Atomistic description of the OCTN1 recognition mechanism via in silico methods.

The Organic Cation Transporter Novel 1 (OCTN1), also known as SLC22A4, is widely expressed in various human tissues, and involved in numerous physiological and pathological processes remains. It facilitates the transport of organic cations, zwitterions, with selectivity for positively charged solutes. Ergothioneine, an antioxidant compound, and acetylcholine (Ach) are among its substrates. Given the lack of experimentally solved structures of this protein, this study aimed at generating a reliable 3D model of OCTN1 to shed light on its substrate-binding preferences and the role of sodium in substrate recognition and transport. A chimeric model was built by grafting the large extracellular loop 1 (EL1) from an AlphaFold-generated model onto a homology model. Molecular dynamics simulations revealed domain-specific mobility, with EL1 exhibiting the highest impact on overall stability. Molecular docking simulations identified cytarabine and verapamil as highest affinity ligands, consistent with their known inhibitory effects on OCTN1. Furthermore, MM/GBSA analysis allowed the categorization of substrates into weak, good, and strong binders, with molecular weight strongly correlating with binding affinity to the recognition site. Key recognition residues, including Tyr211, Glu381, and Arg469, were identified through interaction analysis. Ach demonstrated a low interaction energy, supporting the hypothesis of its one-directional transport towards to outside of the membrane. Regarding the role of sodium, our model suggested the involvement of Glu381 in sodium binding. Molecular dynamics simulations of systems at increasing levels of Na+ concentrations revealed increased sodium occupancy around Glu381, supporting experimental data associating Na+ concentration to molecule transport. In conclusion, this study provides valuable insights into the 3D structure of OCTN1, its substrate-binding preferences, and the role of sodium in the recognition. These findings contribute to the understanding of OCTN1 involvement in various physiological and pathological processes and may have implications for drug development and disease management.

Inflammation and Organic Cation Transporters Novel (OCTNs).

Inflammation is a physiological condition characterized by a complex interplay between different cells handled by metabolites and specific inflammatory-related molecules. In some pathological situations, inflammation persists underlying and worsening the pathological state. Over the years, two membrane transporters namely OCTN1 (SLC22A4) and OCTN2 (SLC22A5) have been shown to play specific roles in inflammation. These transporters form the OCTN subfamily within the larger SLC22 family. The link between these proteins and inflammation has been proposed based on their link to some chronic inflammatory diseases such as asthma, Crohn's disease (CD), and rheumatoid arthritis (RA). Moreover, the two transporters show the ability to mediate the transport of several compounds including carnitine, carnitine derivatives, acetylcholine, ergothioneine, and gut microbiota by-products, which have been specifically associated with inflammation for their anti- or proinflammatory action. Therefore, the absorption and distribution of these molecules rely on the presence of OCTN1 and OCTN2, whose expression is modulated by inflammatory cytokines and transcription factors typically activated by inflammation. In the present review, we wish to provide a state of the art on OCTN1 and OCTN2 transport function and regulation in relationships with inflammation and inflammatory diseases focusing on the metabolic signature collected in different body districts and gene polymorphisms related to inflammatory diseases.

A Rhodanese-Like Enzyme that Catalyzes Desulfination of Ergothioneine Sulfinic Acid.

Many actinobacterial species contain structural genes for iron-dependent enzymes that consume ergothioneine by way of O2-dependent dioxygenation. The resulting product ergothioneine sulfinic acid is stable under physiological conditions unless cleavage to sulfur dioxide and trimethyl histidine is catalyzed by a dedicated desulfinase. This report documents that two types of ergothioneine sulfinic desulfinases have evolved by convergent evolution. One type is related to metal-dependent decarboxylases while the other belongs to the superfamily of rhodanese-like enzymes. Pairs of ergothioneine dioxygenases (ETDO) and ergothioneine sulfinic acid desulfinase (ETSD) occur in thousands of sequenced actinobacteria, suggesting that oxidative ergothioneine degradation is a common activity in this phylum.

Uncovering the roles of Mycobacterium tuberculosis melH in redox and bioenergetic homeostasis: implications for antitubercular therapy.

