Enhancement of Cordycepin Production through Liquid Static Fermentation of Caterpillar Medicinal Mushroom Cordyceps militaris (Ascomycetes).

Cordycepin (3'-deoxyadenosine) is a bioactive nucleoside analog synthesized by Cordyceps militaris. Liquid fermentation of C. militaris by addition in different concentrations of five additives singly was evaluated. Glycine at 15.00 g/L after 20 d enhanced the cordycepin of 1773.33 mg/L (15-fold increment over control). Adenine at 4.00 g/L and 6.00 g/L in the liquid media showed significantly higher cordycepin i.e.1596.66 mg/L and 1550.00 mg/L (3-fold increment over control) after 40 d. Tryptone supplementation 14.00 g/L significantly higher cordycepin 784.33 mg/L (6.70-fold increment over control) and 912.66 mg/L production after 20 and 40 d of inoculation. Peanut oil at 10.00 g/L produced 585.66 mg/L (5-fold increment over control) cordycepin after 20 d and after 40 d, also addition of peanut oil at 20.00 g/L and 30.00 g/L in the media showed 631.66 and 624.31 mg/L cordycepin content. Supplementation of mono-sodium glutamate at 0.30 g/L produced significantly highest cordycepin i.e. 614 mg/L and 635.00 mg/L cordycepin after 20 and 40 d, respectively.

Evidence for Regulation of Cordycepin Biosynthesis by Transcription Factors Krüppel-Like Factor 4 and Retinoid X Receptor Alpha in Caterpillar Medicinal Mushroom Cordyceps militaris (Ascomycetes).

Cordyceps militaris, Chinese traditional medicinal fungus, has many bioactive properties. Cordycepin (3'-deoxyadenosine) is a major bioactive component of C. militaris. Various methods can significantly elevate cordycepin production, which suggests a diverse set of metabolic regulatory mechanisms. Thus, we aimed to identify transcription factors that regulate cordycepin biosynthesis pathways. Transcriptome analysis of wild-type C. militaris, C. militaris GYS60, a cordycepin high-producing strain, and C. militaris GYS80, a low-producing strain, were used to measure expression and function of genes related to cordycepin biosynthesis. The transcriptome expression data were confirmed by quantitative real-time polymerase chain reaction. We identified 155 relevant transcription factors in 19 families that included Fork head/winged helix factors, other C4 zinc finger-type factors, C2H2 zinc finger factors, tryptophan cluster factors, nuclear receptors with C4 zinc fingers, homeodomain factors, and Rel homology region factors. Energy generation and amino acid conversion pathways were activated in GYS60 so that abundance of cordycepin precursors was increased. Genes and transcription factors for rate-limiting enzymes in these pathways were identified. Overexpression of two key transcription factors, Kruppel-like factor 4 (Klf4) and Retinoid X receptor alpha (Rxra), promoted high cordycepin production in GYS60. In GYS60, Klf4 and Rxra were responsible for upregulation of genes in cordycepin biosynthesis, namely an oxidoreductase, 3',5'-cyclic AMP phosphodiesterase, a transferase, and adenylate cyclase. Upregulation of these genes increased 3'-AMP content, thereby elevating cordycepin synthesis.

Holistic transcriptional responses of Cordyceps militaris to different culture temperatures.

Cordyceps militaris is a medicinal entomopathogenic fungus containing valuable biometabolites for pharmaceutical applications. Its genetic inheritance and environmental factors play a crucial role in the production of biomass enriched with cordycepin. While temperature is a crucial controlled parameter for fungal cultivation, its impacts on growth and metabolite biosynthesis remains poorly characterized. This study aimed to investigate the metabolic responses and cordycepin production of C. militaris strain TBRC6039 under various temperature conditions through transcriptome analysis. Among 9599 expressed genes, 576 genes were significantly differentially expressed at culture temperatures of 15 and 25 °C. The changes in the transcriptional responses induced by these temperatures were found in several metabolisms involved in nutrient assimilation and energy source, including amino acids metabolism (e.g., glycine, serine and threonine metabolism) and lipid metabolism (e.g., biosynthesis of unsaturated fatty acids and steroid biosynthesis). At the lower temperature (15 °C), the biosynthetic pathways of lipids, specifically ergosterol and squalene, were the target for maintaining membrane function by transcriptional upregulation. Our study revealed the responsive mechanisms of C. militaris in acclimatization to temperature conditions that provide an insight on physiological manipulation for the production of metabolites by C. militaris.

