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. 2026 May 17;123(8):2080–2091. doi: 10.1002/bit.70242

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

Xin‐Yi Chen 1, Shuai Yan 2, Hang‐Cheng Li 1, Hai‐Ping Liu 1, Hui‐Lei Yu 1, Jian‐He Xu 1, Zhi‐Jun Zhang 1,✉
PMCID: PMC13397196  PMID: 42144779

ABSTRACT

Ergothioneine (EGT) offers diverse physiological benefits and potential applications. Production of EGT by engineered bacterial strain has emerged as an eco‐friendly and cost‐effective alternative to traditional extraction and chemical synthesis methods. The fungal pathway requires only two steps and is simpler than the bacterial pathway, which typically requires three or five steps depending on the catalytic characteristics of EgtB. However, heterologous overexpression of fungal enzymes in Escherichia coli still represents a great challenge, which would limit the practical application of fungal pathway for EGT biosynthesis. In this study, a bacterial‐type Egt1 with catalytic function similar to that of the fungal Egt1 was identified by genome data mining, enabling the construction of the simplest bacterial EGT biosynthetic pathway. The pathway was introduced into E. coli resulting in an EGT titer of 426 mg/L. Cip‐based self‐assemble of enzymes requires only one scaffold protein and occurs spontaneously, which has been employed to aggregate the rate‐limiting enzymes of biosynthetic pathway to speed up the desired reaction via substrate channeling effects. The pathway enzymes were self‐assembled using the CipB scaffold protein. The optimal engineered strain E48 produced 925 mg/L of EGT in shake flask after 72 h cultivation, with a productivity of 13 mg/(L·h). Finally, the production of EGT was scaled up in a 5 L fermenter, with an EGT titer reaching 1934 mg/L and a productivity of 36.5 mg/(L·h) after 55 h of fermentation. This study provides new approach for the efficient synthesis of EGT and other value‐added bioactive molecules.

Keywords: biosynthesis, ergothioneine, fermentation, genome mining, protein self‐assemble

Genome mining identified the first bacterial‐type Egt1 enabling the construction of the simplest bacterial pathway for ergothioneine biosynthesis. Efficient synthesis of ergothioneine in E. coli was achieved through protein expression optimization, self‐assemble of pathway enzymes and fed‐batch fermentation.

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Abbreviations

ATP

adenosine triphosphate

Cys

cysteine

Cys‐HER

hercynylcysteine sulfoxide

EGT

ergothioneine

Glu

glutamate

HEPES

4‐(2‐hydroxyethyl)‐1‐piperazineethanesulfonic acid

HER

hercynine

His

histidine

HPLC

high performance liquid chromatography

IPTG

isopropyl‐β‐d‐thiogalactopyranoside

LC‐MS

liquid chromatograph mass spectrometer

Met

methionine

RBS

ribosome‐binding site

SAH

S‐adenosylhomocysteine

SAM

S‐adenosyl‐L‐methionine disulfate tosylate

TCEP

tris(2‐carboxyethyl)phosphine

γGC

γ‐glutamycysteine

γGC‐HER

hercynyl‐γ‐glutamylcysteine sulfoxide

1. Introduction

Ergothioneine (EGT), a sulfur‐containing non‐protein amino acid, exhibits diverse physiological functions such as scavenging free radicals, anti‐inflammatory properties, regulation of cell growth, anti‐aging effects, and protection against radiation (Akanmu et al. 1991; Zhu et al. 2011; Obayashi et al. 2005; Aruoma, et al. 1999). As a result, EGT has found extensive applications in the fields of food, agriculture, cosmetics, and medicine (Han et al. 2021). Currently, the preparation of EGT includes extraction, chemical synthesis, and biosynthesis. Traditional extraction and chemical synthesis have notable drawbacks, such as low product yield and environmental pollution, while the benign reaction conditions and high selectivity characteristics of biocatalysis (Zhu et al. 2019; Su et al. 2022) make the biosynthetic approach more favorable. To meet the fast‐growing market demand for EGT, the utilization of genetic engineering, protein engineering, and metabolic engineering for the development of EGT producing strain has attracted significant attention due to its cost‐effectiveness and eco‐friendly manner (Han et al. 2021).

Due to the diversity of EgtB enzyme, the biosynthesis of EGT in most aerobic microorganisms can be categorized into three distinct pathways (Figure 1A) (Stampfli et al. 2020). In the pathway involving Type Ⅰ EgtB, EGT biosynthesis is primarily mediated by five enzymes (Genghof and Van Damme 1968; Seebeck 2010). The natural substrate for Type Ⅰ EgtB is γ‐glutamylcysteine (γGC), which is produced by EgtA enzyme using Cys and glutamate as substrates. Hercynyl‐γ‐glutamylcysteine sulfoxide (γGC‐HER) formed by Type Ⅰ EgtB is further deprotonated by EgtC producing hercynylcysteine sulfoxide (Cys‐HER). In contrast, type Ⅱ EgtB can directly utilize Cys as the substrate and catalyze the synthesis of Cys‐HER from Cys and hercynine (HER) (Goncharenko et al. 2015; Kamide et al. 2020). Consequently, only three enzymes are necessary for EGT synthesis in this pathway. Both type Ⅰ and type Ⅱ EgtB are involved in the bacterial EGT biosynthetic pathways. It is noteworthy that type III EgtB (referred to as Egt1 in fungal EGT biosynthetic pathway) can catalyze two consecutive reactions: (1) methylation of His to HER and (2) conversion of HER and Cys to Cys‐HER. Therefore, in the fungal EGT biosynthetic pathway, only two enzymes (type III EgtB/Egt1 and EgtE/Egt2) are required (Bello et al. 2012).

