Introduction

An ancient Chinese medicinal fungus, Cordyceps contains a variety of bioactive components, like cordycepin, adenosine, polysaccharide, and ergosterol¹. Cordycepin, an adenosine derivative also known as 3′-deoxyadenosine, is a natural chemical majorly found in some species of the genus Cordyceps and is one of the most important active components. It offers several pharmacological activities, including anti-cancer, anti-inflammatory, anti-viral properties and many more. It has a wide range of mode of action as in case of cancer it inhibits the proliferation of the cancerous tissue by inducing apoptosis and in cases of viral diseases, it inhibits the replication process of the viruses that can cause the disease ^1,2. It also is proven to play a crucial role in cosmeceutical in anti-photoaging and anti-pigmentation ¹. However, the market price of this essential secondary metabolite has increased exponentially, thus the production of cordycepin has been achieved from the extracts of the fruiting body of C. militaris by solid state or liquid fermentation where the production period is comparatively shorter, and the conditions can be controlled ³. In the below review we are going to discuss about media optimization to produce high cordycepin in which we optimize temperature, pH, nitrogen source and carbon source and strain improvement techniques like irradiation with physical mutagens like UV rays, protoplast fusion and chemical mutagenesis. We are also going to discuss the modern strain improvement approaches involving transcriptomics, proteomics, and metabolomics.

Synthesis of Cordycepin

Various techniques used to synthesise cordycepin involve chemical, microbial, in vitro processes and biosynthesis The biosynthesis of cordycepin, in Cordyceps militaris is affected by elements, like the components of the operational factors and gene expression profiles ¹

Biosynthesis

The process of creating cordycepin through biosynthesis includes refining both masuds techniques along with to boost the production of cordycepin.

Liquid Fermentation

The promising method, for increasing cordycepin production is through fermentation of C. militaris although productivity levels remain relatively low at present. The content of Cordycepin (COR)  varies among strains making it crucial to prioritize screening for strains with COR content in COR production. Various optimization techniques have been explored to enhance the COR content in fermentation. These optimization options include screening for strains with COR content, ion beam irradiation, integrating additives in the medium, optimising composition, using two-stage shaking static culture methods, and using LED irradiation ¹

The goal was to reduce costs by around half and increase output by over 30% using a new reasonably priced liquid fermentation combination. Furthermore, by combining reflux extraction and resin adsorption, cordycepin was purified to 95.23%. Pupa powder and wheat bran were used in the liquid fermentation medium ⁴.

In another approach various methods such as single factor experiments Placket–Burman design, central composite design and response surface methodology were used to enhance the fermentation medium and culture conditions for boosting cordycepin content. The study focused on seven variables, with temperature, yeast extract and tryptone concentrations singled out as factors influencing cordycepin yield. After optimization the ideal medium consisted of 9.00 g/L of yeast extract and 17.10 g/L of tryptone resulting in a yield of 7.35 g/L in a 5 L fermenter, under culture conditions ⁵.

Optimizing cordycepin production, in Cordyceps militaris batch fermentation involved a two phase strategy that combined shaking and static fermentation. The growth of hyphae and conidiophores plays a role in enhancing cordycepin production with growth and production rates varying across the shake and static fermentation phases. By implementing the two step shake fermentation method there was an increase in C. militaris cell concentration, during the shaking phase, followed by successful cordycepin production during the static phase resulting in a final yield of 2.62 g/L ⁶.

Solid Fermentation

The process of producing COR through solid state fermentation (SSF) typically takes longer compared to fermentation lasting around 30 days. Despite the duration SSF can yield the fruiting body for obtaining COR. In contrast, to fermentation the COR content is primarily detected in the fruiting body when produced through SSF. Various optimizations have been applied to enhance COR content, such, as utilizing UVB and LED radiation with wavelengths adjusting pH levels controlling temperature, managing culture time and optimizing the composition of substrates .

The researchers experimented with materials, like brown rice, millet, corn, wheat and glutinous rice to see how well they supported the growth of fruiting bodies and the yield of cordycepin in solid state fermentation. They also studied the impact of carbon and nitrogen sources, mineral salts and several growth factors, on fruiting body development and cordycepin production using experimental methods ⁷

Enhancement of cordycepin using media optimizing techniques

Culture condition optimization

Strain selection

To extract cordycepin in high amounts, strains like KYL05 can be used which has a high cordycepin productivity. Various other strains were studied to produce cordycepin in high amount with an outstanding mycelial growth like KCTC 6064, KCTC 6862, KSP8 and G81-3⁸. Thus, for commercialization the highest cordycepin producing strain KYL05 is usually mutated using UV radiation and growth were observed on agar after 10-15 days ^2,9. In many studies, media optimization is carried out on the strain KSP8 using solid fermentation on Brown rice medium and Silkworm pupae medium or submerged cultivation in media of different compositions¹.