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.

Improvement of cordycepin production by an isolated Paecilomyces hepiali mutant from combinatorial mutation breeding and medium screening.

Cordycepin is a major bioactive compound found in Cordyceps sinensis that exhibits a broad spectrum of biological activities. Here a Paecilomyces hepiali OR-1 strain was initially isolated from plateau soil for the bioproduction of cordycepin. Subsequently, strain modification including 60Co γ-ray and ultraviolet irradiation were employed to increase the cordycepin titer, resulted in a high-yield mutant strain P. hepiali ZJB18001 with the cordycepin content of 0.61 mg/gDCW, showing a 2.3-fold to that from the wild strain (0.26 mg/gDCW). Furthermore, medium screening based on Box-Behnken design and the response surface methodology facilitated the enhancement of cordycepin yield to the value of 0.96 mg/gDCW at 25 °C for 5 days in submerged cultivation with an optimized medium composition. The high cordycepin yield, rapid growth rate and stable genetic characteristics of P. hepiali ZJB18001 are beneficial in terms of costs and time for the industrialization of cordycepin production.

Radical Dehalogenation and Purine Nucleoside Phosphorylase E. coli: How Does an Admixture of 2',3'-Anhydroinosine Hinder 2-fluoro-cordycepin Synthesis.

During the preparative synthesis of 2-fluorocordycepin from 2-fluoroadenosine and 3'-deoxyinosine catalyzed by E. coli purine nucleoside phosphorylase, a slowdown of the reaction and decrease of yield down to 5% were encountered. An unknown nucleoside was found in the reaction mixture and its structure was established. This nucleoside is formed from the admixture of 2',3'-anhydroinosine, a byproduct in the preparation of 3-'deoxyinosine. Moreover, 2',3'-anhydroinosine forms during radical dehalogenation of 9-(2',5'-di-O-acetyl-3'-bromo- -3'-deoxyxylofuranosyl)hypoxanthine, a precursor of 3'-deoxyinosine in chemical synthesis. The products of 2',3'-anhydroinosine hydrolysis inhibit the formation of 1-phospho-3-deoxyribose during the synthesis of 2-fluorocordycepin. The progress of 2',3'-anhydroinosine hydrolysis was investigated. The reactions were performed in D2O instead of H2O; this allowed accumulating intermediate substances in sufficient quantities. Two intermediates were isolated and their structures were confirmed by mass and NMR spectroscopy. A mechanism of 2',3'-anhydroinosine hydrolysis in D2O is fully determined for the first time.

Investigation of 2'-Deoxyadenosine-Derived Adducts Specifically Formed in Rat Liver and Lung DNA by N'-Nitrosonornicotine Metabolism.

