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Heterologous and high production of ergothioneine in Escherichia coli Ryo Osawa, Tomoyuki Kamide, Yasuharu Satoh, Yusuke Kawano, Iwao Ohtsu, and Tohru Dairi J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04924 • Publication Date (Web): 25 Dec 2017 Downloaded from http://pubs.acs.org on December 26, 2017

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Journal of Agricultural and Food Chemistry

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Heterologous and high production of ergothioneine in Escherichia coli

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Ryo Osawa1, Tomoyuki Kamide1, Yasuharu Satoh2*, Yusuke Kawano3, Iwao Ohtsu3, and Tohru

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Dairi2*

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1

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060-8628, Japan.

Graduate School of Chemical Science and Engineering, Hokkaido University, Hokkaido

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2

Graduate School of Engineering, Hokkaido University, Hokkaido 060-8628, Japan.

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3

Innovation Medical Research Institute, University of Tsukuba, Ibaraki 305-8550, Japan.

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*Corresponding authors:

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Graduate School of Engineering, Hokkaido University, Hokkaido 060-8628, Japan. Tel.

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+81-11-706-7815; Fax. +81-11-706-7118

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Email: [email protected] & [email protected]

1 ACS Paragon Plus Environment


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ABSTRACT

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Ergothioneine (ERG) is a histidine-derived thiol compound suggested to function as an

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antioxidant and cytoprotectant in humans. Therefore, experimental trials have been conducted

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applying ERG from mushrooms in dietary supplements and as a cosmetic additive. However, this

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method of producing ERG is expensive, so alternative methods for ERG supply are required.

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Five Mycobacterium smegmatis genes, egtABCDE, have been confirmed to be responsible for

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ERG biosynthesis. This enabled us to develop practical fermentative ERG production by

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microorganisms. In this study, we carried out heterologous and high-level production of ERG in

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Escherichia coli using the egt genes from M. smegmatis. By high production of each of the Egt

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enzymes and elimination of bottlenecks in the substrate supply, we succeeded in constructing a

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production system that yielded 24 mg/L (104 µM) of secreted ERG.

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Key words: ergothioneine; heterologous production; Escherichia coli

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INTRODUCTION

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Ergothioneine (ERG), a histidine-derived thiol compound, was isolated from an ergot

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fungus, Claviceps purpurea, more than a century ago. ERG is also known to be synthesized in

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actinobacteria, cyanobacteria, and a fission yeast.1,2,3 Recent studies show that ERG functions as

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an antioxidant like glutathione, mycothiol, and bacillithiol. There is no direct evidence for

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biosynthesis of ERG in humans. However, ERG has been reported to be accumulated in various

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cells and tissues at high concentrations, probably by intake from diets such as mushrooms and red

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beans that contain relatively large amounts of ERG, through an ERG-specific organic cation

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transporter, OCTN1.4,5

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The presence of the ERG-specific transporter and the extensive accumulation of ERG in

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tissues suggest that ERG should have significant biological functions in humans. Although the

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true physiological role of ERG in humans has yet to be fully understood, ERG has been shown by

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in vitro experiments to function as an antioxidant and a cytoprotectant. Therefore, applications of

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ERG in dietary supplements and as a cosmetic additive have been explored, and there is an

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increasing demand for ERG.6 Mushrooms have traditionally been the source of ERG.7 However,



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slow growth, low content and time-consuming purification procedures lead to a high

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manufacturing cost. Therefore, alternative and sustainable sources of ERG are necessary.

