Efficient Synthesis of Ergothioneine in Escherichia coli Through Identification of Bacterial-Type Egt1 and Self-Assemble of Pathway Enzymes.

Ergothioneine (ERG), a food-derived antioxidant and cytoprotectant, has broad application prospects in food, nutraceuticals, cosmetics, and biomedical fields. The growing market demand for ERG has driven the development of microbial cell factories as a sustainable production platform. However, the biosynthetic production of ERG faces challenges including poor substrate channeling, limited precursor supply, and potential inhibition by intracellular product accumulation. To construct a promising cell factory for high-level ERG production, firstly, the ERG biosynthetic pathway from Trichoderma reesei was heterologously expressed in Y. lipolytica Po1f (Δku70). Secondly, the application of Tregt1-Tregt2 fusion with a flexible (GGGGS)₂ linker and multi-copy integration led to an ERG production of 618.97 mg/L, an 83% improvement. Then, carbon flux was systematically redirected toward ERG production via modular engineering to enhance the precursor supply. Finally, fermentation conditions were optimized, resulting in an ERG production of 1075.27 mg/L in shake flask after 120 h fermentation. When scaled up to 5 L bioreactor, the strain achieved a final ERG production of 8.03 g/L within 168 h (47.79 mg/L/h), the highest reported to date without the supplementation of exogenous precursors. This study provides an effective strategy for utilizing Y. lipolytica as a microbial cell factory for ERG biosynthesis.

Ergothioneine (EGT) is a rare amino acid with potent antioxidant and anti-inflammatory properties, with a wide range of applications in food, cosmetics, and medicine. In the present study, Aspergillus oryzae, a common edible fungus, was engineered as an optimal host for EGT production. Moreover, two endogenous genes involved in EGT biosynthesis were characterized. The homolog AoEgt1 was shown to be localized in the vacuoles, whereas the homolog AoEgt2 was found in the peroxisomes. Overexpression of EGT biosynthetic genes from different organisms enhanced EGT production, yielding 15.17 mg EGT/g of dry weight. Using glucose as the carbon source and supplementing methionine (Met) as a precursor further increased EGT production to 20.03 mg EGT/g of dry weight, constituting an eight-fold increase compared to the wild-type strain. This study discusses the successful construction of a high-yielding A. oryzae strain for EGT biosynthesis, providing a novel strategy for efficient EGT synthesis.Key points• Two newly described homologs, AoEgt1 and AoEgt2, were identified in A. oryzae.• AoEgt1 and AoEgt2 were found to contribute to EGT biosynthesis.• EGT production was significantly increased by overexpression of Egt1 and Egt2.• Glucose and Met supplementation in the medium increased EGT production.

Ergothioneine (EGT), which exhibits strong antioxidant properties, is an amino acid derivative with a betaine structure. Currently, studies have examined EGT import systems and its physiological roles in various organisms. Despite the broad applicability of EGT, industrial production with high productivity has not yet been achieved. In this study, we aimed to develop fermentative production methods for EGT using Corynebacterium glutamicum as a host and successfully achieved the highest yield of EGT (459 mg L−1) reported to date. A cysteine-producing strain C. glutamicum CYS-2, which was constructed in a previous study, was engineered to enhance the biosynthesis of histidine and S-adenosylmethionine, both of which, along with cysteine, are required for EGT production. Additionally, heterologous metabolic pathways for EGT biosynthesis from Mycolicibacterium smegmatis and Methylobacterium pseudosasicola were introduced into the engineered strain, which was designated CHS2. In batch cultivation, the CHS2 strain produced more EGT than the CYS-2 strain harboring the same EGT biosynthesis pathway. Interestingly, batch cultivation of the CHS2 strain under high osmotic pressure conditions prolonged the time for EGT production and increased the intracellular accumulation of EGT. These results suggest that increasing osmotic pressure together with engineering the biosynthesis of cysteine, histidine, and S-adenosylmethionine is an effective strategy for enhancing EGT production in recombinant C. glutamicum harboring heterologous EGT biosynthesis pathways.Key points• Ergothioneine production in C. glutamicum was enhanced by metabolic engineering.• Osmotic pressure affects ergothioneine production in engineered C. glutamicum.• Ergothioneine may function as a compatible solute in C. glutamicum.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00253-025-13639-3.