Figure 1.

Figure 1

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(A) Overview of EGT biosynthetic pathway. (B) Comparison of the conserved domains between bacterial‐type Egt1 (top three) and fungal‐type Egt1 (bottom five).

Escherichia coli is an excellent host strain for the heterologous production of EGT due to its facile genetic manipulation, rapid growth rate, and cost‐effectiveness. In 2017, the EGT biosynthetic pathway was constructed in E. coli by Osawa et al. by introducing the egtBCDE genes from Mycolicibacterium smegmatis and overexpressing the endogenous gshA gene, giving an EGT titer of 24 mg/L after 72 h of cultivation (Osawa et al. 2018). Thereafter, Tanaka et al. further introduced the egtA gene from Mycolicibacterium smegmatis and knocked out the metJ gene that regulates Met metabolism, the titer of EGT was increased to 1.31 g/L through a fed‐batch fermentation for 216 h (Tanaka et al. 2019). A unique EgtB enzyme capable of accepting Cys as the sulfur donor was identified and employed to engineer an E. coli strain for EGT biosynthesis with a titer of 657 mg/L, highlighting the pivotal role of EgtB enzyme in the efficient synthesis of EGT (Kamide et al. 2020). By introducing the egtBCDE genes from Mycolicibacterium smegmatis and the egt1 gene from Schizosaccharomyces pombe into E. coli, respectively, and engineering the synthetic pathway of precursor amino acids, an EGT titer of 710.53 mg/L was achieved after 108 h fed‐batch fermentation (Wang et al. 2022). Co‐expression of two EGT biosynthetic genes (egt1 and egt2) from Trichoderma reesei in E. coli enabled the recombinant strain to generate 70.59 mg/L EGT at the shake flask level after 48 h of cultivation and 4.34 g/L EGT after 143 h of fed‐batch fermentation in a 2‐L jar fermenter (Chen et al. 2022). By improving the heterologous expression level of Egt1 from Neurospora crassa and protein engineering of EgtD from Mycolicibacterium smegmatis, a record EGT titer of 5.4 g/L was achieved after 94 h fed‐batch fermentation (Zhang et al. 2023). Recently, protein engineering and metabolic engineering strategies were employed to enhance EGT production in E. coli, and the optimal strain was able to produce 431 and 2332 mg/L EGT, respectively, in shake flask and 5‐L bioreactor fermentation (Kang et al. 2025).

Despite significant advancement of EGT production in engineered E. coli, challenges persist in the scarcity of pathway enzymes, for example, the pathway enzymes used are mainly from Mycolicibacterium smegmatis (Osawa et al. 2018; Tanaka et al. 2019; Wang et al. 2022; Zhang et al. 2023). Moreover, the bacterial pathway contains totally five enzymes EgtABCDE, which significantly complicates the pathway construction and engineering. In contrast, the fungal pathway requires only two enzymes, however, soluble expression of fungal enzymes in E. coli still represents a big challenge (Kang et al. 2025). Therefore, there is still in urgent need for the development of bacterial‐derived enzymes with new catalytic properties for EGT production, especially those with similar catalytic functions to those of fungal enzymes (Egt1 and Egt2).

In nature, cascade reactions are usually catalyzed by multiple enzymes assembled into complexes with the benefit of accelerating the reaction rates due to substrate channeling effects, driving unfavorable equilibrium, preventing undesirable competing pathways, and relieving metabolite inhibition (Wang et al. 2017a; Park et al. 2022). Inspired by nature, various artificial multienzyme assemble strategies have been developed and applied for the efficient synthesis of value‐added bioactive molecules (You et al. 2012; Zhao et al. 2021; Sun et al. 2022; Gao et al. 2025; Wang et al. 2025). For example, two small protein molecules (CipA and CipB) from the entomopathogenic bacterium Photorhabdus luminescens, which can form protein crystalline inclusions (PCI) with diverse shapes in luminescent Pseudomonas, were employed as protein self‐assemble tags for multi‐enzyme cascade reactions in vivo, demonstrating their ability to enhance the efficiency of natural product biosynthesis (Wang et al. 2017b; Huo et al. 2018). Therefore, leveraging protein self‐assemble strategy to enhance EGT production in Escherichia coli represents a promising endeavor.

In this study, the simplest and efficient bacterial EGT synthetic pathway was constructed in Escherichia coli. At first, a bacterial‐type Egt1 with high heterologous expression level and similar catalytic function to the fungal Egt1 was identified by genome data mining. Second, the expression level of pathway enzymes was optimized by ribosome binding site and gene copy number engineering. Subsequently, the pathway enzymes were self‐assembled using the CipB protein scaffold to boost EGT production. Finally, the EGT producing efficiency of the recombinant strain was evaluated by fed‐batch fermentation in a 5 L fermenter.

2. Materials and Methods

2.1. Reagents and Materials

l‐Cys, l‐His, and l‐Met were purchased from Shanghai Titan Scientific Co. Ltd. (Shanghai, China). HER was obtained from Shanghai Kaiwei Chemical Technology Co. Ltd. (Shanghai, China). EGT was provided by Shanghai Dibo Chemicals Technology Co. Ltd. (Shanghai, China). Restriction enzymes and kits for DNA manipulation were purchased from Takara Biomedical Technology Co. Ltd. (Beijing, China) or Nanjing Vazyme Biotech Co. Ltd. (Nanjing, China). Unless otherwise specified, all other chemicals and reagents were obtained commercially and of reagent grade. The synthesis of primers and DNA sequencing were performed by Tsingke Biotech Co. Ltd. (Shanghai, China).