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One such reliable and practical method is a fermentative process using microorganisms such

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as actinobacteria and cyanobacteria that are known to produce ERG. However, their ERG

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productivities are very low (1.18 mg/g dry weight after 4 weeks cultivation of Mycobacterium

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avium and 0.8 mg/g dry weight of Oscillatoria sp.)2,8 and thus genetic and metabolic engineering

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are indispensable for industrial production. Until recently, however, there were no reports on

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ERG biosynthesis genes and enzymes. In 2010, five genes in Mycobacterium smegmatis,

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egtABCDE, were confirmed to be responsible for ERG biosynthesis (Figure 1).9 In the

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biosynthetic pathway, EgtD catalyzes the formation of hercynine (HER) by transfer of three

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methyl groups derived from S-adenosylmethionine (SAM) to L-histidine (L-His). Then, EgtB

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catalyzes O2-dependent C–S bond formation between γ-glutamylcysteine (γGC) supplied by

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EgtA and HER to form hercynyl-γ-glutamylcysteine-sulfoxide (γGC-HER). This is followed by

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removal of the

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hercynylcysteine-sulfoxide (Cys-HER). Then, EgtE, a PLP-dependent C–S lyase, catalyzes the

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formation of ERG with concomitant formation of pyruvate and ammonia as side-products.

L-glutamate

(L-Glu) moiety by EgtC, an amidohydrolase, to produce


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In this study, we developed heterologous and high-level production of ERG in Escherichia

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coli using the egt genes from M. smegmatis. By high productionion of each of the Egt enzymes

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and elimination of bottlenecks in substrate supply, the production system yielded 24 mg/L (104

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µM) of secreted ERG.

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MATERIALS AND METHODS

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General. LB broth (Lennox) medium was purchased from Sigma-Aldrich Japan K.K.

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(Tokyo, Japan), L-Met and L-His were obtained from Wako Pure Chemical Industry (Osaka,

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Japan), and HER was purchased from Shinsei Chemical Company Ltd. (Osaka, Japan). Other

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chemicals were of analytical grade and purchased from Wako Pure Chemical Industry or

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Sigma-Aldrich Japan. Primers were obtained from FASMAC Co. Ltd. (Kanagawa, Japan).

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Enzymes and kits for DNA manipulation were purchased from Takara Bio Inc. (Shiga, Japan) or

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New England Biolabs Japan Inc. (Tokyo, Japan). PCR were carried out using a GeneAmp PCR

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System 9700 thermal cycler (Thermo Fisher Scientific Inc., Waltham, MA, USA) with Tks Gflex

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DNA polymerase (Takara Bio). General genetic manipulations of E. coli were performed

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according to standard protocols. High-resolution electrospray ionization Fourier transform mass



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spectrometry (HR-ESI-FT-MS) analysis was performed using an Exactive system (Thermo

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Fisher Scientific Inc.).

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Bacterial Strains and Cultures. Microorganisms used in this study are summarized in Table

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1. E. coli XL1-Blue (Nippon Gene Co. Ltd., Tokyo, Japan), BL21(DE3) (Merck KGaA,

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Darmstadt, Germany), and BW25113 (National Institute of Genetics, Shizuoka, Japan) that is a

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high producer of L-Cys utilized for a sulfur donor of ERG10 were used for plasmid construction,

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protein production, and ERG production, respectively. The media used were LB broth and M9Y

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minimal medium prepared by adding 1% (w/v) glucose, 5 mM MgSO4, 0.1 mM CaCl2, and 0.1%

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(w/v) yeast extract (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) to M9 minimal

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salts (Becton, Dickinson and Company). Ampicillin (Ap), chloramphenicol (Cm), kanamycin

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(Km), streptomycin (Sm), and tetracycline (Tc) were added to the media at concentrations of 100,

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33, 25, 20, and 5 mg/L, if necessary. Optical density (OD) at 600 nm was measured with a

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NanoDrop 2000C spectrophotometer (Thermo Fisher Scientific Inc.).

