years) were identified. Next, seven bacterial strains were isolated from the C. militaris sclerotia, their biochemical characteristics were assessed, and they were co-cultured with C. militaris to study their effects on C. militaris metabolite production and biomass. Furthermore, the effects of NH4, NO3, and peptone media on C. militaris were compared. The results showed that Rhodococcus, Phyllobacterium, Pseudomonas, Achromobacter, Ensifer, Stenotrophomonas, Sphingobacterium, Variovorax, and Acinetobacter were the core microbes. Although co-culture of C. militaris with the seven bacterial strains isolated from the sclerotia did not directly increase the cordycepin level, they all had NO3 reduction ability, and four had urea decomposition ability. Meanwhile, C. militaris in NH4 medium had an increased cordycepin level compared to C. militaris in the other two media. From this, we inferred that bacteria in the sclerotia can convert NO3 to NH4, and then cordycepin is produced using NH4, which was confirmed by RNA-seq and real-time fluorescence quantitative PCR. Thus, bacteria in the sclerotia may indirectly affect the C. militaris metabolite production by regulating nitrogen metabolism. In summary, there are stable core microbes in the C. militaris sclerotia, and they may directly and indirectly affect the growth and metabolite production of C. militaris.
OBJECTIVE: The model Cordyceps species Cordyceps militaris is rich in therapeutic compounds. It has recently been demonstrated that symbiotic microbes in sclerotia affect Cordyceps\' growth, development, and secondary metabolite production. In this study, core microbes were identified based on C. militaris sclerotia samples obtained from the same site over 5 years. Additionally, bacterial strains isolated from C. militaris sclerotia were found to affect metabolite production and nitrogen utilization, based on functional tests. Moreover, based on the bacterial nitrogen metabolism capacity in the sclerotia and its influence on C. militaris metabolite production, we deduced that bacteria in the sclerotia can indirectly affect C. militaris metabolite production by regulating nitrogen metabolism. This is the first report on how bacteria in the sclerotia affect C. militaris metabolite production from the perspective of the nitrogen cycle. The results increase our understanding of microbial functions in C. militaris sclerotia.

Neospora caninum (N. caninum) is an obligate intracellular Apicomplexa parasite that causes abortions in dairy cows and incurs substantial to significant economic losses in the global dairy farming industry. Cordycepin, a nucleoside antibiotic derived from Chinese medicine Cordyceps militaries, exhibits diverse biological activities. However, it remains unclear whether cordycepin possesses inhibitory effects against N. caninum infection. Therefore, this study aimed to establish both in vivo and in vitro models of N. caninum to investigate the potential impact of cordycepin against N. caninum infection. We successfully established an in vitro model of N. caninum infection in RAW264.7 cells, followed by qRT- PCR analysis to detect the content of N. caninum DNA within the cells. The effects of cordycepin on N. caninum was observed using the Giemsa method on RAW264.7, and the rate of cell infection was calculated. Cordycepin exhibited inhibitory effects on N. caninum tachyzoites in vitro, preserving cellular integrity and reducing the rate of cell infection. In mice, we established an in vivo model of N. caninum infection and detected N. caninum presence in tissues using. Real-time fluorescence quantitative PCR. Histopathological changes were observed through Hematoxylin-eosin staining. Liver function was assessed by using glutamic acid aminotransferase (ALT) and aspartic acid aminotransferase (AST) kits. Oxidative stress status was measured using catalase (CAT), malondialdehyde (MDA), and glutathione (GSH) kits. Compared with the model group, mice treated with cordycepin showed reduced clinical symptoms, increased food intake, and their body weight (P=0.0143, P=0.0068) was significantly higher than those in the model group. Furthermore, cordycepin treatment significantly alleviated hepatic cord disorders, hepatocellular swelling, detachment, and vacuolization; duodenal epithelial detachment and shortening of villi caused by N. caninum infection. Cordycepin administration reduced the increase in ALT (P=0.01, P=0.008) and AST (P<0.001) levels caused by N. caninum infection, while ameliorating hepatocyte swelling, necrosis, and detachment as well as inflammatory cell infiltration within mice liver; it also led to shortened or even disappeared duodenal villi along with and oedema of the submucosa. Analysis of oxidative stress showed that cordycepin ameliorated the damage caused by N. caninum by reducing MDA (P=0.03, P=0.02, P=0.005) and increasing CAT (P=0.004, P<0.001) and GSH (P=0.004, P<0.001) levels. In conclusion, this study reports for the first time on cordycepin\'s efficacy against N. caninum infection providing a potential candidate drug for neosporosis treatment.