2.2. Construction of Recombinant Plasmids and Strains

Recombinant plasmids were constructed by homologous recombination using Gibson Assembly kit from Nanjing Vazyme Biotech Co. Ltd. (Nanjing, China). The target genes and vectors were designed using primers with homologous arms and amplified by polymerase chain reaction (PCR). Fragments were gel‐purified and ligated at 50°C. Ligation products were transformed into E. coli BL21(DE3) and the resulting strains were used for protein expression or EGT production. All plasmids constructed in this study are listed in Supporting Information Table S1. All strains constructed are given in Supporting Information Table S2.

2.3. Expression and Purification of Protein

The recombinant plasmid was transformed into E. coli BL21(DE3) competent cells. A single colony was grown overnight in 4 mL LB medium with 50 μg/mL antibiotic at 37°C and 180 rpm. The culture was diluted 1% (v/v) into fresh LB medium with respective antibiotic (50 μg/mL) and grown until the OD600 reached 0.6–0.8. Protein expression was induced with 0.2 mM isopropyl β‐D‐1‐thiogalactopyranoside (IPTG) at 16°C for 20 h. Cells were harvested by centrifugation at 8000 rpm, 4°C for 5 min. Protein expression was confirmed by SDS‐PAGE analysis. For protein purification, cells (1 g wet cells per 10 mL buffer) were resuspended in buffer A (50 mM HEPES, 300 mM NaCl, 10 mM imidazole, pH 8.0) and lysed by ultrasonication (400 W, 1 s on, 2 s off, 15 min). The lysate was centrifuged at 12,000 rpm, 4°C for 45 min, and the supernatant was loaded onto a nickel column pre‐equilibrated with buffer A. Unbound proteins were washed with buffer A, and the target protein was eluted using a gradient of buffer B (50 mM HEPES, 300 mM NaCl, 500 mM imidazole, pH 8.0). Eluted fractions were analyzed by SDS‐PAGE, and the fractions containing target protein were pooled and concentrated using ultrafiltration (10 or 30 kDa cutoff), and dialyzed 3 times against buffer C (50 mM HEPES, 300 mM NaCl, pH 8.0) to remove imidazole.

2.4. Activity Assay of Bacterial‐Type Egt1

The methyl transferase and sulfoxide synthase activity of bacterial‐type Egt1 was assayed separately. For the methyl transferase activity assay: 500 μL reaction mixture containing 50 mM HEPES (pH 8.0), 1 mM His, 5 mM SAM, and 30 μM purified bacterial‐type Egt1 (MsEgtD enzyme as a positive control) was incubated at 25°C and 1000 rpm for 30 min. For the sulfoxide synthase activity assay: 500 μL reaction mixture containing 50 mM HEPES (pH 8.0), 1 mM HER, 1 mM Cys, 2 mM ascorbic acid, 2 mM TCEP, 1 mM FeSO4·7H2O, and 30 μM purified bacterial‐type Egt1 (MpEgtB enzyme as a positive control) was incubated at 25°C and 1000 rpm for 30 min. The amount of HER produced or consumed was determined by HPLC analysis. One unit of enzyme activity (U) was defined as the amount of enzyme required to produce or consume 1 μmol of HER per minute under the standard assay conditions.

2.5. Production of EGT by Enzyme‐Catalyzed Reactions In Vitro

The reaction mixture (500 μL) contained 50 mM HEPES (pH 8.0), 1 mM His, 5 mM SAM, 1 mM Cys, 2 mM ascorbic acid, 2 mM TCEP, 1 mM FeSO4·7H2O, and 1 mM PLP, 30 μM of EgtD (EgtD enzyme was excluded in the case EgtB is a bacterial‐type Egt1), EgtB and EgtE. Reaction was carried out at 25°C and 1000 rpm for 24 h.

2.6. Production of EGT via Microbial Fermentation

Single colony of the engineered E. coli strain producing EGT was cultured in 4 mL of LB medium (containing 50 μg/mL of corresponding antibiotic) at 37°C and 200 rpm for 12 h. The culture was inoculated into 25 mL of fermentation medium (2%, v/v) in 250 mL shake flask and incubated at 37°C and 200 rpm for 3 h. IPTG was added to induce enzyme expression with a final concentration of 0.4 mM, and the cultivation was continued at 25°C for 20 h. Two milliliters of sample were used for EGT production analysis by high‐performance liquid chromatography (HPLC) and OD600 determination by spectrophotometer. The composition of the fermentation medium was as follows (w/v): 2.4% yeast extract, 1.2% tryptone, 0.3% NaCl, 0.2% K2HPO4, 0.05% MgSO4·7H2O, 3% glycerol, 0.006% ammonium ferric citrate, 0.1% l‐Cys, 0.1% l‐His, and 0.1% l‐Met.

2.7. Fed‐Batch Fermentation

Single colony of the engineered E. coli strain producing EGT was cultured in 4 mL LB medium (containing 50 μg/mL chloramphenicol) at 37°C and 200 rpm for 12 h. Subsequently, the culture was inoculated into 80 mL fermentation medium (containing 50 μg/mL chloramphenicol) in a 500 mL shake flask with 1% (v/v) inoculum and incubated at 37°C and 200 rpm for 3 h.