Efficient production of Ergothioneine via an optimized allogenous assembly of the ERG synthesis pathway in Escherichia coli BL21.

Sixteen strains of basidiomycetous yeasts were evaluated for their capability to produce ergothioneine (EGT), an amino acid derivative with strong antioxidant activity. The cells were cultured in either two synthetic media or yeast mold (YM) medium for 72 h, after which cytosolic constituents were extracted from the cells with hot water. After analyzing the extracts via liquid chromatography-mass spectrometry (LC-MS), we found that all strains produced varying amounts of EGT. The EGT-producing strains, including Ustilago siamensis, Anthracocystis floculossa, Tridiomyces crassus, Ustilago shanxiensis, and Moesziomyces antarcticus, were subjected to flask cultivation in YM medium. U. siamensis CBS9960 produced the highest amount of EGT at 49.5 ± 7.0 mg/L after 120 h, followed by T. crassus at 30.9 ± 1.8 mg/L. U. siamensis was also cultured in a jar fermenter and produced slightly higher amounts of EGT than under flask cultivation. The effects of culture conditions, particularly the addition of precursor amino acids, on EGT production by the selected strains were also evaluated. U. siamensis showed a 1.5-fold increase in EGT production with the addition of histidine, while U. shanxiensis experienced a 1.8-fold increase in EGT production with the addition of methionine. These results suggest that basidiomycetous yeasts could serve an abundant source for natural EGT producers.

Development of a plasmid-free Escherichia coli strain for high-yield production of ergothioneine.

Ergothioneine (EGT), an exceptional antioxidant found ubiquitously across diverse living organisms, plays a pivotal role in various vital physiological regulatory functions. Its principal natural sources are mushrooms and animal liver tissues. Ganoderma spp., a traditional Chinese food and medicinal mushroom, boasts high concentrations of EGT. To advance the development of novel Ganoderma spp. strains with enhanced EGT yields, we employed an efficient Ganoderma spp. protoplasmic fusion system. Through molecular and biological characterization, we successfully generated seven novel fusion strains. Notably, fusion strain RS7 demonstrated a remarkable increase in mycelial EGT production (12.70 ± 1.85 mg/L), surpassing the parental strains FQ16 and FQ23 by 34.23% and 39.10%, respectively. Furthermore, in the context of the fruiting body, fusion strain RS11 displayed a notable 53.58% enhancement in EGT production (11.24 ± 1.96 mg/L) compared to its parental strains. Genomic analysis of the RS7, the strain with the highest levels of mycelial EGT production, revealed mutations in the gene EVM0005141 associated with EGT metabolism. These mutations led to a reduction in non-productive shunts, subsequently redirecting more substrate towards the EGT synthesis pathway. This redirection significantly boosted EGT production in the RS7 strain. The insights gained from this study provide valuable guidance for the commercial-scale production of EGT and the selective breeding of Ganoderma spp. strains.

Ergothioneine (EGT) is a powerful and natural antioxidant, which can protect cells in the human body from oxidative damage. Numerous studies have attempted to enhance the heterologous synthesis. However, research on EGT transport out of Escherichia coli cells and the wild-type enzyme from Trichoderma reesei is rare. Here, membrane permeability engineering and protein engineering are combined to improve EGT production in E. coli. After metabolic engineering to enhance precursor supply, we deleted the genes involved in lipopolysaccharide biosynthesis to increase the membrane permeability, which promoted EGT production. Further, a mutant of Tregt2E155C with improved catalytic capacity was created, and the expression level of enzymes involved in EGT synthesis was optimized. Finally, fermentation parameters were systematically tuned to maximize the EGT production. The engineered strain MT9-PET-T1/RSF-T2E155C produced 334.20 ± 6.33 mg/L EGT during 48 h of shake-flask fermentation, corresponding to an 8.4-fold increase compared with wild-type strain MT1. When scaled up to 5 L bioreactors, the strain achieved a final EGT titer of 4.06 g/L within 96 h (42.29 mg/L/h). Our work shows significant implications for EGT synthesis and is also a reference for manipulating E. coli to synthesize other biomolecules.

