Multiplex gene precise editing and large DNA fragment deletion by the CRISPR-Cas9-TRAMA system in edible mushroom Cordyceps militaris.

The medicinal mushroom Cordyceps militaris contains abundant valuable bioactive ingredients that have attracted a great deal of attention in the pharmaceutical and cosmetic industries. However, the development of this valuable mushroom faces the obstacle of lacking powerful genomic engineering tools. Here, by excavating the endogenous tRNA-processed element, introducing the extrachromosomal plasmid and alongside with homologous template, we develop a marker-free CRISPR-Cas9-TRAMA genomic editing system to achieve the multiplex gene precise editing and large synthetic cluster deletion in C. militaris. We further operated editing in the synthetases of cordycepin and ergothioneine to demonstrate the application of Cas9-TRAMA system in protein modification, promoter strength evaluation and 10 kb metabolic synthetic cluster deletion. The Cas9-TRAMA system provides a scalable method for excavating the valuable metabolic resource of medicinal mushrooms and constructing a mystical cellular pathway to elucidate the complex cell behaviours of the edible mushroom.

Enhancement of ergothioneine production by discovering and regulating its metabolic pathway in Cordyceps militaris.

BACKGROUND: Cordyceps militaris is a traditional medicinal fungus contains a variety of functional ingredients and has been developed as an important mushroom food recently. Ergothioneine, one of the antioxidative compounds in C. militaris, is benefits on aging-related diseases and therefore became a novel functional food nutritive fortifier. Currently, the main diet source of ergothioneine is mushroom food. However, the mushroom farming faces the problems such as rather low ingredient yield and spontaneous degeneration associated fruiting body that restricts large scale production of ergothioneine. RESULTS: In this study, we excavated the ergothioneine synthetases in mushroom and modified the genes in C. militaris to construct a new ergothioneine synthesis pathway. By further introducing this pathway into C. militaris genome, we succeeded to increase the ingredients' production of engineering strain, the highest amount of ergothioneine and cordycepin were up to 2.5 g/kg dry weight and 2 g/L, respectively. Additionally, the expression of ergothioneine synthetase genes in the shape-mutated degenerative C. militaris could recover the ability of degenerative strain to produce high amount of ingredients, suggesting the metabolic regulation of ergothioneine might release the symptom of mushroom degeneration. CONCLUSION: This study reveals a new pathway to fulfill the market needs of functional mushroom food and food fortifier ergothioneine. It implied the mycelium of C. militaris could be engineered as a novel medicinal mushroom food which could produce higher amount of valuable ingredients.

Ergothioneine exhibits longevity-extension effect in Drosophila melanogaster via regulation of cholinergic neurotransmission, tyrosine metabolism, and fatty acid oxidation.

Many studies have demonstrated the protective effect of ergothioneine (EGT), the unique sulfur-containing antioxidant found in mushrooms, on several aging-related diseases. Nevertheless, to date, no single study has explored the potential role of EGT in the lifespan of animal models. We show here that EGT consistently extends fly lifespan in diverse genetic backgrounds and both sexes, as well as in a dose and gender-dependent manner. Additionally, EGT is shown to increases the climbing activity of flies, enhance acetylcholinesterase (AchE) activity, and maintain the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG)of aged flies. The increase in lifespan by EGT is gut microorganism dependent. We proposed potential mechanisms of lifespan extension in Drosophila by EGT through RNA-seq analysis: preservation of the normal status of the central nervous system via the coordination of cholinergic neurotransmission, tyrosine metabolism, and peroxisomal proteins, regulation of autophagic activity by altering the lysosomal protein CTSD, and the preservation of normal mitochondrial function through controlled substrate feeding into the tricarboxylic acid (TCA) cycle, the major energy-yielding metabolic process in cells.

Comparative transcriptome and proteome provide new insights into the regulatory mechanisms of the postharvest deterioration of Pleurotus tuoliensis fruitbodies during storage.

The Pleurotus tuoliensis (Pt), a precious edible mushroom with high economic value, is widely popular for its rich nutrition and meaty texture. However, rapid postharvest deterioration depreciates the commercial value of Pt and severely restricts its marketing. By RNA-Seq transcriptomic and TMT-MS MS proteomic, we study the regulatory mechanisms of the postharvest storage of Pt fruitbodies at 25 ℃ for 0, 38, and 76 h (these three-time points recorded as groups A, B, and C, respectively). 2,008 DEGs (Differentially expressed genes) were identified, and all DEGs shared 265 factors with all DEPs (Differentially expressed proteins). Jointly, the DEGs and DEPs of two-omics showed that the category of the metabolic process contained the most DEGs and DEPs in the biological process by GO (Gene Ontology) classification. The top 17 KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways with the highest sum of DEG and DEP numbers in groups B/A (38 h vs. 0 h) and C/A (76 h vs. 0 h) and pathways closely related to energy metabolism were selected for analysis and discussion. Actively expression of CAZymes (Carbohydrate active enzymes), represented by laccase, chitinase, and ß-glucanase, directly leads to the softening of fruitbodies. The transcription factor Rlm1 of 1,3-ß-glucan synthase attracted attention with a significant down-regulation of gene levels in the C/A group. Laccase also contributes, together with phenylalanine ammonia-lyase (PAL), to the discoloration reaction in the first 76 h of the fruitbodies. Significant expression of several crucial enzymes for EMP (Glycolysis), Fatty acid degradation, and Valine, leucine and isoleucine degradation at the gene or protein level supply substantial amounts of acetyl-CoA to the TCA cycle. Citrate synthase (CS), isocitrate dehydrogenase (ICDH), and three mitochondrial respiratory complexes intensify respiration and produce high levels of ROS (Reactive oxygen species) by significant up-regulation. In the ROS scavenging system, only Mn-SOD was significantly up-regulated at the gene level and was probably interacted with Hsp60 (Heat shock protein 60), which was significantly up-regulated at the protein level, to play a dominant role in antioxidation. Three types of stresses - cell wall stress, starvation, and oxidative stress - were suffered by Pt fruitbodies postharvest, resulting in cell cycle arrest and gene expression disorder.

Transcriptome Analysis Reveals the Flexibility of Cordycepin Network in Cordyceps militaris Activated by L-Alanine Addition.

Cordycepin, isolated from the traditional medicinal fungus Cordyceps militaris, has gained much attention due to its various clinical functions. Previous reports of L-alanine addition could significantly improve cordycepin production, but the molecular mechanism remains unknown. In this study, transcriptome analysis of C. militaris with doubled cordycepin production induced by L-alanine addition provides an insight into the flexibility of the cordycepin network. The biopathways of energy generation and amino acid conversion were activated so that cordycepin substrate generation was consequently improved. Specific genes of rate-limiting enzymes in these pathways, as well as related transcription factors, were figured out. Two key Zn2Cys6-type transcription factors CmTf1 and CmTf2 were verified to play the roles of doubling the cordycepin production by overexpression of their coding genes in C. militaris wild type. These results provide a complete map of the cordycepin network in C. militaris with a distinct understanding of the flexibility of joints, giving a better foundation for increasing cordycepin yield and strain breeding in the future.

Cmfhp Gene Mediates Fruiting Body Development and Carotenoid Production in Cordyceps militaris.

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.

Efficient CRISPR-Cas9 Gene Disruption System in Edible-Medicinal Mushroom Cordyceps militaris.

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.