The 80 mL culture was used to inoculate 2 L fermentation medium (containing 50 μg/mL chloramphenicol) in a 5 L fermenter with a 4% (v/v) inoculum and cultured at 37°C. During the fermentation, pH was maintained at 6.8 by feeding a 50% (v/v) ammonia solution, while the glycerol concentration was controlled at 0.5% (v/v) by adding a 75% (v/v) glycerol solution. Dissolved oxygen was regulated in conjunction with agitation (200–750 rpm) at around 30%. When the OD600 of the fermentation broth reached 20, the temperature of the culture was cooled to 25°C, followed by induction using IPTG, pyridoxal phosphate and ferric citrate ammonium at a final concentration of 0.1 mM, 0.02 g/L, and 0.02 g/L, respectively. After 6 h of induction, a mixture of amino acids (l‐His, l‐Met, and l‐Cys sterilized separately and mixed at a final concentration of 12, 12, and 48 g/L, respectively) was added at a rate of 20 mL/h. During cultivation, samples were periodically taken for the determination of OD600 and EGT production.

2.8. Analytic Methods

The culture broth or enzymatic reaction mixture was taken and incubated at 95°C for 10 min to denature the protein, and centrifuged at 13,000 rpm for 15 min. The supernatant was diluted with the corresponding mobile phase by 10‐fold. The resulting samples were filtered through a 0.22 μm filter membrane before HPLC or LC‐MS analysis.

For HER and EGT analysis, the HPLC analytic conditions were as follows: XBridge BEH Amide Column (150 mm × 4.6 mm, 3.5 μm); flow rate: 0.8 mL/min; temperature: 30°C; mobile phase: H2O containing 0.1% ammonia and acetonitrile (v/v, 80:20); injection volume: 10 μL; detection time: 20 min; detection wavelength: 210 nm for HER and 257 nm for EGT.

For Cys‐HER determination, the LC‐MS analytic conditions were as follows: positive ion mode with a scan range of 200–400 m/z; Supersil AQ‐C18 Column (250 mm × 4.6 mm, 5 μm); flow rate: 0.8 mL/min; temperature: 35°C; mobile phase: H2O containing 0.05% heptafluorobutyric acid (HFBA) ammonia and methanol (v/v, 93:7); injection volume: 10 μL; detection time: 20 min; detection wavelength: 250 nm.

3. Results and Discussion

3.1. Screening of Bacterial‐Type Egt1

Although the fungal EGT biosynthetic pathway represents the simplest one, functional overexpression of fungal enzymes in E. coli is still challenging and searching for prokaryotic counterparts is a promising alternative to overcome this issue. The discovery of type Ⅱ EgtB, which could directly utilize Cys as the substrate and catalyze the synthesis of Cys‐HER from Cys and hercynine (HER), successfully reduce the EGT biosynthetic pathway from five steps to three steps (Kamide et al. 2020). However, additional one more reaction step (synthesis of HER from His) catalyzed by EgtD is still required as compared with that of fungal pathway (Figure 1A). More reaction steps in a biosynthetic pathway mean more enzymes required, which would complicate the pathway, especially for balancing the different optimal reaction conditions of various enzymes and lead to higher cost. We therefore envisioned by mining bacterial‐type Egt1, which shows both methyltransferase activity (synthesis of HER from His) and sulfoxide synthase activity (synthesis of Cys‐HER from HER and Cys), as reported for fungal Egt1, it is possible to develop a bacterial two‐step EGT biosynthetic pathway resemble to that of fungi (Figure 1A). EnzymeMiner (https://loschmidt.chemi.muni.cz/enzymeminer/) was used for virtual screening of genome databases with the amino acid sequences of reported EgtB enzymes (Seebeck 2010; Kamide et al. 2020) (primarily Type Ⅰ, Type Ⅱ, and Type Ⅲ) as probes and their key amino acid residues (residues for iron and HER binding and catalytic residue Tyr) (Stampfli et al. 2019) as filter tags. To narrow down the search scope, the source of candidate proteins was limited to bacteria. Subsequently, considering that the amino acid sequence of Egt1 (Chen et al. 2022; Takusagawa et al. 2019; Yu et al. 2020) typically contains ~700 residues and generally encompasses three domains (an S‐adenosylmethionine‐dependent methyltransferase domain Methyltransf_33, a DinB_2 domain, and an FGE‐sulfatase domain), the candidate proteins with sequence length shorter than 690 residues or lacking the three domains were excluded. Furthermore, given that only Type II and III EgtB enzymes can accept Cys as substrate, the candidate proteins originating from Type I EgtB enzymes were removed. Totally, 258 potential candidates were obtained, and their sequences were aligned using ClustalW via neighbor‐joining method and an evolutionary tree was constructed by MEGA 11 (Supporting Information Figure S1). Additionally, besides solubility, the k cat values of the candidate proteins toward four target substrates (including His, SAM, HER, and Cys) were also predicted by DLKcat (Li et al. 2022). Based on the predicted results (Supporting Information Figure S1), three candidate proteins (CpEgtB, PpEgtB, and PoEgtB) sourced from Cupriavidus pauculus, Panacagrimonas perspica, and Pseudomonas otitidis, respectively, were chosen for subsequent investigation.