Media composition optimization

Studies have been done on effects of various carbon sources and carbon/nitrogen ratios on the growth of Cordyceps militaris and the production of cordycepin under a varied controlled condition. Upon investigations on the nutrient sources, glucose was observed to be an effective and economic source of carbon cordycepin production, although similar or even better growth patterns can be observed when culture is carried out in the presence of sucrose or lactose as the carbon source for several strains of C.militaris¹⁰.With an increase in the initial glucose concentration, there was a surge in the growth of C. militaris. An increase in the initial glucose content resulted in the increase in the cell growth of cordyceps¹¹. Furthermore, nitrogen sources in the media also plays a crucial role in the growth of the fungus. Amongst the various organic and inorganic sources of nitrogen like yeast extract, peptone, ammonia, usage of peptone resulted in higher production of cordycepin¹¹. However, in a comparative study, similar enhanced growth was observed by using casein hydrolysate (CH). The result showed that the cordycepin concentration increased with an increase in the CH concentration. In other studies, the use of Corn Steep Liquor Hydrolysate (CSLH) resulted in a lesser peptone consumption, and higher cordycepin yield, thus in future CSLH holds the potential to serve as a nitrogen source or C. militaris cultivation¹².

pH optimization, temp optimization

Along with the media composition, other physical factors like temperature, pH, light significantly affect the growth of the fungus. As most fungus proliferate effectively at a temperature range of 20-25 °C, a study on Japanese C. militaris showed results of enhanced mycelial growth at temperature range of 15-20°C¹³. However for strains collected from other regions, the optimal growth was achieved at 25°C¹⁴. Thus, maximum growth was achieved at 25°C where the cordycepin production were found to be approximately 2 folds higher than other temperatures. The maximum yield of cordycepin was achieved when the culture was maintained at a pH of 6 and a shaking condition of 150 rpm⁸.

Genetic approaches for strain improvement of cordyceps

Strain enhancement is the process of boosting the qualities of microorganisms, commonly bacteria, fungi, or yeast, for a specific purpose in industries or biotechnology. This enhancement might focus on genetic, metabolic, or physiological adjustments aimed at amplifying the microorganism’s productivity, efficiency, or adaptability to conditions or duties ¹⁵. The process often entails employing methods like genetic manipulation, inducing mutations, or selectively breeding to cultivate traits beneficial for the intended use, such as augmenting the yield of a desired output, enhancing resistance to environmental pressures, or optimizing nutrient utilization. It holds significant importance across various domains like pharmaceuticals, agriculture, food manufacturing, and environmental management ¹⁶.

For the strain improvement of cordyceps there are many methods like mutagenesis, protoplast fusion, regeneration, and transformation ¹⁷. Many research ¹⁷ has been done for the improvement in the quality of cordyceps and the quantity of the products obtained from it . Some of the methods are discussed as follows

Transformation System

This technique is used to focus the functions of genes related to various biological processes. The process involves introducing foreign DNA into the cells of host organism, ensuring the transferred DNA is stably maintained and replicated, and ultimately expressing the desired trait as shown in Figure 1. This approach facilitates the manipulation of an organism’s genome, linking laboratory DNA studies with real-world biological results. Numerous techniques are employed to create transformation systems, such as electroporation, polyethylene glycol (PEG) treatment, integration via protoplasts using restriction enzymes, gene transfer facilitated by Agrobacterium tumefaciens, particle bombardment, DNA uptake by cells treated with lithium acetate, protoplast transformation, and nitrate reductase transformation¹⁷.

Figure 1: Transformation method for Cordycepin using Cordyceps militaris and Cordyceps sinensis.Click here to view Figure

In order to identify functional genes in C. militaris, researchers developed and refined a system for transformation by Agrobacterium tumefaciens. They also observed PEG-mediated transformation which involved isolating C. militaris protoplast using enzyme mixtures like Yatalase, Usukizyme, and lysozyme. During transformation, the efficiency of transformants varied from 30 to 600 per 1 × 105 conidia when C. militaris and A. tumefaciens cells were cultured together in the presence of acetosyringone for 2 days ¹⁸. The pH level was discovered to impact transformation efficiency, with the highest number of resistant colonies achieved at pH 5.5. Consequently, a cultivation medium with pH 5.5 was deemed optimal for developing the C. militaris ATMT system ¹⁹.

