Methyl viologen boosts Erinacine A biosynthesis in Hericium erinaceus fermentation: Multi-omics insights

Methyl viologen boosts Erinacine A biosynthesis in Hericium erinaceus fermentation: Multi-omics insights | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Methyl viologen boosts Erinacine A biosynthesis in Hericium erinaceus fermentation: Multi-omics insights Jian Wang, Juncai Wan, Junhao Huang, Ran Ma, Yahya Saud Mohamed Hamed, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8698276/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Erinacine A, a critical secondary metabolite of Hericium erinaceus , has garnered significant interest due to its bioactive properties. This study aimed to investigate the effects of methyl viologen (MV) on the synthesis of Erinacine A during liquid fermentation of Hericium erinaceus , employing a multi-omics approach to elucidate the underlying mechanisms. Our findings demonstrate that the addition of 5 mg/L of MV after four days of fermentation significantly enhanced Erinacine A production, achieving a remarkable yield of 23 mg/g and 414 mg/L, marking the highest yield reported to date. Scanning electron microscopy (SEM) revealed a distinctive wrinkled and collapsed mycelial surface, indicative of stress responses. Moreover, metabolomics and transcriptomics analyses revealed oxidative stress within Hericium erinaceus , characterized by the downregulation of antioxidant-like metabolites and alterations in the oxidoreductase system. MV was found to impact several metabolic pathways, notably those associated with sugars, amino acids, and fatty acids. Comprehensive analysis of the omics data confirmed that MV enhances the expression of the mevalonate pathway and the Eri gene cluster in response to oxidative stress induced by reactive oxygen species (ROS). This process facilitates the conversion of geranylgeranyl pyrophosphate (GGPP) into Erinacine-like substances. This study provides novel insights, demonstrating for the first time that MV can be effectively utilized in the liquid fermentation of Hericium erinaceus to significantly improve the yield of Erinacine A. Hericium erinaceus Erinacine A Diterpenoid Submerged fermentation Methyl viologen Multi-omics Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Key points · MV supplementation achieves record Erinacine A yields (23 mg/g, 414 mg/L) in liquid fermentation. · Oxidative stress from MV activates mevalonate pathway and Eri gene cluster for enhanced biosynthesis. · Multi-omics reveals MV's regulation of sugar, amino acid, and fatty acid metabolism pathways. Introduction Hericium erinaceus (HE), a popular edible and medicinal mushroom species, also known as the Lion's Mane mushroom or Monkey Head mushroom, belongs to the Basidiomycota phylum , Agaricomycetes class , Hericiaceae family , and Hericium erinaceus genus . Erinacine is a unique type of diterpene of HE, which has been concerned for its potential treatment of neurodegenerative diseases. So far, more than 20 types of Erinacines have been identified, mainly from mycelium (Li et al. 2018 ). Erinacine A is also the most concern, and studies have shown that Erinacine A promotes the release of nerve growth factor (Shimbo et al. 2005 ), increase the expression of insulin-degrading enzymes, and reduce β amyloid deposition (Chen et al. 2016 ) and its efficacy in inhibiting cancer cell proliferation in vitro (Prasher et al. 2023 ). Liquid fermentation is a rapid and cost-effective method for producing a substantial quantity of mycelium rich in Erinacine A, which has been identified as the primary pathway for the synthesis of Erinacine A. Although Erinacine A can be chemically synthesized, the synthesis steps are cumbersome, and side products are produced in the process (Li et al. 2018 ). The biosynthesis of diterpenes is the synthesis of the key precursor GGPP through the mevalonate (MVA) pathway and MEP pathway and the modification of diterpene synthase, diterpene cyclase and cytochrome P450 (Zerbe and Bohlmann 2015 ). HE genome sequencing by Chen et al. found that GGPP, a key precursor of synthetic diterpenoids, had only the gene expression of the mevalonate pathway (Chen et al. 2017 ), suggesting that this may be the only route for Erinacine synthesis in HE. In an earlier study by Liu et al., the gene cluster (Eri) responsible for Erinacine synthesis in HE, including one GGPPs (EriE), a diterpenoid ring enzyme (Eri G) responsible for cyclization of the cyathane skeleton, three cytochrome P450 (EriA / C / I), and one UDP-glycosyltransferase (Eri J) (Yang et al. 2017). In subsequent studies, through the expression of gene editing in engineered yeast, the inference of the biosynthesis of Erinacines (Fig. S1 ), and the successful synthesis of various Erinacines (Ma et al. 2021 ). Finally, Erinacine was successfully isolated from the transformant SC-GICAJLM, and Erinacine P was synthesized as Erinacine A by a non-enzymatic spontaneous MICHAEL addition elimination reaction, which also supports the previous conclusion (Kenmoku et al. 2000 ). In the past, two reports have brought the yield of Erinacine A to 192 mg/L and 225.54 mg/L (Chang et al. 2016 ; Krzyczkowski et al. 2010 ), which is almost the limit of the synthesis. We wanted to seek an unusual culture method to prompt HE to synthesize more Erinacine A. Exogenous factors refer to multiple factors coming from outside the organism, which can affect the physiology, biochemical processes or behavior of the organism, and the addition of exogenous factors during fermentation seems to be a good strategy. For example, the addition of 5 mg/L rotenone increased the yield of cordycepin produced by 316.09% (Ma et al. 2023 ), the addition of 1mg/L methyl viologen and rotenone increased the total pigment yield of Monascus purpureus by 39.08% and 40.89%, respectively, and the yield of yellow pigment by 74.62% and 114.06%, respectively (Liu et al. 2021 ). The addition of orange peel essential oil to Taiwanofungus camphoratus fermentation increased the total triterpene yield by more than 10 times (Ma et al. 2014 ). To date, no reports have been identified regarding the use of exogenous factors to enhance the yield of Erinacine A. In this study, we attempted to add several exogenous factors during liquid fermentation of HE and found that methyl viologen (MV) was the most helpful in improving the yield of Erinacine A. Based on the results of the screening, we investigated the properties of MV affecting liquid fermentation, and the addition of 5–10 mg/L MV at the end of the logarithmic growth phase of the mycelium significantly increased the yield of Erinacine A. Furthermore, we investigated the potential mechanisms by which MV affects the biosynthesis of Erinacine A through a combined analysis of transcriptomics and metabolomics. Finally, the relationship between the changes in antioxidant properties of Erinacine A and the synthesis of Erinacine A was explored under MV treatment. This study is the first application of MV to increase the yield of Erinacine A in HE fermentation, providing a new idea to utilize the appropriate oxidative stress to improve Erinacine A synthesis. Materials and methods Strains and reagent materials The edible Hericium erinaceus (Bill.) Pers. used in this study was a proprietary strain developed by Xueyu Biotechnology Co., Ltd (Hangzhou, China), which was identified by the Institute of Microbiology, Chinese Academy of Sciences. The HE strain XY-2 was derived from the revitalization of strain XY-1. The HE strain HW8415 was obtained from the Microbial Research Institute of the Heilongjiang Academy of Sciences. The HE strain CGMCC 5.739 was purchased from the China General Microbiological Culture Collection Center. The strain was statically cultured on a potato dextrose agar (PDA) slant at 28°C. The liquid seed culture medium consisted of 30 g/L glucose, 30 g/L soybean meal, 1 g/L potassium dihydrogen phosphate, and 0.5 g/L magnesium sulfate. The liquid fermentation medium consisted of 30 g/L glucose, 30 g/L yeast powder, 1 g/L potassium dihydrogen phosphate, and 0.5 g/L magnesium sulfate. All culture media materials were provided by Xueyu Biotechnology Co., Ltd (Hangzhou, China). The chemicals used in this study include methyl viologen (> 96%, Aladdin), rotenone (95%, Aladdin), oleic acid (Sinopharm), sweet orange oil (Bonai), chitosan (Rhawn), corn oil (Rhawn), and methyl jasmonate (Macklin). Liquid fermentation and mycelium collection Seed cultures of HE strains XY-1, XY-2, HW8415, and CGMCC 5.739 were prepared and introduced into the fermentation medium. The fermentation was conducted at 27°C and 160 rpm for 7 days, leading to the development of small, uniform mycelial pellets in the seed culture. The seed culture was injected into the liquid fermentation medium at a 10% (v/v) inoculum size, and the fermentation took place at 27°C and 140 rpm. Exogenous factors such as chitosan, corn oil, methyl viologen, rotenone, methyl jasmonate, sweet orange oil, and oleic acid were introduced to the fermentation medium following previously reported protocols. To prepare the culture medium, 0.1 g/L of chitosan was added (Vicente et al. 2024 ), 2.5% (v/v) corn oil was added after 2 days of fermentation (Meng et al. 2021 ), and a 5 mg/L concentration of methyl viologen and rotenone (in water and ethanol solutions, respectively) was added after 4 days of fermentation (Ma et al. 2023 ). Ma (Ma et al. 2014 ) added 50 µL of sweet orange oil and oleic acid after 4 days of incubation. The experimental group that did not receive any exogenous inducer acted as a blank control. At the end of fermentation, the mycelia were separated with an 80-mesh filter cloth and were washed three times with distilled water Afterward, the mycelia were dried to constant weight in a blast drying oven at 60°C and the dried mycelia was prepared as a powder for analysis. Extraction and determination of Erinacine A One gram of HE powder was weighed and combined with 20 mL of 80% methanol for the extraction of Erinacine A. The extraction was carried out using ultrasonic extraction at 60°C and 300 W power for 1 hour. After extraction, the mixture was centrifuged to obtain the supernatant, which was then filtered through a 0.22 µm membrane and subjected to liquid chromatographic analysis. High performance liquid chromatography (HPLC) detection conditions: SHIMAZU LC2030 HPLC system, using a Phenomenex luna C18 column (4.6×250 mm, 5 µm); mobile phase: composed of water and acetonitrile, 0–23 min, 30% acetonitrile-50% acetonitrile; 23–26 min, 50% acetonitrile, 26–32 min, 50% acetonitrile-100% acetonitrile, 32–44 min, 100% acetonitrile, 44–60 min, 30% acetonitrile; column temperature 30°C; flow rate 1.0 mL/s; injection volume 20 µL; detection wavelength 340 nm. The elution time of Erinacine A is around 31 min. The Erinacine A standard (purity > 96%) is provided by Huida Biotech Co., Ltd (Hangzhou, China). Reduction sugar detection The fermentation broth was collected in centrifuge tubes and sterilized in a boiling water bath for 15 min, then stored at 4°C for a few hours for analysis. On the same day, the reducing sugar content of the fermentation broth was measured. Take 1 mL of the fermentation broth after sterilization, dilute it to an appropriate concentration, and use the 2,4-dinitrosalicylic acid (DNS) method to detect the reducing sugars in the fermentation broth. Mycelium morphology observation The mycelial and ultrastructural morphology were observed using a scanning electron microscope (Hitachi Reguius8100, Japan). The mycelium was washed three times with pre-cooled PBS solution, then fixed in 2.5% glutaraldehyde fixative at 4°C in the dark overnight for sample preparation. The sample preparation and observation were carried out by Shiyanjia Lab (Hangzhou, China). Metabolomics analysis The fermentation broth in the shaker flask was centrifuged at 5,000 rpm for 5 min at 4°C. The mycelium was collected and quickly washed 2–3 times with pre-chilled PBS to completely remove the supernatant. The clean mycelium was then snap-frozen in liquid nitrogen and stored at -80°C for subsequent analysis. For the metabolomics analysis, each experimental group was set up with 6 parallel samples. Place 100 mg liquid nitrogen ground tissue sample in an EP tube, add 500 µL of 80% methanol, vortex, sit for 5 min, and centrifuge. A certain amount of supernatant was diluted with mass spectral grade water to 53% methanol, and the supernatant was collected by centrifugation and injected for LC-MS analysis. The metabolic profiles in the positive and negative ion modes were analyzed by the Vanquish UHPLC system (Thermo Fisher, Germany) and the Q Exactive™ HF/Q Exactive™ HF-X mass spectrometer (Thermo Fisher, Germany). Using the Hypersil Gold C18 column (Thermo Fisher, USA). The gradient elution system is shown in Table S1 , mobile phase A is 0.1% formic acid in positive mode, mobile phase B is 5 mM ammonium acetate, pH 9.0, and mobile phase B is methanol in negative mode. Column temperature was 40 ℃, and the flow rate was 0.2 mL/min. Transcriptome analysis Mycelium samples were collected the same as extraction and determination of Erinacine A, and in transcriptome analysis three parallel runs were set for each experimental group. Sample testing, RNA extraction, Library construction, quality detection and sequencing were performed by the Novogene Bioinformatics Institute (Beijing, China). RNA was quantified using Qubit® 2.0 Flurometer (Life Technologies, San Diego, CA, USA), checked for integrity on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). The library was diluted to 1.5 ng/ul by quantification and the effective concentration of the library was accurately quantified by qRT-PCR to ensure the quality of the library (the effective concentration of the library was higher than 1.5 nm). The index-coded samples were clustered after passing the library inspection. After cluster generation, the library preparations were