Sequence homology analysis of the three candidate proteins indicated that they share sequence identity ranging from 37.8% to 51.2% to the bacterial‐derived MsEgtD (Osawa et al. 2018) and MpEgtB (Kamide et al. 2020), respectively (Supporting Information Table S4). Pfam analysis of the conserved structural domains of three bacterial‐type Egt1s indicated that although they are highly similar to those of the fungal‐type Egt1s (Chen et al. 2022; Takusagawa et al. 2019; Yu et al. 2020), the domain positions differ between fungal Egt1s and the newly identified bacterial‐type Egt1s (Figure 1B). In bacterial‐type Egt1s, the DinB_2 domain locates in front of the FGE‐sulfatase superfamily domain and the Methyltransf_33 domain, while in the case of fungal Egt1s, the DinB_2 domain locates between the FGE‐sulfatase domain and the Methyltransf_33 domain. The position difference of conserved domains suggests that the bacterial‐type Egt1 cannot be hit by simple similarity search using fungal Egt1 sequences as queries. However, the conserved domains indicated that the three candidate proteins might have similar catalytic functions to the fungal Egt1s, including methyltransferase activity and sulfoxide synthase activity. Therefore, these three candidate proteins were selected for following catalytic performance characterization.

3.2. Catalytic Activity of Bacterial‐Type Egt1

In principle, the bacterial‐type Egt1 should show both methyltransferase and sulfoxide synthase activity, as reported for fungal Egt1 (Chen et al. 2022; Takusagawa et al. 2019; Yu et al. 2020; Chen et al. 2025). To validate the catalytic activity of candidate enzymes, the newly identified bacterial‐type Egt1 enzymes (CpEgtB, PpEgtB, and PoEgtB) were overexpressed in Escherichia coli, purified by nickel column affinity chromatography. As shown in Supporting Information Figure S2, the overall expression level of CpEgtB was significantly higher than those of PpEgtB and PoEgtB. Moreover, the soluble fraction and the inclusion body accounted for about 50%, respectively, for both CpEgtB and PpEgtB. For PoEgtB, the soluble fraction was only 30%, while the inclusion body was up to 70%.

The activities of candidate enzymes toward l‐His (methyltransferase activity) and HER (sulfoxide synthase activity) were investigated by performing enzymatic reactions in vitro using purified enzymes (Supporting Information Figure S4). The reactions were analyzed by LC‐MS, as expected, both products (HER and Cys‐HER) were formed in the reaction mixtures, respectively, confirming the bifunctional property of the candidate enzymes (Supporting Information Figure S4). The specific activity of the bacterial‐type Egt1 enzymes was then determined and compared with those reported in literatures (Figure 2). Although the methyltransferase activity of the newly identified bacterial‐type Egt1 enzymes was lower than those of NcEgt1 (Chen et al. 2025) and MsEgtD (Osawa et al. 2018), respectively, their sulfoxide synthase activity was significantly higher than that of NcEgt1 (Chen et al. 2025), and comparable to that of MpEgtB (Kamide et al. 2020). Furthermore, the sulfoxide synthase activity of CpEgtB toward HER was about twofold that of MpEgtB (Kamide et al. 2020). The kinetic parameters of CpEgtB toward l‐His and HER were also determined (Table 1 and Supporting Information Figure S5). The K m values of CpEgtB were 0.22 mM for l‐His and 0.47 mM for HER, indicating that CpEgtB shows higher affinity for l‐His than HER. In contrast, the k cat value of CpEgtB toward l‐His was merely 0.005 min–1, while that for HER was 5.68 min–1. Overall, the k cat/K m of CpEgtB toward HER was significantly higher than that of l‐His, which is consistent with the activity assay results.

Figure 2.

Figure 2

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Specific activity of EgtBs in different reaction steps. The yellow column represents methyltransferase activity, while the purple column represents sulfoxide synthase activity. MpEgtB is a single‐function sulfoxide synthase, MsEgtD is a single‐function methyltransferase, while the other enzymes are dual‐function proteins. One unit (U) of enzyme activity is defined as the amount of protein required to generate or consume 1 μmol of HER per minute under the standard reaction conditions. Error bars represent standard deviation of three independent experiments.

Table 1.

Steady state kinetic parameters of CpEgtB.

Substrate K m (mM) k cat (min–1) k cat/K m (min–1 mM–1)
l‐Histidine 0.22 ± 0.03 0.005 ± 0.0001 0.023
Hercynine 0.47 ± 0.07 5.68 ± 0.15 12.1
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Note: Kinetic parameters for L‐Histidine were determined in the presence of 0–16 mM His, 5 mM SAM, 60 μM purified CpEgtB, and 50 mM HEPES (pH 8.0) at 25°C and 1000 rpm for 60 min; Kinetic parameters for Hercynine were determined in the presence of 0–20 mM HER, 1 mM Cys, 2 mM ascorbic acid, 2 mM TCEP, 1 mM FeSO4·7H2O, 8 μM CpEgtB, and 50 mM HEPES (pH 8.0) at 25°C and 1000 rpm for 30 min.

The stability of the bacterial type Egt1 enzymes was also investigated by incubating the enzymes at 30°C and their residual activities were measured at different time periods. As shown in Supporting Information Figure S7, although all three enzymes were not very stable at 30°C, CpEgtB displayed obviously better stability than those of PpEgtB and PoEgtB. The half‐life of CpEgtB was 10 h, which is 7.7‐ and 5.1‐fold those of PpEgtB (1.3 h) and PoEgtB (1.96 h), respectively.