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Preparation of Egt Recombinant Enzymes. Detailed plasmid construction methods are

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described in Supporting Information (Supplementary methods 1) and the plasmids are

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summarized in Table 2. Briefly, EgtB, C, D and E were amplified by PCR using M. smegmatis

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genomic DNA as the template and appropriate primers (Table S1) in which restriction sites were

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introduced at the N- and C-termini. The PCR products were respectively cloned into the

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expression vectors. The plasmids obtained were introduced into E. coli BL21(DE3). A liquid

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culture of the transformant in LB supplied with appropriate antibiotic was induced by adding 0.5

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mM isopropyl β-D-1-thiogalactopyranoside (IPTG) when the optical density at 600 nm reached

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about 0.6. The cultivation was continued for an additional 16 h at 20°C. The production of each

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recombinant enzyme was analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis

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(SDS-PAGE) with Coomassie Brilliant Blue staining. The purification of each recombinant

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protein was carried out using Ni-NTA agarose (QIAGEN K.K., Tokyo, Japan) or amylose resins

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(New England Biolabs) according to the manufacturer’s protocols. Protein concentrations were

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determined using a Bradford protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA)

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with bovine serum albumin as the standard.

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ERG Production. E. coli harboring plasmids carrying ERG biosynthetic genes (Figure 2)

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was cultured in 3 mL of M9Y media at 30°C for 16 h. The cultures (1 mL) were inoculated into

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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

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in 200-mL Erlenmeyer flasks and incubated at 30°C with shaking (200 rpm) for up to 72 h.

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Na2S2O3 was added to media at 20 mM, if needed. IPTG was added to a final concentration of

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0.5 mM after 3 h of cultivation. Samples (1 mL) were collected at appropriate time points and

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analyzed by LC-ESI-MS.

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LC-ESI-MS Analysis of Products. Mixtures of 0.05% (v/v) heptafluorobutyric acid

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(HFBA) solution (180 µL) and culture broth or enzymatic reaction solutions (20 µL) were

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analyzed. The LC-ESI-MS conditions were: Waters ACQUITY UPLC system equipped with a

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photodiode array and a SQ Detector2 (Tokyo, Japan); XBridge BEH C18 XP column (150 mm L

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× 2.0 mm; ID, 2.5 µm; Waters); flow rate, 0.15 mL/min; temperature, 35°C; mobile phase, water

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containing 0.05% HFBA and 7% methanol; injection volume, 2 µL; detection, 210 nm for His

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and HER, 250 nm for Cys-HER and γGC-HER, and 258 nm for ERG.

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RESULTS AND DISCUSSION

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The most reliable strategy for high production of ERG is overproduction of each of the

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enzymes for ERG biosynthesis and then optimization of any inefficient biosynthetic step(s)

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(identified by intermediate accumulation) by molecular genetics and metabolic engineering. If no

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intermediates accumulate, supply of the initial substrates, L-His, L-Glu, L-Cys, and L-Met, should

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be enhanced.

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ERG biosynthetic genes are present in some microorganisms such as actinobacteria,

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cyanobacteria, and α-proteobacteria.11 Among these bacteria, we selected M. smegmatis as the

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gene source, because the ERG biosynthetic pathway was first identified in this strain and the

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biosynthesis genes constitute an operon.9 Since egtB, egtC and egtE are translationally coupled,

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we first tried expressing the egt genes as the operon with tac and T7 promoters. We constructed

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several plasmids utilizing pQE1a-Red and pET-21a, but ERG productivities of transformants

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harboring the plasmids were low (less than 0.2 mg/L). Therefore, we overexpressed egt genes

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stepwise under the control of individual promoters by checking production of recombinant

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enzymes and their activities by production of intermediate compounds (HER, γGC-HER, and

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Cys-HER) and ERG by in vitro experiment (Figure 1). Since E. coli has an egtA ortholog, gshA,



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which is responsible for glutathione biosynthesis,12 we expressed the other genes, egtB, C, D, and

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E. We also prepared the intermediate compounds, which are not commercially available, with the

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recombinant enzymes to obtain standards for quantitative analysis (Supplementary methods 2).

