Submerged Culture of Edible and Medicinal Mushroom Mycelia and Their Applications in Food Products: A Review.

Edible mushrooms have rich nutrition (e.g., proteins, dietary fibers, polysaccharides) and they can be potential sources of important ingredients in food processing. However, the cultivation of mushroom fruiting bodies needs a relatively long time, and they can be easily polluted during the growth process. At the same time, a lot of labor and larger planting areas are also required. As we all know, submerged fermentation is a good way to produce edible mushroom mycelia with less environmental pollution and small footprint, which are also rich in nutrition and bioactive components that are used as dietary supplements or health care products in the food industry. Therefore, it can be considered that the replacement of edible mushroom fruiting bodies with edible mushroom mycelia produced through submerged fermentation has great application potential in food production. At present, most of the research about edible mushroom mycelia focuses on the production of bioactive metabolites in fermentation liquid, but there are few reports that concentrate on their applications in food. This paper reviews the research progress of submerged culture of edible mushroom mycelia and their applications in food products.

Proteomics analysis of enzyme systems and pathway changes during the moromi fermentation of soy sauce mash.

BACKGROUND: During the brewing of soy sauce, the conversion of multiple substances is driven by various microorganisms and their secreted enzyme systems. Soy sauce mash is an important source of enzyme systems during moromi fermentation, but the changes of enzyme systems in soy sauce mash during moromi fermentation are poorly understood. In order to explore the predominant enzyme systems existing during moromi fermentation and to explain the characteristics of the enzyme system changes, an enzymatic activities assay and 4D-label-free proteomics analysis were conducted on soy sauce mash at different stages of fermentation. RESULTS: The activities of hydrolytic enzymes in soy sauce mash decreased continuously throughout the fermentation process, while most of the characteristic physicochemical substances in soy sauce mash supernatant had already accumulated at the early stage of fermentation. Four hydrolytic enzymes were found to be positively correlated with important physicochemical indexes by principal component analysis and Pearson correlation analysis. The proteomics analysis revealed three highly upregulated enzymes and two enzymes that were present in important metabolic pathways throughout the fermentation process. Furthermore, it was found that Aspergillus oryzae was able to accumulate various nutrients in the soy sauce mash by downregulating most of its metabolic pathways. CONCLUSION: Enzymes present with excellent properties during the moromi fermentation period could be obtained from these results. Meanwhile, the characterization of the metabolic pathways of microorganisms during the moromi fermentation period was revealed. The results provide a basis for more scientific and purposeful improvement of moromi fermentation in the future. © 2024 Society of Chemical Industry.

The beneficial potential of protein hydrolysates as prebiotic for probiotics and its biological activity: a review.

Probiotics are not only a food supplement, but they have shown great potential in their nutritional, health and therapeutic effects. To maximize the beneficial effects of probiotics, it is commonly achieved by adding prebiotics. Prebiotics primarily comprise indigestible carbohydrates, specific peptides, proteins, and lipids, with oligosaccharides being the most extensively studied prebiotics. However, these rapidly fermenting oligosaccharides have many drawbacks and can cause diarrhea and flatulence in the body. Hence, the exploration of new prebiotic is of great interest. Besides oligosaccharides, protein hydrolysates have been demonstrated to enhance the expression of beneficial properties of probiotics. Consequently, this paper outlines the mechanism underlying the action of protein hydrolysates on probiotics, as well as the advantageous impacts of proteins hydrolysates derived from various food sources on probiotics. In addition, this paper also reviews the currently reported biological activities of protein hydrolysates. The aim is a theoretical basis for the development and implementation of novel prebiotics.

Progress in biological activities and biosynthesis of edible fungi terpenoids.

The edible fungi have both edible and medicinal functions, in which terpenoids are one of the most important active ingredients. Terpenoids possess a wide range of biological activities and show great potential in the pharmaceutical and healthcare industries. In this review, the diverse biological activities of edible fungi terpenoids were summarized with emphasis on the mechanism of anti-cancer and anti-inflammation. Subsequently, this review focuses on advances in knowledge and understanding of the biosynthesis of terpenoids in edible fungi, especially in the generation of sesquiterpenes, diterpenes, and triterpenes. This paper is aim to provide an overview of biological functions and biosynthesis developed for utilizing the terpenoids in edible fungi.

Amelioration effect of water extract from Ganoderma resinaceum FQ23 solid-state fermentation fungal substance with high-yield ergothioneine on anxiety-like insomnia mice.