The potential of the bacterial‐type Egt1 enzymes for EGT biosynthesis was then evaluated by combining with NcEgt2 (Takusagawa et al. 2019) and MsEgtE (Seebeck 2010), respectively. In contrast to NcEgt2, MsEgtE was mainly expressed as inclusion body in E. coli (Supporting Information Figure S3). Small Ubiquitin‐like Modifier (SUMO) and Maltose Binding Protein (MBP) tags were then fused with MsEgtE aiming to improve its expression, and MBP resulted in significant better expression of MsEgtE as compared with SUMO (Supporting Information Figure S3). The EGT titers ranged from 15.47 to 18.99 mg/L (Supporting Information Figure S6). Of the candidate enzymes, CpEgtB exhibited slightly higher product titer compared to PpEgtB and PoEgtB, likely attributed to its better specific activity and stability (Figure 2 and Supporting Information Figure S7). In addition, the bacterial‐derived MsEgtE was found to be superior to the fungal‐derived NcEgt2 as the component enzyme for the final step of EGT biosynthesis. However, in all cases, the titers of EGT were relatively low, possibly due to the poor stability and low catalytic activity of component enzymes (Figure 2 and Supporting Information Figure S7). Considering the relatively poor stability of free enzymes in the case of in vitro reaction, as well as the high cost and complexity of extracting enzymes from hosting cells, it would be more competitive to construct the EGT biosynthetic pathway in Escherichia coli and use the recombinant Escherichia coli whole cells as catalyst for the synthesis of EGT.

3.3. Construction of Recombinant E. coli for EGT Biosynthesis

The pathway enzymes involved in EGT biosynthesis were cloned into three different vectors, respectively, with varying copy numbers (pACYCDuet‐1: 10, pETDuet‐1: 40, and pRSFDuet‐1: 100), and transformed into Escherichia coli. Shake flask fermentation of the engineered strains showed that their EGT production efficiency was significantly higher than those of in vitro reactions (Figure 3), indicating that the intracellular environment is beneficial for the catalytic efficiency and/or stability of the pathway enzymes. Of the recombinant strains constructed, strain E1 exhibited the highest EGT titer of 221.8 mg/L. SDS‐PAGE analysis of the cell‐free extract of strain E1 confirmed the successful heterologous expression of pathway enzymes CpEgtB and MsEgtE (Supporting Information Figure S8). It is noteworthy that low‐copy number plasmid (pACYCDuet‐1) appeared to be more advantageous for EGT production than high‐ (pRSFDuet‐1) and medium‐copy number (pETDuet‐1) plasmids, likely due to potential cellular metabolic burdens. Moreover, location of the pathway enzymes on the plasmid also had a significant effect on product titer. For instance, the product titer of strain E4 was 5.83‐fold that of strain E3. Ultimately, strain E1 was selected for further optimization due to its best performance in EGT biosynthesis.

Figure 3.

Figure 3

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Ergothioneine production titer of different recombinant E. coli strains. Error bars represent standard deviation of three independent experiments.

3.4. Improve the EGT Titer by Optimizing the Expression of Pathway Enzymes

By optimizing ribosome binding site (RBS) and gene copy number, the expression level of key enzymes in the biosynthetic pathway can be regulated to increase the metabolic flux of the pathway toward product synthesis, thereby achieving high‐efficient production of target chemicals (Zelcbuch et al. 2013; Liu et al. 2018). Starting from strain E1, the RBS of pathway enzymes were systematically optimized. Initially, the RBS of CpEgtB was replaced by RBSs with different translation efficiencies (Supporting Information Table S5), giving the best strain E20 with an EGT titer of 353.1 mg/L (Figure 4C). Subsequently, based on strain E20, the RBS of MsEgtE was replaced with different RBSs (Supporting Information Table S5), leading to strain E28 with an EGT titer of 410.6 mg/L (Figure 4D). SDS‐PAGE analysis indicated a positive correlation between the EGT titer and the expression levels of pathway enzymes (Figure 4A,B). Overall, after optimization of the RBS of pathway enzymes, the EGT titer of strain E28 was improved by 1.39‐fold compared to the starting strain E1.

Figure 4.

Figure 4

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Effects of RBS and gene copy number of pathway enzymes on the titer of EGT. (A) SDS‐PAGE analysis of the pathway enzymes in strains with CpEgtB regulated by different RBSs. S: supernatant of cell‐free extract; P: precipitate of cell‐free extract; M: protein maker. (B) SDS‐PAGE analysis of the pathway enzymes in strains with MsEgtE regulated by different RBSs. (C) EGT titers of strains with CpEgtB regulated by different RBSs. (D) EGT titers of strains with MsEgtE regulated by different RBSs. (E) Schematic diagram for gene copy number optimization. (F) Effect of gene copy number of pathway enzymes on the titer of EGT. ++: the gene copy number was doubled based on a single plasmid; ++*: the gene copy number was doubled based on two plasmids. Error bars represent standard deviation of three independent experiments.

To further improve the titer of EGT, the gene copy number of pathway enzymes in strain E28 was optimized by using single or double plasmids (Figure 4E). Compared with CpEgtB, increasing the gene copy number of MsEgtE is more significant in improving the EGT titer (Figure 4F). When the gene copy number of MsEgtE was doubled by using a single‐plasmid, the EGT titer of strain E32 reached 425.8 mg/L, which was 1.42‐fold higher than that of strain E1 (Figure 4F). Notably, increasing the gene copy number of pathway enzymes by single‐plasmid was superior to dual‐plasmid in improving EGT titer. Meanwhile, the cell growth rate (OD600) of strains carrying two plasmids was not significantly affected as compared with those carrying one plasmid, indicating that the potential metabolic burden of dual plasmid on cell growth is neglectable, and the difference in product titer might be attributed to a higher possibility of plasmid loss in recombinant E. coli strains with more plasmids.