Mycobacterium tuberculosis (Mtb), the pathogenic bacterium that causes tuberculosis, has evolved sophisticated defense mechanisms to counteract the cytotoxicity of reactive oxygen species (ROS) generated within host macrophages during infection. The melH gene in Mtb and Mycobacterium marinum (Mm) plays a crucial role in defense mechanisms against ROS generated during infection. We demonstrate that melH encodes an epoxide hydrolase and contributes to ROS detoxification. Deletion of melH in Mm resulted in a mutant with increased sensitivity to oxidative stress, increased accumulation of aldehyde species, and decreased production of mycothiol and ergothioneine. This heightened vulnerability is attributed to the increased expression of whiB3, a universal stress sensor. The absence of melH also resulted in reduced intracellular levels of NAD+, NADH, and ATP. Bacterial growth was impaired, even in the absence of external stressors, and the impairment was carbon source dependent. Initial MelH substrate specificity studies demonstrate a preference for epoxides with a single aromatic substituent. Taken together, these results highlight the role of melH in mycobacterial bioenergetic metabolism and provide new insights into the complex interplay between redox homeostasis and generation of reactive aldehyde species in mycobacteria. IMPORTANCE: This study unveils the pivotal role played by the melH gene in Mycobacterium tuberculosis and in Mycobacterium marinum in combatting the detrimental impact of oxidative conditions during infection. This investigation revealed notable alterations in the level of cytokinin-associated aldehyde, para-hydroxybenzaldehyde, as well as the redox buffer ergothioneine, upon deletion of melH. Moreover, changes in crucial cofactors responsible for electron transfer highlighted melH's crucial function in maintaining a delicate equilibrium of redox and bioenergetic processes. MelH prefers epoxide small substrates with a phenyl substituted substrate. These findings collectively emphasize the potential of melH as an attractive target for the development of novel antitubercular therapies that sensitize mycobacteria to host stress, offering new avenues for combating tuberculosis.

Effect of Selected Antioxidants on the In Vitro Aging of Human Fibroblasts.

The modification of the replicative lifespan (RLS) of fibroblasts is of interest both from a knowledge point of view and for the attenuation of skin aging. The effect of six antioxidants at a concentration of 1 µM on the replicative lifespan of human dermal fibroblasts was studied. The nitroxide 4-hydroxy-TEMPO (TEMPOL), ergothioneine, and Trolox extended the replicative lifespan (RLS) (40 ± 1 population doublings (PD)) by 7 ± 2, 4 ± 1, and 3 ± 1 PD and lowered the expression of p21 at late passages. Coumaric acid, curcumin and resveratrol did not affect the RLS . The level of reactive oxygen species (ROS) was decreased or not affected by the antioxidants although TEMPOL and coumaric acid decreased the level of glutathione. Only ergothioneine and resveratrol decreased the level of protein carbonylation. The antioxidants that could prolong the RLS elevated the mitochondrial membrane potential. Protecting the activity of mitochondria seems to be important for maintaining the replicative capacity of fibroblasts.

Ergothioneine Prevents Neuronal Cell Death Caused by the Neurotoxin 6-Hydroxydopamine.

Neuronal cell death is a key mechanism involved in the development and exacerbation of Parkinson's disease (PD). The excessive production of reactive oxygen species (ROS) is a major cause leading to neuronal death; therefore, compounds that prevent oxidative stress-dependent neuronal death may be promising as a preventive method for PD. Ergothioneine is a natural amino acid with antioxidant properties, and its protective functions in the body are attracting attention. However, there has been no investigation into the protective functions of ergothioneine using in vivo and in vitro PD models. Thus, in this study, we analyzed the efficacy of ergothioneine against 6-hydroxydopamine (6-OHDA)-dependent neuronal cell death using immortalized hypothalamic neurons (GT1-7 cells). First, we found that ergothioneine prevents 6-OHDA-dependent neuronal cell death by suppressing ROS overproduction in GT1-7 cells. The cytoprotective effect of ergothioneine was partially abolished by verapamil, an inhibitor of OCTN1, which is involved in ergothioneine uptake. Furthermore, ergothioneine-rich Rice-koji (Ergo-koji) showed cytoprotective and antioxidant effects similar to those of ergothioneine. Taken together, these results suggest that ergothioneine or foods containing ergothioneine may be an effective method for preventing the development and progression of PD.