Herein, a solid-state fermentation (SSF) system of Ganoderma resinaceum FQ23 with high-yield ergothioneine (EGT) was established, and the amelioration effect of the water extract from its fungal substance on anxiety-like insomnia mice was studied. The content of EGT in the G. resinaceum FQ23 SSF fungal substance increased to 1.146 ± 0.066 mg g-1 DW in the optimization tests. Besides EGT, the common functional components of the water extract from the G. resinaceum FQ23 SSF fungal substance (GSW) were determined, including triterpenoids, polysaccharides, phenols, proteins and amino acids. The animal experiments showed that GSW could alleviate the anxiety-like behavior, improve the antioxidant capacity and protect the organ structure of the anxiety-like insomnia mice. With an increase in the dose of GSW given to the anxiety-like insomnia mice, their serum 5-HT and GABA levels increased, HPA axis hormone levels significantly decreased, BDNF level notably increased, and the response level of the BDNF/CREB signaling pathway was significantly enhanced, indicating that GSW may improve neuroendocrine regulation and neuroprotection in anxiety-like insomnia mice. A 30-times dose of GSW had no acute toxicity in the normal mice. Therefore, the SSF fungal substance of G. resinaceum FQ23 is a potential dietary source for improving sleep. It can be used as a solid drink to help people who are poor sleepers and as a substitute for tea or coffee to help people who are like to drink tea or coffee and cannot sleep.

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.

Cordyceps militaris Immunomodulatory Protein Promotes the Phagocytic Ability of Macrophages through the TLR4-NF-κB Pathway.

Enhancing the phagocytosis of immune cells with medicines provides benefits to the physiological balance by removing foreign pathogens and apoptotic cells. The fungal immunomodulatory protein (FIP) possessing various immunopotentiation functions may be a good candidate for such drugs. However, the effect and mechanism of FIP on the phagocytic activity is limitedly investigated. Therefore, the present study determined effects of Cordyceps militaris immunomodulatory protein (CMIMP), a novel FIP reported to induce cytokines secretion, on the phagocytosis using three different types of models, including microsphere, Escherichia Coli and Candida albicans. CMIMP not only significantly improved the phagocytic ability (p < 0.05), but also enhanced the bactericidal activity (p < 0.05). Meanwhile, the cell size, especially the cytoplasm size, was markedly increased by CMIMP (p < 0.01), accompanied by an increase in the F-actin expression (p < 0.001). Further experiments displayed that CMIMP-induced phagocytosis, cell size and F-actin expression were alleviated by the specific inhibitor of TLR4 (p < 0.05). Similar results were observed in the treatment with the inhibitor of the NF-κB pathway (p < 0.05). In conclusion, it could be speculated that CMIMP promoted the phagocytic ability of macrophages through increasing F-actin expression and cell size in a TLR4-NF-κB pathway dependent way.

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.

Enhancement of carotenoid production and its regulation in edible mushroom Cordyceps militaris by abiotic stresses.

Cordyceps militaris carotenoids are widely used as food additives, animal feed supplements, and so on. However, the biosynthetic pathway of carotenoids in C. militaris is still obscure. In this paper, changes of mycelial morphology and carotenoid accumulation of C. militaris were investigated under oxidative (KMnO4) and osmotic stress (NaCl). Subsequently, qRT-PCR was employed to detect the expression levels of genes related to carotenogenesis to explore the mechanism of adaptation to abiotic stress. When the concentrations of KMnO4 and NaCl were respectively 0.4 g/L and 2 g/L, carotenoid accumulation reached a maximum of 6616.82 ±â€¯666.43 µg/g and 6416.77 ±â€¯537.02 µg/g. Under the oxidative stress condition of KMnO4, the expressions of psy and hsp70 increased significantly compared with control. Besides, the genes fus3 and hog1 were significantly enriched in the MAPK signal pathway. Compared with the control group, there was no significant difference in expression of psy in the NaCl group. Moreover, the accumulation of triacylglycerols may contribute significantly to the increase in carotenoid accumulation. The increased accumulation of antioxidant carotenoids induced under environmental stress is to resist oxidative conditions. Fus3 and Hog1 signaling in the MAPK pathway was activated and subsequently take effects on the resistance of oxidative condition by regulating related metabolic processes. C. militaris resist the stress of high oxygen by producing a large amount of glycerol and carotenoids when this fungus is cultured in a saline environment for a long time.

Physicochemical, functional and structural properties of the major protein fractions extracted from Cordyceps militaris fruit body.

The physicochemical and functional as well as structural properties of major protein fractions (albumin, globulin, glutelin) sequentially extracted in water, salt, alkaline solution respectively from Cordyceps militaris Minfu20 fruit body were investigated. The glutelin (43.11%, w/w) was the predominant protein component of C. militaris fruit body followed by albumin (36.47%) and globulin (17.94%). The three proteins extracted from different solvents showed different characteristics, which were related to the alternation of amino acid composition, surface hydrophobicity, and structural feature. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that the albumin and globulin mainly consisted of polypeptides with size < 20 kDa. The glutelin showed serious staining on the lane which may have a relatively bigger molecular weight. Intrinsic fluorescence intensity (FI) suggested glutelin contained more unfolding conformations (highest FI) which were probably resulted in a better foaming capacity of 151% and emulsion formation with the smallest size oil droplets (10.410 µm). The protein fractions showed great nutritional quality since they satisfied all recommended essential amino acid allowances for adults of Food & Agriculture Organization (FAO)/World Health Organization (WHO). Therefore, Cordyceps militaris Minfu20 fruit body proteins have potential alternative renewable edible fungi (mushroom) protein and could be used effectively as a food ingredient to improve food nutrition and product diversification compared with plant proteins.