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Overproduction of Recombinant Enzymes. For overproduction of Egt enzymes, we used

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pQE1a-Red and pCF1s-Red, both of which are home-constructed and compatible vectors with

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the tac promoter for protein production (Figure S1). As Figure S2 shows, transformants

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harboring pCF1s-MsD carrying egtD overproduced EgtD in soluble form. To confirm whether

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the recombinant protein had the expected activity, we carried out in vitro experiments with cell

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free extracts containing the recombinant enzyme.9,13 The cell free extracts were incubated with

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L-His

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peak with the same retention time and MS spectrum as the HER standard was clearly detected

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after 1 h of incubation, demonstrating that EgtD converted L-His into HER (Figure S3).

and excess SAM. After the reaction, the product was analyzed by LC-ESI-MS. A specific

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We next overproduced recombinant EgtB using pQE1a-Red (Figure S1) and changing the

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probable start codon TTG into ATG. However, recombinant EgtB formed inclusion bodies in

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multiple culture conditions. Therefore, a plasmid from which a recombinant enzyme is produced


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as a maltose binding protein (MBP)-fused enzyme was examined. We successfully produced a

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MBP-fused EgtB in a soluble form in transformants harboring pQE1a-mMsB. To confirm

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whether the recombinant enzyme had the expected activity, we carried out in vitro experiments

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with purified recombinant enzyme (Figure S4).9 The recombinant MBP-fused EgtB was

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incubated with HER and γGC for 2 h. As Figure S5 shows, HER completely disappeared, and the

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formation of a new product with m/z 462.20 was detected. Based on HR-ESI-FT-MS analysis of

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the purified compound, the molecular formula of the product was determined to be C17H27O8N5S

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(m/z: [M+H]+ calculated for C17H28O8N5S+, 462.16531; observed, 462.16507), which

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corresponded to that of γGC-HER, suggesting the formation of γGC-HER. The thus formed

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intermediate compound γGC-HER was purified by HPLC (Supplementary methods 2) and used

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as a standard for quantitative analysis.

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Finally, overproduction of recombinant EgtC and EgtE was examined. We first used the

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same vector as for EgtB production but no production of either enzyme was observed. We then

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examined a plasmid from which a recombinant enzyme is produced as a His-tagged enzyme. In

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this case, both EgtC and EgtE were successfully overproduced. To confirm whether the

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recombinant enzymes had the expected activity, we carried out in vitro experiments with the



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purified recombinant enzymes (Figure S4).9 Recombinant EgtC was incubated with

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enzymatically prepared γGC-HER. After 17 h of reaction, the product was analyzed by

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LC-ESI-MS. As shown in Figure 3, a new peak with m/z 333.23 was detected and Cys-HER

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formation was suggested. Based on HR-ESI-FT-MS analysis of the purified compound, the

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molecular formula of the product was determined to be C12H20O5N4S (m/z: [M+H]+ calculated for

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C12H21O5N4S+, 333.12272; observed, 333.12290), which corresponded to that of Cys-HER. The

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thus formed Cys-HER was purified (Supplementary methods 2) and used as a standard for

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quantitative analysis.

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By adding recombinant EgtE together with EgtC into the EgtB reaction mixture, we

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confirmed the formation of ERG by LC-ESI-MS analysis (Figure 4), showing that all the

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recombinant Egt enzymes possessed the expected activities.

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Simultaneous production of Egt Enzymes for ERG Production. To reconstruct the

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ERG-producing pathway in E. coli, we optimized the production conditions in a stepwise

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manner: HER, then γGC-HER, and finally ERG production (Figure 1 and 2).


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The first step was HER production. E. coli BW25113 harboring pCF1s-MsD (named strain

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ET1) was cultured in M9Y medium and 91 ± 2 mg/L HER (460 ± 10 µM) was produced in the

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culture broth after 48 h. We then carried out feeding experiments with L-His and L-Met, because

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amino acid biosynthesis in E. coli is strictly regulated by feedback inhibition and/or

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transcriptional repression. L-Met and L-His feeding increased the yield: 164 ± 3 mg/L HER (833

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± 15 µM) was produced after 48 h of cultivation (Figure 5). After this, L-His and L-Met feeding

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was employed in all in vivo production experiments.

