Heterologous and High Production of Ergothioneine in Escherichia coli
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†Graduate School of Chemical Science and Engineering and ‡Graduate School of Engineering, Hokkaido University, Sapporo, Hokkaido 060-8628, Japan
§ Innovation Medical Research Institute, University of Tsukuba, Tsukuba, Ibaraki 305-8550, Japan
*Telephone: +81-11-706-7815. Fax. +81-11-706-7118. E-mail: [email protected]
*Telephone: +81-11-706-7815. Fax. +81-11-706-7118. E-mail: [email protected]
Copyright © 2017 American Chemical Society
Abstract
Ergothioneine (ERG) is a histidine-derived thiol compound suggested to function as an antioxidant and cytoprotectant in humans. Therefore, experimental trials have been conducted applying ERG from mushrooms in dietary supplements and as a cosmetic additive. However, this method of producing ERG is expensive; therefore, alternative methods for ERG supply are required. Five Mycobacterium smegmatis genes, egtABCDE, have been confirmed to be responsible for ERG biosynthesis. This enabled us to develop practical fermentative ERG production by microorganisms. In this study, we carried out heterologous and high-level production of ERG in Escherichia coli using the egt genes from M. smegmatis. By high production of each of the Egt enzymes and elimination of bottlenecks in the substrate supply, we succeeded in constructing a production system that yielded 24 mg/L (104 μM) secreted ERG.
Introduction
ARTICLE SECTIONS
Ergothioneine (ERG), a histidine-derived thiol compound, was isolated from an ergot fungus, Claviceps purpurea, more than a century ago. ERG is also known to be synthesized in actinobacteria, cyanobacteria, and a fission yeast.(1-3) Recent studies show that ERG functions as an antioxidant, such as glutathione, mycothiol, and bacillithiol. There is no direct evidence for biosynthesis of ERG in humans. However, ERG has been reported to be accumulated in various cells and tissues at high concentrations, probably by intake from diets, such as mushrooms and red beans, that contain relatively large amounts of ERG through an ERG-specific organic cation transporter, OCTN1.(4, 5)
The presence of the ERG-specific transporter and the extensive accumulation of ERG in tissues suggest that ERG should have significant biological functions in humans. Although the true physiological role of ERG in humans has yet to be fully understood, ERG has been shown by in vitro experiments to function as an antioxidant and a cytoprotectant. Therefore, applications of ERG in dietary supplements and as a cosmetic additive have been explored, and there is an increasing demand for ERG.(6) Mushrooms have traditionally been the source of ERG.(7) However, slow growth, low content, and time-consuming purification procedures lead to a high manufacturing cost. Therefore, alternative and sustainable sources of ERG are necessary.
One such reliable and practical method is a fermentative process using microorganisms, such as actinobacteria and cyanobacteria, that are known to produce ERG. However, their ERG productivities are very low (1.18 mg/g of dry weight after 4 weeks of cultivation of Mycobacterium avium and 0.8 mg/g of dry weight of Oscillatoria sp.),(2, 8) and thus, genetic and metabolic engineering are indispensable for industrial production. Until recently, however, there were no reports on ERG biosynthesis genes and enzymes. In 2010, five genes in Mycobacterium smegmatis, egtABCDE, were confirmed to be responsible for ERG biosynthesis (Figure 1).(9) In the biosynthetic pathway, EgtD catalyzes the formation of hercynine (HER) by transfer of three methyl groups derived from S-adenosylmethionine (SAM) to l-histidine (l-His). Then, EgtB catalyzes O2-dependent C–S bond formation between γ-glutamylcysteine (γGC) supplied by EgtA and HER to form hercynyl-γ-glutamylcysteine sulfoxide (γGC-HER). This is followed by removal of the l-glutamate (l-Glu) moiety by EgtC, an amidohydrolase, to produce hercynylcysteine sulfoxide (Cys-HER). Then, EgtE, a PLP-dependent C–S lyase, catalyzes the formation of ERG with concomitant formation of pyruvate and ammonia as side products.
Figure 1
Figure 1. ERG biosynthetic pathway.
In this study, we developed heterologous and high-level production of ERG in Escherichia coli using the egt genes from M. smegmatis. By high production of each of the Egt enzymes and elimination of bottlenecks in substrate supply, the production system yielded 24 mg/L (104 μM) secreted ERG.
Materials and Methods
ARTICLE SECTIONS
General Procedures
Lysogeny broth (LB, Lennox) medium was purchased from Sigma-Aldrich Japan K.K. (Tokyo, Japan); l-methionine (l-Met) and l-His were obtained from Wako Pure Chemical Industry (Osaka, Japan); and HER was purchased from Shinsei Chemical Company, Ltd. (Osaka, Japan). Other chemicals were of analytical grade and purchased from Wako Pure Chemical Industry or Sigma-Aldrich Japan. Primers were obtained from FASMAC Co., Ltd. (Kanagawa, Japan). Enzymes and kits for DNA manipulation were purchased from Takara Bio, Inc. (Shiga, Japan) or New England BioLabs Japan, Inc. (Tokyo, Japan). Polymerase chain reaction (PCR) was carried out using a GeneAmp PCR System 9700 thermal cycler (Thermo Fisher Scientific, Inc., Waltham, MA, U.S.A.) with Tks Gflex DNA polymerase (Takara Bio). General genetic manipulations of E. coli were performed according to standard protocols. High-resolution electrospray ionization Fourier transform mass spectrometry (HR-ESI–FT–MS) analysis was performed using an Exactive system (Thermo Fisher Scientific, Inc.).
Bacterial Strains and Cultures
Microorganisms used in this study are summarized in Table 1. E. coli XL1-Blue (Nippon Gene Co., Ltd., Tokyo, Japan), BL21(DE3) (Merck KGaA, Darmstadt, Germany), and BW25113 (National Institute of Genetics, Shizuoka, Japan), which is a high producer of l-cysteine (l-Cys) used for a sulfur donor of ERG,(10) were used for plasmid construction, protein production, and ERG production, respectively. The media used were LB and M9Y minimal medium prepared by adding 1% (w/v) glucose, 5 mM MgSO4, 0.1 mM CaCl2, and 0.1% (w/v) yeast extract (Becton, Dickinson and Company, Franklin Lakes, NJ, U.S.A.) to M9 minimal salts (Becton, Dickinson and Company). Ampicillin (Ap), chloramphenicol (Cm), kanamycin (Km), streptomycin (Sm), and tetracycline (Tc) were added to the media at concentrations of 100, 33, 25, 20, and 5 mg/L, if necessary. Optical density (OD) at 600 nm was measured with a NanoDrop 2000C spectrophotometer (Thermo Fisher Scientific, Inc.).
Table 1. Bacterial Strains Used in This Study
| strain | description | source |
|---|---|---|
| M. smegmatis JCM6386 | ERG producer | JCMa |
| E. coli | ||
| XL1-Blue | hsdR17, recA1, endA1, gyrA96, thi-1, supE44, relA1, lac[F′, proAB, lacIqZΔM15, Tn10(TcR)] | Nippon Gene |
| BL21(DE3) | F–, ompThsdSB(rB– mB–) galdcm (DE3) | Merck |
| BW25113 | rrnB3 ΔlacZ4787hsdR514 Δ(araBAD)567 Δ(rhaBAD)568 rph-1 | NIGb |
| ET1 | BW25113 harboring pCF1s-MsD | this study |
| ET2 | BW25113 harboring pCF1s-MsD, pQE1a-mMsB | this study |
| ET3 | BW25113 harboring pCF1s-MsD, pQE1a-mMsB, pAC1c-hMsC/hMsE | this study |
| ET4 | BW25113 harboring pCF1s-MsD, pQE1a-mMsB/EcA, pAC1c-hMsC/hMsE | this study |
a
Japan Collection of Microorganisms, Riken BioResource Center.
b
National Institute of Genetics.
Preparation of Egt Recombinant Enzymes
Detailed plasmid construction methods are described in Supplementary Methods 1 of the Supporting Information, and the plasmids are summarized in Table 2. Briefly, EgtB, EgtC, EgtD, and EgtE were amplified by PCR using M. smegmatis genomic DNA as the template and appropriate primers (Table S1 of the Supporting Information), in which restriction sites were introduced at the N and C termini. The PCR products were respectively cloned into the expression vectors. The plasmids obtained were introduced into E. coli BL21(DE3). A liquid culture of the transformant in LB supplied with appropriate antibiotics was induced by adding 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) when the optical density at 600 nm reached about 0.6. The cultivation was continued for an additional 16 h at 20 °C. The production of each recombinant enzyme was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) with Coomassie Brilliant Blue staining. The purification of each recombinant protein was carried out using nickel–nitrilotriacetic acid (Ni–NTA) agarose (QIAGEN K.K., Tokyo, Japan) or amylose resins (New England BioLabs) according to the protocols of the manufacturer. Protein concentrations were determined using a Bradford protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA, U.S.A.) with bovine serum albumin as the standard.