The International Agency for Research on Cancer has classified the tobacco-specific nitrosamines N'-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) as "carcinogenic to humans" (Group 1). To exert its carcinogenicity, NNN requires metabolic activation to form reactive intermediates which alkylate DNA. Previous studies have identified cytochrome P450-catalyzed 2'-hydroxylation and 5'-hydroxylation of NNN as major metabolic pathways, with preferential activation through the 5'-hydroxylation pathway in some cultured human tissues and patas monkeys. So far, the only DNA adducts identified from NNN 5'-hydroxylation in rat tissues are 2-[2-(3-pyridyl)-N-pyrrolidinyl]-2'-deoxyinosine (Py-Py-dI), 6-[2-(3-pyridyl)-N-pyrrolidinyl]-2'-deoxynebularine (Py-Py-dN), and N6-[4-hydroxy-1-(pyridine-3-yl)butyl]-2'-deoxyadenosine (N6-HPB-dAdo) after reduction. To expand the DNA adduct panel formed by NNN 5'-hydroxylation and identify possible activation biomarkers of NNN metabolism, we investigated the formation of dAdo-derived adducts using a new highly sensitive and specific liquid chromatography-nanoelectrospray ionization-high-resolution tandem mass spectrometry method. Two types of NNN-specific dAdo-derived adducts, N6-[5-(3-pyridyl)tetrahydrofuran-2-yl]-2'-deoxyadenosine (N6-Py-THF-dAdo) and 6-[2-(3-pyridyl)-N-pyrrolidinyl-5-hydroxy]-2'-deoxynebularine (Py-Py(OH)-dN), were observed for the first time in calf thymus DNA incubated with 5'-acetoxyNNN. More importantly, Py-Py(OH)-dN was also observed in relatively high abundance in the liver and lung DNA of rats treated with racemic NNN in the drinking water for 3 weeks. These new adducts were characterized using authentic synthesized standards. Both NMR and MS data agreed well with the proposed structures of N6-Py-THF-dAdo and Py-Py(OH)-dN. Reduction of Py-Py(OH)-dN by NaBH3CN led to the formation of Py-Py-dN both in vitro and in vivo, which was confirmed by its isotopically labeled internal standard [pyridine-d4]Py-Py-dN. The NNN-specific dAdo adducts Py-THF-dAdo and Py-Py(OH)-dN formed by NNN 5'-hydroxylation provide a more comprehensive understanding of the mechanism of DNA adduct formation by NNN.

[Advances in biosynthesis of cordycepin from Cordyceps militaris].

Cordycepin as the main active ingredient of Cordyceps militaris, a traditional medicinal fungus in China, has many physiological functions such as anti-cancer, anti-tumor and anti-virus activity. The most potential route for effective cordycepin production has been considered as liquid fermentation of C. militaris though with low productivity at present. Thus, it is urgent to apply both process engineering strategy and metabolic engineering strategy to enhance the productivity of cordycepin. In this review, the effects of medium components (i.e. the carbon/nitrogen source, precursor substances and metal ions) and operation factors (i.e. pH, dissolved oxygen and light) on cordycepin biosynthesis in liquid fermentation system are summarized. Besides, separation of cordycepin, the gene cluster involved and predicted biosynthesis pathways of cordycepin are also discussed, providing possible solutions of finally realizing efficient production of cordycepin.

Synthesis of cordycepin: Current scenario and future perspectives.

Cordyceps genus, such as C. militaris and C. kyushuensis, is a source of a rare traditional Chinese medicine that has been used for the treatment of numerous chronic and malignant diseases. Cordycepin, 3'-deoxyadenosine, is a major active compound found in most Cordyceps. Cordycepin exhibits a variety of biological activities, including anti-tumor, immunomodulation, antioxidant, and anti-aging, among others, which could be applied in health products, medicine, cosmeceutical etc. fields. This review focuses on the synthesis methods for cordycepin. The current methods for cordycepin synthesis involve chemical synthesis, microbial fermentation, in vitro synthesis and biosynthesis; however, some defects are unavoidable and the production is still far from the demand of cordycepin. For the future study of cordycepin synthesis, based on the illumination of cordycepin biosynthesis pathway, genetical engineering of the Cordyceps strain or introducing microbes by virtue of synthetic biology will be the great potential strategies for cordycepin synthesis. This review will aid the future synthesis of the valuable cordycepin.

Overexpression of ribonucleotide reductase small subunit, RNRM, increases cordycepin biosynthesis in transformed Cordyceps militaris.