To validate stable vector integration in transformants, researchers conducted mitotic stability analysis after monosporic purification. Later by southern bloting and then analysing the data of transformants showed vector hybridization, with integration potentially being directed or random. Random integrations might occur due to low similarity between the genes of the two integrated genomes.

Protoplast Fusion

Protoplast fusion has emerged as a strategy to overcome the limitations associated with traditional breeding methods, enabling the transfer of genes across barriers. This technique plays a pivotal role in enhancing strains through genetic recombination, fostering the development of hybrid strains in both micro and macro fungi²⁰. By allowing the amalgamation of genes from disparate organisms, protoplast fusion facilitates the creation of strains possessing desired traits. Notably, it has been particularly effective in augmenting mushroom strains by the help of certain enzymes and osmotic stabilizers.

In a specific study, protoplast fusion was conducted using two distinct species of cordyceps (CM1 and CM2) to generate a hybrid ².While the resulting hybrids exhibited high biomass production, their stability across subsequent generations was not sustained, prompting further investigation. Protoplast fusion becomes necessary when two mating types are unavailable²¹.

This technique can be applied intra-specifically, inter-specifically, inter-generically, and even inter-heterogenerically. For example, protoplasts from two different species within the Cordyceps genus, namely Cm029B and CS, were fused, thus resulting hybrid strains were looked via RAPD-PCR methods¹⁷.

To isolate protoplasts from the mycelia of fungus, an specific technique based on Hashiba ²² was utilized, involving lytic enzyme treatment followed by protoplast separation via filtration. The isolated protoplasts were then washed and suspended in an osmotic stabilizer solution. Protoplast fusion was induced using polyethylene glycol (PEG 30%), CaCl2 (0.05 M), and glycine (0.05 M) at pH 7.5. The fused protoplasts were cultured on regeneration medium under controlled conditions to observe and record colony growth and fusion rates ¹⁷.

Characterization studies, including analysis of mycelial growth, hyphal size, and isozyme patterns, were conducted to evaluate the hybrids. Furthermore, RAPD-PCR analysis confirmed the stable formation of hybrids between Cordyceps militaris and Cordyceps sinensis. 

UV irradiation

Ultraviolet (UV) irradiation involves exposing an object or substance to electromagnetic radiation within the ultraviolet spectrum, which has a very short wavelengths and high energy, typically ranging from about 10 nanometers (nm) to 400 nm ²³. This method is utilized for strain improvement by inducing mutations in the wild type organism.

To induce sporulation, strains were cultured on CMA plates at a temperature of 20ºC and further  subcultured. After subculturing, a conidial suspension was made in 0.85% NaCl solution with 0.1% Tween 80. This suspension solution was further introduced with sodium nitrate at a concentration of 500μg/ml and exposed to positioned 50 cm away. Following irradiation, the suspension was incubated at a temperature of 20ºC for 60 minutes in darkness ²⁴. Subsequently, 0.1 ml of the suspension was plated on solidified PMP medium to inhibit fungal colony growth. The culture was then incubated at 20ºC for 6 days until colonies became visible. Any isolates displaying altered characteristics after incubation were recognised as mutants.

After incubation for 6-8 days, only an average of five viable colonies were observed. These colonies were then inoculated into 150ml of PMP broth and incubated for 10 days at 20°C. Following incubation, the cultures were plated on CMA plates. From each parental strain, MTCC-3936 and CCM-1 were selected for further analysis. Notably, the CCM-1 isolate exhibited a change in colony color from white to black. Additionally, MTCC-3936 displayed slow growth, indicating a mutation. However, this method resulted in a reduced harvest of live spores and decreased cell viability. Moreover, with repeated subculturing, it was noted that mutant colonies gradually lost their distinctive characteristics and viability over time²⁴.

In another study two hybrids are taken and irradiated with UV, the two mutants from each hybrid were selected and analyzed for further development ² giving sub-lethal doses of UV radiation increasing the production of cordycepin ²⁵ which is stable through many subcultures.

Chemical mutagenesis 

A variety of chemicals or compounds can be used as mutagens to alter the structure of genome permanetly. These mutagens are of mainly three types -base modifiers, base analogues, intercalating agents.