Microbial Cell Factory of Baccatin III Preparation in Escherichia coli by Increasing DBAT Thermostability and in vivo Acetyl-CoA Supply.

Given the rapid development of genome mining in this decade, the substrate channel of paclitaxel might be identified in the near future. A robust microbial cell factory with gene dbat, encoding a key rate-limiting enzyme 10-deacetylbaccatin III-10-O-transferase (DBAT) in paclitaxel biosynthesis to synthesize the precursor baccatin III, will lay out a promising foundation for paclitaxel de novo synthesis. Here, we integrated gene dbat into the wild-type Escherichia coli BW25113 to construct strain BWD01. Yet, it was relatively unstable in baccatin III synthesis. Mutant gene dbat S189V with improved thermostability was screened out from a semi-rational mutation library of DBAT. When it was over-expressed in an engineered strain N05 with improved acetyl-CoA generation, combined with carbon source optimization of fermentation engineering, the production level of baccatin III was significantly increased. Using this combination, integrated strain N05S01 with mutant dbat S189V achieved a 10.50-fold increase in baccatin III production compared with original strain BWD01. Our findings suggest that the combination of protein engineering and metabolic engineering will become a promising strategy for paclitaxel production.

TMT-MS/MS proteomic analysis of the carbohydrate-active enzymes in the fruiting body of Pleurotus tuoliensis during storage.

BACKGROUND: The fruiting body of Pleurotus tuoliensis deteriorates rapidly after harvest, causing a decline in its commercial value and a great reduction in its shelf life. According to the present research, carbohydrate-active enzymes (CAZymes) may cause the softening, liquefaction and autolysis of mature mushrooms after harvest. To further understand the in vivo molecular mechanism of CAZymes affecting the postharvest quality of P. tuoliensis fruiting bodies, a tandem mass tags labelling combined liquid chromatography-tandem mass spectrometry (TMT-MS/MS) proteomic analysis was performed on P. tuoliensis fruiting bodies during storage at 25 °C. RESULTS: A total of 4737 proteins were identified, which had at least one unique peptide and had a confidence level above 95%. Consequently, 1307 differentially expressed proteins (DEPs) were recruited using the criteria of abundance fold change (FC) >1.5 or < 0.67 and P < 0.05. The identified proteins were annotated by dbCAN2, a meta server for automated CAZymes annotation. Subsequently, 222 CAZymes were obtained. Several CAZymes participating in the cell wall degradation process, including ß-glucosidase, glucan 1,3-ß-glucosidase, endo-1,3(4)-ß-glucanase and chitinases, were significantly upregulated during storage. The protein expression level of CAZymes, such as xylanase, amylase and glucoamylase, were upregulated significantly, which may participate in the P. tuoliensis polysaccharide degradation. CONCLUSIONS: The identified CAZymes degraded the polysaccharides and lignin, destroying the cell wall structure, preventing cell wall remodeling, causing a loss of nutrients and the browning phenomenon, accelerating the deterioration of P. tuoliensis fruiting body. © 2020 Society of Chemical Industry.

[Genotype Analysis of Pregnant Women with α- and ß- Thalassemia in Fuzhou Area of Fujian Province in China].

OBJECTIVE: To analyze the genotype of pregnant women with α- and ß- thalassemia in Fuzhou area of Fujian province in China. METHODS: Blood routine examination and hemoglobin electrophoresis were performed for pregnant women, and positive samples were examined by gap polymerase chain reaction and reverse dot blot hybridization. RESULTS: 412 cases were diagnosed as α-thalassemia (63.9%); 201 cases were diagnosed as ß-thalassemia (31.2%); 32 cases were diagnosed as α and ß-composite thalassemia. There were 12 genotypes in α-thalassemia, whose major genotypes were --SEA/αα, α3.7/αα, -α4.2/αα and αQSα/αα, with carrying rate of 64.32%, 20.14%, 7.77% and 1.94%, respectively. There were 10 genotypes in ß- thalassemia, whose major genotypes were CD41-42/N, CD17/N, IVS-II-654/N and -28/N, with carrying rate of 30.84%, 27.86%, 15.92% and 10.45%, respectively. There were 9 genotypes in α and ß-composite thalassemia, whose major genotypes were --SEA/αα composited CD41-42/N, -α3.7/αα composited CD41-42/N, --SEA/αα composited CD17/N, with carrying rate of 18.75%, 15.62%, 15.62% respectively. CONCLUSION: The major genotypes of pregnant women with α- and ß- thalassemia in Fuzhou area of Fujian province in China are --SEA/αα, α3.7/αα, CD41-42/N and CD17/N. Thalassemia screening and prenatal gene diagnosis should be strengthened in Fuzhou area of Fujian province in China.