Table 2. Plasmids Used in This Study
| plasmid | description | source |
|---|---|---|
| pQE1a-Red | protein production plasmid, tac promoter, ColE1 ori, ApR | lab stock |
| pCF1s-Red | protein production plasmid, tac promoter, CDF ori, SmR | lab stock |
| pET-21a | protein production plasmid, T7 promoter, pBR322 ori, ApR | Merck |
| pACYCDuet-1 | protein production plasmid, T7 promoter, p15A ori, CmR | Merck |
| pQE1a-MsB | pQE1a-Red derivative, production of EgtB | this study |
| pQE1a-mMsB | pQE1a-MsB derivative, production of MBP-fused EgtB | this study |
| pQE1a-mMsB/EcA | pQE1a-mMsB derivative, co-production of MBP-fused EgtB and GshA | this study |
| pQE1a-MsC | pQE1a-Red derivative, production of EgtC | this study |
| pQE1a-hMsC | pQE1a-MsC derivative, production of His-tagged EgtC | this study |
| pQE1a-MsE | pQE1a-Red derivative, production of EgtE | this study |
| pQE1a-hMsE | pQE1a-Red derivative, production of His-tagged EgtE | this study |
| pAC1c-hMsC/hMsE | pACYCDuet-1 derivative, co-production of His-tagged EgtC and EgtE | this study |
| pCF1s-MsD | pCF1s-Red derivative, production of EgtD | this study |
ERG Production
E. coli harboring plasmids carrying ERG biosynthetic genes (Figure 2) was cultured in 3 mL of M9Y media at 30 °C for 16 h. The cultures (1 mL) were inoculated into 50 mL of M9Y media supplemented with 0.5 g/L l-His, 0.5 g/L l-Met, and 20 mg/L FeSO4·7H2O in 200 mL Erlenmeyer flasks and incubated at 30 °C with shaking (200 rpm) for up to 72 h. Na2S2O3 was added to media at 20 mM, if needed. IPTG was added to a final concentration of 0.5 mM after 3 h of cultivation. Samples (1 mL) were collected at appropriate time points and analyzed by liquid chromatography–electrospray ionization–mass spectrometry (LC–ESI–MS).
Figure 2
Figure 2. Plasmids for ERG production.
LC–ESI–MS Analysis of Products
Mixtures of 0.05% (v/v) heptafluorobutyric acid (HFBA) solution (180 μL) and culture broth or enzymatic reaction solutions (20 μL) were analyzed. The LC–ESI–MS conditions were as follows: Waters ACQUITY UPLC system equipped with a photodiode array and a SQ Detector2 (Tokyo, Japan); XBridge BEH C18 XP column (150 mm length × 2.0 mm internal diameter, 2.5 μm, Waters); flow rate, 0.15 mL/min; temperature, 35 °C; mobile phase, water containing 0.05% HFBA and 7% methanol; injection volume, 2 μL; and detection, 210 nm for His and HER, 250 nm for Cys-HER and γGC-HER, and 258 nm for ERG.
Results and Discussion
ARTICLE SECTIONS
The most reliable strategy for high production of ERG is overproduction of each of the enzymes for ERG biosynthesis and then optimization of any inefficient biosynthetic step(s) (identified by intermediate accumulation) by molecular genetics and metabolic engineering. If no intermediates accumulate, supply of the initial substrates, l-His, l-Glu, l-Cys, and l-Met, should be enhanced.
ERG biosynthetic genes are present in some microorganisms, such as actinobacteria, cyanobacteria, and α-proteobacteria.(11) Among these bacteria, we selected M. smegmatis as the gene source, because the ERG biosynthetic pathway was first identified in this strain and the biosynthesis genes constitute an operon.(9) Because egtB, egtC, and egtE are translationally coupled, we first tried expressing the egt genes as the operon with tac and T7 promoters. We constructed several plasmids using pQE1a-Red and pET-21a, but ERG productivities of transformants harboring the plasmids were low (less than 0.2 mg/L). Therefore, we overexpressed egt genes stepwise under the control of individual promoters by checking production of recombinant enzymes and their activities by production of intermediate compounds (HER, γGC-HER, and Cys-HER) and ERG by in vitro experiment (Figure 1). Because E. coli has an egtA orthologue, gshA, which is responsible for glutathione biosynthesis,(12) we expressed the other genes, egtB, egtC, egtD, and egtE. We also prepared the intermediate compounds, which are not commercially available, with the recombinant enzymes to obtain standards for quantitative analysis (Supplementary Methods 2 of the Supporting Information).
Overproduction of Recombinant Enzymes
For overproduction of Egt enzymes, we used pQE1a-Red and pCF1s-Red, both of which are home-constructed and compatible vectors with the tac promoter for protein production (Figure S1 of the Supporting Information). As Figure S2 of the Supporting Information shows, transformants harboring pCF1s-MsD carrying egtD overproduced EgtD in soluble form. To confirm whether the recombinant protein had the expected activity, we carried out in vitro experiments with cell-free extracts containing the recombinant enzyme.(9, 13) The cell-free extracts were incubated with l-His and excess SAM. After the reaction, the product was analyzed by LC–ESI–MS. A specific peak with the same retention time and mass spectrometry (MS) spectrum as the HER standard was clearly detected after 1 h of incubation, demonstrating that EgtD converted l-His into HER (Figure S3 of the Supporting Information).
We next overproduced recombinant EgtB using pQE1a-Red (Figure S1 of the Supporting Information) and changed the probable start codon TTG into ATG. However, recombinant EgtB formed inclusion bodies in multiple culture conditions. Therefore, a plasmid from which a recombinant enzyme is produced as a maltose binding protein (MBP)-fused enzyme was examined. We successfully produced a MBP-fused EgtB in a soluble form in transformants harboring pQE1a-mMsB. To confirm whether the recombinant enzyme had the expected activity, we carried out in vitro experiments with purified recombinant enzyme (Figure S4 of the Supporting Information).(9) The recombinant MBP-fused EgtB was incubated with HER and γGC for 2 h. As Figure S5 of the Supporting Information shows, HER completely disappeared and the formation of a new product with m/z 462.20 was detected. On the basis of HR-ESI–FT–MS analysis of the purified compound, the molecular formula of the product was determined to be C17H27O8N5S (m/z: [M + H]+ calculated for C17H28O8N5S+, 462.165 31; observed, 462.165 07), which corresponded to that of γGC-HER, suggesting the formation of γGC-HER. The thus formed intermediate compound γGC-HER was purified by high-performance liquid chromatography (HPLC) (Supplementary Methods 2 of the Supporting Information) and used as a standard for quantitative analysis.
Finally, overproduction of recombinant EgtC and EgtE was examined. We first used the same vector as for EgtB production, but no production of either enzyme was observed. We then examined a plasmid from which a recombinant enzyme is produced as a His-tagged enzyme. In this case, both EgtC and EgtE were successfully overproduced. To confirm whether the recombinant enzymes had the expected activity, we carried out in vitro experiments with the purified recombinant enzymes (Figure S4 of the Supporting Information).(9) Recombinant EgtC was incubated with enzymatically prepared γGC-HER. After 17 h of reaction, the product was analyzed by LC–ESI–MS. As shown in Figure 3, a new peak with m/z 333.23 was detected and Cys-HER formation was suggested. On the basis of HR-ESI–FT–MS analysis of the purified compound, the molecular formula of the product was determined to be C12H20O5N4S (m/z: [M + H]+ calculated for C12H21O5N4S+, 333.122 72; observed, 333.122 90), which corresponded to that of Cys-HER. The thus formed Cys-HER was purified (Supplementary Methods 2 of the Supporting Information) and used as a standard for quantitative analysis.
Figure 3
Figure 3. LC–ESI–MS analysis of EgtC reaction products. (a) Traces at 250 nm of reaction products. The reaction (40 μL) was carried out by adding (i) purified recombinant EgtC (3.9 μM) or (ii) boiled EgtC to the EgtB reaction solution at 25 °C for 17 h. (b) MS spectrum of the EgtC reaction product (ESI positive mode).