Co-augmentation of a transport gene mfsT1 in Mycolicibacterium neoaurum with genome engineering to enhance ergothioneine production.

Ergothioneine (EGT) is a rare thiohistidine derivative with exceptional antioxidant properties. The blood level of EGT is considered highly reliable predictors for cardiovascular diseases and mortality, yet animals lack the ability to synthesize this compound. Free plasmids have been previously used to overexpress genes involved in the EGT biosynthetic pathway of Mycolicibacterium neoaurum. Here, we tentatively introduced a putative transporter gene mfsT1 into high-copy plasmids and sharply increased the ratio of extracellular EGT concentration from 18.7% to 44.9%. Subsequently, an additional copy of egtABCDE, hisG, and mfsT1 was inserted into the genome with a site-specific genomic integration tool of M. neoaurum, leading a 2.7 times increase in EGT production. Co-enhancing the S-adenosyl-L-methionine regeneration pathway, or alternatively, the integration of three copies of egtABCDE, hisG and mfsT1 into the genome further increased the total EGT yield by 16.1% (64.6 mg/L) and 21.7% (67.7 mg/L), respectively. After 168-h cultivation, the highest titer reached 85.9 mg/L in the latter strain with three inserted copies. This study provided a solid foundation for genome engineering to increase the production of EGT in M. neoaurum.

Enzyme-Catalyzed Oxidative Degradation of Ergothioneine.

Ergothioneine is a sulfur-containing metabolite that is produced by bacteria and fungi, and is absorbed by plants and animals as a micronutrient. Ergothioneine reacts with harmful oxidants, including singlet oxygen and hydrogen peroxide, and may therefore protect cells against oxidative stress. Herein we describe two enzymes from actinobacteria that cooperate in the specific oxidative degradation of ergothioneine. The first enzyme is an iron-dependent thiol dioxygenase that produces ergothioneine sulfinic acid. A crystal structure of ergothioneine dioxygenase from Thermocatellispora tengchongensis reveals many similarities with cysteine dioxygenases, suggesting that the two enzymes share a common mechanism. The second enzyme is a metal-dependent ergothioneine sulfinic acid desulfinase that produces Nα-trimethylhistidine and SO2 . The discovery that certain actinobacteria contain the enzymatic machinery for O2 -dependent biosynthesis and O2 -dependent degradation of ergothioneine indicates that these organisms may actively manage their ergothioneine content.

Structure of mycobacterial ergothioneine-biosynthesis C-S lyase EgtE.

L-ergothioneine is widely distributed among various microbes to regulate their physiology and pathogenicity within complex environments. One of the key steps in the ergothioneine-biosynthesis pathway, the C-S bond cleavage reaction, uses the pyridoxal 5'-phosphate dependent C-S lyase to produce the final product L-ergothioneine. Here, we present the crystallographic structure of the ergothioneine-biosynthesis C-S lyase EgtE from Mycobacterium smegmatis (MsEgtE) represents the first published structure of ergothioneine-biosynthesis C-S lyases in bacteria and shows the effects of active site residues on the enzymatic reaction. The MsEgtE and the previously reported ergothioneine-biosynthesis C-S lyase Egt2 from Neurospora crassa (NcEgt2) fold similarly. However, discrepancies arise in terms of substrate recognition, as observed through sequence and structure comparison of MsEgtE and NcEgt2. The structural-based sequence alignment of the ergothioneine-biosynthesis C-S lyase from fungi and bacteria shows clear distinctions among the recognized substrate residues, but Arg348 is critical and an extremely conserved residue for substrate recognition. The α14 helix is exclusively found in the bacteria EgtE, which represent the most significant difference between bacteria EgtE and fungi Egt2, possibly resulting from the convergent evolution of bacteria and fungi.

Structural insights into a novel nonheme iron-dependent oxygenase in selenoneine biosynthesis.