sequenced on Illumina with 150 bp paired-end reads. De novo Transcriptome assembly After the sequencing of the cDNA library, the sequencing fragments of the image data measured by the high-throughput sequencer are converted into sequence reads by CASAVA bases identification, and the raw reads are obtained after quality filtering. To ensure the reliability of data analysis, we removed some low-quality and unavailable reads to obtain clean reads. Meanwhile, Q20, Q30 and GC content were calculated for clean reads. All subsequent analyses were based on the high-quality analysis performed by clean reads. Since HE lacks a suitable reference genome, De novo assembly was applied to construct transcripts from the clean reads, and the Trinity assembler was used for transcriptome assembly of the clean reads (Haas et al. 2013 ). Based on Trinity splicing, the transcripts are aggregated into many clusters according to the Shared Reads between transcripts, combined with the transcript expression level between different samples and the H-Cluster algorithm, the transcripts with the expression difference between samples are separated from the original cluster to establish a new cluster, and finally each cluster is defined as "Gene". The BUSCO (Benchmarking Universal Single-Copy Orthologs) assessment evaluates the integrity of the assembled transcripts using a single-copy direct orthologous gene library combined with software such as tblastn, augustus and hmmer. Gene functional annotation and differential gene enrichment We annotated the spliced transcripts with seven major databases (Nr, Nt, PFAM, KOG / COG, Swiss-Prot, KEGG, GO), and performed differential expression analysis in two conditions/groups using the DESeq2 R package (1.20.0). DESeq2 provides statistical procedures for determining differential expression in digital gene expression data using a model based on a negative binomial distribution. The resulting P-values were adjusted to control the false discovery rate using the method of Benjamini and Hochberg method. Genes identified by DESeq2 with an adjusted P-value < 0.05 were designated as differentially expressed. Statistical analysis All experiments were carried out at least three times, and data were expressed as the mean ± standard deviation. One-way ANOVA and independent t-test were carried out using GraphPadPrism software version 8 (La Jolla, USA) for statistical analysis. Results Effect of exogenous factors on the growth and development of different HE and the yield of Erinacine A The effect of methyl viologen on the production of Erinacine A in the liquid fermentation of different HE strains. As shown in Fig. 1 A, methyl viologen had a good stimulatory effect on the four HE strains, significantly improving the production of Erinacine A, indicating that methyl viologen has a universal effect on enhancing the biosynthesis of Erinacine A. The results show that the inductive effect of methyl viologen was most significant on HE strain XY-2 and HE strain CGMCC 5.739. Although the Erinacine A production of the blank group of HE XY-2 was relatively low, it can be observed that it has a high growth potential under the stimulation of the exogenous inducing factor. During the seed culture process, it was found that the growth rate of HE CGMCC 5.739 was slower than that of HE XY-2. Therefore, HE XY-2 was selected for further research. The effect of seven exogenous inducers on liquid fermentation of HE after 4 d fermentation is shown in Fig. 1 B. Although the blank group showed a rapid growth rate and the yield of dry mycelium increased (24.52 ± 0.68 g/L). However, the synthesis capacity of Erinacine A was significantly reduced (4.48 ± 0.96 mg/L), the result may be due to the change of species characteristics, and the subsequent characteristics of different HE induced fermentations will be carefully studied. The results showed that stimulated of methyl viologen and rotenone, reaching 37.79 ± 7.96 mg/L and 13.71 ± 0.24 mg/L, respectively, and the yield of Erinacine A in the methyl viologen experiment group increased approximately 8.44-fold compared with the blank group. However, chitosan, corn oil, methyl jasmonate, sweet orange oil and oleic acid did not positively affect the biosynthesis of Erinacine A, which may be that the mechanism of action could not stimulate the activity of diterpene synthase. According to this result, methyl viologen was selected as an exogenous induction factor for subsequent studies. Methyl viologen has a significant inhibitory effect on the growth of HE mycelium, which may be a negative effect caused by oxidative damage, such as protein and lipid oxidation, or even DNA damage(Wang et al. 2022 ). Therefore, it is necessary to study the optimal time for the addition of MV and consider the improvement of Erinacine A yield without affecting mycelium development. As shown in Fig. 1 C, the first 3 d of fermentation was the rapid growth period of HE in liquid culture. The addition of methyl viologen previously caused a significant decrease in the yield of HE mycelium, which was not conducive to the synthesis of Erinacine A. The best addition time for the induction effect is 4 d after fermentation, at which time the HE mycelium is almost full and tends to mature. During this period, the metabolic enzyme activity in HE bacteria may be high. After 5 d of fermentation, the effect of adding methyl viologen will gradually decrease, and at this stage, the nutrients in the fermentation system will be gradually exhausted, and the exchangeability of HE and external substances will also decrease. Therefore, 4 d after fermentation was chosen as the optimal time for methyl viologen supplementation. A yield of 103.99 mg/L of Erinacine A was obtained when the total fermentation time reached 8 d. After determining the optimal induction process of ME, the additive amount of MYE was investigated separately, and the results are shown in Fig. 1 D. The results showed that the yield of Erinacine A reached the maximum at 5 mg/L and 10 mg/L, reaching 414.84 ± 30.03 mg/L and 400.44 ± 69.30 mg/L, respectively, with no significant difference. Therefore, the optimal additive amount of methyl viologen was determined to be 5 mg/L. This result was significantly higher than the yield and content of Erinacine A reported by Chang (Chang et al. 2016 ), who achieved 225.54 mg and 13.39 mg/g dry mycelium, respectively, after optimizing the BCRC35669 medium. Effect of methyl viologen on liquid fermentation characteristics of HE To observe the effect of MV on the fermentation characteristics of Erinacine A during fermentation, we drew the growth curve (Fig. 2 ) and observed the optimal duration of MV treatment. MV after 4 d of fermentation showed delayed growth (Fig. 2 A). Due to the high-temperature sterilization of the culture medium, the loss of about 2 g/L of the reducing sugar will occur. Fermentation for 12 d may not be enough for mycelium autolysis, because the excess yeast powder in the culture medium can also be used by HE to decompose a small amount of reducing sugars to maintain basic life activities, but it seems that when mycelium is under MV stress, slowing down some cell autolysis trend (Fig. 2 A-B). On the fifth day of fermentation, a buffer period was observed for MV bulb growth in the MV group, during which it reduced nutrient intake to adapt to the strong stimulation of foreign stress (Fig. 2 B). However, after the buffer period, the mycelium will still consume the carbon source at a faster rate, synthesizing more secondary metabolites rather than supporting the increase in mycelium volume. Finally, the highest level of Erinacine A was reached after 10d of fermentation, yielding about 414.84 mg/L (Fig. 2 C). Obtaining such a high yield of Erinacine A was surprising, indicating that promoting the expression of the Erinacine A synthesis gene by administering appropriate exogenous stress is feasible and the effect is extremely remarkable. After determining the optimal period of MV stress, we optimized the concentration of added MV and found that the MV concentration of 5–10 mg/L kept the yield of Erinacine A at the highest level, far ahead of the positive effects of other concentrations, because the fungus was sensitive to MV and giving too high concentration led to its death (Liu et al. 2021 ). MV on the micromorphology of HE mycelium To observe the effect of MV on the morphology of the mycelium, mycelial samples were taken after 10 days of fermentation and observed under a scanning electron microscope (SEM). At 1000x magnification (Fig. 3 A), The mycelium of the blank group showed denser mycelium, and the tips of the mycelium had more branches, still showing a tendency to grow. The mycelium of the methyl viologen group was more slender, more twisted and broken, with less sporulation at the ends of the mycelium (Fig. 3 D). Under 5000x and 10000x ultra-microscopic observation (Fig. 3 B-C), the surface of the mycelium in the blank group appeared smooth, plump, and with a dense structure and intact tubular morphology. However, the mycelium of the methyl viologen group showed obvious shrinkage and even different degrees of collapse (Fig. 3 E-F). Summary of transcription To ensure the quality and reliability of the data analysis, the raw data needs to be filtered. After excluding low-quality reads, the proportion of clean reads in each sample exceeded 98%, with Q20 and Q30 greater than 97.6% and 93.5%, respectively (Table S2). By comparing with the Nr library annotation, we can obtain the similarity of the species and the functional information of the species gene, after annotation alignment, in the reference species distribution map (Fig. 4 A), the genes from the Nr library mainly from ( Hericium alpestre ) and small spores ( Deptipellis sp .). The former is a related species of HE, while the latter has a high evolutionary affinity (Chen et al. 2017 ). Furthermore, no species more distantly related to HE was found on the distribution map, and the results show that the transcriptomic data were highly reliable. In setting conditions | Log2Foldchange | 1 and padj < 0.005. As shown in Fig. 4 B, 651 differentially significantly expressed genes (differentially expressed genes, DEGs) were selected, of which 356 were upregulated and 295 were downregulated. GO enrichment analysis and KEGG pathway analysis of differentially expressed genes To study the changes of MV-induced in response to the biological functions of HE, the enriched DEGs were subjected to GO function annotation analysis, which found that MV mainly activated the oxidative stress response in the HE organism, resulting in the most significant changes in oxidoreductase activity, whether up or downregulation. However, in the upregulated GO enrichment, only significant differential expression of oxidoreductase activity was observed (Fig. 4 C). This phenomenon may be caused by the oxidative stress generated by MV, which stalls the metabolic activities of the growth and development to improve the antioxidant capacity. The remaining down-regulated DEGs were mainly enriched in the extracellular matrix, transmembrane transport, and basal metabolic activities such as lipids and carbohydrates (Fig. 4 D). The enriched DEGs were projected into KEGG map to study the effect of MV on metabolic activity of HE (Fig. 4 E-F). In the down-regulated KEGG metabolism pathway, the methane metabolism pathway and the fatty acid synthesis pathway are significantly down-regulated, found in the specific analysis (Table 1 ), in the methane metabolism pathway it is mainly methanol to formaldehyde, the formic acid carbon dioxide reaction pathway is inhibited, this may be that MV stress affects mitochondrial aerobic respiration, must regulate other carbon dioxide pathways to maintain balance of life. Under oxidative stress, the growth was arrested. Fatty acids are closely related to the growth and development of mycelium, and the significant down-regulation of the fatty acid enzyme, fatty acid synthase (FAS), is found in the fatty acid synthesis pathway, which may be related to the growth inhibition of HE (Wernig et al. 2020 ). In the upregulated KEGG metabolic pathways, the significant upregulation of the terpenoid backbone biosynthesis pathway was observed, and it clearly showed that HE enhanced the expression of the mevalonate acid pathway. By integrating the KEGG pathways, it was found that in addition to the pathways mentioned above, many carbohydrate metabolism pathways were also significantly changed. After 10 days of fermentation, the glucose was already depleted, and HE will utilize the glucan, mannan, and chitin in the yeast powder in the culture medium to maintain its metabolic activity (Gao et al. 2024 ). Table 1 KEGG pathway enrichment analysis of significantly affected DEGs. Term Sample Number Background Number P-value Unigene KO Gene name Methane metabolism 8 30 0.0004 Cluster-310.7244 Cluster-310.3705 Cluster-310.3946 Cluster-310.7922 Cluster-310.6962 Cluster-310.8604 Cluster-310.6545 Cluster-310.6552 K00122 K01070 K00863 K00121 K17066 K00863 K00925 K00122 FDH/frmB, ESD, fghA/DAK, TKFC/ frmA, ADH5, adh/ MOX/DAK, TKFC/ ackA/ FDH Starch and sucrose metabolism 10 64 0.0031 Cluster-310.8207 Cluster-310.5885 Cluster-310.6788 Cluster-310.5278 Cluster-310.3664 Cluster-310.4681 Cluster-310.10422 Cluster-310.6399 Cluster-310.9901 Cluster-310.7095 K01187 K01179 K05349 K05349 K00700 K01179 K01210 K01835 K00700 K00688 MalZ/E3.2.1.4/bglX/ bglX/GBE1,glgB/ E3.2.1.58/pgm/ GBE1,glgB/PYG,glgP/ E3.2.1.4 Fatty acid biosynthesis 4 15 0.0127 Cluster-310.7093 Cluster-310.9052 Cluster-310.7516 Cluster-310.10200 K00668 K00668 K00668 K00668 FAS1/FAS1/FAS1/ FAS1 Glyoxylate and dicarboxylate metabolism 6 35 0.0146 Cluster-310.7244 Cluster-310.6711 Cluster-310.6552 Cluster-310.7834 K00122 K00626 K00122 K01637 FDH/ACAT,atoB/FDH/aceA/GLDC,gcvP/ katE, CAT, catB, srpA Table 1 ( continued ) Term Sample Number Background Number P-value Unigene KO Gene name Cluster-310.8309 