Cordycepin was the first adenosine analogue used as an anticancer and antiviral agent, which is extracted from Cordyceps militaris and hasn't been biosynthesized until now. This study was first conducted to verify the role of ribonucleotide reductases (RNRs, the two RNR subunits, RNRL and RNRM) in the biosynthesis of cordycepin by over expressing RNRs genes in transformed C. militaris. Quantitative real-time PCR (qRT-PCR) and western blotting results showed that the mRNA and protein levels of RNR subunit genes were significantly upregulated in transformant C. militaris strains compared to the control strain. The results of the HPLC assay indicated that the cordycepin was significantly higher in the C. militaris transformants carrying RNRM than in the wild-type strain, whereas the RNRML was preferentially downregulated. For the C. militaris transformant carrying RNRL, the content of cordycepin wasn't remarkably changed. Furthermore, we revealed that inhibiting RNRs with Triapine (3-AP) almost abrogated the upregulation of cordycepin. Therefore, our results suggested that RNRM can probably directly participate in cordycepin biosynthesis by hydrolyzing adenosine, which is useful for improving cordycepin synthesis and helps to satisfy the commercial demand of cordycepin in the field of medicine.

Expression, purification and characterization of 5'-nucleotidase from caterpillar fungus by efficient genome-mining.

5'- nucleotidase (5'-NT) is a key enzyme in nucleoside/nucleotide metabolic pathway, it plays an important role in the biosynthesis of cordycepin in caterpillar fungus. In this study, a 5'-NT gene was identified and mined from genomic DNA of caterpillar fungus, which was 1968 bp in length and encoded 656 amino acid residues. The recombinant 5'-NT was first time heterologously expressed in Pichia pastoris GS115, subsequently purified and functionally characterized. The optimal reaction temperature for 5'-NT was 35 °C, and it retained 52.8% of its residual activity after incubation at 50 °C for 1 h. The optimal reaction pH was 6.0 and it exhibited high activity over a neutral pH range. Furthermore, 5'-NT exhibited excellent Km (1.107 mM), Vmax (0.113 µmol/mg·min) and kcat (4.521 S-1) values compared with other typical 5'-nucleotidase. Moreover, substrate specificity analyses indicated that 5'-NT exhibited different phosphatase activity towards the substrates containing different basic groups. The work presented here could be useful to 5'-NT applications and provide more scientific basis and new ideas for the biosynthesis of artificial control cordycepin.

Alternative metabolic routes in channeling xylose to cordycepin production of Cordyceps militaris identified by comparative transcriptome analysis.

The responsive mechanism of C. militaris TBRC7358 on xylose utilization was investigated by comparative analysis of transcriptomes, growth kinetics and cordycepin productions. The result showed that the culture grown on xylose exhibited high production yield of cordycepin on dry biomass. Comparing xylose to other carbon sources, a set of significantly up-regulated genes in xylose were enriched in pentose and glucuronate interconversion, and cordycepin biosynthesis. After validating up-regulated genes using quantitative real-time PCR, interestingly, putative alternative 3'-AMP-associated metabolic route on cordycepin biosynthesis was identified. Through reporter metabolites analysis of C. militaris, significant metabolites (e.g., AMP, glycine and L-glutamate) were identified guiding involvement of growth and cordycepin production. These findings suggested that there was a cooperative mechanism in transcriptional control of the supplying precursors pool directed towards the cordycepin biosynthesis through main and putative alternative metabolic routes for leverage of cell growth and cordycepin production on xylose of C. militaris strain TBRC7358.

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%.

An Efficient Strategy for Enhancement of Bioactive Compounds in the Fruit Body of Caterpillar Medicinal Mushroom, Cordyceps militaris (Ascomycetes), by Spraying Biotic Elicitors.