Ergothioneine (ERG) is an unusual sulfur-containing amino acid with antioxidant activity that can be synthesized by certain bacteria and fungi. Microbial fermentation is a promising method for ERG production. In this study, the bifunctional enzyme methyltransferase-sulfoxide synthase NcEgt1 from Neurospora crassa was truncated to obtain sulfoxide synthase TNcEgt1, which showed a higher expression level in Escherichia coli BL21(DE3). Then, the genes egtD encoding methyltransferase EgtD and egtE encoding C-S lyase EgtE from Mycobacterium smegmatis were cloned with TncEgt1 into E. coli BL21(DE3) to produce 70 mg/L ERG. To improve ERG production, TNcEgt1 and EgtD were modified, and the resulting mutants were screened with an established high-throughput method which could directly analyze the ERG content in culture broths. After several rounds of mutation and screening, the optimal mutant MD4 was obtained and produced 290 mg/L ERG. Furthermore, a fed-batch culture was conducted in a 5 L bioreactor. After optimizing the fermentation process, the ERG yield reached 5.4 g/L after 94 h of cultivation supplemented with amino acids and glycerol, which is the highest ERG yield reported to date. The results showed that ERG production was significantly improved by modifying the key enzymes, and the engineered strains constructed in this study have potential industrial application prospects.

The naturally occurring sulphur-containing histidine derivative, ergothioneine (EGT), exhibits potent antioxidant properties and has been proposed to confer human health benefits. Although it is only produced by select fungi and prokaryotes, likely to protect against environmental stress, the GRAS organism Saccharomyces cerevisiae does not produce EGT naturally. Herein, it is demonstrated that the recombinant expression of a single gene, Aspergillus fumigatus egtA, in S. cerevisiae results in EgtA protein presence which unexpectedly confers complete EGT biosynthetic capacity. Both High Performance Liquid Chromatography (HPLC) and LC–mass spectrometry (MS) analysis were deployed to detect and confirm EGT production in S. cerevisiae. The localisation and quantification of the resultant EGT revealed a significantly (p < 0.0001) larger quantity of EGT was extracellularly present in culture supernatants than intracellularly accumulated in 96 h yeast cultures. Methionine addition to cultures improved EGT production. The additional expression of two candidate cysteine desulfurases from A. fumigatus was thought to be required to complete EGT biosynthesis, namely AFUA_2G13295 and AFUA_3G14240, termed egt2a and egt2b in this study. However, the co-expression of egtA and egt2a in S. cerevisiae resulted in a significant decrease in the observed EGT levels (p < 0.05). The AlphaFold prediction of A. fumigatus EgtA 3-Dimensional structure illuminates the bidomain structure of the enzyme and the opposing locations of both active sites. Overall, we clearly show that recombinant S. cerevisiae can biosynthesise and secrete EGT in an EgtA-dependent manner which presents a facile means of producing EGT for biotechnological and biomedical use.

Ergothioneine (EGT) is a unique naturally occurring amino acid that is usually biosynthesized by bacteria and fungi. As a food-derived antioxidant and cytoprotectant, it has several physiological benefits and has a wide range of applications in food, medicine, and cosmetics. Traditional production of EGT is mainly through biological extraction or chemical synthesis; however, these methods are inefficient, making large-scale production to meet the growing market demand difficult. Nowadays, the rapid development of synthetic biology has greatly accelerated the research on the EGT production by microbial fermentation. In this paper, the biological characteristics, applications, biosynthesis, separation, and detection methods of EGT were fully reviewed. Furthermore, the approaches and challenges for engineering microbial cells to efficiently synthesize EGT were also discussed. This work provides new ideas and future research potentials in EGT production.

Ergothioneine (EGT) is a unique natural chiral compound endowed with potent antioxidative, anti-inflammatory, and cytoprotective properties. Currently, EGT is primarily produced via bioextraction from mushrooms and chemical synthesis; however, the low efficiency and high costs associated with these methods hinder their ability to meet the growing market demand. Consequently, heterologous EGT production in non-native host strains (e.g., Escherichia coli and Corynebacterium glutamicum) has garnered increasing attention. With the rapid advancement of synthetic biology and metabolomics, remarkable progress has been achieved in EGT production in recent years, with the high titers have reached 7.2 g/L in E. coli and 9.3 g/L in Yarrowia lipolytica. Meanwhile, the development of a "chemoenzymatic catalytic cascade″ route has achieved the highest titer: 47.3 g/L. This review focuses on the latest advances in the discovery and identification of key enzymes involved in EGT biosynthetic and catabolic pathways and metabolic engineering strategies for EGT production. Additionally, the multifunctional roles and practical applications of EGT in the food, cosmetics, and pharmaceutical industries are summarized.