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We next carried out in vivo coproduction of EgtD and EgtB for γGC-HER production.

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Transformants of E. coli BW25113 carrying both pCF1s-MsD and pQE1a-mMsB (strain ET2)

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were cultivated in the medium supplemented with L-His and L-Met. As Figure 6 shows, we

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confirmed 24 ± 1 mg/L γGC-HER (52 ± 2 µM) production after 24-h cultivation, indicating that

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EgtB converted HER to γGC-HER using endogenous γGC in E. coli. However, accumulation of

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3-fold higher amounts of HER (110 ± 5 mg/L) than Cys-HER (36 ± 2 mg/L) was detected,

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suggesting that γGC-HER production was rate limiting.

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For simultaneous expression of all egt genes, egtC and egtE, both of which were expressed

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from the tac promoter of pQE1a, were recloned into plasmid pACYCDuet-1, which is compatible



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with pQE1a-Red and pCF1s-Red, to construct pAC1c-hMsC/hMsE (Figure 2 and Supplementary

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methods 1). The plasmid was successfully constructed and production of both enzymes in soluble

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forms in E. coli was confirmed by SDS-PAGE (Figure S6). The plasmid was introduced into

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strain ET2 to construct strain ET3 and recombinant enzyme production was examined. As Figure

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S7 shows, all the enzymes were produced in soluble forms. We then examined ERG production.

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Strain ET3 produced 19 ± 2 mg/L (83 ± 8 µM) ERG together with 73 ± 15 mg/L (370 ± 76 µM)

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HER in the culture broth after 72 h of cultivation (Figure 7 and Table 3). These results suggested

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that the EgtB-catalyzed reaction is a bottleneck.

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ERG Production and Improvement of Rate-limiting Steps. Considering that EgtB was

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overproduced in a soluble form in the producing strain ET3, and that it showed enough activity in

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in vitro experiments, we considered that insufficient supply of γGC, the substrate of EgtB, might

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cause the accumulation of HER. To test this hypothesis, overproduction of γGC synthetase was

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carried out. To produce γGC synthetase, the gshA gene from E. coli12 was cloned and inserted

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into pQE1a-mMsB to construct pQE1a-mMsB/EcA. Although MBP-fused EgtB and GshA were

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produced in soluble forms in E. coli BW25113 harboring the three plasmids (strain ET4) (Figure


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S8), ERG productivity was decreased compared to that of strain ET3, to 17 ± 1 mg/L (72 ± 2

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µM) after 72-h cultivation (Table 3). Comparing SDS-PAGE data for strains ET3 and ET4

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(Figures S7 and S8), soluble MBP-fused EgtB was found to be decreased in strain ET4. This

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could be the reason for the reduced productivity.

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We then employed another strategy to enhance the γGC supply. In ERG biosynthesis, γGC is

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used as a sulfur donor. In particular, L-Cys is a net sulfur donor, because the L-Glu moiety of

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γGC is released by EgtC. Therefore, reinforcement of L-Cys flux may be effective in enhancing

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γGC flux and ERG production. Since L-Cys addition into media was reported to be toxic to E.

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coli cells,14 we employed another strategy. We have been studying a L-Cys biosynthetic pathway

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in E. coli and demonstrated that thiosulfate (S2O32–) was a better sulfur source than sulfate

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(SO42–) for high L-Cys production.10 We therefore fed thiosulfate into the growth media. When

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strain ET3 was cultured in M9Y supplemented with

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productivity was increased to 24 ± 4 mg/L (104 ± 17 µM) (Table 3), indicating that

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reinforcement of L-Cys flux was very effective in enhancing the ERG production.

L-Met, L-His,

and thiosulfate, ERG

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In conclusion, to establish a reliable and practical method for ERG supply, fermentative ERG

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production by microorganisms was investigated. We heterologously overexpressed genes



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egtBCDE of M. smegmatis in E. coli and succeeded in the production of ERG (19 mg/L).