Cluster-310.4672 K00281 K03781 Amino sugar and nucleotide sugar metabolism 7 51 0.0234 Cluster-310.7652 Cluster-310.4791 Cluster-310.3531 Cluster-310.3088 Cluster-310.7254 Cluster-310.6399 Cluster-310.5974 K08678 K01183 K01183 K01809 K01183 K01835 K01183 UXS1,uxs/E3.2.1.14/E3.2.1.14/manA,MPI/E3.2.1.14/pgm/ E3.2.1.14 Terpenoid backbone biosynthesis 4 19 0.0249 Cluster-310.4346 Cluster-310.6953 Cluster-310.6981 Cluster-310.6711 K00787 K00804 K01641 K00626 FDPS GGPS1 HMGCS ACAT, atoB Fructose and mannose metabolism 4 24 0.0475 Cluster-310.8604 Cluster-310.3088 Cluster-310.7468 Cluster-310.3946 K00863 K01809 K19029 K00863 DAK,TKFC/manA,MPI/PFKFB2/DAK,TKFC/ Note: "Unigene" refers to the transcript number assembled in transcriptome analysis; "KO" indicates the entry of the gene in the KO annotation; "Gene Name" indicates the gene encoding the corresponding enzyme, and "E3.2.1.4" etc. indicates the enzyme node to which the transcript gene is annotated in the KEGG pathway, but there is no explicit gene name; when a Gene Name appears multiple times, it means that the gene is a node in multiple reactions in the pathway. Effect of MV-induced on the metabolic profile of HE A non-targeted LC-MS metabolomics analysis method was used to detect the metabolites of HE under different treatment groups, and a positive and negative merged analysis was performed. Principal component analysis (PCA) showed that the MV group and the MOCK group exhibited distinct metabolic patterns, and the QC samples were well clustered, indicating that the addition of MV changed the abundance of HE metabolites, and the experimental results were reliable (Fig. 5 A). The metabolites were clustered, and the heat map analysis showed good clustering effects of different metabolic groups (Fig. S2). After partial least squares-discriminant analysis (PLS-DA), the results in Fig. 5 B showed that the PLS-DA model could distinguish the different experimental groups, and the inter-group differences were significant. In this experiment, the Q2 of the model was 0.8, which is generally considered to indicate that the model has predictive ability when Q2 > 0.5, and this result proves that the model is very reliable and can be used for subsequent identification of differential metabolites. A total of 483 differentially accumulated metabolites (DAMs) were screened between the two groups of samples, with 367 DAMs showing downregulation and 116 DAMs showing upregulation (Fig. 5 C). The number of DAMs was much higher than the number of upregulated DAMs, but in the transcriptome analysis, the number of upregulated DEGs was slightly higher than the number of downregulated DEGs. This phenomenon suggests that in response to the oxidative stress caused by MV, most metabolites were involved in antioxidant reactions and were consumed. Based on the DAMs obtained from the metabolomics analysis, the 20 most significantly downregulated and upregulated DAMs were screened according to the FC values and plotted in a volcano plot (Fig. 5 D). Among them, substances that are unlikely to exist in HE was excluded, as these substances may be misidentified during the metabolite database matching process in mass spectrometry. From the 20 most significantly downregulated DAMs, the main components were antioxidants (such as soy isoflavones, soy lutein, ergothioneine (Halliwell et al. 2023 ), 2-hydroxycinnamic aldehyde (Gupta et al. 2023 ), and dye wood (Zhao et al. 2016 )), amino acids and their derivatives (such as cysteine, serine, 2-hydroxy-L-phenylalanine, and N-acetyl-D-tryptophan), and lipids. The appearance of soy isoflavones and soy lutein may be due to the absorption of nutrients from the soy meal in the seed culture medium into the fermentation system. The significant decrease in the content of antioxidant substances may be due to the consumption of antioxidant precursor substances or direct participation in antioxidant reactions. Furthermore, a significant decrease in the key reduced glutathione (GSH) and L-glutathione was observed, indicating that the GSH system is the main way to scavenge free radicals in HE. Among the top 20 upregulated DAMs, most substances were hormones (mevalonate, bilirubin, and sitosterol), antimicrobials ( simvastatin and 1-naphthol), and vitamin derivatives (isotretinoin and retinoic acid), indicating that HE synthesized more hormonal substances to regulate metabolic activities under adverse environmental conditions (Escalante et al. 2023 ). To explore the effects of metabolites on the metabolic activities of HE, all the DAMs that could be mapped to KEGG pathways were subjected to enrichment analysis. The KEGG enrichment results are shown in Fig. 5 E. Among the enriched KEGG pathways, only seven had significant changes in DAMs (P < 0.5), which were sorted in ascending order of P-value: Starch and sucrose metabolism, ABC transporters, Arginine and proline metabolism, Phenylalanine metabolism, beta-Alanine metabolism, Arachidonic acid metabolism, and Pyrimidine metabolism. The significant changes in starch and sucrose metabolism indicate that the utilization of carbohydrates was altered in the mycelium under the influence of methyl viologen. The changes in the ABC transporter pathway suggest that HE achieves multidrug efflux reactions (Ongley et al. 2016 ) by altering the transmembrane transport mechanism to resist the stress of foreign toxins. Furthermore, multiple pathways for amino acid metabolism were enriched, indicating that amino acid metabolism plays a major role in the combat of intracellular oxidative stress. Furthermore, the significant changes in pyrimidine metabolism suggest that RNA metabolism in the organism has been altered. It is worth noting that the accumulation of mevalonate was observed in the DAMs, and the transcriptome analysis also showed enhanced expression of the mevalonate pathway in the terpenoid synthesis pathway, indicating that under the stress of methyl viologen, HE enhances the expression of the mevalonate pathway to provide the necessary precursors for the synthesis of more diterpene-based antitoxins. Discussion Studies have pointed out that methyl viologen is a reactive oxygen species (ROS) inducer that produces oxidative stress in cells(Shimada et al. 2016 ). The addition of reagents to induce oxidative stress during liquid fermentation can regulate the yield of secondary metabolites(Hu et al. 2020 ; Miranda et al. 2014 ), a defense mechanism promoting antioxidants to combat damage is generated when organisms are subjected to foreign stress-induced oxidative stress(Huang et al. 2018 ), Erinacine A may be an important metabolite involved in the oxidative damage system. The morphological damage indicates that methyl viologen treatment significantly affects the surface structure of the mycelium and disrupts the normal growth of the mycelium. The deformation of the cell wall may affect the exchange pattern between the mycelium and the external substances, and in this case, it may promote the synthesis of metabolites within the mycelium(Wang et al. 2018 ), this phenomenon is consistent with the case of Jiang (Jiang et al. 2022 ), similar results were obtained for the treatment of Aspergillus carbanus using eugenol. Extracellular matrix (ECM) organization plays an important role in supporting the mycelium structure, such as the cell wall and EPS (Domozych and LoRicco 2023 ). From the perspective of microstructure, the cell wall of the mycelium was also destroyed to varying degrees, which was confirmed by the downregulation of ECM expression. However, when microorganisms are stressed by MV, they will achieve the exclusion of toxic compounds through a series of transporters, in which ABC transporters play a crucial role, which is consistent with the results of metabolomics analysis (Ongley et al. 2016 ), in terms of molecular function, in addition to the DEGs enriched in oxidoreductase activity, there are also some hydrolases concentrated in catalytic activity, which is related to the enzymatic antioxidant system adopted by HE in response to ROS. The obtained differentially expressed genes and differentially accumulated metabolites were separately mapped to the KEGG pathway database to obtain their common pathway information, and the major biochemical pathways and signal transduction pathways jointly participated by the differentially accumulated metabolites and differentially expressed genes were determined (Fig. 6 A). The results indicate that the synthesis of Erinacine A may be regulated by multiple secondary metabolic pathways. Furthermore, in the terpene synthesis backbone pathway, the gene expression for the conversion of acetyl-CoA to mevalonate was observed (Table 1 ), and the significant accumulation of mevalonate was detected in the metabolites, indicating that HE will promote the synthesis of mevalonate when subjected to MV stress. In subsequent studies, the significant expression of farnesyl diphosphate synthase and geranylgeranyl diphosphate synthase was observed, but no significant accumulation of farnesyl diphosphate and geranylgeranyl diphosphate (GGPP) was observed, while GGPP is the key precursor for the synthesis of diterpene compounds. In this case, the additionally synthesized GGPP may be converted into more diterpene-type secondary metabolites. Benefiting from previous studies, Yang (Yang et al. 2017) identified the enzymes responsible for the cyclization of the skeleton in HE, characterized the function of EriG in catalyzing the formation of the cyclooctane skeleton, and Ma et al revealed the biosynthetic pathway of GGPP being converted into various erinacine-containing diterpenoids (Ma et al. 2021 ), including a gene cluster containing various diterpene cyclase such as EriE, EriG, EriA/C/I (three P450 hydroxylases), and EriJ (glycosyltransferase). In this study, these gene clusters were successfully annotated and found to be significantly expressed (Table 2 ), which explains the significant increase in the content of Erinacine A. However, due to the scarcity of standard materials, information on other Erinacines, such as Erinacine Q and Erinacine P, cannot be obtained by HPLC test. Table 2 The differentially expressed genes (DEGs) associated with the biosynthesis of Erinacines. Gene id Log2Fc P-value Description Cluster-310.6711 1.192 7.65E-10 acetyl-CoA C-acetyltransferase (KO) Cluster-310.6981 1.7066 1.25E-32 hydroxymethylglutaryl-CoA synthase (KO) Cluster-310.4346 1.2376 4.91E-06 farnesyl diphosphate synthase (KO) Cluster-310.6953 4.2522 9.22E-54 Hericium erinaceus isolate EriE Geranylgeranyl pyrophosphate synthase mRNA(NT) Geranylgeranyl pyrophosphate synthase AN1592 OS= Hericium erinaceus (Swissprot) Cluster-310.6793 2.9629 6.79E-28 Polyprenyl transferase eriG(NR) Cluster-310.7938 1.402 1.69E-07 Cytochrome P450 monooxyhenase eriI(NR) obsolete peroxidase reaction//response to oxidative stress(BP Description) Cluster-310.6053 1.7654 7.39E-19 Cytochrome P450 monooxyhenase eriA(NR) Cluster-310.5426 2.0262 1.30E-20 Cytochrome P450 monooxyhenase eriC(NR) Cluster-310.6761 2.8633 6.20E-28 Short-chain dehydrogenase/reductase eriH(NR) Cluster-310.7544 2.1592 2.90E-29 Dehydrogenase eriK(NR) Cluster-310.7111 4.2973 2.88E-15 Dehydrogenase eriK(Swissprot) Cluster-310.8343 1.933 7.34E-11 Dehydrogenase eriK (Swissprot) Cluster-310.8932 -1.2411 7.92E-08 aryl-alcohol oxidase 11(NR) Dehydrogenase eriK(Swissprot) Cluster-310.5279 2.1096 8.46E-09 Acetyltransferase eriL(NR) Cluster-310.7576 1.5127 3.50E-06 UDP-glycosyltransferase eriJ(NR) Cluster-310.5207 1.2632 7.69E-06 Short-chain dehydrogenase/reductase eriH(NR) Based on the transcriptomics and metabolomics data, the data was integrated into the biosynthetic pathway of Erinacine A, as shown in Fig. 6 B. From the overall perspective of mycelial metabolic activity, the key to the synthesis of erinacines lies in the expression of the mevalonate pathway, and the only precursor for mevalonate synthesis is acetyl-CoA, which may be provided by fatty acid degradation, carbohydrate metabolism, energy metabolism, and amino acid metabolism. However, it was evident that by day 10 of fermentation, glucose had almost been depleted, which resulted in the inhibition of glycolysis/gluconeogenesis, thereby allowing acetyl-CoA to flow into the mevalonate pathway through the metabolism of other sugars. When stress occurs, it will drive the significant expression of the mevalonate pathway, especially the activation of GGPP synthase (19-fold higher expression than the MOCK group) in this pathway, and mevalonate will be more converted to GGPP, which will form the cyclooctane skeleton under the action of EriG. In the modification of the diterpene skeleton, the oxygenation behavior is more realized through cytochrome P450 monooxygenases (Wang et al. 2018 ). Under oxidative stress, cytochrome P450 enzymes will be activated (such as EriI responding to oxidative stress, Table 2 ), synthesizing a series of Erinacine-like compounds, and being converted to Erinacine A through non-enzymatic reactions, enhancing HE's ability to resist unfavorable growth environments. In summary, diterpene compounds such as Erinacines may be important substances for HE to resist oxidative stress, providing appropriate oxidative stress behavior or increasing cytochrome P450 activity during fermentation may effectively improve the synthesis of HE diterpene compounds. Conclusion This study demonstrates that Methyl Viologen (MV) acts as a powerful elicitor, dramatically boosting the production of the neuroprotective diterpenoid Erinacine A in the liquid fermentation of HE, achieving an unprecedented yield of 414 mg/L - the highest reported fermentation broth titer to date. Mechanistically, MV-induced oxidative stress activates interconnected metabolic rewiring (sugar, amino acid, and fatty acid pathways) and specifically amplifies the ROS-responsive Erinacine A biosynthetic machinery. Multi-omics integration revealed that MV upregulates both the mevalonate pathway and the Eri gene cluster, redirecting geranylgeranyl pyrophosphate (GGPP) toward Erinacine A synthesis. Critically, while MV’s toxicity necessitates caution in direct industrial application, this work uncovers critical stress-responsive regulatory nodes for targeted metabolic engineering. These insights provide essential targets (e.g., specific genes, pathways, stress responses) for the future development of safe and sustainable strategies, such as food-grade elicitors or metabolic engineering approaches, aimed at harnessing this potential for the efficient and scalable production of Erinacine A as a valuable functional food ingredient or nutraceutical. Declarations Funding This work was supported by the China Postdoctoral Science Foundation [Grant Number 2025M773001], the Joint Fund of Zhejiang Provincial Natural Science Foundation of China [Grant Number LLSSZ25C200001], and the Research project Foundation of Zhejiang University of Technology [Grant Number KYY-HX-20240435]. Acknowledgement The authors gratefully acknowledge Xueyu Biotechnology Co., Ltd (Hangzhou, China) for providing the proprietary Hericium erinaceus (Bill.) Pers. strain used in this study. Author Contributions Jian Wang: Conceptualization, Funding acquisition, Investigation, Supervision, Writing – original draft, Writing – review & editing. Juncai Wan: Data curation, Formal analysis, Investigation, Visualization, Writing – original draft. Junhao Huang: Formal analysis, Investigation, Methodology, Software, Visualization, Writing – original draft. Ran Ma: Data curation, Validation, Visualization. Yahya Saud Mohamed Hamed: Writing - Reviewing and Editing. Hynek Roubík: Writing - Reviewing and Editing. Ming Cai: Funding acquisition, Resources. Lingli Li: Funding acquisition, Resources, Writing – review & editing. Peilong Sun: Funding acquisition, Resources, Supervision. Kai Yang: Funding acquisition, Project administration, Resources, Supervision. Competing interest The authors declared that they have no conflicts of interest to this work. 