Selenoneine (SEN) is a natural histidine derivative with radical-scavenging activity and shows higher antioxidant potential than its sulfur-containing isolog ergothioneine (EGT). Recently, the SEN biosynthetic pathway in Variovorax paradoxus was reported. Resembling EGT biosynthesis, the committed step of SEN synthesis is catalyzed by a nonheme Fe-dependent oxygenase termed SenA. This enzyme catalyzes oxidative carbon­selenium (C-Se) bond formation to conjugate N-α-trimethyl histidine (TMH) and selenosugar to yield selenoxide; the process parallels the EGT biosynthetic route, in which sulfoxide synthases known as EgtB members catalyze the conjugation of TMH and cysteine or γ-glutamylcysteine to afford sulfoxides. Here, we report the crystal structures of SenA and its complex with TMH and thioglucose (SGlc), an analog of selenoglucose (SeGlc) at high resolution. The overall structure of SenA adopts the archetypical fold of EgtB, which comprises a DinB-like domain and an FGE-like domain. While the TMH-binding site is highly conserved to that of EgtB, a various substrate-enzyme interaction network in the selenosugar-binding site of SenA features a number of water-mediated hydrogen bonds. The obtained structural information is beneficial for understanding the mechanism of SenA-mediated C-Se bond formation.

L-ergothioneine reduces mitochondrial-driven NLRP3 activation in gestational diabetes mellitus.

BACKGROUND: Maternal hyperglycaemia has a significant impact on placental metabolism and mitochondrial function. The NLRP3 inflammasome is responsive to endogenous signals of mitochondrial dysfunction. We tested our hypothesis that mitochondrial dysfunction orchestrates activation of the NLRP3 inflammasome and contributes to inflammation in gestational diabetes mellitus (GDM). METHODS: Fasting blood, omental and placental tissue were collected on the day of caesarean section from nulliparous women with normal glucose tolerant (NGT) (n = 30) and GDM (n = 27) pregnancies. Cell-free mitochondrial DNA (cf-mtDNA) copy number was quantified by real-time PCR. M1-like (CD14+CD86+CD206-) and M2-like (CD14+CD86+CD206+) macrophage populations were characterized by flow cytometry. Immunoblotting for protein expression of NLRP3, ASC and caspase-1 was performed in maternal BMI and age-matched tissue samples. IL-1ß and IL-18 were measured by multiplex ELISA. Placental explants from GDM participants were cultured for 24 h with 1 mM L-ergothioneine (antioxidant) and 1 µM MCC950 (NLRP3 inhibitor). RESULTS: Cf-mtDNA copy numbers were significantly higher in GDM compared to NGT participants (p = 0.002). Placental populations of CD14+ (p = 0.02) and CD14+CD86+CD206- (p = 0.03) macrophages produced significantly increased levels of mitochondrial superoxide in GDM compared to NGT participants. Placental production of IL-18 (p = 0.04) was significantly increased in GDM. This increase in placental IL-18 was attenuated by treatment with 1 µM MCC950 (p = 0.0005), and 1 mM L-ergothioneine (p = 0.007). CONCLUSION: Placental inflammation is significantly increased in women with GDM. Furthermore, this increase may be initiated by elevated production of mitochondrial superoxide by macrophage subpopulations and orchestrated by the NLRP3 inflammasome. The mitochondrial antioxidant, L-ergothioneine, ameliorates NLRP3-induced placental inflammation in GDM, identifying a potential therapeutic role.

Ergothioneine: an underrecognised dietary micronutrient required for healthy ageing?

