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Cordyceps militaris fruiting bodies contain a variety of bioactive components that are beneficial to the human body. However, the low yield of fruiting bodies and the low carotenoid content in C. militaris have seriously hindered the development of the C. militaris industry. To elucidate the developmental mechanism of the fruiting bodies of C. militaris and the biosynthesis mechanism of carotenoids, the function of the flavohemoprotein-like Cmfhp gene of C. militaris was identified for the first time. The Cmfhp gene was knocked out by the split-marker method, and the targeted gene deletion mutant ΔCmfhp was obtained. An increased nitric oxide (NO) content, no fruiting body production, decreased carotenoid content, and reduced conidial production were found in the mutant ΔCmfhp. These characteristics were restored when the Cmfhp gene expression cassette was complemented into the ΔCmfhp strain by the Agrobacterium tumefaciens-mediated transformation method. Nonetheless, the Cmfhp gene had no significant effect on the mycelial growth rate of C. militaris. These results indicated that the Cmfhp gene regulated the biosynthesis of NO and carotenoids, the development of fruiting bodies, and the formation of conidia. These findings potentially pave the way to reveal the developmental mechanism of fruiting bodies and the biosynthesis mechanism of carotenoids in C. militaris.

Cordyceps militaris, a valuable edible and medicinal fungus, has attracted increasing attention because of its various bioactive ingredients. However, the biosynthetic pathway of C. militaris carotenoids is still unknown due to lack of transcriptome information. To uncover genes related to the biosynthesis of C. militaris carotenoids, the transcriptomes of mycelia CM10_D cultured under dark conditions and mycelia CM10_L cultured under light exposure conditions were sequenced. Compared with mycelia CM10_D, 866 up-regulated genes and 856 down-regulated genes were found in mycelia CM10_L. Gene ontology (GO) analysis of differentially expressed genes (DEGs) indicated that DEGs were mainly classified into the “metabolic process,” “membrane,” and “catalytic activity” terms. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of DEGs suggested that DEGs were mainly enriched in “metabolic pathways,” “MAPK signaling pathway-yeast,” and “biosynthesis of secondary metabolites.” In addition, the carotenoid content of the Cmtns gene deletion mutant (ΔCmtns) was significantly lower than that of the wild-type C. militaris CM10, while the carotenoid content of the complementary strain (ΔCmtns-c) of the Cmtns gene was not significantly different from that of C. militaris CM10, suggesting that the Cmtns gene significantly affected the biosynthesis of carotenoids in C. militaris. These results potentially pave the way for revealing the biosynthetic pathway of carotenoids and improving carotenoids production in C. militaris.

The macrofungus Cordyceps militaris contains many kinds of bioactive ingredients that are regulated by functional genes, but the functions of many genes in C. militaris are still unknown. In this study, to improve the frequency of homologous integration, a genetic transformation system based on a split-marker approach was developed for the first time in C. militaris to knock out a gene encoding a terpenoid synthase (Tns). The linear and split-marker deletion cassettes were constructed and introduced into C. militaris protoplasts by PEG-mediated transformation. The transformation of split-marker fragments resulted in a higher efficiency of targeted gene disruption than the transformation of linear deletion cassettes did. The color phenotype of the Tns gene deletion mutants was different from that of wild-type C. militaris. Moreover, a PEG-mediated protoplast transformation system was established, and stable genetic transformants were obtained. This method of targeted gene deletion represents an important tool for investigating the role of C. militaris genes.

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