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Trends Biotechnol 33(7):419–428. 10.1016/j.tibtech.2015.04.006 Zhao L, Wang Y, Liu J, Wang K, Guo X, Ji B, Wu W, Zhou F (2016) Protective Effects of Genistein and Puerarin against Chronic Alcohol-Induced Liver Injury in Mice via Antioxidant, Anti-inflammatory, and Anti-apoptotic Mechanisms. J Agric Food Chem 64(38):7291–7297. 10.1021/acs.jafc.6b02907 Additional Declarations No competing interests reported. Supplementary Files TitleTitlePagewithSupplementaryTableandFigure1085.pdf GRAPHICALABSTRACT.png Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8698276","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":588380179,"identity":"a9137496-29e5-48c6-aedd-34f2ea674d97","order_by":0,"name":"Jian Wang","email":"","orcid":"","institution":"Zhejiang University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Jian","middleName":"","lastName":"Wang","suffix":""},{"id":588380180,"identity":"6f9fad2a-ac85-4d11-ae7d-06ac9179551e","order_by":1,"name":"Juncai Wan","email":"","orcid":"","institution":"Zhejiang University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Juncai","middleName":"","lastName":"Wan","suffix":""},{"id":588380183,"identity":"a1d952c9-a542-447d-b0c6-0002aa82cf07","order_by":2,"name":"Junhao Huang","email":"","orcid":"","institution":"Zhejiang University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Junhao","middleName":"","lastName":"Huang","suffix":""},{"id":588380184,"identity":"490d07e7-17b3-4869-81a5-ab56c645e8e6","order_by":3,"name":"Ran Ma","email":"","orcid":"","institution":"Zhejiang University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Ran","middleName":"","lastName":"Ma","suffix":""},{"id":588380185,"identity":"4dad0bee-a7c5-4669-9714-ed3a00c50bba","order_by":4,"name":"Yahya Saud Mohamed Hamed","email":"","orcid":"","institution":"Zhejiang University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Yahya","middleName":"Saud Mohamed","lastName":"Hamed","suffix":""},{"id":588380186,"identity":"1adeccde-8eeb-4242-b976-2f954904119f","order_by":5,"name":"Hynek Roubík","email":"","orcid":"","institution":"Czech University of Life Sciences Prague","correspondingAuthor":false,"prefix":"","firstName":"Hynek","middleName":"","lastName":"Roubík","suffix":""},{"id":588380187,"identity":"8e8b0c29-5b91-4c21-b67b-b1095bae425a","order_by":6,"name":"Ming Cai","email":"","orcid":"","institution":"Zhejiang University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Ming","middleName":"","lastName":"Cai","suffix":""},{"id":588380188,"identity":"8aea0867-3d94-4b86-adeb-792b14c75595","order_by":7,"name":"Peilong Sun","email":"","orcid":"","institution":"Zhejiang University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Peilong","middleName":"","lastName":"Sun","suffix":""},{"id":588380189,"identity":"98b4c639-bcec-4de4-8dc8-c1aad959cc03","order_by":8,"name":"Lingli Li","email":"","orcid":"","institution":"Lishui Institute of Agriculture and Forestry Sciences","correspondingAuthor":false,"prefix":"","firstName":"Lingli","middleName":"","lastName":"Li","suffix":""},{"id":588380190,"identity":"9e6365bb-6732-4b8f-ae65-00cd7a5637e1","order_by":9,"name":"Kai Yang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4klEQVRIie3LoQrCUBTG8TOEWYbWY9D5CI7BVHyZrbgyBZtB5IiwJFpn8hX0DS4MZrliNWoUFmYxitfZr7MJ3n/5Tjg/AJXqR9MIsAHAXnepOLEFEfsFAY/eW4C09of4Gk06/mbG22cY9zwqH5ic8GG/u01wMCNuEXDfI2PoygkLHOus42AOgmhh7BEaLTk5poI80Ndz8ihCToF92YXoGjmhAqR2Sh1tvUQrgmQUuYlvh0YgJ5VjYN8W96lpRvE2yya9+qrM5aTJQMf8QgbgitWl/yKToJTlV5U+/apUKtW/9gRdr0twIHU1nAAAAABJRU5ErkJggg==","orcid":"","institution":"Zhejiang University of Technology","correspondingAuthor":true,"prefix":"","firstName":"Kai","middleName":"","lastName":"Yang","suffix":""}],"badges":[],"createdAt":"2026-01-26 08:41:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8698276/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8698276/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102412280,"identity":"3a2a7f64-f020-4769-93e1-a77a184b3a3d","added_by":"auto","created_at":"2026-02-11 12:14:47","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":605959,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of exogenous factors on the growth and development of different HE and the yield of Erinacine A. (A) Effects of different HE strains on the production of Erinacine A. (B) Effects of different exogenous inducible factors on the production of Erinacine A by liquid fermentation of HE. (C) Effect of MV addition time on the production of Erinacine A by liquid fermentation of HE. (D) Effect of different concentrations of MV on the growth of HE and the yield of Erinacine A. Note: different letters indicate significant differences (p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Fig.1..png","url":"https://assets-eu.researchsquare.com/files/rs-8698276/v1/468f0c132cadfa4676eff3a3.png"},{"id":102412279,"identity":"1c4d089c-619f-4a13-804f-10505dd7b119","added_by":"auto","created_at":"2026-02-11 12:14:47","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":270950,"visible":true,"origin":"","legend":"\u003cp\u003eTime curve of mycelium yield (A), residual reducing sugar (B) and Erinacine A (C) in liquid fermentation of HE induced by methyl viologen.\u003c/p\u003e","description":"","filename":"Fig.2..png","url":"https://assets-eu.researchsquare.com/files/rs-8698276/v1/075b306f54e9bbdd5c266306.png"},{"id":102412282,"identity":"98281949-d0b1-4cd6-8770-0d4e90653a6a","added_by":"auto","created_at":"2026-02-11 12:14:47","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4531322,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of methyl viologen on micromorphology of HE mycelium. Electron microscope images of blank group mycelium samples magnified 1000 (A), 5000 (B), 10000 (C) times; Electron microscope images of mycelium samples of methyl viologen treatment group magnified 1000 (D), 5000 (E), 10000 (F) times.\u003c/p\u003e","description":"","filename":"Fig.3..png","url":"https://assets-eu.researchsquare.com/files/rs-8698276/v1/b2a8f4ebc16524f5ceb4f98b.png"},{"id":104808125,"identity":"b6fd6fd6-b0dc-46e9-87fc-cc6eaf20db77","added_by":"auto","created_at":"2026-03-17 12:17:02","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1143366,"visible":true,"origin":"","legend":"\u003cp\u003eSummary of transcription; GO functional annotation enrichment analysis and KEGG pathway annotation for DEGs. (A) 3D pie chart of species classification by gene comparison. (B)Volcano map of differentially expressed gene in MOCK group and MV group. (C) Up-regulated 20 most significant concentrations of GO function. (D) Down-regulated 20 of the most significant GO functional enrichment. (E) The 20 most significant KEGG pathways down-regulated. (F) Upregulated to the 20 most significant KEGG pathways.\u003c/p\u003e","description":"","filename":"Fig.4..png","url":"https://assets-eu.researchsquare.com/files/rs-8698276/v1/671b896ce058dd4d420c462e.png"},{"id":102962364,"identity":"da947b1d-af1b-450a-9617-7a95893e2a4b","added_by":"auto","created_at":"2026-02-19 04:07:31","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1011819,"visible":true,"origin":"","legend":"\u003cp\u003eMetabolome PCA (A) and PLS-DA (B) in MOCK group and MV group. (C) MOCK group and MV group sample comparison of volcano diagram. (D) 20 most significantly down-regulated and most significantly up-regulated metabolites valve stem diagram. (E)Enrichment analysis of KEGG pathway of DAMs.\u003c/p\u003e","description":"","filename":"Fig.5..png","url":"https://assets-eu.researchsquare.com/files/rs-8698276/v1/2945db5c2eb6c3a44f0e1814.png"},{"id":102412284,"identity":"5749495f-1028-4982-8c26-7ada208d0785","added_by":"auto","created_at":"2026-02-11 12:14:47","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":982955,"visible":true,"origin":"","legend":"\u003cp\u003eCombined transcriptomic and metabolomics analysis of the reasons for increased the yield of Erinacine A. (A) KEGG pathway integration analysis of metabolome and transcriptome. (B) Map of the diterpenoid synthesis pathway in the mevalonate pathway of HE; DEGs and DAMs in the pathway of Erinacine A biosynthesis.\u003c/p\u003e\n\u003cp\u003eNote: The letter on the arrow is the abbreviation of the enzyme involved in the reaction in the Pathway. The red font indicates that the gene encoding the enzyme is significantly up-regulated at the transcriptional level; The orange font indicates that the gene encoding the enzyme is not significantly up-regulated at the transcriptional level; The gray font indicates that the gene encoding the enzyme was not detected in this transcriptome sequencing; Metabolites in the red box indicate significant upregulation.\u003c/p\u003e","description":"","filename":"Fig.6..png","url":"https://assets-eu.researchsquare.com/files/rs-8698276/v1/bfe4d57ef2ff29f4adcfcb15.png"},{"id":102412283,"identity":"da53eca4-9d53-4039-8a82-678367a6ab3b","added_by":"auto","created_at":"2026-02-11 12:14:47","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":563548,"visible":true,"origin":"","legend":"","description":"","filename":"TitleTitlePagewithSupplementaryTableandFigure1085.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8698276/v1/e7b118eb9cd2740893bddf17.pdf"},{"id":102412285,"identity":"1dd35d69-2bbe-45c1-834c-bb1e3ccd3747","added_by":"auto","created_at":"2026-02-11 12:14:47","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2507799,"visible":true,"origin":"","legend":"","description":"","filename":"GRAPHICALABSTRACT.png","url":"https://assets-eu.researchsquare.com/files/rs-8698276/v1/03e0e8145022669664ef4bf9.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Methyl viologen boosts Erinacine A biosynthesis in Hericium erinaceus fermentation: Multi-omics insights","fulltext":[{"header":"Key points","content":"\u003cp\u003e\u0026middot; MV supplementation achieves record Erinacine A yields (23 mg/g, 414 mg/L) in liquid fermentation.\u003c/p\u003e\u003cp\u003e\u0026middot; Oxidative stress from MV activates mevalonate pathway and Eri gene cluster for enhanced biosynthesis.\u003c/p\u003e\u003cp\u003e\u0026middot; Multi-omics reveals MV's regulation of sugar, amino acid, and fatty acid metabolism pathways.