3.5. Improve the EGT Titer by Self‐Assemble of Pathway Enzymes

Recently, self‐assemble of pathway enzymes has found widespread applications in cascade reactions and microbial cell factories for the efficient synthesis of chemicals owing to its advantages of substrate channeling effects, driving unfavorable equilibrium, preventing undesirable competing pathways, stabilizing pathway enzymes, and relieving metabolite inhibition (Wang et al. 2017a; Park et al. 2022). In this study, the effect of self‐assemble of pathway enzymes in the engineered strain on EGT production was investigated using the Cip protein scaffold (Wang et al. 2017b; Huo et al. 2018) (Figure 5A).

Figure 5.

Figure 5

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Effect of self‐assemble of pathway enzymes on the titer of EGT. (A) Schematic diagram of self‐assemble process of Cip scaffold protein with enzyme in E. coli. (B) Different self‐assemble strategies used for the pathway enzymes. (C) Fluorescence microscope analysis of the engineered strains with their pathway enzymes self‐assembled. (D) Time courses of EGT production in different strains. Error bars represent standard deviation of three independent experiments.

First, to investigate whether the location of Cip scaffold would impact enzyme activity, scaffold proteins CipA and CipB were fused to the N‐ and C‐termini of CpEgtB and MsEgtE in strain E28, respectively, and the EGT titers of the engineered strains were determined. Higher EGT titer was observed when the scaffold protein was located at the C‐terminus of CpEgtB or the N‐terminus of MsEgtE, for instance, an EGT titer of 424.9 mg/L was produced by strain E37 (Supporting Information Figure S9A). Similarly, significant enhancements of EGT production were also observed in strains E36, E40, and E41. These findings highlight the importance of self‐assemble of pathway enzymes for efficient EGT production.

Based on the above results, different self‐assemble strategies were employed to the pathway enzymes in strain E32 using the Cip protein scaffold (Figure 5B). In addition to the fusion of CipA or CipB with the same pathway enzyme, they were also fused with different pathway enzymes from the same strain to achieve co‐assemble of different enzymes. Moreover, CipA and CipB were also fused with CpEgtB and MsEgtE, respectively, to accomplish self‐assemble of the two enzymes separately in the same strain. The EGT production efficiency of these engineered strains was then evaluated (Supporting Information Figure S9B). Notably, the highest EGT titer of 460.5 mg/L was observed with strain E48 using CipB as a self‐assemble protein scaffold, which is 1.5‐fold enhancement compared to strain E1. Furthermore, self‐assemble of pathway enzymes using CipA and CipB independently also contributed to enhanced EGT production, as evidenced by strains E49 and E50 with product titers of 410.3 and 414.3 mg/L, respectively.

To confirm the self‐assemble of pathway enzymes within the cellular environment, strains E51 and E52 were constructed by fusing green fluorescent protein (GFP) to CpEgtB and red fluorescent protein (RFP) to MsEgtE, based on strain E48 and strain E32 without the CipB scaffold, respectively. Subsequently, the self‐assembled pathway enzymes in strains E51 and E52 were visualized by fluorescence microscopy (Figure 5C). The elongated rod‐shaped green and red fluorescence patterns observed in strain E51 indicated that the pathway enzymes were freely distributed in the cells. In contrast, punctate clustering of green and red fluorescence observed in strain E52 suggested that the two pathway enzymes (CpEgtB and MsEgtE) were self‐assembled in the cells. It should be noted that the resulting self‐assemble protein complex mediated by CipB scaffold is stably and homogeneously formed across the E. coli population, similar results were also observed in previous studies (Zhao et al. 2021; Wang et al. 2017b). We propose that the better performance of the engineered strain with its pathway enzymes self‐assembled in EGT production can be attributed to enzyme aggregation effects. Enzyme aggregation not only facilitate formation of high‐concentration catalytic centers in localized regions, thereby increasing collision frequency with substrate molecules and improving catalytic efficiency but also enhance mass transfer efficiency of intermediate products between different cascade enzymes. Furthermore, enzyme aggregation may serve as a means of immobilization, affording protection against adverse microenvironment such as temperature fluctuations, pH changes, or proteolytic degradation.

The product accumulation of strains E28 and E48 during the cultivation process was monitored. After 72 h of cultivation, strains E28 and E48 produced EGT with titers of 600 and 925 mg/L, respectively (Figure 5D). The EGT titer of strain E48 was 1.54‐fold that of strain E28, with a specific productivity reaching 13 mg/(L·h), demonstrating significant potential of strain E48 for practical EGT production in the future.