Ergothioneine (ERG) is an unusual sulfur-containing amino acid. It is a potent antioxidant, which shows great potential for ameliorating neurodegenerative and cardiovascular diseases. L-ergothioneine is rare in nature, with mushrooms being the primary dietary source. The chemical synthesis process is complex and expensive. Alternatively, ERG can be produced by fermentation of recombinant microorganisms engineered for ERG overproduction. Here, we describe the engineering of S. cerevisiae for high-level ergothioneine production on minimal medium with glucose as the only carbon source. To this end, metabolic engineering targets in different layers of the amino acid metabolism were selected based on literature and tested. Out of 28 targets, nine were found to improve ERG production significantly by 10%–51%. These targets were then sequentially implemented to generate an ergothioneine-overproducing yeast strain capable of producing 106.2 ± 2.6 mg/L ERG in small-scale cultivations. Transporter engineering identified that the native Aqr1 transporter was capable of increasing the ERG production in a yeast strain with two copies of the ERG biosynthesis pathway, but not in the strain that was further engineered for improved precursor supply. Medium optimization indicated that additional supplementation of pantothenate improved the strain's productivity further and that no supplementation of amino acid precursors was necessary. Finally, the engineered strain produced 2.39 ± 0.08 g/L ERG in 160 h (productivity of 14.95 ± 0.49 mg/L/h) in a controlled fed-batch fermentation without supplementation of amino acids. This study paves the way for the low-cost fermentation-based production of ergothioneine.

Ergothioneine (ERG), a sulfur-containing histidine derivative recognized for its high stability, is of significant value across multiple sectors, including food, cosmetics, and medicine. In comparison to chemical synthesis, the establishment of microbial cell factories for ERG production represents a more efficient, environmentally friendly, and sustainable strategy. In this study, we achieved de novo synthesis of ERG in Escherichia coli by introducing genes from Trichoderma reesei. Protein engineering was subsequently employed to enable the soluble expression of the key genes Tr1 and Tr2, which resulted in a 198.1% increase in ERG production. Furthermore, strain modifications, including the knockout of competing pathways and optimization of key gene copies, were used to enhance ERG production. Following strategic combinations and medium optimization, strain E25 produced 430.9 mg/L ERG in an Erlenmeyer flask and 2331.58 mg/L via fed-batch fermentation in a 5 L bioreactor. This study not only establishes a solid foundation for the efficient and sustainable scale-up production of ERG and its derivatives but also provides valuable insights and references for its industrial production.

Ergothioneine (ERG), a unique thiol compound, is suggested to function as an antioxidant and cytoprotectant. Despite several recent attempts to produce ERG using various organisms, its yield was still very low and the costs remained high. Since the level of ERG produced depends strictly on the availability of three distinct precursor amino acids (l-cysteine (Cys), l-histidine, and l-methionine (Met)), metabolic engineering for enhancement of the flux toward ERG biosynthesis is required. Herein, we took advantage of a high-Cys production system using Escherichia coli cells, in which Cys biosynthesis and excretion were activated, and applied it to the fermentative production of ERG from glucose. The Cys overproduction in E. coli cells carrying the egtBCDE genes from Mycobacterium smegmatis was effective for ERG production. Furthermore, coexpression of the egtA gene, which encodes γ-glutamylcysteine synthetase that synthesizes the γ-glutamylcysteine used as a sulfur source of ERG biosynthesis, enhanced ERG production even though E. coli intrinsically has γ-glutamylcysteine synthetase. Additionally, disruption of the metJ gene that encodes the transcriptional repressor involved in Met metabolism was effective in further increasing the production of ERG. Finally, we succeeded in the high-level production of 1.31 g/L ERG in a fed-batch culture process using a jar fermenter.