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eHericium erinaceus\u003c/em\u003e (HE), a popular edible and medicinal mushroom species, also known as the Lion's Mane mushroom or Monkey Head mushroom, belongs to the \u003cem\u003eBasidiomycota phylum\u003c/em\u003e, \u003cem\u003eAgaricomycetes class\u003c/em\u003e, \u003cem\u003eHericiaceae family\u003c/em\u003e, and \u003cem\u003eHericium erinaceus genus\u003c/em\u003e. Erinacine is a unique type of diterpene of HE, which has been concerned for its potential treatment of neurodegenerative diseases. So far, more than 20 types of Erinacines have been identified, mainly from mycelium (Li et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Erinacine A is also the most concern, and studies have shown that Erinacine A promotes the release of nerve growth factor (Shimbo et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), increase the expression of insulin-degrading enzymes, and reduce β amyloid deposition (Chen et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) and its efficacy in inhibiting cancer cell proliferation in vitro (Prasher et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Liquid fermentation is a rapid and cost-effective method for producing a substantial quantity of mycelium rich in Erinacine A, which has been identified as the primary pathway for the synthesis of Erinacine A. Although Erinacine A can be chemically synthesized, the synthesis steps are cumbersome, and side products are produced in the process (Li et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe biosynthesis of diterpenes is the synthesis of the key precursor GGPP through the mevalonate (MVA) pathway and MEP pathway and the modification of diterpene synthase, diterpene cyclase and cytochrome P450 (Zerbe and Bohlmann \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). HE genome sequencing by Chen et al. found that GGPP, a key precursor of synthetic diterpenoids, had only the gene expression of the mevalonate pathway (Chen et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), suggesting that this may be the only route for Erinacine synthesis in HE. In an earlier study by Liu et al., the gene cluster (Eri) responsible for Erinacine synthesis in HE, including one GGPPs (EriE), a diterpenoid ring enzyme (Eri G) responsible for cyclization of the cyathane skeleton, three cytochrome P450 (EriA / C / I), and one UDP-glycosyltransferase (Eri J) (Yang et al. 2017). In subsequent studies, through the expression of gene editing in engineered yeast, the inference of the biosynthesis of Erinacines (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), and the successful synthesis of various Erinacines (Ma et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Finally, Erinacine was successfully isolated from the transformant SC-GICAJLM, and Erinacine P was synthesized as Erinacine A by a non-enzymatic spontaneous MICHAEL addition elimination reaction, which also supports the previous conclusion (Kenmoku et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2000\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the past, two reports have brought the yield of Erinacine A to 192 mg/L and 225.54 mg/L (Chang et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Krzyczkowski et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), which is almost the limit of the synthesis. We wanted to seek an unusual culture method to prompt HE to synthesize more Erinacine A. Exogenous factors refer to multiple factors coming from outside the organism, which can affect the physiology, biochemical processes or behavior of the organism, and the addition of exogenous factors during fermentation seems to be a good strategy. For example, the addition of 5 mg/L rotenone increased the yield of cordycepin produced by 316.09% (Ma et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), the addition of 1mg/L methyl viologen and rotenone increased the total pigment yield of \u003cem\u003eMonascus purpureus\u003c/em\u003e by 39.08% and 40.89%, respectively, and the yield of yellow pigment by 74.62% and 114.06%, respectively (Liu et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The addition of orange peel essential oil to \u003cem\u003eTaiwanofungus camphoratus\u003c/em\u003e fermentation increased the total triterpene yield by more than 10 times (Ma et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). To date, no reports have been identified regarding the use of exogenous factors to enhance the yield of Erinacine A.\u003c/p\u003e \u003cp\u003eIn this study, we attempted to add several exogenous factors during liquid fermentation of HE and found that methyl viologen (MV) was the most helpful in improving the yield of Erinacine A. Based on the results of the screening, we investigated the properties of MV affecting liquid fermentation, and the addition of 5\u0026ndash;10 mg/L MV at the end of the logarithmic growth phase of the mycelium significantly increased the yield of Erinacine A. Furthermore, we investigated the potential mechanisms by which MV affects the biosynthesis of Erinacine A through a combined analysis of transcriptomics and metabolomics. Finally, the relationship between the changes in antioxidant properties of Erinacine A and the synthesis of Erinacine A was explored under MV treatment. This study is the first application of MV to increase the yield of Erinacine A in HE fermentation, providing a new idea to utilize the appropriate oxidative stress to improve Erinacine A synthesis.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStrains and reagent materials\u003c/h2\u003e \u003cp\u003eThe edible \u003cem\u003eHericium erinaceus\u003c/em\u003e (Bill.) Pers. used in this study was a proprietary strain developed by Xueyu Biotechnology Co., Ltd (Hangzhou, China), which was identified by the Institute of Microbiology, Chinese Academy of Sciences. The HE strain XY-2 was derived from the revitalization of strain XY-1. The HE strain HW8415 was obtained from the Microbial Research Institute of the Heilongjiang Academy of Sciences. The HE strain CGMCC 5.739 was purchased from the China General Microbiological Culture Collection Center. The strain was statically cultured on a potato dextrose agar (PDA) slant at 28\u0026deg;C. The liquid seed culture medium consisted of 30 g/L glucose, 30 g/L soybean meal, 1 g/L potassium dihydrogen phosphate, and 0.5 g/L magnesium sulfate. The liquid fermentation medium consisted of 30 g/L glucose, 30 g/L yeast powder, 1 g/L potassium dihydrogen phosphate, and 0.5 g/L magnesium sulfate. All culture media materials were provided by Xueyu Biotechnology Co., Ltd (Hangzhou, China). The chemicals used in this study include methyl viologen (\u0026gt;\u0026thinsp;96%, Aladdin), rotenone (95%, Aladdin), oleic acid (Sinopharm), sweet orange oil (Bonai), chitosan (Rhawn), corn oil (Rhawn), and methyl jasmonate (Macklin).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eLiquid fermentation and mycelium collection\u003c/h3\u003e\n\u003cp\u003eSeed cultures of HE strains XY-1, XY-2, HW8415, and CGMCC 5.739 were prepared and introduced into the fermentation medium. The fermentation was conducted at 27\u0026deg;C and 160 rpm for 7 days, leading to the development of small, uniform mycelial pellets in the seed culture. The seed culture was injected into the liquid fermentation medium at a 10% (v/v) inoculum size, and the fermentation took place at 27\u0026deg;C and 140 rpm. Exogenous factors such as chitosan, corn oil, methyl viologen, rotenone, methyl jasmonate, sweet orange oil, and oleic acid were introduced to the fermentation medium following previously reported protocols. To prepare the culture medium, 0.1 g/L of chitosan was added (Vicente et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), 2.5% (v/v) corn oil was added after 2 days of fermentation (Meng et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and a 5 mg/L concentration of methyl viologen and rotenone (in water and ethanol solutions, respectively) was added after 4 days of fermentation (Ma et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Ma (Ma et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) added 50 \u0026micro;L of sweet orange oil and oleic acid after 4 days of incubation. The experimental group that did not receive any exogenous inducer acted as a blank control.\u003c/p\u003e \u003cp\u003eAt the end of fermentation, the mycelia were separated with an 80-mesh filter cloth and were washed three times with distilled water Afterward, the mycelia were dried to constant weight in a blast drying oven at 60\u0026deg;C and the dried mycelia was prepared as a powder for analysis.\u003c/p\u003e\n\u003ch3\u003eExtraction and determination of Erinacine A\u003c/h3\u003e\n\u003cp\u003eOne gram of HE powder was weighed and combined with 20 mL of 80% methanol for the extraction of Erinacine A. The extraction was carried out using ultrasonic extraction at 60\u0026deg;C and 300 W power for 1 hour. After extraction, the mixture was centrifuged to obtain the supernatant, which was then filtered through a 0.22 \u0026micro;m membrane and subjected to liquid chromatographic analysis. High performance liquid chromatography (HPLC) detection conditions: SHIMAZU LC2030 HPLC system, using a Phenomenex luna C18 column (4.6\u0026times;250 mm, 5 \u0026micro;m); mobile phase: composed of water and acetonitrile, 0\u0026ndash;23 min, 30% acetonitrile-50% acetonitrile; 23\u0026ndash;26 min, 50% acetonitrile, 26\u0026ndash;32 min, 50% acetonitrile-100% acetonitrile, 32\u0026ndash;44 min, 100% acetonitrile, 44\u0026ndash;60 min, 30% acetonitrile; column temperature 30\u0026deg;C; flow rate 1.0 mL/s; injection volume 20 \u0026micro;L; detection wavelength 340 nm. The elution time of Erinacine A is around 31 min. The Erinacine A standard (purity\u0026thinsp;\u0026gt;\u0026thinsp;96%) is provided by Huida Biotech Co., Ltd (Hangzhou, China).\u003c/p\u003e\n\u003ch3\u003eReduction sugar detection\u003c/h3\u003e\n\u003cp\u003eThe fermentation broth was collected in centrifuge tubes and sterilized in a boiling water bath for 15 min, then stored at 4\u0026deg;C for a few hours for analysis. On the same day, the reducing sugar content of the fermentation broth was measured. Take 1 mL of the fermentation broth after sterilization, dilute it to an appropriate concentration, and use the 2,4-dinitrosalicylic acid (DNS) method to detect the reducing sugars in the fermentation broth.\u003c/p\u003e\n\u003ch3\u003eMycelium morphology observation\u003c/h3\u003e\n\u003cp\u003eThe mycelial and ultrastructural morphology were observed using a scanning electron microscope (Hitachi Reguius8100, Japan). The mycelium was washed three times with pre-cooled PBS solution, then fixed in 2.5% glutaraldehyde fixative at 4\u0026deg;C in the dark overnight for sample preparation. The sample preparation and observation were carried out by Shiyanjia Lab (Hangzhou, China).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMetabolomics analysis\u003c/h2\u003e \u003cp\u003eThe fermentation broth in the shaker flask was centrifuged at 5,000 rpm for 5 min at 4\u0026deg;C. The mycelium was collected and quickly washed 2\u0026ndash;3 times with pre-chilled PBS to completely remove the supernatant. The clean mycelium was then snap-frozen in liquid nitrogen and stored at -80\u0026deg;C for subsequent analysis. For the metabolomics analysis, each experimental group was set up with 6 parallel samples.\u003c/p\u003e \u003cp\u003ePlace 100 mg liquid nitrogen ground tissue sample in an EP tube, add 500 \u0026micro;L of 80% methanol, vortex, sit for 5 min, and centrifuge. A certain amount of supernatant was diluted with mass spectral grade water to 53% methanol, and the supernatant was collected by centrifugation and injected for LC-MS analysis.\u003c/p\u003e \u003cp\u003eThe metabolic profiles in the positive and negative ion modes were analyzed by the Vanquish UHPLC system (Thermo Fisher, Germany) and the Q Exactive\u0026trade; HF/Q Exactive\u0026trade; HF-X mass spectrometer (Thermo Fisher, Germany). Using the Hypersil Gold C18 column (Thermo Fisher, USA). The gradient elution system is shown in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, mobile phase A is 0.1% formic acid in positive mode, mobile phase B is 5 mM ammonium acetate, pH 9.0, and mobile phase B is methanol in negative mode. Column temperature was 40 ℃, and the flow rate was 0.2 mL/min.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTranscriptome analysis\u003c/h3\u003e\n\u003cp\u003eMycelium samples were collected the same as extraction and determination of Erinacine A, and in transcriptome analysis three parallel runs were set for each experimental group.\u003c/p\u003e \u003cp\u003eSample testing, RNA extraction, Library construction, quality detection and sequencing were performed by the Novogene Bioinformatics Institute (Beijing, China). RNA was quantified using Qubit\u0026reg; 2.0 Flurometer (Life Technologies, San Diego, CA, USA), checked for integrity on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). The library was diluted to 1.5 ng/ul by quantification and the effective concentration of the library was accurately quantified by qRT-PCR to ensure the quality of the library (the effective concentration of the library was higher than 1.5 nm). The index-coded samples were clustered after passing the library inspection. After cluster generation, the library preparations were sequenced on Illumina with 150 bp paired-end reads.\u003c/p\u003e\n\u003ch3\u003eDe novo Transcriptome assembly\u003c/h3\u003e\n\u003cp\u003eAfter the sequencing of the cDNA library, the sequencing fragments of the image data measured by the high-throughput sequencer are converted into sequence reads by CASAVA bases identification, and the raw reads are obtained after quality filtering. To ensure the reliability of data analysis, we removed some low-quality and unavailable reads to obtain clean reads. Meanwhile, Q20, Q30 and GC content were calculated for clean reads. All subsequent analyses were based on the high-quality analysis performed by clean reads.\u003c/p\u003e \u003cp\u003eSince HE lacks a suitable reference genome, De novo assembly was applied to construct transcripts from the clean reads, and the Trinity assembler was used for transcriptome assembly of the clean reads (Haas et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Based on Trinity splicing, the transcripts are aggregated into many clusters according to the Shared Reads between transcripts, combined with the transcript expression level between different samples and the H-Cluster algorithm, the transcripts with the expression difference between samples are separated from the original cluster to establish a new cluster, and finally each cluster is defined as \"Gene\". The BUSCO (Benchmarking Universal Single-Copy Orthologs) assessment evaluates the integrity of the assembled transcripts using a single-copy direct orthologous gene library combined with software such as tblastn, augustus and hmmer.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eGene functional annotation and differential gene enrichment\u003c/h2\u003e \u003cp\u003eWe annotated the spliced transcripts with seven major databases (Nr, Nt, PFAM, KOG / COG, Swiss-Prot, KEGG, GO), and performed differential expression analysis in two conditions/groups using the DESeq2 R package (1.20.0). DESeq2 provides statistical procedures for determining differential expression in digital gene expression data using a model based on a negative binomial distribution. The resulting P-values were adjusted to control the false discovery rate using the method of Benjamini and Hochberg method. Genes identified by DESeq2 with an adjusted P-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were designated as differentially expressed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll experiments were carried out at least three times, and data were expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. One-way ANOVA and independent t-test were carried out using GraphPadPrism software version 8 (La Jolla, USA) for statistical analysis.