By adding recombinant EgtE together with EgtC into the EgtB reaction mixture, we confirmed the formation of ERG by LC–ESI–MS analysis (Figure 4), showing that all of the recombinant Egt enzymes possessed the expected activities.
Figure 4
Figure 4. LC–ESI–MS analysis of EgtE reaction products. (a) Traces at 250 nm of (i) ERG standard and (ii) reaction products. The reaction (40 μL) was carried out by adding purified recombinant EgtE (2.5 μM) and EgtC (3.9 μM) (ii) to the boiled supernatant of the EgtB reaction mixture containing 2 mM dithiothreitol at 25 °C for 17 h. (b) MS spectra of (i) ERG standard and (ii) EgtE reaction product (ESI positive mode).
Simultaneous Production of Egt Enzymes for ERG Production
To reconstruct the ERG-producing pathway in E. coli, we optimized the production conditions in a stepwise manner: HER, then γGC-HER, and finally ERG production (Figures 1 and 2).
The first step was HER production. E. coli BW25113 harboring pCF1s-MsD (named strain ET1) was cultured in M9Y medium, and 91 ± 2 mg/L HER (460 ± 10 μM) was produced in the culture broth after 48 h. We then carried out feeding experiments with l-His and l-Met, because amino acid biosynthesis in E. coli is strictly regulated by feedback inhibition and/or transcriptional repression. l-Met and l-His feeding increased the yield: 164 ± 3 mg/L HER (833 ± 15 μM) was produced after 48 h of cultivation (Figure 5). After this, l-His and l-Met feeding was employed in all in vivo production experiments.
Figure 5
Figure 5. Culture profiles of strain ET1. E. coli BW25113 harboring pCF1s-MsD (strain ET1) was cultured in M9Y medium supplemented with l-His and l-Met. After 3 h of cultivation, 0.5 mM IPTG was added to the medium. Data are presented as mean values with standard errors from three independent experiments.
We next carried out in vivo co-production of EgtD and EgtB for γGC-HER production. Transformants of E. coli BW25113 carrying both pCF1s-MsD and pQE1a-mMsB (strain ET2) were cultivated in the medium supplemented with l-His and l-Met. As Figure 6 shows, we confirmed 24 ± 1 mg/L γGC-HER (52 ± 2 μM) production after 24 h of cultivation, indicating that EgtB converted HER to γGC-HER using endogenous γGC in E. coli. However, accumulation of 3-fold higher amounts of HER (110 ± 5 mg/L) than Cys-HER (36 ± 2 mg/L) was detected, suggesting that γGC-HER production was rate-limiting.
Figure 6
Figure 6. Culture profiles of strain ET2. E. coli BW25113 harboring pCF1s-MsD and pQE1a-mMsB (strain ET2) was cultured in M9Y medium supplemented with l-His and l-Met. After 3 h of cultivation, 0.5 mM IPTG was added to the medium. Data are presented as mean values with standard errors from three independent experiments.
For simultaneous expression of all egt genes, egtC and egtE, both of which were expressed from the tac promoter of pQE1a, were recloned into plasmid pACYCDuet-1, which is compatible with pQE1a-Red and pCF1s-Red, to construct pAC1c-hMsC/hMsE (Figure 2 and Supplementary Methods 2 of the Supporting Information). The plasmid was successfully constructed, and production of both enzymes in soluble forms in E. coli was confirmed by SDS–PAGE (Figure S6 of the Supporting Information). The plasmid was introduced into strain ET2 to construct strain ET3, and recombinant enzyme production was examined. As Figure S7 of the Supporting Information shows, all of the enzymes were produced in soluble forms. We then examined ERG production. Strain ET3 produced 19 ± 2 mg/L (83 ± 8 μM) ERG together with 73 ± 15 mg/L (370 ± 76 μM) HER in the culture broth after 72 h of cultivation (Figure 7 and Table 3). These results suggested that the EgtB-catalyzed reaction is a bottleneck.
Figure 7
Figure 7. Culture profiles of strain ET3. E. coli BW25113 harboring pCF1s-MsD, pQE1a-mMsB, and pAC1c-hMsC/hMsE (strain ET3) was cultured in M9Y medium supplemented with l-His and l-Met. After 3 h of cultivation, 0.5 mM IPTG was added to the medium. Data are presented as mean values with standard errors from three independent experiments.
Table 3. Culture Profiles of ET3 and ET4 Strainsa
| strain | OD | HER (mg/L) | γGC-HER (mg/L) | Cys-HER (mg/L) | ERG (mg/L) |
|---|---|---|---|---|---|
| ET3b | 10.1 ± 0.5 | 73 ± 15 | NDc | 10 ± 2 | 19 ± 2 |
| ET4b | 8.7 ± 0.2 | 121 ± 12 | 1 ± 1 | 9 ± 1 | 17 ± 1 |
| ET3d | 11.1 ± 0.5 | 48 ± 17 | ND | 9 ± 0 | 24 ± 4 |
a
Data after 72 h of cultivation are presented as mean values with standard error from three independent experiments.
b
ET3 and ET4 were cultured in M9Y media supplemented with l-His and l-Met.
c
ND = not detected.
d
ET3 was cultured in M9Y media supplemented with l-His, l-Met, and thiosulfate.
ERG Production and Improvement of Rate-Limiting Steps
Considering that EgtB was overproduced in a soluble form in the producing strain ET3 and that it showed enough activity in in vitro experiments, we considered that insufficient supply of γGC, the substrate of EgtB, might cause the accumulation of HER. To test this hypothesis, overproduction of γGC synthetase was carried out. To produce γGC synthetase, the gshA gene from E. coli(12) was cloned and inserted into pQE1a-mMsB to construct pQE1a-mMsB/EcA. Although MBP-fused EgtB and GshA were produced in soluble forms in E. coli BW25113 harboring the three plasmids (strain ET4) (Figure S8 of the Supporting Information), ERG productivity was decreased in comparison to that of strain ET3 to 17 ± 1 mg/L (72 ± 2 μM) after 72 h of cultivation (Table 3). In comparison of SDS–PAGE data for strains ET3 and ET4 (Figures S7 and S8 of the Supporting Information), soluble MBP-fused EgtB was found to be decreased in strain ET4. This could be the reason for the reduced productivity.
We then employed another strategy to enhance the γGC supply. In ERG biosynthesis, γGC is used as a sulfur donor. In particular, l-Cys is a net sulfur donor, because the l-Glu moiety of γGC is released by EgtC. Therefore, reinforcement of l-Cys flux may be effective in enhancing γGC flux and ERG production. Because l-Cys addition into media was reported to be toxic to E. coli cells,(14) we employed another strategy. We have been studying a l-Cys biosynthetic pathway in E. coli and demonstrated that thiosulfate (S2O32–) was a better sulfur source than sulfate (SO42–) for high l-Cys production.(10) We therefore fed thiosulfate into the growth media. When strain ET3 was cultured in M9Y supplemented with l-Met, l-His, and thiosulfate, ERG productivity was increased to 24 ± 4 mg/L (104 ± 17 μM) (Table 3), indicating that reinforcement of l-Cys flux was very effective in enhancing the ERG production.
In conclusion, to establish a reliable and practical method for ERG supply, fermentative ERG production by microorganisms was investigated. We heterologously overexpressed genes egtBCDE of M. smegmatis in E. coli and succeeded in the production of ERG (19 mg/L). Reinforcement of the γGC supply by feeding of thiosulfate, a suitable sulfur donor for high l-Cys production, resulted in higher ERG production (24 mg/L). Considering that the reported ERG contents of mushrooms were from 0.15 to 7.27 mg/g of dry weight,(7) our system might become an alternative method for ERG supply. However, significant amounts of HER still accumulated, suggesting that more supply of γGC and l-Cys by metabolic engineering and use of another EgtB with higher activity are indispensable for high-level production of ERG.
ARTICLE SECTIONS
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b04924.