From the previous fusion hybrids, CMF5U1 is taken and further mutated with ethidium bromide by incorporating the ethidium bromide and another batch which are just physically exposed which leads to high biomass production ². Cordycepin production from wild type increased many folds in suspension culture rather than using a normal substrate ²⁶.

In another study, the hybrids were stabilized using abrin which is a toxic compound extracted from a plant called rosary pea shows the elevated production of cordycepin ²⁴. In another study, scientists carried out sexual reproduction between two compatible parental strains, spores were isolated from them which gave rise to eight different mated strains. 32 crosses were made in which the strain (ksp8) showed high cordycepin production. It is concluded that the increase in cordycepin production is due to recombination in the genes ²⁷.

Recent research on cordycepin and related compounds focuses on optimizing yields through various strategies. Table 1 summarizes key strategies, main optimizations, and resulting cordycepin yields. Approaches include solid fermentation, liquid fermentation, protoplast fusion, and proton beam irradiation showing significant yield improvements for commercial and therapeutic applications.

Table 1: Optimization Strategies and Cordycepin Yields

Omics-Guided Strategies for Cordycepin Enhancement

Omics technologies, like transcriptomics, proteomics, and metabolomics, are making it simpler to understand biological systems from tissue to the molecular level providing a detailed understanding of gene expression and regulation, protein interactions, and metabolite profiles ³¹.

Transcriptomic studies can be very helpful for understanding into the transcriptional level of the genes about how they are getting involved in biosynthetic pathways, as well as regulatory mechanisms for Cordycepin production ³². Differential gene expression analysis can also tell us the regulatory genes and factors, like transcription factors and signalling pathways, that modify Cordycepin production concerning environmental factors and media availability ²⁸.

Proteomic approaches, like one-dimensional or two-dimensional gel electrophoresis coupled with the use of mass spectrometry (MS), are extensively utilised for identifying proteins and enzymes in fungi and other closely related species ³³. To find the protein expression between wild-type and genetically engineered strains a Comparative proteomic study can be done and this can also be done to identify potential protein molecules related to raised Cordycepin production ³⁴.

A comprehensive study of small-molecule metabolites in biological systems can be also done via Metabolomics to gain knowledge of metabolic pathways and understand cellular metabolism³⁵. Metabolomic techniques provide a significant understanding of metabolic flux as well as the regulation of Cordycepin biosynthesis pathways ³⁶.

The cointegration of all the omics technology mainly transcriptomic, proteomic, and metabolomic databases makes it possible for systems-level modelling of Cordycepin biosynthetic pathways, finding critical regulating points and metabolic constraints ³⁵. By understanding the molecular mechanisms guiding Cordycepin synthesis, omics-guided advancements can also help us to understand and apply targeted genetic and metabolic engineering strategies that will enhance Cordycepin yield ³⁶.

Economic Importance

Cordycepin, a compound found in certain fungi species like Cordyceps, has long been revered in traditional medicine across cultures. In traditional Chinese medicine (TCM), it’s been utilized for centuries to enhance vitality, boost immunity, and improve respiratory function³⁷. Similarly, in Tibetan medicine, known as “yartsa gunbu,” or ‘summer grass winter worm’ Cordycepin is esteemed as a potent tonic for treating respiratory issues and enhancing overall well-being³⁸. Beyond its medicinal use, cordycepin holds a significant place in folklore, particularly in the Himalayan regions where it’s referred to as “Himalayan Gold”. This rich tapestry of traditional use and folklore underscores cordycepin’s enduring cultural significance and its continued exploration in modern science for its pharmacological potential³⁹.

The global market for cordycepin is experiencing robust growth, driven by rising consumer awareness about its health benefits and increasing demand for natural remedies. According to market research reports, the demand for cordycepin is expected to continue growing steadily in the coming years, propelled by factors such as the growing aging population, rising prevalence of chronic diseases, and shifting consumer preferences towards natural and organic products³⁹. Pharmaceutical companies are exploring the development of cordycepin-based drugs and formulations for various therapeutic purposes. Clinical trials are underway to evaluate the safety and efficacy of cordycepin in humans, with promising results reported in preclinical studies. The successful commercialization of cordycepin-based pharmaceuticals could have a significant 16 economic impact, not only in terms of revenue generation but also in improving public health outcomes⁴⁰. In addition to standalone supplements, cordycepin is also being incorporated into functional foods and beverages to enhance their nutritional value and health-promoting properties. Products like energy bars, protein shakes, and herbal teas fortified with cordycepin are gaining popularity among fitness enthusiasts and consumers seeking holistic approaches to wellness. The growing demand for functional foods and beverages presents lucrative opportunities for manufacturers to capitalize on cordycepin’s market potential⁴¹.