Successful biosynthesis of natural antioxidant ergothioneine in Saccharomyces cerevisiae required only two genes from Grifola frondosa.

BACKGROUND: Ergothioneine (EGT) has a unique antioxidant ability and diverse beneficial effects on human health. But the content of EGT is very low in its natural producing organisms such as Mycobacterium smegmatis and mushrooms. Therefore, it is necessary to highly efficient heterologous production of EGT in food-grade yeasts such as Saccharomyces cerevisiae. RESULTS: Two EGT biosynthetic genes were cloned from the mushroom Grifola frondosa and successfully heterologously expressed in Saccharomyces cerevisiae EC1118 strain in this study. By optimization of the fermentation conditions of the engineered strain S. cerevisiae EC1118, the 11.80 mg/L of EGT production was obtained. With daily addition of 1% glycerol to the culture medium in the fermentation process, the EGT production of the engineered strain S. cerevisiae EC1118 can reach up to 20.61 mg/L. CONCLUSION: A successful EGT de novo biosynthetic system of S. cerevisiae containing only two genes from mushroom Grifola frondosa was developed in this study. This system provides promising prospects for the large scales production of EGT for human health.

Structural Analysis and Antioxidant Activity of Extracellular Polysaccharides Extracted from Culinary-Medicinal White Jelly Mushroom Tremella fuciformis (Tremellomycetes) Conidium Cells.

Using Tremella fuciformis conidium cells for submerged fermentation is a cost-effective way to harvest bioactive compounds. In this study, we emphasized the structural and functional analysis of extracellular polysaccharides (EPS) extracted from T. fuciformis conidium cells. An EPS high-yield strain tyc63 was selected and a 6-day optimum fermentation period was determined. Crude EPS was extracted and three high molecular weight (5189, 171.6, and 661 kDa) polysaccharides TFP-1, TFP-2, and TFP-3 were isolated and purified. TFP-1 is mainly composed of glucose, xylose, mannose, and fucose, while both TFP-2 and TFP-3 are mainly composed of rhamnose, arabinose, mannose, galactose, and glucose. FT-IR analysis revealed that TFP-1, TFP-2, and TFP-3 have typical polysaccharide structure. The antioxidant assay revealed that the crude EPS, TFP-1, TFP-2, and TFP-3 presented high free radical scavenging activities but low ferric reducing power, suggesting that the EPS produced by liquid fermentation could be used as a potent radical scavenger.

The Biosynthetic Pathway of Ergothioneine in Culinary-Medicinal Winter Mushroom, Flammulina velutipes (Agaricomycetes).

Ergothioneine is a natural 2-thiol-amidazole amino acid that plays an important role in inflammation, depression, and cardiovascular disease. Flammulina velutipes is a common basidiomycete mushroom rich in ergothioneine (EGT). However, the biosynthetic pathway of EGT in F. velutipes is still unclear. In this study, the F. velutipes ergothioneine biosynthetic gene 1 (Fvegtl), F. velutipes ergothioneine biosynthetic gene 2 (Fvegt2), and F. velutipes ergothioneine biosynthetic gene 3 (Fvegt3) were cloned and expressed, and the activities of the proteins encoded by these three genes (FvEgt1, F. velutipes ergothioneine biosynthase 1; FvEgt2, F. velutipes ergothioneine biosynthase 2; and FvEgt3, F. velutipes ergothioneine biosynthase 3) were identified. The results showed that FvEgtl not only has the function of methyltransferase, but also has the function of hercynlcysteineteine sulfoxide (Hersul) synthase, which can catalyze the production of Hersul from histidine and cysteine in F. velutipes. FvEgt2 and FvEgt3 are two functionally different cysteine desulfurase enzymes. Among them, FvEgt2 is a cysteine-cysteine desulfurase-which catalyzes the activation of the S-H bond on cysteine, while FvEgt3 is a pyridoxal phosphate (PLP)-dependent cysteine desulfurase responsible for catalyzing the production of ketimine complex. Our results show that FvEgt1/FvEgt2/FvEgt3 can simultaneously catalyze the production of EGT by histidine, cysteine, and pyridoxal phosphate. Collectively, the in vitro synthesis of EGT in the edible fungus F. velutipes was first achieved, which laid the foundation for the biological production of EGT.

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.