Supplementary Methods 1 and 2, vectors pQE1a-Red and pCF1s-Red (Figure S1), SDS–PAGE analysis of EgtD production (Figure S2), LC–ESI–MS analysis of EgtD reaction products (Figure S3), SDS–PAGE analysis of purified recombinant EgtB, EgtC, and EgtE (Figure S4), LC–ESI–MS analysis of EgtB reaction products (Figure S5), SDS–PAGE analysis of recombinant EgtC and EgtE production (Figure S6), SDS–PAGE analysis of production of recombinant Egt enzymes in strain ET3 (Figure S7), SDS–PAGE analysis of production of recombinant Egt enzymes and GshA in strain ET4 (Figure S8), and primers used in this study (Table S1) (PDF)
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Author Information
ARTICLE SECTIONS
- Yasuharu Satoh - †Graduate School of Chemical Science and Engineering and ‡Graduate School of Engineering, Hokkaido University, Sapporo, Hokkaido 060-8628, Japan; Innovation Medical Research Institute, University of Tsukuba, Tsukuba, Ibaraki 305-8550, Japan;
http://orcid.org/0000-0001-6671-7758;
- Tohru Dairi - †Graduate School of Chemical Science and Engineering and ‡Graduate School of Engineering, Hokkaido University, Sapporo, Hokkaido 060-8628, Japan; Innovation Medical Research Institute, University of Tsukuba, Tsukuba, Ibaraki 305-8550, Japan;http://orcid.org/0000-0002-3406-7970;
This work was supported by the Science and Technology Research Promotion Program for Agriculture, Forestry, Fisheries and Food Industry (26027AB) from Ministry of Agriculture, Forestry and Fisheries (MAFF), Japan (to Iwao Ohtsu).
The authors declare no competing financial interest.
Acknowledgment
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The authors are grateful to Professor Tsutomu Sato (Graduate School of Science and Technology, Niigata University, Niigata, Japan) for providing M. smegmatis genomic DNA. The authors thank James Allen from Edanz Group (www.edanzediting.com) for editing a draft of this manuscript.
| ERG | ergothioneine |
| HER | hercynine |
| γGC | γ-glutamylcysteine |
| γGC-HER | hercynyl-γ-glutamylcysteine sulfoxide |
| Cys-HER | hercynylcysteine sulfoxide |
| SAM | S-adenosylmethionine |
| SAH | S-adenosylhomocysteine |
| l-His | l-histidine |
| l-Glu | l-glutamate |
| l-Cys | l-cysteine |
| l-Met | l-methionine |
| HR-ESI–FT–MS | high-resolution electrospray ionization Fourier transform mass spectrometry |
| MBP | maltose-binding protein |
| SDS–PAGE | sodium dodecyl sulfate polyacrylamide gel electrophoresis |
References
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This article references 14 other publications.
- 1Genghof, D. S. Biosynthesis of ergothioneine and hercynine by fungi and Actinomycetales J. Bacteriol. 1970, 103, 475– 478[Crossref], [PubMed], [CAS], Google Scholar1https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE3cXks1ensb0%253D&md5=23a99b25c8ef231727da9b475ea549bfBiosynthesis of ergothioneine and hercynine by fungi and ActinomycetalesGenghof, Dorothy S.Journal of Bacteriology (1970), 103 (2), 475-8CODEN: JOBAAY; ISSN:0021-9193.Unlike other bacteria, aerobic members of the order Actinomycetales show a close biochem. relation to the fungi by their capacity to synthesize hercynine and ergothioneine. The myxomycete Physarum polycephalum, possessing the same synthetic ability, also shows this relation. The unusual position of yeasts as fungi is indicated by the inability of all yeastlike ascomycetes and all except a few false yeasts to synthesize these 2 betaines.
- 2Pfeiffer, C.; Bauer, T.; Surek, B.; Schömig, E.; Gründemann, D. Cyanobacteria produce high levels of ergothioneine Food Chem. 2011, 129, 1766– 1769 DOI: 10.1016/j.foodchem.2011.06.047[Crossref], [CAS], Google Scholar2https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtVWjs7bK&md5=a1ac1860f6bcf39c025d15e153b5b53bCyanobacteria produce high levels of ergothioneinePfeiffer, Carolin; Bauer, Tim; Surek, Barbara; Schoemig, Edgar; Gruendemann, DirkFood Chemistry (2011), 129 (4), 1766-1769CODEN: FOCHDJ; ISSN:0308-8146. (Elsevier Ltd.)Ergothioneine (ET) is a unique natural antioxidant. We have examd. the origin of ET in zebrafish. There was virtually no ET, measured by LC-MS, in most tank vegetation (plant, green and red alga). However, ET was detected in a Phormidium sample, a cyanobacterium. In com. fish feed prepns., ET content increased with the content of cyanobacteria Arthrospira platensis or Arthrospira maxima (Spirulina). High levels of ET (up to 0.8 mg per g dry mass) were measured in cyanobacteria prepns. sold as dietary supplements for humans and in fresh Scytonema and Oscillatoria cultures. Cyanobacteria contained as much ET as King Oyster mushrooms (Pleurotus eryngii). All samples with substantial ET content also contained the biosynthesis intermediate hercynine; this strongly suggests that cyanobacteria synthesize ET de novo. In conclusion, our data establish that cyanobacteria can produce high levels of ergothioneine. Spirulina is a novel, safe, accessible, and affordable source of ergothioneine for humans.
- 3Pluskal, T.; Ueno, M.; Yanagida, M. Genetic and metabolomic dissection of the ergothioneine and selenoneine biosynthetic pathway in the fission yeast, S. pombe, and construction of an overproduction system. PLoS One 2014, 9, e97774 DOI: 10.1371/journal.pone.0097774
- 4Gründemann, D.; Harlfinger, S.; Golz, S.; Geerts, A.; Lazar, A.; Berkels, R.; Jung, N.; Rubbert, A.; Schömig, E. Discovery of the ergothioneine transporter Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 5256– 5261 DOI: 10.1073/pnas.0408624102[Crossref], [PubMed], [CAS], Google Scholar4https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXjsFemurs%253D&md5=7b6838c9dfaf25916ba77a31ad1d2cf4Discovery of the ergothioneine transporterGruendemann, Dirk; Harlfinger, Stephanie; Golz, Stefan; Geerts, Andreas; Lazar, Andreas; Berkels, Reinhard; Jung, Norma; Rubbert, Andrea; Schoemig, EdgarProceedings of the National Academy of Sciences of the United States of America (2005), 102 (14), 5256-5261CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Variants of the SLC22A4 gene are assocd. with susceptibility to rheumatoid arthritis and Crohn's disease. SLC22A4 codes for an integral membrane protein, OCTN1, that has been presumed to carry org. cations like tetraethylammonium across the plasma membrane. Here, we show that the key substrate of this transporter is in fact ergothioneine (ET). Human OCTN1 was expressed in 293 cells. A substrate lead, stachydrine (alias proline betaine), was identified by liq. chromatog. MS difference shading, a new substrate search strategy. Anal. of transport efficiency of stachydrine-related solutes, affinity, and Na+ dependence indicates that the physiol. substrate is ET. Efficiency of transport of ET was as high as 195 μl per min per mg of protein. By contrast, the carnitine transporter OCTN2 from rat did not transport ET at all. Because ET is transported >100 times more efficiently than tetraethylammonium and carnitine, we propose the functional name ETT (ET transporter) instead of OCTN1. ET, all of which is absorbed from food, is an intracellular antioxidant with metal ion affinity. Its particular purpose is unresolved. Cells with expression of ETT accumulate ET to high levels and avidly retain it. By contrast, cells lacking ETT do not accumulate ET, because their plasma membrane is virtually impermeable for this compd. The real-time PCR expression profile of human ETT, with strong expression in CD71+ cells, is consistent with a pivotal function of ET in erythrocytes. Moreover, prominent expression of ETT in monocytes and SLC22A4 polymorphism assocns. suggest a protective role of ET in chronic inflammatory disorders.
- 5Cheah, I. K.; Halliwell, B. Ergothioneine; antioxidant potential, physiological function and role in disease Biochim. Biophys. Acta, Mol. Basis Dis. 2012, 1822, 784– 793 DOI: 10.1016/j.bbadis.2011.09.017[Crossref], [PubMed], [CAS], Google Scholar5https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xjs1GltL8%253D&md5=c225636c91ecc8c3a12d91e8aef75518Ergothioneine; antioxidant potential, physiological function and role in diseaseCheah, Irwin K.; Halliwell, BarryBiochimica et Biophysica Acta, Molecular Basis of Disease (2012), 1822 (5), 784-793CODEN: BBADEX; ISSN:0925-4439. (Elsevier B. V.)A review. Since its discovery, the unique properties of the naturally occurring amino acid, L-ergothioneine (EGT; 2-mercaptohistidine trimethylbetaine), have intrigued researchers for more than a century. This widely distributed thione is only known to be synthesized by non-yeast fungi, mycobacteria and cyanobacteria but accumulates in higher organisms at up to millimolar levels via an org. cation transporter (OCTN1). The physiol. role of EGT has yet to be established. Numerous in vitro assays have demonstrated the antioxidant and cytoprotective capabilities of EGT against a wide range of cellular stressors, but an antioxidant role has yet to be fully verified in vivo. Nevertheless the accumulation, tissue distribution and scavenging properties, all highlight the potential for EGT to function as a physiol. antioxidant. This article reviews our current state of knowledge. This article is part of a Special Issue entitled: Antioxidants and Antioxidant Treatment in Disease.