Challenges and Future Directions

In the fields of medical research and biotechnology, cordycepin has huge potential.  However, there are several challenges but there are mainly technological limitations and regulatory issues that limit its comprehensive application of cordycepin.

First of all, achieving superior and better yield is still a major challenge as there are many technological limitations in the current production processes ⁴². Researchers in a study bring focus to the ongoing issue of poor yield, which limits Cordycepin’s commercial viability.  This is correlated with a complex biosynthetic pathway, which is characterised by multiple stages of enzymatic reactions and regulatory complications ¹. Several attempts have been there to understand and modify this pathway for increased production but still, these experience significant challenges. Furthermore, Cordycepin biosynthesis is dependent upon specific substrate availability, which further complicates the manufacturing process.

Along with technical hurdles, regulatory challenges dominate more. Regulatory agencies impose strict limitations based on the safety and efficacy of bioactive compounds. Regulatory approval of enhanced Cordycepin products requires proof of their safety profile and therapeutic efficacy ⁴³. Further complicating regulatory matters of intellectual property is a major challenge, which is especially significant when talking about genetic engineering methods and synthetic biology strategies ⁴⁴. The management of intellectual property is a major challenge for researchers and commercialisation initiatives. Moreover, regulatory norms are different in different countries which additionally adds a logistical issue, requiring special attention to production procedures, product quality, and labelling specifications.

Dealing with such challenges requires an interdisciplinary approach.  Modern genetic engineering techniques, like Cas9 technology, offer an opportunity to enable precise alterations that improve biosynthetic pathways and thus improving yield ⁴⁵. The use of novel bioprocess strategies like fed-batch and continuous fermentation systems can be used to improve the culture conditions for increased Cordycepin production ⁴⁶. Optimising substrate supply and rerouting metabolic flux towards Cordycepin biosynthesis pathways are also some strategies to improve overall production efficiency through metabolic engineering.

Looking ahead, future research offers potential solutions. Exploration of some new biosynthetic pathways and enzyme cascades in heterologous hosts could open new avenues for enhancing production efficiency. Integrating multi-omics approaches, such as transcriptomics, proteomics, and metabolomics analyses, promises comprehensive insights into molecular mechanisms governing Cordycepin biosynthesis, identifying potential metabolic engineering targets. The development of sustainable production methods, such as fermentation technology using renewable feedstocks or bioremediation approaches, holds great promise in minimizing environmental impact and resource consumption. Furthermore, establishing standardised protocols for the production and quality control of enhanced Cordycepin formulations across diverse therapeutic areas and are essential steps towards realising the full potential of Cordycepin in medicine and biotechnology.

Conclusion

In summary, Cordycepin, derived from the medicinal fungus Cordyceps, displays a wide range of pharmacological effects spanning from anti-inflammatory to anti-cancer properties. Traditionally sourced from the fruiting body of Cordyceps militaris through solid-state or liquid fermentation, contemporary methods strive to enhance production through media composition adjustments and strain enhancement techniques. Further research can explore the scalability of the optimized media formulation for industrial scale production. Additionally, investigations into the impact of media composition on the production of specific bioactive compounds within Cordyceps militaris like cordycepin. Nevertheless, challenges persist, encompassing technological constraints, regulatory complexities, and the management of intellectual property. Addressing these hurdles necessitates interdisciplinary collaboration, utilizing modern genetic engineering tools, refining bioprocesses, and exploring novel biosynthetic pathways. Future research endeavors are geared towards developing sustainable production methods, establishing standardized protocols, and gaining comprehensive understanding of Cordycepin’s molecular mechanisms, thereby fostering its broad application in medicine and biotechnology.

Acknowledgement

We would like to express our sincere gratitude to the department of Molecular Biology and Genetic Engineering of Lovely Professional University for providing us the sources and our mentors Dr. Vikas Sharma and Dr. Shivika Sharma for their invaluable mentorship throughout our work.

Conflict of Interest

There is no conflict of interest.

Funding Source

There is no funding sources.