Ergothioneine is a naturally occurring amino acid and thiol antioxidant found in high amounts in mushrooms and fermented foods. Humans and animals acquire ergothioneine from the diet through the pH-dependent activity of a membrane transporter, the large solute carrier 22A member 4 (SLC22A4), expressed on the apical membrane of the small intestine. The SLC22A4 transporter also functions in the renal reabsorption of ergothioneine in the kidney, with avid absorption and retention of ergothioneine from the diet observed in both animals and humans. Ergothioneine is capable of scavenging a diverse range of reactive oxygen and nitrogen species, has metal chelation properties, and is predicted to directly regulate nuclear factor erythroid 2-related factor 2 (Nrf2) activity. Although not lethal, the genetic knockout of the SLC22A4 gene in multiple organisms increases susceptibility to oxidative stress, damage and inflammation; in agreement with a large body of preclinical data suggesting the physiological function of ergothioneine is as a cellular antioxidant and cytoprotectant agent. In humans, blood levels of ergothioneine decline after the age of 60 years, and lower levels of ergothioneine are associated with more rapid cognitive decline. Conversely, high plasma ergothioneine levels have been associated with significantly reduced cardiovascular mortality and overall mortality risks. In this horizon's manuscript, we review evidence suggesting critical roles for dietary ergothioneine in healthy ageing and the prevention of cardiometabolic disease. We comment on some of the outstanding research questions in the field and consider the question of whether or not ergothioneine should be considered a conditionally essential micronutrient.

Antioxidant effect of ergothioneine on in vitro maturation of porcine oocytes.

BACKGROUND: Ergothioneine (EGT) is a natural amino acid derivative in various animal organs and is a bioactive compound recognized as a food and medicine. OBJECTIVES: This study examined the effects of EGT supplementation during the in vitro maturation (IVM) period on porcine oocyte maturation and subsequent embryonic development competence after in vitro fertilization (IVF). METHODS: Each EGT concentration (0, 10, 50, and 100 µM) was supplemented in the maturation medium during IVM. After IVM, nuclear maturation, intracellular glutathione (GSH), and reactive oxygen species (ROS) levels of oocytes were investigated. In addition, the genes related to cumulus function and antioxidant pathways in oocytes or cumulus cells were investigated. Finally, this study examined whether EGT could affect embryonic development after IVF. RESULTS: After IVM, the EGT supplementation group showed significantly higher intracellular GSH levels and significantly lower intracellular ROS levels than the control group. Moreover, the expression levels of hyaluronan synthase 2 and Connexin 43 were significantly higher in the 10 µM EGT group than in the control group. The expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and NAD(P)H quinone dehydrogenase 1 (NQO1) were significantly higher in the oocytes of the 10 µM EGT group than in the control group. In the assessment of subsequent embryonic development after IVF, the 10 µM EGT treatment group improved the cleavage and blastocyst rate significantly than the control group. CONCLUSIONS: Supplementation of EGT improved oocyte maturation and embryonic development by reducing oxidative stress in IVM oocytes.

What Is the Redox Potential of Ergothioneine?

Commonly given citations for the redox potential of ergothioneine are incorrect. In addition, the value of -0.06 V should be viewed with skepticism since the method used to determine the redox potential of ergothioneine was also used to incorrectly determine the redox potential of glutathione. Antioxid. Redox Signal. 38, 1212-1213.

Potential Cytoprotective and Regulatory Effects of Ergothioneine on Gene Expression of Proteins Involved in Erythroid Adaptation Mechanisms and Redox Pathways in K562 Cells.

This study aimed to establish the importance of ergothioneine (ERT) in the erythroid adaptation mechanisms by appraising the expression levels of redox-related genes associated with the PI3K/AKT/FoxO3 and Nrf2-ARE pathways using K562 cells induced to erythroid differentiation and H2O2-oxidative stress. Cell viability and gene expression were evaluated. Two concentrations of ERT were assessed, 1 nM (C1) and 100 µM (C2), with and without stress induction (100 µM H2O2). Assessments were made in three periods of the cellular differentiation process (D0, D2, and D4). The C1 treatment promoted the induction of FOXO3 (D0 and 2), PSMB5, and 6 expressions (D4); C1 + H2O2 treatment showed the highest levels of NRF2 transcripts, KEAP1 (D0), YWHAQ (D2 and 4), PSMB5 (D2) and PSMB6 (D4); and C2 + H2O2 (D2) an increase in FOXO3 and MST1 expression, with a decrease of YWHAQ and NRF2 was observed. in C2 + H2O2 (D2) an increase in FOXO3 and MST1, with a decrease in YWHAQ and NRF2 was observed All ERT treatments increased gamma-globin expression. Statistical multivariate analyzes highlighted that the Nrf2-ARE pathway presented a greater contribution in the production of PRDX1, SOD1, CAT, and PSBM5 mRNAs, whereas the PI3K/AKT/FoxO3 pathway was associated with the PRDX2 and TRX transcripts. In conclusion, ERT presented a cytoprotective action through Nrf2 and FoxO3, with the latter seeming to contribute to erythroid proliferation/differentiation.