- 6Aruoma, O. I.; Coles, L. S.; Landes, B.; Repine, J. E. Functional benefits of ergothioneine and fruit- and vegetable-derived nutraceuticals: Overview of the supplemental issue contents Prev. Med. 2012, 54, S4– S8 DOI: 10.1016/j.ypmed.2012.04.001
- 7Kalaras, M. D.; Richie, J. P.; Calcagnotto, A.; Beelman, R. B. Mushrooms: A rich source of the antioxidants ergothioneine and glutathione Food Chem. 2017, 233, 429– 433 DOI: 10.1016/j.foodchem.2017.04.109[Crossref], [PubMed], [CAS], Google Scholar7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXmvVGju78%253D&md5=7f756f1f10835ed37b2eef8f6e466606Mushrooms: A rich source of the antioxidants ergothioneine and glutathioneKalaras, Michael D.; Richie, John P.; Calcagnotto, Ana; Beelman, Robert B.Food Chemistry (2017), 233 (), 429-433CODEN: FOCHDJ; ISSN:0308-8146. (Elsevier Ltd.)While mushrooms are the highest dietary source for the unique sulfur-contg. antioxidant ergothioneine, little is known regarding levels of the major biol. antioxidant glutathione. Thus, our objectives were to det. and compare levels of glutathione, as well as ergothioneine, in different species of mushrooms. Glutathione levels varied >20-fold (0.11-2.41 mg/g dw) with some varieties having higher levels than reported for other foods. Ergothioneine levels also varied widely (0.15-7.27 mg/g dw) and were highly correlated with those of glutathione (r = 0.62, P < 0.001). Both antioxidants were more concd. in pileus than stipe tissues in selected mushrooms species. Agaricus bisporus harvested during the third cropping flush contained higher levels of ergothioneine and glutathione compared to the first flush, possibly as a response to increased oxidative stress. This study demonstrated that certain mushroom species are high in glutathione and ergothioneine and should be considered an excellent dietary source of these important antioxidants.
- 8Genghof, D. S.; Van Damme, O. Biosynthesis of ergothioneine and hercynine by mycobacteria J. Bacteriol. 1964, 87, 852– 862[PubMed], [CAS], Google Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaF2cXntVajsg%253D%253D&md5=083ba4a03a4cf2fd7bb561975a2c061fBiosynthesis of ergothioneine and hercynine by mycobacteriaGenghof, Dorothy S.; van Damme, OlgaJournal of Bacteriology (1964), 87 (4), 852-62CODEN: JOBAAY; ISSN:0021-9193.Ergothioneine and hercynine were synthesized by a wide variety of mycobacteria grown in chem. defined media free from these compds. The cultures included 53 recently isolated and lab. strains of Mycobacterium tuberculosis, 26 unclassified mycobacteria (Runyon groups I to IV), and representatives of most other species in the genus. Purification and sepn. of the betaines was achieved by means of chromatography on 2 successive alumina columns. Photometric measurement of the diazotized effluents from the 2nd column permitted amts. of each compd. to be detd. Measurement of hercynine by this method was made possible by the development of a standard curve. The pathway of ergothioneine biosynthesis in mycobacteria, as judged by the use of sulfate-35S and L-histidine-2-14C as tracers, appears similar to that found in Neurospora crassa and Claviceps purpurea, that is, from histidine to ergothioneine via hercynine. None of a small group of bacteria other than mycobacteria produced ergothioneine. Two strains of group A streptococci and one of Escherichia coli produced hercyninelike material, as yet unidentified.
- 9Seebeck, F. P. In vitro reconstitution of Mycobacterial ergothioneine biosynthesis J. Am. Chem. Soc. 2010, 132, 6632– 6633 DOI: 10.1021/ja101721e[ACS Full Text
], [CAS], Google Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXltFyktLo%253D&md5=81e0cfadeb008ebcd7210df63f523409In Vitro Reconstitution of Mycobacterial Ergothioneine BiosynthesisSeebeck, Florian P.Journal of the American Chemical Society (2010), 132 (19), 6632-6633CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Ergothioneine is a histidine-derived thiol of bacterial and fungal origin that has also been isolated from animal and human tissue. Recent findings point to crit. functions of ergothioneine in human physiol., but its role in microbial life is poorly understood. This report describes the identification of the ergothioneine biosynthetic gene cluster from mycobacteria and in vitro reconstitution of this process using recombinant proteins from Mycobacterium smegmatis. The key reactions are catalyzed by a methyltransferase that transfers three Me groups to the α-amino moiety of histidine and an iron(II)-dependent enzyme that catalyzes oxidative sulfurization of trimethylhistidine. A search for homologous genes indicated that ergothioneine prodn. is a frequent trait among fungi, actinobacteria, and cyanobacteria but also occurs in numerous bacteroidetes and proteobacteria. - 10Kawano, Y.; Onishi, F.; Shiroyama, M.; Miura, M.; Tanaka, N.; Oshiro, S.; Nonaka, G.; Nakanishi, T.; Ohtsu, I. Improved fermentative l-cysteine overproduction by enhancing a newly identified thiosulfate assimilation pathway in Escherichia coli Appl. Microbiol. Biotechnol. 2017, 101, 6879– 6889 DOI: 10.1007/s00253-017-8420-4[Crossref], [PubMed], [CAS], Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXht1CmtL7L&md5=73c5f5a54ff9312e07f07220d9b42808Improved fermentative L-cysteine overproduction by enhancing a newly identified thiosulfate assimilation pathway in Escherichia coliKawano, Yusuke; Onishi, Fumito; Shiroyama, Maeka; Miura, Masashi; Tanaka, Naoyuki; Oshiro, Satoshi; Nonaka, Gen; Nakanishi, Tsuyoshi; Ohtsu, IwaoApplied Microbiology and Biotechnology (2017), 101 (18), 6879-6889CODEN: AMBIDG; ISSN:0175-7598. (Springer)Sulfate (SO42-) is an often-utilized and well-understood inorg. sulfur source in microorganism culture. Recently, another inorg. sulfur source, thiosulfate (S2O32-), was proposed to be more advantageous in microbial growth and biotechnol. applications. Although its assimilation pathway is known to depend on O-acetyl-L-serine sulfhydrylase B (CysM in Escherichia coli), its metab. has not been extensively investigated. Therefore, we aimed to explore another yet-unidentified CysM-independent thiosulfate assimilation pathway in E. coli. ΔcysM cells could accumulate essential L-cysteine from thiosulfate as the sole sulfur source and could grow, albeit slowly, demonstrating that a CysM-independent thiosulfate assimilation pathway is present in E. coli. This pathway is expected to consist of the initial part of the thiosulfate to sulfite (SO32-) conversion, and the latter part might be shared with the final part of the known sulfate assimilation pathway [sulfite → sulfide (S2-) → L-cysteine]. This is because thiosulfate-grown ΔcysM cells could accumulate a level of sulfite and sulfide equiv. to that of wild-type cells. The catalysis of thiosulfate to sulfite is at least partly mediated by thiosulfate sulfurtransferase (GlpE), because its overexpression could enhance cellular thiosulfate sulfurtransferase activity in vitro and complement the slow-growth phenotype of thiosulfate-grown ΔcysM cells in vivo. GlpE is therefore concluded to function in the novel CysM-independent thiosulfate assimilation pathway by catalyzing thiosulfate to sulfite. We applied this insight to L-cysteine overprodn. in E. coli and succeeded in enhancing it by GlpE overexpression in media contg. glucose or glycerol as the main carbon source, by up to ∼1.7-fold (1207 mg/l) or ∼1.5-fold (1529 mg/l), resp.