Author Contribution Statement

The authors confirm contribution to the paper as follows: Conceptualize and design of the review paper: R.K, N.M; Introduction and Enhancement of Cordycepin using media optimisation section:N.M; Synthesis of Cordycepin section,Economic importance and Table 1: R.K.; Genetic approaches for strain improvement of Cordyceps: A.S., S.N.R; Omics-guided strategies, challenges, and future directions: A.C.; Conclusion, Figure 1: A.S;Overall supervision of review paper: Dr. S.S. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement

Not Applicable

Ethics Approval Statement

Not Applicable

References

1. Wang L, Yan H, Zeng B, Hu Z. Research Progress on Cordycepin Synthesis and Methods for Enhancement of Cordycepin Production in Cordyceps militaris. Bioengineering. 2022;9(2):69. doi:10.3390/bioengineering9020069
   CrossRef
2. Rathod LBL, Pasha SM, Rajan A, . M, Pasha C. Sequential Strain Improvement of Cordyceps militaris. J Adv Microbiol. 2023;23(3):13-20. doi:10.9734/jamb/2023/v23i3711
   CrossRef
3. Cai X, Jin JY, Zhang B, Liu ZQ, Zheng YG. Improvement of cordycepin production by an isolated Paecilomyces hepiali mutant from combinatorial mutation breeding and medium screening. Bioprocess Biosyst Eng. 2021;44(11):2387-2398. doi:10.1007/s00449-021-02611-w
   CrossRef
4. Luo QY, Cao HF, Liu SK, et al. Novel liquid fermentation medium of Cordyceps militaris and optimization of hydrothermal reflux extraction of cordycepin. J Asian Nat Prod Res. 2020;22(2):167-178. doi:10.1080/10286020.2018.1539080
   CrossRef
5. Tang J, Liu Y, Zhu L. Optimization of fermentation conditions and purification of cordycepin from Cordyceps militaris. Prep Biochem Biotechnol. 2014;44(1):90-106. doi:10.1080/10826068.2013.833111
   CrossRef
6. Tang J, Qian Z, Zhu L. Two-step shake-static fermentation to enhance cordycepin production by Cordyceps militaris. Chem Eng Trans. 2015;46:19-24. doi:10.3303/CET1546004
7. Wen TC, Kang JC, David Hyde K, et al. Optimization of Solid-State Fermentation for Fruiting Body Growth and Cordycepin Production by Cordyceps militaris. Vol 41.; 2014. http://epg.science.cmu.ac.th/ejournal/.
8. Lee SK, Lee JH, Kim HR, et al. Improved cordycepin production by Cordyceps militaris KYL05 using casein hydrolysate in submerged conditions. Biomolecules. 2019;9(9). doi:10.3390/biom9090461
   CrossRef
9. Lee SK, Lee JH, Kim HR, et al. Improved Cordycepin Production by Cordyceps militaris KYL05 Using Casein Hydrolysate in Submerged Conditions. Biomolecules. 2019;9(9):461. doi:10.3390/biom9090461
   CrossRef
10. Kang C, Wen TC, Kang JC, Meng ZB, Li GR, Hyde KD. Optimization of Large-Scale Culture Conditions for the Production of Cordycepin with Cordyceps militaris by Liquid Static Culture. The Scientific World Journal. 2014;2014:1-15. doi:10.1155/2014/510627
    CrossRef
11. Mao XB, Eksriwong T, Chauvatcharin S, Zhong JJ. Optimization of carbon source and carbon/nitrogen ratio for cordycepin production by submerged cultivation of medicinal mushroom Cordyceps militaris. Process Biochemistry. 2005;40(5):1667-1672. doi:10.1016/j.procbio.2004.06.046
    CrossRef
12. Chang Y, Liu X, Jiao Y, Zheng X. Improved Cordycepin Production by Cordyceps militaris Using Corn Steep Liquor Hydrolysate as an Alternative Protein Nitrogen Source. Foods. 2024;13(5):813. doi:10.3390/foods13050813
    CrossRef
13. Hung LT, Keawsompong S, Hanh VT, Sivichai S, Hywel-Jones NL. Effect of Temperature on Cordycepin Production in Cordyceps militaris. Vol 42.; 2009. www.thaiagj.org.