A microbial transporter of the dietary antioxidant ergothioneine.

Low-molecular-weight (LMW) thiols are small-molecule antioxidants required for the maintenance of intracellular redox homeostasis. However, many host-associated microbes, including the gastric pathogen Helicobacter pylori, unexpectedly lack LMW-thiol biosynthetic pathways. Using reactivity-guided metabolomics, we identified the unusual LMW thiol ergothioneine (EGT) in H. pylori. Dietary EGT accumulates to millimolar levels in human tissues and has been broadly implicated in mitigating disease risk. Although certain microorganisms synthesize EGT, we discovered that H. pylori acquires this LMW thiol from the host environment using a highly selective ATP-binding cassette transporter-EgtUV. EgtUV confers a competitive colonization advantage in vivo and is widely conserved in gastrointestinal microbes. Furthermore, we found that human fecal bacteria metabolize EGT, which may contribute to production of the disease-associated metabolite trimethylamine N-oxide. Collectively, our findings illustrate a previously unappreciated mechanism of microbial redox regulation in the gut and suggest that inter-kingdom competition for dietary EGT may broadly impact human health.

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

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

First evidence of dermo-protective activity of marine sulfur-containing histidine compounds.

Among natural products, ovothiol (ovo), produced by marine invertebrates, bacteria, and microalgae, is receiving increasing interest for its unique antioxidant properties. Recently, ovo has been shown to exhibit anti-inflammatory activity in an in vitro model of endothelial dysfunction and in an in vivo model of liver fibrosis. The aim of this study was to evaluate the effect of ovo and its precursor 5-thiohistidine (5-thio) in comparison with ergothioneine (erg), in human skin cells and tissues upon inflammation. We used both an in vitro and ex vivo model of human skin, represented by a keratinocytes cell line (HaCaT) and skin biopsies, respectively. We observed that ovo, 5-thio, and erg were not cytotoxic in HaCaT cells, but instead exerted a protective function against TNF-α -induced inflammation. In order to get insights on their mechanism of action, we performed western blot analysis of ERK and JNK, as well as sub-cellular localization of Nrf2, a key mediator of the anti-inflammatory response. The results indicated that the pre-treatment with ovo, 5-thio, and erg differently affected the phosphorylation of ERK and JNK. However, all the three molecules promoted the accumulation of Nrf2 in the nucleus of HaCaT cells. In addition, gene expression analysis by RTqPCR and ELISA assays performed in ex vivo human skin tissues pre-treated with thiohistidines and then inflamed with IL-1ß revealed a significant downregulation of IL-8, TNF-α and COX-2 genes and a concomitant significant decrease in the cytokines IL-6, IL-8 and TNF-α production. Moreover, the protective action of ovo and 5-thio resulted to be stronger when compared with dexamethasone, a corticosteroid drug currently used to treat skin inflammatory conditions. Our findings suggest that ovo and 5-thio can ameliorate skin damage and may be used to develop natural skin care products to prevent the inflammatory status induced by environmental stressors and aging.

[Recent progress in ergothioneine biosynthesis: a review].

Ergothioneine is a multifunctional physiological cytoprotector, with broad application in foods, beverage, medicine, cosmetics and so on. Biosynthesis is an increasingly favored method in the production of ergothioneine. This paper summarizes the new progress in the identification of key pathways, the mining of key enzymes, and the development of natural edible mushroom species and high-yield engineering strains for ergothioneine biosynthesis in recent years. Through this review, we aim to reveal the molecular mechanism of ergothioneine biosynthesis and then employ the methods of fermentation engineering, metabolic engineering, and synthetic biology to greatly increase the yield of ergothioneine.