C. militaris retains a conserved metabolism for efficiently producing ATP by mitochondrial oxidative phosphorylation in response to high-altitude environments. Moreover, it was found that C. militaris contains genes converting various hormones (e.g., serotonin, adrenaline, and dopamine) into amino acids; this is in accordance with the fact that in nature, C. militaris assimilates nitrogen and carbon from the larval corpus with high content in juvenile and neuronal hormones such as serotonin and dopamine.

3. Genetics, Genomics, and Genetic Engineering of C. militaris

Genetic improvement of C. militaris is of high importance in order to create new strains with high commercial potential. So far, C. militaris can be selectively improved by using traditional breeding techniques, and these are mainly restricted to reversing degenerative phenotypes to ascomata-producing strains. Polyethylene glycol (PEG)-mediated transformation of C. militaris mononuclear protoplasts has been performed in order to knock-down a terpenoid synthase (Tns) gene using a glufosinate ammonium selection marker [48,49]. Moreover, Agrobacterium tumefaciens-mediated transformation of C. militaris has been used as a tool for insertional mutagenesis [81] and complementation of ΔCmfhp mutants with the wild-type Cmfhp gene [50]. Agrobacterium-mediated transformation of C. militaris has also been used as a technique for deleting MAT genes in single mating-type isolates of this fungus [37]. Efficient CRISPR in C. militaris was achieved by creating a Cas9-stably transformed strain via Agrobacterium-mediated transformation and then by delivering of a presynthesized sgRNA targeting the ura3 gene by PEG-mediated protoplast transformation [51]. In the same study it was reported that this method was superior to co-transformation of a single vector expressing a sgRNA-cmcas9 cassette, providing encouraging results for future interventions (genetic engineering) in the cordycepin biosynthesis pathway (Figure 1).

Figure 1. Schematic overview of genetic engineering approaches commonly used in C. militaris.

4. Molecular, Nutritional, and Environmental Aspects of Cordycepin Biosynthesis and Ascomata Formation

C. militaris produces a large number of bioactive metabolites, including ergothioneine, ergosterol, adenosine, polysaccharides, and cordycepin [14,91,92,93,94]. Among them, cordycepin exhibits a large range of health beneficial effects, such as broad-spectrum antibiotic activity and the ability to inhibit cell proliferation and to induce cell apoptosis, as well as having antioxidant, anticancer, and anti-inflammatory properties [95,96,97,98,99]. It is currently considered to be one of the most promising fungal compounds with respect to its pharmacological and therapeutic potential; therefore, the study of aspects related to cordycepin biosynthesis and ascomata production is essential to the commercial exploitation of this species. The basic metabolic route for cordycepin biosynthesis has been recently explored using a combination of in silico analyses and functional genomics approaches [100]. Four genes for cordycepin synthetase were identified in C. militaris genome and were designated as cns1–cns4; these genes contain different conserved domains, such as the oxidoreductase/dehydrogenase domain in Cns1, the HDc family of metal-dependent phosphohydrolase domain in Cns2, and the N-terminal nucleoside/nucleotide kinase (NK) and C-terminal HisG domains in Cns3, while Cns4 is a putative ATP-binding cassette type of transporter. Using Agrobacterium-mediated single and joint gene deletion mutants of the aforementioned genes, Xia et al. [100] demonstrated that Cns1 and Cns2 are required for cordycepin biosynthesis, while Cns3 is implicated in the biosynthesis of an additional “safeguard” molecule, i.e., pentostatin. In addition, the same authors reported that cordycepin biosynthesis (starting from adenosine and proceeding through the stepwise reactions of phosphorylation, dephosphorylation, and reduction) is catalyzed by the Cns1/Cns2 complex in parallel with the biosynthesis of pentostatin. This dual production is similar to the bacterial “protector-protégé strategy” of purine metabolism in which the safeguarded cordycepin can be deaminated to 30-deoxyinosine once the former reaches a self-toxic level in fungal cells [100].

5. Conclusions

The ethnopharmacological importance of C. militaris has been widely analyzed in the past by many researchers [2,10,13,70,122,123,124] since this fungus is considered to be a valuable source of metabolites that act directly on various human metabolic pathways. For example, the methanolic extract of C. militaris presented antioxidant, antibacterial, antifungal, and antiproliferative properties in different human tumor cell lines [122], while cordycepin, the major bioactive compound of C. militaris, presented potent anti-inflammatory, anticancer, anti-metastatic, and immune-modulatory activities [123,124,125,126,127]. Although C. militaris can be easily cultivated in rice-based media, particular requirements related to the physiology and genetic makeup of this fungus impose serious obstacles in commercial applications. Hence, strain degeneration can completely hinder fruit body production at a commercial-scale, while improper cultivation techniques can downgrade metabolite production. Because of the entomopathogenic nature of C. militaris, the use of insect-based substrates in large-scale cultivation projects must be taken into consideration. During the past few years, insect mass production systems have become a contemporary trend in organic waste treatment and biotransformation [128], while the insect-based pet feed industry is growing exponentially. Considering the increasing availability of such by-products, the cultivation practices of C. militaris could exploit this type of waste material in the frame of the circular economy concept.