**4. Unknown Mycotoxins May be Produced by** *Cordyceps* **Fungi**

Apart from the metabolites described above, many more can be expected after genomic analysis of *Cordyceps* fungi that identifies an array of BGCs encoded in each fungus (Figure 3A). For example, eight non-ribosomal peptide synthetase (NRPS), seven polyketide synthase (PKS), five NRPS-PKS and four terpene synthase (TS) BGCs are encoded in the genome of *C. militaris* [25,51]. Likewise, the BGCs of eight NRPSs, eight PKSs, six NRPS-PKSs and six TSs are encoded in *C. cicadae* [23]. Consistently, metabolomic analysis also suggested that diverse compounds can be produced by these fungi [23,52]. Due to the frequent occurrence of gene silence of fungal BGCs under experimental conditions [2], many unknown metabolites remain to be determined. Genome-wide phylogenetic, gene cluster content, and core enzyme structure analyses of NRPS, PKS, and NRPS-PKS BGCs suggest that the *Cordyceps* fungi may not produce carcinogenic mycotoxins such as aflatoxins [23,25]. However, our further analysis indicated that the putative aristolochen synthase CCM\_03050 is highly similar (65% identity at amino acid level) to PRX2, and the clustered CCM\_03051 is similar to the short chain dehydrogenase (SDR) PRX4 of *Penicillium roqueforti* involved in PR-toxin production (Figure 3B). The bicyclic sesquiterpene PR-toxin can cause significant damage to liver and kidney, induce abortions and reduce fertility in cattle. However, its derivatives such as PR-acid and PR-imine are largely non-toxic and unstable [53]. The biosynthesis of PR-toxin requires at least four genes, i.e., *Prx1-Prx4* in *P. roqueforti* [54]. Considering that the tailoring enzyme genes *Prx1* (for a SDR) and *Prx3* (for a quinone oxidoreductase) are missing in *C. militaris* whereas the clustered *CCM\_03049* encodes a putative dioxygenase/oxygenase and *CCM\_03052* encodes a cytochrome P450 enzyme, this TS BGC may not biosynthesize PR-toxin but its analog(s) requiring further investigation. Intriguingly, these conserved genes are absent in the genomes of the closely-related *C. cicadae* and other Cordycipitaceae fungi, based on the survey of their genome contents.

**Figure 3.** Conservation analysis of the gene clusters between *Cordyceps* and other fungi involved in toxin production. (**A**) Comparative analysis of the biosynthetic gene clusters (BGCs) encoded in the genomes of the selected Cordycipitaceae fungi. NRPS, non-ribosomal peptide synthetase; PKS, polyketide synthase; TS, terpene synthase. (**B**) Conservation between the gene clusters of *C. militaris* and those of *P. roqueforti* involved in PR-toxin production. (**C**) Conservation between the gene clusters of the Cordycipitaceae fungi *C. cicadae* and *A. lecanii* and that of *F. graminearum* involved in the biosynthesis of trichothecenes. (**D**) Conservation between the gene clusters of *C. militaris* and *C. cicadae* and that of *U. virens* involved in the production of ustilaginoidins. The genes labeled in the same color within the panels (**B**–**D**) represent orthologous relationships. The compound(s) shown in the right of the panels (**B**–**D**) indicates the structures of the representative metabolites produced by the known BGCs in the related or reference fungal species.

We also found that the Cordycipitaceae fungi *C. cicadae* and *Akanthomyces lecanii* (anamorph: *Lecanicillium lecanii*) each encode a gene cluster that shows conservation with the BGC involved in the production of trichothecene (Tri) mycotoxins in wheat head blight *Fusarium graminearum* (Figure 3C). Phytotoxic trichothecenes are a class of sesquiterpenes that contain more than 150 chemically-related compounds such as type A mycotoxins T-2, HT-2, and NX-2, and type B deoxynivalenol (DON), acetylated DON, and nivalenol toxins that can inhibit protein synthesis and are neurotoxic, immunosuppressive, and nephrotoxic in mammals [55,56]. The biosynthesis of trichothecenes has been clarified with the involvement of 15 *Tri* genes located at three different loci including a 12-gene core cluster, a single gene *Tri101* and two genes *Tri1* and *Tri16* loci on different chromosomes of *F. graminearum* [57,58]. We found that the homologs of the core gene *Tri5* for trichodiene synthase are conservedly present in *C. cicadae* (CCAD\_05859, 70% identity) and *A. lecanii* (LEL\_0770, 79% identity). The homologs of the essential biosynthetic genes *Tri3*, *Tri4*, *Tri11* and *Tri14* are also present in the genomes of these two fungi. In addition, interestingly, the homologs of the isolated gene *Tri101* (encoding an acetyltransferase) in *F. graminearum* are present in the core cluster of *Cordyceps* fungi, i.e., *LEL\_04769* (40% identity) and *CCAD\_05860* (37% identity) (Figure 3C). Likewise, the homologs of the isolated gene *Tri1* are also present in the separated loci of two fungi (*CCAD\_02575*, 42% identity; *LEL\_06734*, 41% identity). Taken together, it is considerably likely that *C. cicadae* and *A. lecanii* may produce trichothecene-like mycotoxin(s) that requires clarification and toxicity tests. In support, the trichothecene derivative 4-β-acetoxyscirpendiol (4-acetyl-12,13-epoxyl-9-trichothecene-3,15-diol) was once isolated from the fruit-body samples of *Isaria japonica* (syn. *Cordyceps tenuipes*) and the compound could induce apoptosis of human leukemia cells [59]. This *Tri*-like BGC is not present in *C. militaris* but is present in the closely-related biocontrol fungus *B. bassiana* (TRI5 vs. BBA\_08696, 69% identity; TRI101 vs. BBA\_08697, 41%) after genome survey [56]. A *Tri*-like BGC has also been identified in diverse fungal species including *Trichoderma* spp., which is responsible for the production of the trichothecene derivatives harzianums A and B with antimicrobial activities [60,61].

