Cordyceps militaris is a well-known edible medicinal mushroom in East Asia that contains abundant and diverse bioactive compounds. Since traditional genome editing systems in C. militaris were inefficient and complicated, here, we show that the codon-optimized cas9, which was used with the newly reported promoter Pcmlsm3 and terminator Tcmura3, was expressed. Furthermore, with the help of the negative selection marker ura3, a CRISPR-Cas9 system that included the Cas9 DNA endonuclease, RNA presynthesized in vitro and a single-strand DNA template efficiently generated site-specific deletion and insertion. This is the first report of a CRISPR-Cas9 system in C. militaris, and it could accelerate the genome reconstruction of C. militaris to meet the need for rapid development in the fungi industry.
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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.