14. Kryukov VY, Tomilova OG, Yaroslavtseva ON, et al. Temperature adaptations of Cordyceps militaris, impact of host thermal biology and immunity on mycosis development. Fungal Ecol. 2018;35:98-107. doi:10.1016/j.funeco.2018.07.003
    CrossRef
15. Lee SY, Lee DY, Kim TY. Systems biotechnology for strain improvement. Trends Biotechnol. 2005;23(7):349-358. doi:10.1016/j.tibtech.2005.05.003
    CrossRef
16. Crook N, Alper HS. Classical Strain Improvement. In: Engineering Complex Phenotypes in Industrial Strains. Wiley; 2012:1-33. doi:10.1002/9781118433034.ch1
    CrossRef
17. Deshmukh L, Sharma AK, Sandhu SS. Contrive Himalayan Soft Gold Cordyceps Species: a Lineage of Eumycota Bestowing Tremendous Pharmacological and Therapeutic Potential. Curr Pharmacol Rep. 2020;6(4):155-166. doi:10.1007/s40495-020-00223-8
    CrossRef
18. Zheng Z, Huang C, Cao L, Xie C, Han R. Agrobacterium tumefaciens-mediated transformation as a tool for insertional mutagenesis in medicinal fungus Cordyceps militaris. Fungal Biol. 2011;115(3):265-274. doi:10.1016/j.funbio.2010.12.011
    CrossRef
19. Sandhu SS, Kinghorn JR, Rajak RC, Unkles SE. Transformation system of Beauveria bassiana and Metarhizium anisopliae using nitrate reductase gene of Aspergillus nidulans. Indian J Exp Biol. 2001;39(7):650-653.
20. Aswini L, Arunagirinathan N, Kavitha M. Strain Improvement of Pleurotus Species by Protoplast Fusion. International Journal of Advancements in Research & Technology. 2014;3(8).
    CrossRef
21. Holliday JC, Cleaver P, Loomis-Powers M, Patel D. Analysis of Quality and Techniques for Hybridization of Medicinal Fungus Cordyceps sinensis (Berk.)Sacc. (Ascomycetes). Int J Med Mushrooms. 2004;6(2):151-164. doi:10.1615/IntJMedMushr.v6.i2.60
22. HASHIBA K. Sex Differences in Phenotypic Manifestation and Gene Transmission in the Romano‐Ward Syndrome. Ann N Y Acad Sci. 1992;644(1):142-156. doi:10.1111/j.1749-6632.1992.tb31008.x
    CrossRef
23. Das SK, Masuda M, Hatashita M, Sakurai A, Sakakibara M. A new approach for improving cordycepin productivity in surface liquid culture of Cordyceps militaris using high-energy ion beam irradiation. Lett Appl Microbiol. 2008;47(6):534-538. doi:10.1111/j.1472-765X.2008.02456.x
    CrossRef
24. Mani A. An effective approach of strain improvement in Cordyceps militaris using abrin. Current Research in Environmental & Applied Mycology. 2016;6(3):166-172. doi:10.5943/cream/6/3/3
    CrossRef
25. Pasha C, Kuhad RC, Rao LV. Strain improvement of thermotolerant Saccharomyces cerevisiae VS3 strain for better utilization of lignocellulosic substrates. J Appl Microbiol. 2007;103(5):1480-1489. doi:10.1111/j.1365-2672.2007.03375.x
    CrossRef
26. Jędrejko K, Kała K, Sułkowska-Ziaja K, et al. Cordyceps militaris—Fruiting Bodies, Mycelium, and Supplements: Valuable Component of Daily Diet. Antioxidants. 2022;11(10):1861. doi:10.3390/antiox11101861
    CrossRef
27. Kang N, Lee HH, Park I, Seo YS. Development of High Cordycepin-Producing Cordyceps militaris Mycobiology. 2017;45(1):31-38. doi:10.5941/MYCO.2017.45.1.31
    CrossRef
28. Kaushik V, Singh A, Arya A, Sindhu SC, Sindhu A, Singh A. Enhanced production of cordycepin in Ophiocordyceps sinensis using growth supplements under submerged conditions. Biotechnology Reports. 2020;28:e00557. doi:10.1016/j.btre.2020.e00557
    CrossRef
29. Lai WK, Wu YC, Yeh TS, et al. The protoplast two-way fusions and fusant characteristics of Antrodia cinnamomea and Cordyceps militaris. Food Science and Human Wellness. 2022;11(5):1240-1251. doi:10.1016/j.fshw.2022.04.032
    CrossRef
30. Masuda M, Das SK, Fujihara S, Hatashita M, Sakurai A. Production of cordycepin by a repeated batch culture of a Cordyceps militaris mutant obtained by proton beam irradiation. J Biosci Bioeng. 2011;111(1):55-60. doi:10.1016/j.jbiosc.2010.08.018