3.6. Production of EGT by 5 L Bioreactor Fermentation

Prior to 5 L fermentation, the culture conditions of the engineered EGT‐producing strain E48 were systematically optimized in shake flask, including medium loading, precursor supply, cell membrane permeability, and induction conditions (Supporting Information Figures S10–S13). Based on the optimized culture conditions (10% v/v medium loading, 1 g/L His, 1 g/L Cys, 4 g/L Met, 0.2 g/L CaCl2, inoculum size 4% v/v, temperature 25°C, and IPTG concentration 0.1 mM), a 5 L bioreactor fermentation experiment was carried out to further assess the practical production potential of strain E48. According to the fermentation results, in the initial phase of fermentation, strain E48 exhibited rapid growth, reaching an OD600 value of 70 in 15 h (Figure 6). At the same time, there was a swift accumulation of EGT, displaying a positive correlation with the growth of strain E48. However, from 15 to 55 h of fermentation, strain growth was slightly inhibited as the OD600 decreased from 70 to 65, possibly due to the inhibitory effect of high concentration of substrates (12 g/L His, 12 g/L Met, and 48 g/L Cys) in the feeding solution. Accordingly, the accumulation of EGT also slowed down, therefore, we did not further extend the fermentation time. The peak titer of EGT was achieved at 55 h of fermentation, giving 1934 mg/L with a specific productivity of 36.5 mg/(L·h), demonstrating the great potential of the engineered strain E48 for practical production of EGT in the future.

Figure 6.

Figure 6

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Fermentation process of the engineered strain E48 for EGT production in 5 L fermenter.

4. Conclusions

In summary, a bacterial‐type Egt1 enzyme with similar catalytic function to the fungal Egt1 was identified by genome data mining, enabling the establishment of the simplest bacterial synthetic pathway of EGT. Production of EGT by the newly constructed pathway was confirmed by in vitro and in vivo reactions, respectively. Considering the product titer, stability of pathway enzymes and production cost, biocatalyst in the form of whole cell was selected for further optimization. The EGT titer of the engineered strain was significantly improved by RBS, gene copy number optimization and self‐assemble of pathway enzymes. Up to 1013 mg/L EGT was produced by the best strain E48 in shake flask after 72 h cultivation under the optimal conditions, with a productivity of 14 mg/(L∙h). Finally, fermentation of the engineered strain E48 in a 5 L bioreactor resulted in an EGT titer of 1934 mg/L and a productivity of 36.5 mg/(L·h), respectively. Future research will focus on improving the catalytic efficiency of the pathway enzymes by protein engineering strategies to develop more efficient microbial cell factories for EGT production.

Author Contributions

Xin‐Yi Chen: conceptualization, methodology, investigation, writing – original draft. Shuai Yan: validation, writing – review and editing. Hang‐Cheng Li: investigation. Hai‐Ping Liu: investigation. Hui‐Lei Yu: supervision. Jian‐He Xu: supervision. Zhi‐Jun Zhang: supervision, writing – review and editing, funding acquisition.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Figure S1: Phylogenetic tree and heat map analysis of bacterial‐type Egt1. Figure S2: SDS‐PAGE analysis of candidate bacterial‐type Egt1s. Figure S3: SDS‐PAGE analysis of other pathway enzymes for EGT biosynthesis. Figure S4: LC‐MS analysis of products formed by candidate bacterial‐type Egt1 enzymes. Figure S5: Steady‐state kinetic parameters of CpEgtB towards L‐Histidine (A) and Hercynine (B). Figure S6: EGT titers of different pathway enzyme combinations in vitro. Figure S7: Comparison of the residual activities of candidate bacterial‐type Egt1 enzymes at 30 ℃. Figure S8: SDS‐PAGE analysis of protein expression levels of pathway enzymes in strain E1. Figure S9: Effect of self‐assemble strategies of pathway enzymes on the titer of EGT. Figure S10: Effect of media volume in 250 mL shake flask on the titer of EGT. Figure S11: Effect of precursor supply on the titer of EGT. Figure S12: Effect of CaCl2 concentration on the titer of EGT. Figure S13: Effect of cultivation conditions on the titer of EGT. Table S1: Plasmids used in this study. Table S2: Strains used in this study. Table S3: Primers used in this study. Table S4: Detail information of EgtBs identified in this study. Table S5: Sequences of different RBSs used in this study.

BIT-123-2080-s001.docx (2.2MB, docx)

Acknowledgments

This work was financially supported by the National Key Research and Development Program of China (2024YFA0917800) and the National Natural Science Foundation of China (22578119).

Data Availability Statement

The data that support the findings of this study are available in the Supporting Material of this article.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1: Phylogenetic tree and heat map analysis of bacterial‐type Egt1. Figure S2: SDS‐PAGE analysis of candidate bacterial‐type Egt1s. Figure S3: SDS‐PAGE analysis of other pathway enzymes for EGT biosynthesis. Figure S4: LC‐MS analysis of products formed by candidate bacterial‐type Egt1 enzymes. Figure S5: Steady‐state kinetic parameters of CpEgtB towards L‐Histidine (A) and Hercynine (B). Figure S6: EGT titers of different pathway enzyme combinations in vitro. Figure S7: Comparison of the residual activities of candidate bacterial‐type Egt1 enzymes at 30 ℃. Figure S8: SDS‐PAGE analysis of protein expression levels of pathway enzymes in strain E1. Figure S9: Effect of self‐assemble strategies of pathway enzymes on the titer of EGT. Figure S10: Effect of media volume in 250 mL shake flask on the titer of EGT. Figure S11: Effect of precursor supply on the titer of EGT. Figure S12: Effect of CaCl2 concentration on the titer of EGT. Figure S13: Effect of cultivation conditions on the titer of EGT. Table S1: Plasmids used in this study. Table S2: Strains used in this study. Table S3: Primers used in this study. Table S4: Detail information of EgtBs identified in this study. Table S5: Sequences of different RBSs used in this study.

BIT-123-2080-s001.docx (2.2MB, docx)

Data Availability Statement

The data that support the findings of this study are available in the Supporting Material of this article.

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