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eEffect of exogenous factors on the growth and development of different HE and the yield of Erinacine A\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe effect of methyl viologen on the production of Erinacine A in the liquid fermentation of different HE strains. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, methyl viologen had a good stimulatory effect on the four HE strains, significantly improving the production of Erinacine A, indicating that methyl viologen has a universal effect on enhancing the biosynthesis of Erinacine A. The results show that the inductive effect of methyl viologen was most significant on HE strain XY-2 and HE strain CGMCC 5.739. Although the Erinacine A production of the blank group of HE XY-2 was relatively low, it can be observed that it has a high growth potential under the stimulation of the exogenous inducing factor. During the seed culture process, it was found that the growth rate of HE CGMCC 5.739 was slower than that of HE XY-2. Therefore, HE XY-2 was selected for further research.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe effect of seven exogenous inducers on liquid fermentation of HE after 4 d fermentation is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB. Although the blank group showed a rapid growth rate and the yield of dry mycelium increased (24.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68 g/L). However, the synthesis capacity of Erinacine A was significantly reduced (4.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.96 mg/L), the result may be due to the change of species characteristics, and the subsequent characteristics of different HE induced fermentations will be carefully studied. The results showed that stimulated of methyl viologen and rotenone, reaching 37.79\u0026thinsp;\u0026plusmn;\u0026thinsp;7.96 mg/L and 13.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24 mg/L, respectively, and the yield of Erinacine A in the methyl viologen experiment group increased approximately 8.44-fold compared with the blank group. However, chitosan, corn oil, methyl jasmonate, sweet orange oil and oleic acid did not positively affect the biosynthesis of Erinacine A, which may be that the mechanism of action could not stimulate the activity of diterpene synthase. According to this result, methyl viologen was selected as an exogenous induction factor for subsequent studies.\u003c/p\u003e \u003cp\u003eMethyl viologen has a significant inhibitory effect on the growth of HE mycelium, which may be a negative effect caused by oxidative damage, such as protein and lipid oxidation, or even DNA damage(Wang et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Therefore, it is necessary to study the optimal time for the addition of MV and consider the improvement of Erinacine A yield without affecting mycelium development. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, the first 3 d of fermentation was the rapid growth period of HE in liquid culture. The addition of methyl viologen previously caused a significant decrease in the yield of HE mycelium, which was not conducive to the synthesis of Erinacine A. The best addition time for the induction effect is 4 d after fermentation, at which time the HE mycelium is almost full and tends to mature. During this period, the metabolic enzyme activity in HE bacteria may be high. After 5 d of fermentation, the effect of adding methyl viologen will gradually decrease, and at this stage, the nutrients in the fermentation system will be gradually exhausted, and the exchangeability of HE and external substances will also decrease. Therefore, 4 d after fermentation was chosen as the optimal time for methyl viologen supplementation. A yield of 103.99 mg/L of Erinacine A was obtained when the total fermentation time reached 8 d. After determining the optimal induction process of ME, the additive amount of MYE was investigated separately, and the results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD. The results showed that the yield of Erinacine A reached the maximum at 5 mg/L and 10 mg/L, reaching 414.84\u0026thinsp;\u0026plusmn;\u0026thinsp;30.03 mg/L and 400.44\u0026thinsp;\u0026plusmn;\u0026thinsp;69.30 mg/L, respectively, with no significant difference. Therefore, the optimal additive amount of methyl viologen was determined to be 5 mg/L. This result was significantly higher than the yield and content of Erinacine A reported by Chang (Chang et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), who achieved 225.54 mg and 13.39 mg/g dry mycelium, respectively, after optimizing the BCRC35669 medium.\u003c/p\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eEffect of methyl viologen on liquid fermentation characteristics of HE\u003c/h2\u003e \u003cp\u003eTo observe the effect of MV on the fermentation characteristics of Erinacine A during fermentation, we drew the growth curve (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) and observed the optimal duration of MV treatment. MV after 4 d of fermentation showed delayed growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Due to the high-temperature sterilization of the culture medium, the loss of about 2 g/L of the reducing sugar will occur. Fermentation for 12 d may not be enough for mycelium autolysis, because the excess yeast powder in the culture medium can also be used by HE to decompose a small amount of reducing sugars to maintain basic life activities, but it seems that when mycelium is under MV stress, slowing down some cell autolysis trend (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-B). On the fifth day of fermentation, a buffer period was observed for MV bulb growth in the MV group, during which it reduced nutrient intake to adapt to the strong stimulation of foreign stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). However, after the buffer period, the mycelium will still consume the carbon source at a faster rate, synthesizing more secondary metabolites rather than supporting the increase in mycelium volume. Finally, the highest level of Erinacine A was reached after 10d of fermentation, yielding about 414.84 mg/L (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Obtaining such a high yield of Erinacine A was surprising, indicating that promoting the expression of the Erinacine A synthesis gene by administering appropriate exogenous stress is feasible and the effect is extremely remarkable. After determining the optimal period of MV stress, we optimized the concentration of added MV and found that the MV concentration of 5\u0026ndash;10 mg/L kept the yield of Erinacine A at the highest level, far ahead of the positive effects of other concentrations, because the fungus was sensitive to MV and giving too high concentration led to its death (Liu et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eMV on the micromorphology of HE mycelium\u003c/h2\u003e \u003cp\u003eTo observe the effect of MV on the morphology of the mycelium, mycelial samples were taken after 10 days of fermentation and observed under a scanning electron microscope (SEM). At 1000x magnification (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), The mycelium of the blank group showed denser mycelium, and the tips of the mycelium had more branches, still showing a tendency to grow. The mycelium of the methyl viologen group was more slender, more twisted and broken, with less sporulation at the ends of the mycelium (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Under 5000x and 10000x ultra-microscopic observation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB-C), the surface of the mycelium in the blank group appeared smooth, plump, and with a dense structure and intact tubular morphology. However, the mycelium of the methyl viologen group showed obvious shrinkage and even different degrees of collapse (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE-F).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eSummary of transcription\u003c/h2\u003e \u003cp\u003eTo ensure the quality and reliability of the data analysis, the raw data needs to be filtered. After excluding low-quality reads, the proportion of clean reads in each sample exceeded 98%, with Q20 and Q30 greater than 97.6% and 93.5%, respectively (Table S2). By comparing with the Nr library annotation, we can obtain the similarity of the species and the functional information of the species gene, after annotation alignment, in the reference species distribution map (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), the genes from the Nr library mainly from (\u003cem\u003eHericium alpestre\u003c/em\u003e) and small spores (\u003cem\u003eDeptipellis sp\u003c/em\u003e.). The former is a related species of HE, while the latter has a high evolutionary affinity (Chen et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Furthermore, no species more distantly related to HE was found on the distribution map, and the results show that the transcriptomic data were highly reliable. In setting conditions | Log2Foldchange | 1 and padj\u0026thinsp;\u0026lt;\u0026thinsp;0.005. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, 651 differentially significantly expressed genes (differentially expressed genes, DEGs) were selected, of which 356 were upregulated and 295 were downregulated.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eGO enrichment analysis and KEGG pathway analysis of differentially expressed genes\u003c/h2\u003e \u003cp\u003eTo study the changes of MV-induced in response to the biological functions of HE, the enriched DEGs were subjected to GO function annotation analysis, which found that MV mainly activated the oxidative stress response in the HE organism, resulting in the most significant changes in oxidoreductase activity, whether up or downregulation. However, in the upregulated GO enrichment, only significant differential expression of oxidoreductase activity was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). This phenomenon may be caused by the oxidative stress generated by MV, which stalls the metabolic activities of the growth and development to improve the antioxidant capacity. The remaining down-regulated DEGs were mainly enriched in the extracellular matrix, transmembrane transport, and basal metabolic activities such as lipids and carbohydrates (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eThe enriched DEGs were projected into KEGG map to study the effect of MV on metabolic activity of HE (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE-F). In the down-regulated KEGG metabolism pathway, the methane metabolism pathway and the fatty acid synthesis pathway are significantly down-regulated, found in the specific analysis (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e1\u003c/span\u003e), in the methane metabolism pathway it is mainly methanol to formaldehyde, the formic acid carbon dioxide reaction pathway is inhibited, this may be that MV stress affects mitochondrial aerobic respiration, must regulate other carbon dioxide pathways to maintain balance of life. Under oxidative stress, the growth was arrested. Fatty acids are closely related to the growth and development of mycelium, and the significant down-regulation of the fatty acid enzyme, fatty acid synthase (FAS), is found in the fatty acid synthesis pathway, which may be related to the growth inhibition of HE (Wernig et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In the upregulated KEGG metabolic pathways, the significant upregulation of the terpenoid backbone biosynthesis pathway was observed, and it clearly showed that HE enhanced the expression of the mevalonate acid pathway. By integrating the KEGG pathways, it was found that in addition to the pathways mentioned above, many carbohydrate metabolism pathways were also significantly changed. After 10 days of fermentation, the glucose was already depleted, and HE will utilize the glucan, mannan, and chitin in the yeast powder in the culture medium to maintain its metabolic activity (Gao et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eKEGG pathway enrichment analysis of significantly affected DEGs.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTerm\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003cp\u003eNumber\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBackground\u003c/p\u003e \u003cp\u003eNumber\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eP-value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eUnigene\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eKO\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eGene name\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMethane\u003c/p\u003e \u003cp\u003emetabolism\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCluster-310.7244\u003c/p\u003e \u003cp\u003eCluster-310.3705 Cluster-310.3946 Cluster-310.7922 Cluster-310.6962 Cluster-310.8604 Cluster-310.6545 Cluster-310.6552\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eK00122 K01070 K00863 K00121 K17066 K00863 K00925 K00122\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eFDH/frmB, ESD, fghA/DAK, TKFC/ frmA, ADH5, adh/ MOX/DAK, TKFC/\u003c/p\u003e \u003cp\u003eackA/ FDH\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStarch and sucrose metabolism\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0031\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCluster-310.8207 Cluster-310.5885 Cluster-310.6788 Cluster-310.5278 Cluster-310.3664 Cluster-310.4681 Cluster-310.10422 Cluster-310.6399 Cluster-310.9901 Cluster-310.7095\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eK01187 K01179 K05349 K05349 K00700 K01179 K01210 K01835 K00700 K00688\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eMalZ/E3.2.1.4/bglX/ bglX/GBE1,glgB/ E3.2.1.58/pgm/\u003c/p\u003e \u003cp\u003eGBE1,glgB/PYG,glgP/ E3.2.1.