    CrossRef
31. Ma YC, Huang P, Wang XL, Liu GQ. Multi-omics analysis unravels positive effect of rotenone on the cordycepin biosynthesis in submerged fermentation of Cordyceps militaris. Bioresour Technol. 2023;373:128705. doi:10.1016/j.biortech.2023.128705
    CrossRef
32. Yin Y, Yu G, Chen Y, et al. Genome-Wide Transcriptome and Proteome Analysis on Different Developmental Stages of Cordyceps militaris. PLoS One. 2012;7(12):e51853. doi:10.1371/journal.pone.0051853
    CrossRef
33. Gonzalez-Fernandez R, Jorrin-Novo J V. Contribution of Proteomics to the Study of Plant Pathogenic Fungi. J Proteome Res. 2012;11(1):3-16. doi:10.1021/pr200873p
    CrossRef
34. Zhang X, Liu Q, Zhou W, et al. A comparative proteomic characterization and nutritional assessment of naturally- and artificially-cultivated Cordyceps sinensis. J Proteomics. 2018;181:24-35. doi:10.1016/j.jprot.2018.03.029
    CrossRef
35. Chen L, Liu Y, Guo Q, Zheng Q, Zhang W. Metabolomic comparison between wild Ophiocordyceps sinensis and artificial cultured Cordyceps militaris. Biomedical Chromatography. 2018;32(9). doi:10.1002/bmc.4279
    CrossRef
36. Liu Y, Xiao K, Wang Z, Wang S, Xu F. Comparison of metabolism substances in Cordyceps sinensis and Cordyceps militaris cultivated with tussah pupa based on LC‐MS. J Food Biochem. 2021;45(6). doi:10.1111/jfbc.13735
    CrossRef
37. Paterson RRM. Cordyceps – A traditional Chinese medicine and another fungal therapeutic biofactory? Phytochemistry. 2008;69(7):1469-1495. doi:10.1016/j.phytochem.2008.01.027
    CrossRef
38. Winkler D. Caterpillar Fungus (Ophiocordyceps sinensis) Production and Sustainability on the Tibetan Plateau and in the Himalayas. Asian Medicine. 2009;5(2):291-316. doi:10.1163/157342109X568829
    CrossRef
39. Dong C, Guo S, Wang W, Liu X. Cordyceps industry in China. Mycology. 2015;6(2):121-129. doi:10.1080/21501203.2015.1043967
    CrossRef
40. Zhou X, Gong Z, Su Y, Lin J, Tang K. Cordyceps fungi: natural products, pharmacological functions and developmental products. Journal of Pharmacy and Pharmacology. 2009;61(3):279-291. doi:10.1211/jpp/61.03.0002
    CrossRef
41. Phull AR, Ahmed M, Park HJ. Cordyceps militaris as a Bio Functional Food Source: Pharmacological Potential, Anti-Inflammatory Actions and Related Molecular Mechanisms. Microorganisms. 2022;10(2):405. doi:10.3390/microorganisms10020405
    CrossRef
42. Manabe N, Azuma Y, Sugimoto M, et al. Effects of the mycelial extract of cultured Cordyceps sinensis on in vivo hepatic energy metabolism and blood flow in dietary hypoferric anaemic mice. British Journal of Nutrition. 2000;83(2):197-204. doi:10.1017/S0007114500000258
    CrossRef
43. Oh J, Yoon DH, Shrestha B, Choi HK, Sung GH. Metabolomic profiling reveals enrichment of cordycepin in senescence process of Cordyceps militaris fruit bodies. Journal of Microbiology. 2019;57(1):54-63. doi:10.1007/s12275-019-8486-z
    CrossRef
44. Shweta, Abdullah S, Komal, Kumar A. A brief review on the medicinal uses of Cordyceps militaris. Pharmacological Research – Modern Chinese Medicine. 2023;7:100228. doi:10.1016/j.prmcm.2023.100228
    CrossRef
45. Tavakoli K, Pour-Aboughadareh A, Kianersi F, Poczai P, Etminan A, Shooshtari L. Applications of CRISPR-Cas9 as an Advanced Genome Editing System in Life Sciences. BioTech. 2021;10(3):14. doi:10.3390/biotech10030014
    CrossRef
46. Paterson RRM. Cordyceps – A traditional Chinese medicine and another fungal therapeutic biofactory? Phytochemistry. 2008;69(7):1469-1495. doi:10.1016/j.phytochem.2008.01.027
    CrossRef