Plastic pollution is an emerging environmental issue, with microplastics and nanoplastics raising health concerns due to bioaccumulation. This work explored the impact of polystyrene nanoparticle (PS-NPs) exposure during prepuberty on male reproductive function post maturation in rats. Rats were gavaged with PS-NPs (80 nm) at 0, 3, 6, 12 mg/kg/day from postnatal day 21 to 95. PS-NPs accumulated in the testes and reduced sperm quality, serum reproductive hormones, and testicular coefficients. HE staining showed impaired spermatogenesis. PS-NPs disrupted the blood-testis barrier (BTB) by decreasing junction proteins, inducing inflammation and apoptosis. Transcriptomics identified differentially expressed genes related to metabolism, lysosome, apoptosis, and TLR4 signaling. Molecular docking revealed Cordycepin could compete with polystyrene for binding to TLR4. Cordycepin alleviated oxidative stress and improved barrier function in PS-NPs treated Sertoli cells. In conclusion, prepubertal PS-NPs exposure induces long-term reproductive toxicity in male rats, likely by disrupting spermatogenesis through oxidative stress and BTB damage. Cordycepin could potentially antagonize this effect by targeting TLR4 and warrants further study as a protective agent. This study elucidates the mechanisms underlying reproductive toxicity of PS-NPs and explores therapeutic strategies.

Glioma is a serious primary malignant tumor of the human central nervous system with a poor prognosis and a high recurrence rate; however, inhibition of immune checkpoints can greatly improve the survival rate of patients. The purpose of this study was to investigate the regulation of PD-L1 by cordycepin and the mechanism of its anti-tumor action. The results of previous studies indicate that cordycepin has good anti-proliferative and anti-migratory activities and can induce apoptosis in U251 and T98G cells in vitro. Here, transcriptome sequencing showed that cordycepin may exert anti-tumor effects through the NOD-like receptor signaling pathway. Further intervention with BMS-1, a small molecule inhibitor of PD-L1, was used to explore whether inhibition of PD-L1 affected the regulation of the NOD-like receptor signaling pathway by cordycepin. Mechanistically, on the one hand, cordycepin regulated the expression of NFKB1 and STAT1 through the NOD-like receptor signaling pathway, thereby inhibiting the expression of PD-L1. In addition, inhibition of PD-L1 enhanced the regulation by cordycepin of the NOD-like receptor signaling pathway. On the other hand, cordycepin directly upregulated expression of STAT1 and downregulated that of PD-L1. In vivo studies further showed that cordycepin could downregulate expression of PD-L1 and NFKB1 and upregulate that of STAT1 in glioma xenograft tumor tissues, consistent with the results of in vitro studies. The results suggest that cordycepin may down-regulate the expression of PD-L1 through NOD-like receptor signaling pathway and NFKB signaling pathway, thereby inhibiting the immune escape of glioma, and can be developed as a PD-L1 inhibitor. Our results therefore provide a theoretical foundation for the use of cordycepin in treatment of glioma and enrich our understanding of its pharmacological mechanism.

Colon cancer has a poor clinical response to anti-PD1 therapy. This study aimed to evaluate the effect of cordycepin on the efficacy of anti-PD1 treatment in colon cancer. The viability of CT26 mouse colon carcinoma cells, cell-cycle progression, morphology, and the expression of mRNA and protein were assessed. A syngeneic animal model was established by implanting CT26 cells into BALB/c mice for in vivo experiments. Multi-parameter flow cytometry was used to analyze the splenic cell lineages and tumor microenvironment (TME). The in vitro data revealed that cordycepin, but not adenosine, inhibited CT26 cell viability. The protein, but not mRNA, expression levels of A2AR and A2BR were suppressed by cordycepin but not by adenosine in CT26 cells. The combination of cordycepin, but not adenosine, with anti-PD1 exhibited a greater tumor-inhibitory effect than anti-PD1 alone as well as inhibited the expression of A2AR and A2BR in splenic macrophages. In the TME, the combination of cordycepin and anti-PD1 increased the number of CD3+ T cells and neutrophils and decreased the number of natural killer (NK) cells. Overall, cordycepin augmented the antitumor effects of anti-PD1 against mouse colon carcinoma cells and inhibited the expression of the adenosine receptors A2AR and A2BR in splenic macrophages and intratumoral NK cells.

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.