Cordyceps militaris is a mushroom species with high nutritive and medicinal values based on diverse bioactive metabolites. The contents of bioactive ingredients are indicative of the quality of commercially available fruit body of this fungus. Although the application of biotic elicitors has been an efficient strategy to induce the accumulation of valuable bioactive compounds in vivo, related research in C. militaris is rarely reported. In this study, five biotic elicitors in different concentrations (0.05, 0.5, 1, and 2 mg/mL), including chitosan (CHT), 2,4-dichlorophenoxyacetic acid (2,4-D), methyl jasmonate (MeJA), gibberellic acid (GA), and triacontanol (TRIA), were first introduced to enhance the production of 10 kinds of major bioactive components in the fruit body of C. militaris. Results showed that the effect of biotic elicitors on bioactive compounds in the fruit body of C. militaris was elicitor-specific and concentration-dependent. Overall, 1 mg/L CHT was considered the most favorable for the production of 10 bioactive ingredients in C. militaris fruit body, which could increase the content of protein, polysaccharides, polyphenol, triterpenoids, flavonoids, cordyceps acid, cordycepin, and anthocyanins by 20.38-, 1.41-, 0.7-, 0.47-, 11.90-, 1.09-, 0.34-, and 2.64-fold, respectively, compared with the control. The results of this study would provide an efficient strategy for the production of a superior quality fruit body of and contribute to further elucidation of the effects of biotic elicitors on metabolite accumulation in C. militaris.

Neutral Lipid Content in Lipid Droplets: Potential Biomarker of Cordycepin Accumulation in Cordycepin-Producing Fungi.

To clarify the relationship between neutral lipid content and cordycepin accumulation in Cordyceps militaris, mutants were generated from mixed spores of two C. militaris strains with varying cordycepin-producing capacities. Fifteen stable mutants producing from 0.001 to 2.363 mg/mL cordycepin were finally selected. The relative fluorescence intensities of the 15 mutants, two C. militaris strains and an Aspergillus nidulans strain at different concentrations of lyophilized mycelium powder were then investigated using the Nile red method. The mutant CM1-1-1 with the highest relative fluorescence intensity among the eighteen strains was selected for optimizing the Nile red method. Relative fluorescence intensity was linearly correlated with cordycepin concentration in liquid broth (R2 = 0.9514) and in lyophilized mycelium powder (R2 = 0.9378) for the 18 cordycepin-producing strains under identical culture conditions and with cordycepin concentration in liquid broth (R2 = 0.9727) and in lyophilized mycelium powder (R2 = 0.9613) for CM1-1-1 under eight different sets of conditions. In addition, the cordycepin content in lyophilized mycelium powder measured by the Nile red method was linearly correlated with that determined by an HPLC method (R2 = 0.9627). In conclusion, neutral lipids in lipid droplets are required during cordycepin accumulation; these neutral lipids are potential biomarkers of cordycepin biosynthesis.

Improved Cordycepin Production by Cordyceps militaris KYL05 Using Casein Hydrolysate in Submerged Conditions.

Cordycepin, a beneficial bioactive product specifically found in Cordyceps, has received attention in various bioindustrial applications such as in pharmaceuticals, functional foods, and cosmetics, due to its significant functions. However, low productivity of cordycepin is a barrier to commercialization. In this study, Cordyceps militaris was mutated by UV irradiation to improve the cordycepin production. The highest producer KYL05 strain was finally selected and its cordycepin production was increased about 1.5-fold compared to wild type. In addition, the effects of culture conditions were fundamentally investigated. Optimal conditions were as follows: pH 6, temperature of 25 °C, shaking speed of 150 rpm, and culture time of 6 days. Effects of medium component on cordycepin production were also investigated by using various carbon and nitrogen sources. It was found that glucose and casein hydrolysate (CH) were most effective as carbon and nitrogen sources in cordycepin production (2.3-fold improvement) with maximum cordycepin production of about 445 mg/L. In particular, production was significantly affected by CH. These results should be of value in improving the efficiency of mass production of cordycepin.

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.

Streptococcus suis synthesizes deoxyadenosine and adenosine by 5'-nucleotidase to dampen host immune responses.