- 11Jones, G. W.; Doyle, S.; Fitzpatrick, D. A. The evolutionary history of the genes involved in the biosynthesis of the antioxidant ergothioneine Gene 2014, 549, 161– 170 DOI: 10.1016/j.gene.2014.07.065[Crossref], [PubMed], [CAS], Google Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhtlCit7%252FL&md5=b25e432276b3dfcf29e84a0afc290b5dThe evolutionary history of the genes involved in the biosynthesis of the antioxidant ergothioneineJones, Gary W.; Doyle, Sean; Fitzpatrick, David A.Gene (2014), 549 (1), 161-170CODEN: GENED6; ISSN:0378-1119. (Elsevier B.V.)Ergothioneine (EGT) is a histidine betaine deriv. that exhibits antioxidant action in humans. EGT is primarily synthesized by fungal species and a no. of bacterial species. A five-gene cluster (egtA, egtB, egtC, egtD & egtE) responsible for EGT prodn. in Mycobacteria smegmatis has recently been identified. The first fungal biosynthetic EGT gene (NcEgt-1) has also been identified in Neurospora crassa. NcEgt-1 contains domains similar to those found in M. smegmatis egtB and egtD. EGT is biomembrane impermeable. Here the authors inferred the evolutionary history of the EGT cluster in prokaryotes as well as examg. the phyletic distribution of Egt-1 in the fungal kingdom. A genomic survey of 2509 prokaryotes showed that the five-gene EGT cluster is only found in the Actinobacteria. Our survey identified more than 400 diverse prokaryotes that contain genetically linked orthologs of egtB and egtD. Phylogenetic analyses of Egt proteins show a complex evolutionary history and multiple incidences of horizontal gene transfer. Our anal. also identified two independent incidences of a fusion event of egtB and egtD in bacterial species. A genomic survey of over 100 fungal genomes shows that Egt-1 is found in all fungal phyla, except species that belong to the Saccharomycotina subphylum. This anal. provides a comprehensive anal. of the distribution of the key genes involved in the synthesis of EGT in prokaryotes and fungi. Our phylogenetic inferences illuminate the complex evolutionary history of the genes involved in EGT synthesis in prokaryotes. The potential to synthesize EGT is a fungal trait except for species belonging to the Saccharomycotina subphylum.
- 12Watanabe, K.; Yamano, Y.; Murata, K.; Kimura, A. The nucleotide sequence of the gene for γ-glutamylcysteine synthetase of Escherichia coli Nucleic Acids Res. 1986, 14, 4393– 4400 DOI: 10.1093/nar/14.11.4393
- 13Vit, A.; Misson, L.; Blankenfeldt, W.; Seebeck, F. P. Ergothioneine biosynthetic methyltransferase EgtD reveals the structural basis of aromatic amino acid betaine biosynthesis ChemBioChem 2015, 16, 119– 125 DOI: 10.1002/cbic.201402522[Crossref], [PubMed], [CAS], Google Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhvFantLnK&md5=3bca85854c34be9658d0e8030a90b5e1Ergothioneine Biosynthetic Methyltransferase EgtD Reveals the Structural Basis of Aromatic Amino Acid Betaine BiosynthesisVit, Allegra; Misson, Laetitia; Blankenfeldt, Wulf; Seebeck, Florian P.ChemBioChem (2015), 16 (1), 119-125CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)Ergothioneine is an N-α-trimethyl-2-thiohistidine deriv. that occurs in human, plant, fungal, and bacterial cells. Biosynthesis of this redox-active betaine starts with trimethylation of the α-amino group of histidine. The three consecutive Me transfers are catalyzed by the S-adenosylmethionine-dependent methyltransferase EgtD. Three crystal structures of this enzyme in the absence and in the presence of N-α-dimethylhistidine and S-adenosylhomocysteine implicate a preorganized array of hydrophilic interactions as the determinants for substrate specificity and apparent processivity. We identified two active site mutations that change the substrate specificity of EgtD 107-fold and transform the histidine-methyltransferase into a proficient tryptophan-methyltransferase. Finally, a genomic search for EgtD homologues in fungal genomes revealed tyrosine and tryptophan trimethylation activity as a frequent trait in ascomycetous and basidomycetous fungi.
- 14Kari, C.; Nagy, Z.; Kovács, P.; Hernádi, F. Mechanism of the growth inhibitory effect of cysteine on Escherichia coli J. Gen. Microbiol. 1971, 68, 349– 356 DOI: 10.1099/00221287-68-3-349
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Abstract
Figure 1
Figure 1. ERG biosynthetic pathway.
Figure 2
Figure 2. Plasmids for ERG production.
Figure 3
Figure 3. LC–ESI–MS analysis of EgtC reaction products. (a) Traces at 250 nm of reaction products. The reaction (40 μL) was carried out by adding (i) purified recombinant EgtC (3.9 μM) or (ii) boiled EgtC to the EgtB reaction solution at 25 °C for 17 h. (b) MS spectrum of the EgtC reaction product (ESI positive mode).
Figure 4
Figure 4. LC–ESI–MS analysis of EgtE reaction products. (a) Traces at 250 nm of (i) ERG standard and (ii) reaction products. The reaction (40 μL) was carried out by adding purified recombinant EgtE (2.5 μM) and EgtC (3.9 μM) (ii) to the boiled supernatant of the EgtB reaction mixture containing 2 mM dithiothreitol at 25 °C for 17 h. (b) MS spectra of (i) ERG standard and (ii) EgtE reaction product (ESI positive mode).
Figure 5
Figure 5. Culture profiles of strain ET1. E. coli BW25113 harboring pCF1s-MsD (strain ET1) was cultured in M9Y medium supplemented with l-His and l-Met. After 3 h of cultivation, 0.5 mM IPTG was added to the medium. Data are presented as mean values with standard errors from three independent experiments.
Figure 6
Figure 6. Culture profiles of strain ET2. E. coli BW25113 harboring pCF1s-MsD and pQE1a-mMsB (strain ET2) was cultured in M9Y medium supplemented with l-His and l-Met. After 3 h of cultivation, 0.5 mM IPTG was added to the medium. Data are presented as mean values with standard errors from three independent experiments.
Figure 7
Figure 7. Culture profiles of strain ET3. E. coli BW25113 harboring pCF1s-MsD, pQE1a-mMsB, and pAC1c-hMsC/hMsE (strain ET3) was cultured in M9Y medium supplemented with l-His and l-Met. After 3 h of cultivation, 0.5 mM IPTG was added to the medium. Data are presented as mean values with standard errors from three independent experiments.
References
ARTICLE SECTIONSThis article references 14 other publications.
- 1Genghof, D. S. Biosynthesis of ergothioneine and hercynine by fungi and Actinomycetales J. Bacteriol. 1970, 103, 475– 478[Crossref], [PubMed], [CAS], Google Scholar1https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE3cXks1ensb0%253D&md5=23a99b25c8ef231727da9b475ea549bfBiosynthesis of ergothioneine and hercynine by fungi and ActinomycetalesGenghof, Dorothy S.Journal of Bacteriology (1970), 103 (2), 475-8CODEN: JOBAAY; ISSN:0021-9193.Unlike other bacteria, aerobic members of the order Actinomycetales show a close biochem. relation to the fungi by their capacity to synthesize hercynine and ergothioneine. The myxomycete Physarum polycephalum, possessing the same synthetic ability, also shows this relation. The unusual position of yeasts as fungi is indicated by the inability of all yeastlike ascomycetes and all except a few false yeasts to synthesize these 2 betaines.
- 2Pfeiffer, C.; Bauer, T.; Surek, B.; Schömig, E.; Gründemann, D. Cyanobacteria produce high levels of ergothioneine Food Chem. 2011, 129, 1766– 1769 DOI: 10.1016/j.foodchem.2011.06.047[Crossref], [CAS], Google Scholar2https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtVWjs7bK&md5=a1ac1860f6bcf39c025d15e153b5b53bCyanobacteria produce high levels of ergothioneinePfeiffer, Carolin; Bauer, Tim; Surek, Barbara; Schoemig, Edgar; Gruendemann, DirkFood Chemistry (2011), 129 (4), 1766-1769CODEN: FOCHDJ; ISSN:0308-8146. (Elsevier Ltd.)Ergothioneine (ET) is a unique natural antioxidant. We have examd. the origin of ET in zebrafish. There was virtually no ET, measured by LC-MS, in most tank vegetation (plant, green and red alga). However, ET was detected in a Phormidium sample, a cyanobacterium. In com. fish feed prepns., ET content increased with the content of cyanobacteria Arthrospira platensis or Arthrospira maxima (Spirulina). High levels of ET (up to 0.8 mg per g dry mass) were measured in cyanobacteria prepns. sold as dietary supplements for humans and in fresh Scytonema and Oscillatoria cultures. Cyanobacteria contained as much ET as King Oyster mushrooms (Pleurotus eryngii). All samples with substantial ET content also contained the biosynthesis intermediate hercynine; this strongly suggests that cyanobacteria synthesize ET de novo. In conclusion, our data establish that cyanobacteria can produce high levels of ergothioneine. Spirulina is a novel, safe, accessible, and affordable source of ergothioneine for humans.