The bis-naphthopyrone-type pigments are widely produced by different fungi that can protect filamentous fungi from fungivores [62] or abiotic stress factors like UV radiation and high temperatures [63,64]. Genome survey indicated that both *C. militaris* and *C. cicadae* contain a conserved PKS BGC like that of rice false smut *Ustilaginoidea virens* (Figure 3D), which is responsible for the production of ustilaginoidins [65]. Different analogs of ustilaginoidins have varied level of antibacterial, cytotoxic, and phytotoxic activities [66]. The exact compound(s) produced by this PKS gene cluster remains to be determined in *Cordyceps* fungi.

In contrast to other *Cordyceps* fungi, the caterpillar fungus *O. sinensis* has a highly repetitive genome with a limited number of genes and BGCs (Figure 3A) [24]. Besides the reported bioactive constituents like nucleosides, sterols and polysaccharides [18,67–69], a few compounds have been identified from this fungus with different activities such as the aurantiamides cordyceamides A and B with unclear activity/toxicity [70], the cyclodipeptide cordycedipeptide A with cytotoxicity [71], and the cyclodipeptide cordysinins with antioxidant activity [69,72]. Overall, the reputed health benefits of this fungus are still unclear [73]. Despite the mass production of sexual fruiting bodies being recently successful [74], the slow growing nature of this fungus makes it technically problematic to perform transgenic and chemical genetic investigations [73]. With the help of heterologous expression systems, future efforts can be taken to explore the production, if any, of mycotoxin as well as the pharmaceutical potential of this fungus.

### **5. Requirement of Safety Assessments**

As indicated above, toxicological concerns about consuming *Cordyceps* fungi as food supplements or using their byproducts as animal feed are raised based on the evaluations of either the known compounds they produce or those previously unreported toxins being putatively produced by the conserved BGCs of *Cordyceps* fungi. Obviously, the production of the avian-gout toxin oosporein by *C. cicadae* suggests that mass-production byproducts cannot be used as feed for poultry. In particular, except for the unknown metabolites biosynthesized by other BGCs, the potential of the production of analogous PR-toxin in *C. militaris* and Tri-like toxin(s) in *C. cicadae* raises substantial concerns that require further investigation and safety assessments. Traditionally, instead of using the purified compounds, the fruiting bodies or mycelium samples of *Cordyceps* fungi were used for direct safety assessments, including bacterial Ames tests, different cell- and/or animal-model tests for mutagenic, clastogenic, genotoxic, and (sub-)accurate toxic effects, which suggested that consumption of *Cordyceps* fungi might be safe [75,76]. However, considering the magnitude of the mass production of *Cordyceps* fungi in China and other countries, in-depth monitoring and assessment are still required, given the daily consumption of *Cordyceps* fruiting bodies or related products as tonics or food/health additives and the use of culture substrates as feed. In particular, aside from the feature of culture stability, high titer of cordycepin and pentostatin production is being used as the key standard for screening of *C. militaris* strains for industrial mass production. Consistent with the side effects of pentostatin and adenosine analogs [33,38], the negative effects of nausea and diarrhea have been anecdotally reported by enthusiasts after consuming products with enriched contents of cordycepin/pentostatin [14]. Indeed, it is common and typical that mycotoxin production and accumulation are dependent on the fungal culturing media and stage, and the side effects of daily consumptions are dose- and even consumer-dependent [20]. For example, trichothecene production was not detected in *A. lecanii* and *B. bassiana* after inductions in multiple artificial media used for the successful induction of toxin formations in other fungi [56]. It is critical at least that toxin production is clarified for the *Cordyceps* fungi under mass production conditions.

### **6. Conclusions and Prospective**

Along with the increasing level of the mass production of *Cordyceps* fungi such as *C. militaris* and *C. cicadae*, there is increasing consumption of the fruiting bodies or related products as food supplements or health additives, and use of the byproducts as animal feed. To alleviate safety concerns, full elucidation of the BGCs' capacities in production of different compounds is critically needed. In addition to promoting toxicological tests with the known and newly-identified compounds, in-depth investigations may also benefit the exploration of these fungi for pharmaceutical potential.

**Author Contributions:** Conceptualization, C.W.; methodology, B.C. and C.W.; data curation, B.C., Y.S., F.L., and C.W.; manuscript writing, C.W.; supervision, C.W.; funding acquisition, C.W. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the National Natural Science Foundation of China (31530001) and the Shanghai Academic/Technology Research Leader Program (grant 18XD1404500).

**Acknowledgments:** The authors thank Chunru Li from BioAsia Pharmaceuticals for provision of the mass production pictures of *C. cicadae*.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**


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