Streptococcus suis is a major porcine bacterial pathogen and emerging zoonotic agent. S. suis 5'-nucleotidase is able to convert adenosine monophosphate to adenosine, resulting in inhibiting neutrophil functions in vitro and it is an important virulence factor. Here, we show that S. suis 5'-nucleotidase not only enables producing 2'-deoxyadenosine from 2'-deoxyadenosine monophosphate by the enzymatic assay and reversed-phase high performance liquid chromatography (RP-HPLC) analysis in vitro, but also synthesizes both 2'-deoxyadenosine and adenosine in mouse blood in vivo by RP-HPLC and liquid chromatography with tandem mass spectrometry analyses. Cellular cytotoxicity assay and Western blot analysis indicated that the production of 2'-deoxyadenosine by 5'-nucleotidase triggered the death of mouse macrophages RAW 264.7 in a caspase-3-dependent way. The in vivo infection experiment showed that 2'-deoxyadenosine synthesized by 5'-nucleotidase caused monocytopenia in mouse blood. The in vivo transcriptome analysis in mouse blood showed the inhibitory effect of 5'-nucleotidase on neutrophil functions and immune responses probably mediated through the generation of adenosine. Taken together, these findings indicate that S. suis synthesizes 2'-deoxyadenosine and adenosine by 5'-nucleotidase to dampen host immune responses, which represents a new mechanism of S. suis pathogenesis.

Uncovering global metabolic response to cordycepin production in Cordyceps militaris through transcriptome and genome-scale network-driven analysis.

The cellular metabolic adaptations of Cordyceps militaris have been progressively studied. In particular, the cordycepin pathway is of interest in medicinal applications. Even though the metabolic pathways for cordycepin production are known to be related to different carbon sources, the regulatory mechanisms at a systems level are poorly characterized. To explore the regulatory mechanisms, this study therefore aimed to investigate the global metabolic response to cordycepin production in C. militaris through transcriptome analysis and genome-scale network-driven analysis. Here, transcriptome analysis of 16,805 expressed genes in C. militaris strain TBRC6039 grown on different carbon sources was performed. Of these genes, 2,883 were significantly differentially expressed genes, uncovering sucrose- and glucose-mediated changes in the transcriptional regulation of central carbon metabolism in C. militaris, which was shown using the CmSNF1 mechanism as an example. After applying genome-scale metabolic network-driven analysis, reporter metabolites and key metabolic subnetworks involving adenosine, cordycepin and methionine were proposed through the up-regulation of cordycepin biosynthetic genes. Our findings suggest that the transcriptional regulation of these pathways is a ubiquitous feature in response to specific culture conditions during cordycepin overproduction.

Optimal conditions for cordycepin production in surface liquid-cultured Cordyceps militaris treated with porcine liver extracts for suppression of oral cancer.

Cordycepin is one of the most crucial bioactive compounds produced by Cordyceps militaris and has exhibited antitumor activity in various cancers. However, industrial production of large amounts of cordycepin is difficult. The porcine liver is abundant in proteins, vitamins, and adenosine, and these ingredients may increase cordycepin production and bioconversion during C. militaris fermentation. We observed that porcine liver extracts increased cordycepin production. In addition, air supply (2 h/d) significantly increased the cordycepin level in surface liquid-cultured C. militaris after 14 days. Moreover, blue light light-emitting diode irradiation (16 h/d) increased cordycepin production. These findings indicated that these conditions are suitable for increasing cordycepin production. We used these conditions to obtain water extract from the mycelia of surface liquid-cultured C. militaris (WECM) and evaluated the anti-oral cancer activity of this extract in vitro and in vivo. The results revealed that WECM inhibited the cell viability of SCC-4 oral cancer cells and arrested the cell cycle in the G2/M phase. Oxidative stress and mitochondrial dysfunction (mitochondrial fission) were observed in SCC-4 cells treated with WECM for 12 hours. Furthermore, WECM reduced tumor formation in 7,12-dimethylbenz[a]anthracene-induced hamster buccal pouch carcinogenesis through the downregulation of proliferating cell nuclear antigen, vascular endothelial growth factor, and c-fos expression. The results indicated that porcine liver extracts irradiated with blue light light-emitting diode and supplied with air can be used as a suitable medium for the growth of mycelia and production of cordycepin, which can be used in the treatment of oral cancer.