- 3Pluskal, T.; Ueno, M.; Yanagida, M. Genetic and metabolomic dissection of the ergothioneine and selenoneine biosynthetic pathway in the fission yeast, S. pombe, and construction of an overproduction system. PLoS One 2014, 9, e97774 DOI: 10.1371/journal.pone.0097774
- 4Gründemann, D.; Harlfinger, S.; Golz, S.; Geerts, A.; Lazar, A.; Berkels, R.; Jung, N.; Rubbert, A.; Schömig, E. Discovery of the ergothioneine transporter Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 5256– 5261 DOI: 10.1073/pnas.0408624102[Crossref], [PubMed], [CAS], Google Scholar4https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXjsFemurs%253D&md5=7b6838c9dfaf25916ba77a31ad1d2cf4Discovery of the ergothioneine transporterGruendemann, Dirk; Harlfinger, Stephanie; Golz, Stefan; Geerts, Andreas; Lazar, Andreas; Berkels, Reinhard; Jung, Norma; Rubbert, Andrea; Schoemig, EdgarProceedings of the National Academy of Sciences of the United States of America (2005), 102 (14), 5256-5261CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Variants of the SLC22A4 gene are assocd. with susceptibility to rheumatoid arthritis and Crohn's disease. SLC22A4 codes for an integral membrane protein, OCTN1, that has been presumed to carry org. cations like tetraethylammonium across the plasma membrane. Here, we show that the key substrate of this transporter is in fact ergothioneine (ET). Human OCTN1 was expressed in 293 cells. A substrate lead, stachydrine (alias proline betaine), was identified by liq. chromatog. MS difference shading, a new substrate search strategy. Anal. of transport efficiency of stachydrine-related solutes, affinity, and Na+ dependence indicates that the physiol. substrate is ET. Efficiency of transport of ET was as high as 195 μl per min per mg of protein. By contrast, the carnitine transporter OCTN2 from rat did not transport ET at all. Because ET is transported >100 times more efficiently than tetraethylammonium and carnitine, we propose the functional name ETT (ET transporter) instead of OCTN1. ET, all of which is absorbed from food, is an intracellular antioxidant with metal ion affinity. Its particular purpose is unresolved. Cells with expression of ETT accumulate ET to high levels and avidly retain it. By contrast, cells lacking ETT do not accumulate ET, because their plasma membrane is virtually impermeable for this compd. The real-time PCR expression profile of human ETT, with strong expression in CD71+ cells, is consistent with a pivotal function of ET in erythrocytes. Moreover, prominent expression of ETT in monocytes and SLC22A4 polymorphism assocns. suggest a protective role of ET in chronic inflammatory disorders.
- 5Cheah, I. K.; Halliwell, B. Ergothioneine; antioxidant potential, physiological function and role in disease Biochim. Biophys. Acta, Mol. Basis Dis. 2012, 1822, 784– 793 DOI: 10.1016/j.bbadis.2011.09.017[Crossref], [PubMed], [CAS], Google Scholar5https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xjs1GltL8%253D&md5=c225636c91ecc8c3a12d91e8aef75518Ergothioneine; antioxidant potential, physiological function and role in diseaseCheah, Irwin K.; Halliwell, BarryBiochimica et Biophysica Acta, Molecular Basis of Disease (2012), 1822 (5), 784-793CODEN: BBADEX; ISSN:0925-4439. (Elsevier B. V.)A review. Since its discovery, the unique properties of the naturally occurring amino acid, L-ergothioneine (EGT; 2-mercaptohistidine trimethylbetaine), have intrigued researchers for more than a century. This widely distributed thione is only known to be synthesized by non-yeast fungi, mycobacteria and cyanobacteria but accumulates in higher organisms at up to millimolar levels via an org. cation transporter (OCTN1). The physiol. role of EGT has yet to be established. Numerous in vitro assays have demonstrated the antioxidant and cytoprotective capabilities of EGT against a wide range of cellular stressors, but an antioxidant role has yet to be fully verified in vivo. Nevertheless the accumulation, tissue distribution and scavenging properties, all highlight the potential for EGT to function as a physiol. antioxidant. This article reviews our current state of knowledge. This article is part of a Special Issue entitled: Antioxidants and Antioxidant Treatment in Disease.
- 6Aruoma, O. I.; Coles, L. S.; Landes, B.; Repine, J. E. Functional benefits of ergothioneine and fruit- and vegetable-derived nutraceuticals: Overview of the supplemental issue contents Prev. Med. 2012, 54, S4– S8 DOI: 10.1016/j.ypmed.2012.04.001
- 7Kalaras, M. D.; Richie, J. P.; Calcagnotto, A.; Beelman, R. B. Mushrooms: A rich source of the antioxidants ergothioneine and glutathione Food Chem. 2017, 233, 429– 433 DOI: 10.1016/j.foodchem.2017.04.109[Crossref], [PubMed], [CAS], Google Scholar7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXmvVGju78%253D&md5=7f756f1f10835ed37b2eef8f6e466606Mushrooms: A rich source of the antioxidants ergothioneine and glutathioneKalaras, Michael D.; Richie, John P.; Calcagnotto, Ana; Beelman, Robert B.Food Chemistry (2017), 233 (), 429-433CODEN: FOCHDJ; ISSN:0308-8146. (Elsevier Ltd.)While mushrooms are the highest dietary source for the unique sulfur-contg. antioxidant ergothioneine, little is known regarding levels of the major biol. antioxidant glutathione. Thus, our objectives were to det. and compare levels of glutathione, as well as ergothioneine, in different species of mushrooms. Glutathione levels varied >20-fold (0.11-2.41 mg/g dw) with some varieties having higher levels than reported for other foods. Ergothioneine levels also varied widely (0.15-7.27 mg/g dw) and were highly correlated with those of glutathione (r = 0.62, P < 0.001). Both antioxidants were more concd. in pileus than stipe tissues in selected mushrooms species. Agaricus bisporus harvested during the third cropping flush contained higher levels of ergothioneine and glutathione compared to the first flush, possibly as a response to increased oxidative stress. This study demonstrated that certain mushroom species are high in glutathione and ergothioneine and should be considered an excellent dietary source of these important antioxidants.
- 8Genghof, D. S.; Van Damme, O. Biosynthesis of ergothioneine and hercynine by mycobacteria J. Bacteriol. 1964, 87, 852– 862[PubMed], [CAS], Google Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaF2cXntVajsg%253D%253D&md5=083ba4a03a4cf2fd7bb561975a2c061fBiosynthesis of ergothioneine and hercynine by mycobacteriaGenghof, Dorothy S.; van Damme, OlgaJournal of Bacteriology (1964), 87 (4), 852-62CODEN: JOBAAY; ISSN:0021-9193.Ergothioneine and hercynine were synthesized by a wide variety of mycobacteria grown in chem. defined media free from these compds. The cultures included 53 recently isolated and lab. strains of Mycobacterium tuberculosis, 26 unclassified mycobacteria (Runyon groups I to IV), and representatives of most other species in the genus. Purification and sepn. of the betaines was achieved by means of chromatography on 2 successive alumina columns. Photometric measurement of the diazotized effluents from the 2nd column permitted amts. of each compd. to be detd. Measurement of hercynine by this method was made possible by the development of a standard curve. The pathway of ergothioneine biosynthesis in mycobacteria, as judged by the use of sulfate-35S and L-histidine-2-14C as tracers, appears similar to that found in Neurospora crassa and Claviceps purpurea, that is, from histidine to ergothioneine via hercynine. None of a small group of bacteria other than mycobacteria produced ergothioneine. Two strains of group A streptococci and one of Escherichia coli produced hercyninelike material, as yet unidentified.
- 9Seebeck, F. P. In vitro reconstitution of Mycobacterial ergothioneine biosynthesis J. Am. Chem. Soc. 2010, 132, 6632– 6633 DOI: 10.1021/ja101721e[ACS Full Text], [CAS], Google Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXltFyktLo%253D&md5=81e0cfadeb008ebcd7210df63f523409In Vitro Reconstitution of Mycobacterial Ergothioneine BiosynthesisSeebeck, Florian P.Journal of the American Chemical Society (2010), 132 (19), 6632-6633CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Ergothioneine is a histidine-derived thiol of bacterial and fungal origin that has also been isolated from animal and human tissue. Recent findings point to crit. functions of ergothioneine in human physiol., but its role in microbial life is poorly understood. This report describes the identification of the ergothioneine biosynthetic gene cluster from mycobacteria and in vitro reconstitution of this process using recombinant proteins from Mycobacterium smegmatis. The key reactions are catalyzed by a methyltransferase that transfers three Me groups to the α-amino moiety of histidine and an iron(II)-dependent enzyme that catalyzes oxidative sulfurization of trimethylhistidine. A search for homologous genes indicated that ergothioneine prodn. is a frequent trait among fungi, actinobacteria, and cyanobacteria but also occurs in numerous bacteroidetes and proteobacteria.