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFatty acid biosynthesis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0127\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCluster-310.7093 Cluster-310.9052 Cluster-310.7516 Cluster-310.10200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eK00668 K00668 K00668 K00668\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eFAS1/FAS1/FAS1/\u003c/p\u003e \u003cp\u003eFAS1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGlyoxylate and dicarboxylate metabolism\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0146\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCluster-310.7244 Cluster-310.6711 Cluster-310.6552 Cluster-310.7834\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eK00122 K00626 K00122 K01637\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eFDH/ACAT,atoB/FDH/aceA/GLDC,gcvP/\u003c/p\u003e \u003cp\u003ekatE, CAT, catB, srpA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e(\u003cem\u003econtinued\u003c/em\u003e)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTerm\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003cp\u003eNumber\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBackground\u003c/p\u003e \u003cp\u003eNumber\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eP-value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eUnigene\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eKO\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eGene name\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCluster-310.8309\u003c/p\u003e \u003cp\u003eCluster-310.4672\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eK00281\u003c/p\u003e \u003cp\u003eK03781\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmino sugar and nucleotide sugar metabolism\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0234\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCluster-310.7652 Cluster-310.4791 Cluster-310.3531 Cluster-310.3088 Cluster-310.7254 Cluster-310.6399 Cluster-310.5974\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eK08678 K01183 K01183 K01809 K01183 K01835 K01183\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eUXS1,uxs/E3.2.1.14/E3.2.1.14/manA,MPI/E3.2.1.14/pgm/\u003c/p\u003e \u003cp\u003eE3.2.1.14\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTerpenoid backbone biosynthesis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0249\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCluster-310.4346 Cluster-310.6953 Cluster-310.6981 Cluster-310.6711\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eK00787 K00804 K01641 K00626\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eFDPS GGPS1 HMGCS ACAT, atoB\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFructose and mannose metabolism\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0475\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCluster-310.8604 Cluster-310.3088 Cluster-310.7468 Cluster-310.3946\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eK00863 K01809 K19029 K00863\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eDAK,TKFC/manA,MPI/PFKFB2/DAK,TKFC/\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eNote: \"Unigene\" refers to the transcript number assembled in transcriptome analysis; \"KO\" indicates the entry of the gene in the KO annotation; \"Gene Name\" indicates the gene encoding the corresponding enzyme, and \"E3.2.1.4\" etc. indicates the enzyme node to which the transcript gene is annotated in the KEGG pathway, but there is no explicit gene name; when a Gene Name appears multiple times, it means that the gene is a node in multiple reactions in the pathway.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eEffect of MV-induced on the metabolic profile of HE\u003c/h2\u003e \u003cp\u003eA non-targeted LC-MS metabolomics analysis method was used to detect the metabolites of HE under different treatment groups, and a positive and negative merged analysis was performed. Principal component analysis (PCA) showed that the MV group and the MOCK group exhibited distinct metabolic patterns, and the QC samples were well clustered, indicating that the addition of MV changed the abundance of HE metabolites, and the experimental results were reliable (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). The metabolites were clustered, and the heat map analysis showed good clustering effects of different metabolic groups (Fig. S2). After partial least squares-discriminant analysis (PLS-DA), the results in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB showed that the PLS-DA model could distinguish the different experimental groups, and the inter-group differences were significant. In this experiment, the Q2 of the model was 0.8, which is generally considered to indicate that the model has predictive ability when Q2\u0026thinsp;\u0026gt;\u0026thinsp;0.5, and this result proves that the model is very reliable and can be used for subsequent identification of differential metabolites. A total of 483 differentially accumulated metabolites (DAMs) were screened between the two groups of samples, with 367 DAMs showing downregulation and 116 DAMs showing upregulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). The number of DAMs was much higher than the number of upregulated DAMs, but in the transcriptome analysis, the number of upregulated DEGs was slightly higher than the number of downregulated DEGs. This phenomenon suggests that in response to the oxidative stress caused by MV, most metabolites were involved in antioxidant reactions and were consumed.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBased on the DAMs obtained from the metabolomics analysis, the 20 most significantly downregulated and upregulated DAMs were screened according to the FC values and plotted in a volcano plot (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Among them, substances that are unlikely to exist in HE was excluded, as these substances may be misidentified during the metabolite database matching process in mass spectrometry. From the 20 most significantly downregulated DAMs, the main components were antioxidants (such as soy isoflavones, soy lutein, ergothioneine (Halliwell et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), 2-hydroxycinnamic aldehyde (Gupta et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), and dye wood (Zhao et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2016\u003c/span\u003e)), amino acids and their derivatives (such as cysteine, serine, 2-hydroxy-L-phenylalanine, and N-acetyl-D-tryptophan), and lipids. The appearance of soy isoflavones and soy lutein may be due to the absorption of nutrients from the soy meal in the seed culture medium into the fermentation system. The significant decrease in the content of antioxidant substances may be due to the consumption of antioxidant precursor substances or direct participation in antioxidant reactions. Furthermore, a significant decrease in the key reduced glutathione (GSH) and L-glutathione was observed, indicating that the GSH system is the main way to scavenge free radicals in HE. Among the top 20 upregulated DAMs, most substances were hormones (mevalonate, bilirubin, and sitosterol), antimicrobials ( simvastatin and 1-naphthol), and vitamin derivatives (isotretinoin and retinoic acid), indicating that HE synthesized more hormonal substances to regulate metabolic activities under adverse environmental conditions (Escalante et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo explore the effects of metabolites on the metabolic activities of HE, all the DAMs that could be mapped to KEGG pathways were subjected to enrichment analysis. The KEGG enrichment results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE. Among the enriched KEGG pathways, only seven had significant changes in DAMs (P\u0026thinsp;\u0026lt;\u0026thinsp;0.5), which were sorted in ascending order of P-value: Starch and sucrose metabolism, ABC transporters, Arginine and proline metabolism, Phenylalanine metabolism, beta-Alanine metabolism, Arachidonic acid metabolism, and Pyrimidine metabolism. The significant changes in starch and sucrose metabolism indicate that the utilization of carbohydrates was altered in the mycelium under the influence of methyl viologen. The changes in the ABC transporter pathway suggest that HE achieves multidrug efflux reactions (Ongley et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) by altering the transmembrane transport mechanism to resist the stress of foreign toxins. Furthermore, multiple pathways for amino acid metabolism were enriched, indicating that amino acid metabolism plays a major role in the combat of intracellular oxidative stress. Furthermore, the significant changes in pyrimidine metabolism suggest that RNA metabolism in the organism has been altered. It is worth noting that the accumulation of mevalonate was observed in the DAMs, and the transcriptome analysis also showed enhanced expression of the mevalonate pathway in the terpenoid synthesis pathway, indicating that under the stress of methyl viologen, HE enhances the expression of the mevalonate pathway to provide the necessary precursors for the synthesis of more diterpene-based antitoxins.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eStudies have pointed out that methyl viologen is a reactive oxygen species (ROS) inducer that produces oxidative stress in cells(Shimada et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The addition of reagents to induce oxidative stress during liquid fermentation can regulate the yield of secondary metabolites(Hu et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Miranda et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), a defense mechanism promoting antioxidants to combat damage is generated when organisms are subjected to foreign stress-induced oxidative stress(Huang et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), Erinacine A may be an important metabolite involved in the oxidative damage system.\u003c/p\u003e \u003cp\u003eThe morphological damage indicates that methyl viologen treatment significantly affects the surface structure of the mycelium and disrupts the normal growth of the mycelium. The deformation of the cell wall may affect the exchange pattern between the mycelium and the external substances, and in this case, it may promote the synthesis of metabolites within the mycelium(Wang et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), this phenomenon is consistent with the case of Jiang (Jiang et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), similar results were obtained for the treatment of \u003cem\u003eAspergillus carbanus\u003c/em\u003e using eugenol.\u003c/p\u003e \u003cp\u003eExtracellular matrix (ECM) organization plays an important role in supporting the mycelium structure, such as the cell wall and EPS (Domozych and LoRicco \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). From the perspective of microstructure, the cell wall of the mycelium was also destroyed to varying degrees, which was confirmed by the downregulation of ECM expression. However, when microorganisms are stressed by MV, they will achieve the exclusion of toxic compounds through a series of transporters, in which ABC transporters play a crucial role, which is consistent with the results of metabolomics analysis (Ongley et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), in terms of molecular function, in addition to the DEGs enriched in oxidoreductase activity, there are also some hydrolases concentrated in catalytic activity, which is related to the enzymatic antioxidant system adopted by HE in response to ROS.\u003c/p\u003e \u003cp\u003eThe obtained differentially expressed genes and differentially accumulated metabolites were separately mapped to the KEGG pathway database to obtain their common pathway information, and the major biochemical pathways and signal transduction pathways jointly participated by the differentially accumulated metabolites and differentially expressed genes were determined (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). The results indicate that the synthesis of Erinacine A may be regulated by multiple secondary metabolic pathways. Furthermore, in the terpene synthesis backbone pathway, the gene expression for the conversion of acetyl-CoA to mevalonate was observed (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e1\u003c/span\u003e), and the significant accumulation of mevalonate was detected in the metabolites, indicating that HE will promote the synthesis of mevalonate when subjected to MV stress. In subsequent studies, the significant expression of farnesyl diphosphate synthase and geranylgeranyl diphosphate synthase was observed, but no significant accumulation of farnesyl diphosphate and geranylgeranyl diphosphate (GGPP) was observed, while GGPP is the key precursor for the synthesis of diterpene compounds. In this case, the additionally synthesized GGPP may be converted into more diterpene-type secondary metabolites. Benefiting from previous studies, Yang (Yang et al. 2017) identified the enzymes responsible for the cyclization of the skeleton in HE, characterized the function of EriG in catalyzing the formation of the cyclooctane skeleton, and Ma et al revealed the biosynthetic pathway of GGPP being converted into various erinacine-containing diterpenoids (Ma et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), including a gene cluster containing various diterpene cyclase such as EriE, EriG, EriA/C/I (three P450 hydroxylases), and EriJ (glycosyltransferase). In this study, these gene clusters were successfully annotated and found to be significantly expressed (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e2\u003c/span\u003e), which explains the significant increase in the content of Erinacine A. However, due to the scarcity of standard materials, information on other Erinacines, such as Erinacine Q and Erinacine P, cannot be obtained by HPLC test.