Ergothioneine (ERG), also referred to as 2-mercaptohistidine trimethylbetaine, was initially discovered and isolated from Claviceps purpurea found in wheat grains in 1909. ERG, as an essential physiological compound, is widely utilized in the fields of food, cosmetics, medicine, and other sectors due to its potent antioxidant properties and diverse biological effects, including anti-inflammatory, anti-aging, neuroprotective, and oxidative stress-preventive properties (Hartman, 1990, Han et al., 2021, Liu et al., 2023, Cheah and Halliwell, 2012). ERG is primarily synthesized in nature by specific bacteria and fungi and cannot be synthesized by plants, animals, or humans, making it a rare natural chiral amino acid (Alamgir et al., 2015, Dubost et al., 2007, Kitsanayanyong and Ohshima, 2022, Takusagawa et al., 2019). Currently, the development of engineered strains and microbial fermentation techniques for ERG are anticipated to address the current challenges of low yield and high production costs of ERG, which cannot meet market demand (Han et al., 2021; Q. Liu et al., 2022; Qiu et al., 2021; Xiong et al., 2023).

The biosynthesis pathway of ERG in various microorganisms has been extensively elucidated (Li et al., 2024). To date, four ERG biosynthetic pathways have been reported,(Q. Liu et al., 2022) all of which utilize L-His, L-Cys, and L-Met as precursors (Li et al., 2025). The two most extensively studied ERG biosynthetic pathways are the bacterial pathway, represented by Mycobacterium smegmatis with the egtA/B/C/D/E gene cluster (which encodes the five ERG synthases EgtA, EgtB, EgtC, EgtD, and EgtE), and the fungal pathway, represented by Neurospora crassa with the Egt1 and Egt2 enzymes (Bello et al., 2012, Hu et al., 2014, Seebeck, 2010). Both pathways initially use SAM to transfer three methyl groups to L-His via either the histidine-specific methyltransferase encoded by the egtD gene or the SAM-dependent methyltransferase encoded by the Egt1 gene, resulting in HER (Bello et al., 2012, Seebeck, 2010). In the bacterial pathway, HER and γ-GC (synthesized from cysteine and glutamate by the glutamate cysteine ligase encoded by the egtA gene) are catalyzed by the Fe2 + -dependent oxidase encoded by the egtB gene to produce γGC-HER (Goncharenko et al., 2015). The product is then catalyzed by the aminotransferase encoded by the egtC gene to remove L-Glu, producing Cys-HER (Vit et al., 2015). In contrast, in the fungal pathway, HER directly generates Cys-HER via a single Egt1 enzyme (Bello et al., 2012). Finally, ammonium pyruvate is removed from Cys-HER by the PLP-dependent C-S lyase encoded by the egtE gene or the PLP-dependent cysteine desulfurase encoded by the Egt2 gene (Bello et al., 2012, Hu et al., 2014), resulting in the target product, ERG. The Egt1 enzyme in the N. crassa pathway contains two functional domains, corresponding to the roles of EgtB and EgtD, respectively. The biosynthesis of ERG in N. crassa is more efficient than that in M. smegmatis, primarily due to the absence of L-Glu involvement. Additionally, the elimination of the intermediate γGC in the N. crassa pathway removes the biosynthetic competition between ERG and GSH, significantly enhancing the biosynthetic efficiency of ERG. The discovery of multiple ERG microbial synthesis pathways opens the possibility of utilizing common strains as chassis for integrating ERG biosynthesis pathways. Moreover, the key enzymes involved in ERG synthesis and precursor amino acid biosynthesis can be strategically modified using synthetic biology techniques to optimize their combined expression (Xiong et al., 2024, Zhang et al., 2024).

Escherichia coli, with its well-defined genetic background and rapid growth in inexpensive media, stands as one of the most prevalent chassis organisms for metabolic engineering and synthetic biology (Waegeman and Soetaert, 2011, Xu et al., 2023). E. coli BL21(DE3) enables robust genetic engineering due to its well-characterized genome and compatibility with advanced synthetic biology tools. For instance: Zhang et al. demonstrated that introducing EgtE, modifying EgtD, and utilizing the bifunctional enzyme NcEgt1 in E. coli BL21(DE3) achieved an ERG yield of 5.4 g/L within 96 h of batch fermentation (Zhang et al., 2023). This exemplifies E. coli's capacity for rapid pathway assembly and optimization. While Kim et al. enhanced ERG production in Corynebacterium glutamicum by overexpressing cysEKR and deleting sdaA to accumulate L-His and L-Cys, the final yield remained limited to 264 mg/L after 36 h of fermentation (Kim et al., 2022). Such metabolic rewiring in C. glutamicum necessitates laborious strain engineering compared to E. coli's native metabolic flexibility.

Yeast, as a kind of eukaryotic microorganism with simple genetic system and perfect expression system, is also used to produce ERG (Fujitani et al., 2018, Van der Hoek et al., 2022b, Van der Hoek et al., 2022a, Van der Hoek et al., 2019, Yu et al., 2020). Recent studies demonstrated that in Saccharomyces cerevisiae, ERG production was increased to 1.14 g/L after 10 days of fermentation through optimization of ERG biosynthesis genes, overexpression of upstream substrate synthesis genes, and the glycerol transporter STL1 (Yu et al., 2024). In comparison, Chen et al. achieved 4.34 g/L ERG in fed-batch fermentation using E. coli expressing fungal genes (TrEgt1/TrEgt2 from Trichoderma reesei) (Chen et al., 2022). Kang et al. employed protein engineering to achieve soluble expression of TrEgt12 in E. coli BL21(DE3). By knocking out competing pathways and optimizing the copy number of key genes, the engineered strain E25 produced 2.33 g/L ERG within 80 h in a 5-L bioreactor (Kang et al., 2025). These findings highlight that E. coli BL21(DE3), as an expression system, enables higher yields in shorter fermentation periods compared to yeast-based approaches, positioning it as a superior chassis strain for ERG biosynthesis.

In this study, a novel strategy involving the heterologous assembly of the ERG synthesis pathway was designed and implemented in E. coli BL(DE3). Using fed-batch fermentation, the optimal strain achieved an ERG titer of 790 mg/L within 53 h. This study offers a more efficient approach for ERG expression and establishes both theoretical and experimental foundations for further genetic modifications and yield enhancement in ERG production.