- 10Kawano, Y.; Onishi, F.; Shiroyama, M.; Miura, M.; Tanaka, N.; Oshiro, S.; Nonaka, G.; Nakanishi, T.; Ohtsu, I. Improved fermentative l-cysteine overproduction by enhancing a newly identified thiosulfate assimilation pathway in Escherichia coli Appl. Microbiol. Biotechnol. 2017, 101, 6879– 6889 DOI: 10.1007/s00253-017-8420-4[Crossref], [PubMed], [CAS], Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXht1CmtL7L&md5=73c5f5a54ff9312e07f07220d9b42808Improved fermentative L-cysteine overproduction by enhancing a newly identified thiosulfate assimilation pathway in Escherichia coliKawano, Yusuke; Onishi, Fumito; Shiroyama, Maeka; Miura, Masashi; Tanaka, Naoyuki; Oshiro, Satoshi; Nonaka, Gen; Nakanishi, Tsuyoshi; Ohtsu, IwaoApplied Microbiology and Biotechnology (2017), 101 (18), 6879-6889CODEN: AMBIDG; ISSN:0175-7598. (Springer)Sulfate (SO42-) is an often-utilized and well-understood inorg. sulfur source in microorganism culture. Recently, another inorg. sulfur source, thiosulfate (S2O32-), was proposed to be more advantageous in microbial growth and biotechnol. applications. Although its assimilation pathway is known to depend on O-acetyl-L-serine sulfhydrylase B (CysM in Escherichia coli), its metab. has not been extensively investigated. Therefore, we aimed to explore another yet-unidentified CysM-independent thiosulfate assimilation pathway in E. coli. ΔcysM cells could accumulate essential L-cysteine from thiosulfate as the sole sulfur source and could grow, albeit slowly, demonstrating that a CysM-independent thiosulfate assimilation pathway is present in E. coli. This pathway is expected to consist of the initial part of the thiosulfate to sulfite (SO32-) conversion, and the latter part might be shared with the final part of the known sulfate assimilation pathway [sulfite → sulfide (S2-) → L-cysteine]. This is because thiosulfate-grown ΔcysM cells could accumulate a level of sulfite and sulfide equiv. to that of wild-type cells. The catalysis of thiosulfate to sulfite is at least partly mediated by thiosulfate sulfurtransferase (GlpE), because its overexpression could enhance cellular thiosulfate sulfurtransferase activity in vitro and complement the slow-growth phenotype of thiosulfate-grown ΔcysM cells in vivo. GlpE is therefore concluded to function in the novel CysM-independent thiosulfate assimilation pathway by catalyzing thiosulfate to sulfite. We applied this insight to L-cysteine overprodn. in E. coli and succeeded in enhancing it by GlpE overexpression in media contg. glucose or glycerol as the main carbon source, by up to ∼1.7-fold (1207 mg/l) or ∼1.5-fold (1529 mg/l), resp.
- 11Jones, G. W.; Doyle, S.; Fitzpatrick, D. A. The evolutionary history of the genes involved in the biosynthesis of the antioxidant ergothioneine Gene 2014, 549, 161– 170 DOI: 10.1016/j.gene.2014.07.065[Crossref], [PubMed], [CAS], Google Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhtlCit7%252FL&md5=b25e432276b3dfcf29e84a0afc290b5dThe evolutionary history of the genes involved in the biosynthesis of the antioxidant ergothioneineJones, Gary W.; Doyle, Sean; Fitzpatrick, David A.Gene (2014), 549 (1), 161-170CODEN: GENED6; ISSN:0378-1119. (Elsevier B.V.)Ergothioneine (EGT) is a histidine betaine deriv. that exhibits antioxidant action in humans. EGT is primarily synthesized by fungal species and a no. of bacterial species. A five-gene cluster (egtA, egtB, egtC, egtD & egtE) responsible for EGT prodn. in Mycobacteria smegmatis has recently been identified. The first fungal biosynthetic EGT gene (NcEgt-1) has also been identified in Neurospora crassa. NcEgt-1 contains domains similar to those found in M. smegmatis egtB and egtD. EGT is biomembrane impermeable. Here the authors inferred the evolutionary history of the EGT cluster in prokaryotes as well as examg. the phyletic distribution of Egt-1 in the fungal kingdom. A genomic survey of 2509 prokaryotes showed that the five-gene EGT cluster is only found in the Actinobacteria. Our survey identified more than 400 diverse prokaryotes that contain genetically linked orthologs of egtB and egtD. Phylogenetic analyses of Egt proteins show a complex evolutionary history and multiple incidences of horizontal gene transfer. Our anal. also identified two independent incidences of a fusion event of egtB and egtD in bacterial species. A genomic survey of over 100 fungal genomes shows that Egt-1 is found in all fungal phyla, except species that belong to the Saccharomycotina subphylum. This anal. provides a comprehensive anal. of the distribution of the key genes involved in the synthesis of EGT in prokaryotes and fungi. Our phylogenetic inferences illuminate the complex evolutionary history of the genes involved in EGT synthesis in prokaryotes. The potential to synthesize EGT is a fungal trait except for species belonging to the Saccharomycotina subphylum.
- 12Watanabe, K.; Yamano, Y.; Murata, K.; Kimura, A. The nucleotide sequence of the gene for γ-glutamylcysteine synthetase of Escherichia coli Nucleic Acids Res. 1986, 14, 4393– 4400 DOI: 10.1093/nar/14.11.4393
- 13Vit, A.; Misson, L.; Blankenfeldt, W.; Seebeck, F. P. Ergothioneine biosynthetic methyltransferase EgtD reveals the structural basis of aromatic amino acid betaine biosynthesis ChemBioChem 2015, 16, 119– 125 DOI: 10.1002/cbic.201402522[Crossref], [PubMed], [CAS], Google Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhvFantLnK&md5=3bca85854c34be9658d0e8030a90b5e1Ergothioneine Biosynthetic Methyltransferase EgtD Reveals the Structural Basis of Aromatic Amino Acid Betaine BiosynthesisVit, Allegra; Misson, Laetitia; Blankenfeldt, Wulf; Seebeck, Florian P.ChemBioChem (2015), 16 (1), 119-125CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)Ergothioneine is an N-α-trimethyl-2-thiohistidine deriv. that occurs in human, plant, fungal, and bacterial cells. Biosynthesis of this redox-active betaine starts with trimethylation of the α-amino group of histidine. The three consecutive Me transfers are catalyzed by the S-adenosylmethionine-dependent methyltransferase EgtD. Three crystal structures of this enzyme in the absence and in the presence of N-α-dimethylhistidine and S-adenosylhomocysteine implicate a preorganized array of hydrophilic interactions as the determinants for substrate specificity and apparent processivity. We identified two active site mutations that change the substrate specificity of EgtD 107-fold and transform the histidine-methyltransferase into a proficient tryptophan-methyltransferase. Finally, a genomic search for EgtD homologues in fungal genomes revealed tyrosine and tryptophan trimethylation activity as a frequent trait in ascomycetous and basidomycetous fungi.
- 14Kari, C.; Nagy, Z.; Kovács, P.; Hernádi, F. Mechanism of the growth inhibitory effect of cysteine on Escherichia coli J. Gen. Microbiol. 1971, 68, 349– 356 DOI: 10.1099/00221287-68-3-349
Supporting Information
ARTICLE SECTIONSThe Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b04924.
Supplementary Methods 1 and 2, vectors pQE1a-Red and pCF1s-Red (Figure S1), SDS–PAGE analysis of EgtD production (Figure S2), LC–ESI–MS analysis of EgtD reaction products (Figure S3), SDS–PAGE analysis of purified recombinant EgtB, EgtC, and EgtE (Figure S4), LC–ESI–MS analysis of EgtB reaction products (Figure S5), SDS–PAGE analysis of recombinant EgtC and EgtE production (Figure S6), SDS–PAGE analysis of production of recombinant Egt enzymes in strain ET3 (Figure S7), SDS–PAGE analysis of production of recombinant Egt enzymes and GshA in strain ET4 (Figure S8), and primers used in this study (Table S1) (PDF)
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