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe differentially expressed genes (DEGs) associated with the biosynthesis of Erinacines.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGene id\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLog2Fc\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eP-value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDescription\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCluster-310.6711\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.192\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.65E-10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eacetyl-CoA C-acetyltransferase (KO)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCluster-310.6981\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.7066\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.25E-32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ehydroxymethylglutaryl-CoA synthase (KO)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCluster-310.4346\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.2376\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.91E-06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003efarnesyl diphosphate synthase (KO)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCluster-310.6953\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.2522\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9.22E-54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eHericium erinaceus\u003c/em\u003e isolate EriE Geranylgeranyl pyrophosphate synthase mRNA(NT)\u003c/p\u003e \u003cp\u003eGeranylgeranyl pyrophosphate synthase AN1592 OS= \u003cem\u003eHericium erinaceus\u003c/em\u003e(Swissprot)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCluster-310.6793\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.9629\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.79E-28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePolyprenyl transferase eriG(NR)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCluster-310.7938\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.402\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.69E-07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCytochrome P450 monooxyhenase eriI(NR)\u003c/p\u003e \u003cp\u003eobsolete peroxidase reaction//response to oxidative stress(BP Description)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCluster-310.6053\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.7654\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.39E-19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCytochrome P450 monooxyhenase eriA(NR)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCluster-310.5426\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.0262\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.30E-20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCytochrome P450 monooxyhenase eriC(NR)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCluster-310.6761\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.8633\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.20E-28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eShort-chain dehydrogenase/reductase eriH(NR)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCluster-310.7544\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.1592\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.90E-29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDehydrogenase eriK(NR)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCluster-310.7111\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.2973\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.88E-15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDehydrogenase eriK(Swissprot)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCluster-310.8343\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.933\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.34E-11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDehydrogenase eriK (Swissprot)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCluster-310.8932\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-1.2411\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.92E-08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003earyl-alcohol oxidase 11(NR)\u003c/p\u003e \u003cp\u003eDehydrogenase eriK(Swissprot)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCluster-310.5279\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.1096\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.46E-09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAcetyltransferase eriL(NR)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCluster-310.7576\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.5127\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.50E-06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eUDP-glycosyltransferase eriJ(NR)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCluster-310.5207\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.2632\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.69E-06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eShort-chain dehydrogenase/reductase eriH(NR)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eBased on the transcriptomics and metabolomics data, the data was integrated into the biosynthetic pathway of Erinacine A, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB. From the overall perspective of mycelial metabolic activity, the key to the synthesis of erinacines lies in the expression of the mevalonate pathway, and the only precursor for mevalonate synthesis is acetyl-CoA, which may be provided by fatty acid degradation, carbohydrate metabolism, energy metabolism, and amino acid metabolism. However, it was evident that by day 10 of fermentation, glucose had almost been depleted, which resulted in the inhibition of glycolysis/gluconeogenesis, thereby allowing acetyl-CoA to flow into the mevalonate pathway through the metabolism of other sugars. When stress occurs, it will drive the significant expression of the mevalonate pathway, especially the activation of GGPP synthase (19-fold higher expression than the MOCK group) in this pathway, and mevalonate will be more converted to GGPP, which will form the cyclooctane skeleton under the action of EriG. In the modification of the diterpene skeleton, the oxygenation behavior is more realized through cytochrome P450 monooxygenases (Wang et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Under oxidative stress, cytochrome P450 enzymes will be activated (such as EriI responding to oxidative stress, Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e2\u003c/span\u003e), synthesizing a series of Erinacine-like compounds, and being converted to Erinacine A through non-enzymatic reactions, enhancing HE's ability to resist unfavorable growth environments. In summary, diterpene compounds such as Erinacines may be important substances for HE to resist oxidative stress, providing appropriate oxidative stress behavior or increasing cytochrome P450 activity during fermentation may effectively improve the synthesis of HE diterpene compounds.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study demonstrates that Methyl Viologen (MV) acts as a powerful elicitor, dramatically boosting the production of the neuroprotective diterpenoid Erinacine A in the liquid fermentation of HE, achieving an unprecedented yield of 414 mg/L - the highest reported fermentation broth titer to date. Mechanistically, MV-induced oxidative stress activates interconnected metabolic rewiring (sugar, amino acid, and fatty acid pathways) and specifically amplifies the ROS-responsive Erinacine A biosynthetic machinery. Multi-omics integration revealed that MV upregulates both the mevalonate pathway and the Eri gene cluster, redirecting geranylgeranyl pyrophosphate (GGPP) toward Erinacine A synthesis.\u003c/p\u003e \u003cp\u003eCritically, while MV\u0026rsquo;s toxicity necessitates caution in direct industrial application, this work uncovers critical stress-responsive regulatory nodes for targeted metabolic engineering. These insights provide essential targets (e.g., specific genes, pathways, stress responses) for the future development of safe and sustainable strategies, such as food-grade elicitors or metabolic engineering approaches, aimed at harnessing this potential for the efficient and scalable production of Erinacine A as a valuable functional food ingredient or nutraceutical.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the China Postdoctoral Science Foundation [Grant Number 2025M773001], the Joint Fund of Zhejiang Provincial Natural Science Foundation of China [Grant Number LLSSZ25C200001], and the Research project Foundation of Zhejiang University of Technology [Grant Number KYY-HX-20240435].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors gratefully acknowledge Xueyu Biotechnology Co., Ltd (Hangzhou, China) for providing the proprietary \u003cem\u003eHericium erinaceus\u003c/em\u003e (Bill.) Pers. strain used in this study. \u003c/p\u003e\n\n\n\u003cp\u003eAuthor Contributions\u003c/p\u003e\n\u003cp\u003eJian Wang: Conceptualization, Funding acquisition, Investigation, Supervision, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing. Juncai Wan: Data curation, Formal analysis, Investigation, Visualization, Writing \u0026ndash; original draft. Junhao Huang: Formal analysis, Investigation, Methodology, Software, Visualization, Writing \u0026ndash; original draft. Ran Ma: Data curation, Validation, Visualization. Yahya Saud Mohamed Hamed: Writing - Reviewing and Editing. Hynek Roub\u0026iacute;k: Writing - Reviewing and Editing. Ming Cai: Funding acquisition, Resources. Lingli Li: Funding acquisition, Resources, Writing \u0026ndash; review \u0026amp; editing. Peilong Sun: Funding acquisition, Resources, Supervision. Kai Yang: Funding acquisition, Project administration, Resources, Supervision.\u003c/p\u003e\n\n\n\u003cp\u003eCompeting interest\u003c/p\u003e\n\u003cp\u003eThe authors declared that they have no conflicts of interest to this work. We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted.\u003c/p\u003e\n\n\n\u003cp\u003eData availability\u003c/p\u003e\n\u003cp\u003eThe authors confirm that the data supporting the findings of this study are available within the article [and/or] its supplementary materials.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eChang C-H, Chen Y, Yew X-X, Chen H-X, Kim J-X, Chang C-C, Peng C-C, Peng RY (2016) Improvement of erinacine A productivity in \u003cem\u003eHericium erinaceus\u003c/em\u003e mycelia and its neuroprotective bioactivity against the glutamate-insulted apoptosis. 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J Agric Food Chem 64(38):7291\u0026ndash;7297. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1021/acs.jafc.6b02907\u003c/span\u003e\u003cspan address=\"10.1021/acs.jafc.6b02907\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Hericium erinaceus, Erinacine A, Diterpenoid, Submerged fermentation, Methyl viologen, Multi-omics","lastPublishedDoi":"10.21203/rs.3.rs-8698276/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8698276/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eErinacine A, a critical secondary metabolite of \u003cem\u003eHericium erinaceus\u003c/em\u003e, has garnered significant interest due to its bioactive properties. This study aimed to investigate the effects of methyl viologen (MV) on the synthesis of Erinacine A during liquid fermentation of \u003cem\u003eHericium erinaceus\u003c/em\u003e, employing a multi-omics approach to elucidate the underlying mechanisms. Our findings demonstrate that the addition of 5 mg/L of MV after four days of fermentation significantly enhanced Erinacine A production, achieving a remarkable yield of 23 mg/g and 414 mg/L, marking the highest yield reported to date. Scanning electron microscopy (SEM) revealed a distinctive wrinkled and collapsed mycelial surface, indicative of stress responses. Moreover, metabolomics and transcriptomics analyses revealed oxidative stress within \u003cem\u003eHericium erinaceus\u003c/em\u003e, characterized by the downregulation of antioxidant-like metabolites and alterations in the oxidoreductase system. MV was found to impact several metabolic pathways, notably those associated with sugars, amino acids, and fatty acids. Comprehensive analysis of the omics data confirmed that MV enhances the expression of the mevalonate pathway and the Eri gene cluster in response to oxidative stress induced by reactive oxygen species (ROS). This process facilitates the conversion of geranylgeranyl pyrophosphate (GGPP) into Erinacine-like substances. This study provides novel insights, demonstrating for the first time that MV can be effectively utilized in the liquid fermentation of \u003cem\u003eHericium erinaceus\u003c/em\u003e to significantly improve the yield of Erinacine A.\u003c/p\u003e","manuscriptTitle":"Methyl viologen boosts Erinacine A biosynthesis in Hericium erinaceus fermentation: Multi-omics insights","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-11 12:14:42","doi":"10.21203/rs.3.rs-8698276/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"edf425a6-40cf-4649-9ab2-58bba226a463","owner":[],"postedDate":"February 11th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-17T13:42:13+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-11 12:14:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8698276","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8698276","identity":"rs-8698276","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}