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Reinforcement of the γGC supply by feeding of thiosulfate, a suitable sulfur donor for high L-Cys

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production, resulted in higher ERG production (24 mg/L). Considering the reported ERG

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contents of mushrooms were from 0.15 to 7.27 mg/g dry weight,7 our system might become an

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alternative method for ERG supply. However, significant amounts of HER still accumulated,

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suggesting that more supply of γGC and L-Cys by metabolic engineering and use of another EgtB

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with higher activity are indispensable for high-level production of ERG.

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ABBREVIATIONS USED ERG,

ergothioneine;

HER,

hercynine;

γ-glutamylcysteine;

γGC-HER,

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hercynyl-γ-glutamylcysteine-sulfoxide;

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S-adenosylmethionine; SAH, S-adenosylhomocysteine; L-histidine, L-His; L-glutamate, L-Glu;

249

L-cysteine, L-Cys; L-methionine, L-Met;

250

mass

251

dodecylsulfate-polyacrylamide gel electrophoresis, SDS-PAGE.

spectrometry,

Cys-HER,

γGC,

hercynylcysteine-sulfoxide;

SAM,

high-resolution electrospray ionization Fourier transform

HR-ESI-FT-MS;

maltose

binding

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protein,

MBP;

sodium

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ACKNOWLEDGMENTS

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We are grateful to Professor Tsutomu Sato (Graduate School of Science and Technology,

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Niigata University, Japan) for providing M. smegmatis genomic DNA. This work was supported

256

by the Technology Research Promotion Program for Agriculture, Forestry, Fisheries and Food

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Industry (26027AB) from MAFF, Japan (to I.O.). We thank James Allen, DPhil, from Edanz

258

Group (www.edanzediting.com) for editing a draft of this manuscript.

259 260

SUPPORTING INFORMATION

261 262 263

The Supporting information is available free of charge on the ACS Publications website at xxx. Supplementary methods. Figure S1. Vectors pQE1a-Red and pCF1s-Red. Figure S2.

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SDS-PAGE analysis of EgtD production. Figure S3. LC-ESI-MS analysis of EgtD reaction

265

products. Figure S4. SDS-PAGE analysis of purified recombinant EgtB, EgtC, and EgtE. Figure

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S5. LC-ESI-MS analysis of EgtB reaction products. Figure S6. SDS-PAGE analysis of

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recombinant EgtC and EgtE production. Figure S7. SDS-PAGE analysis of production of



268

recombinant Egt enzymes in strain ET3. Figure S8. SDS-PAGE analysis of production of

269

recombinant Egt enzymes and GshA in strain ET4. Table S1. Primers used in this study.

270 271

REFERENCES

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(1) Genghof, D.S. Biosynthesis of ergothioneine and hercynine by fungi and Actinomycetales. J.

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Bacteriol. 1970, 103, 475–478.

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(2) Pfeiffer, C.; Bauer, T.; Surek, B.; Schömig, E.; Gründemann, D. Cyanobacteria produce high

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levels of ergothioneine. Food Chem. 2011, 129, 1766–1769.

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(3) Pluskal, T.; Ueno M.; Yanagida, M. Genetic and metabolomic dissection of the ergothioneine

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and selenoneine biosynthetic pathway in the fission yeast, S. pombe, and construction of an

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overproduction system. PLoS One, 2014, 9, e97774.

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(4) Gründemann, D.; Harlfinger, S.; Golz, S.; Geerts, A.; Lazar, A.; Berkels, R.; Jung, N.;

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Rubbert, A.; Schömig, E. Discovery of the ergothioneine transporter. Proc. Natl. Acad. Sci. USA,

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2005, 102, 5256–5261.

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(5) Cheah, I.K.; Halliwell, B. Ergothioneine; antioxidant potential, physiological function and role

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in disease. Biochim. Biophys. Acta, 2012, 1822, 784–793.


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(6) Aruoma, O.I.; Coles, L.S.; Landes, B.; Repine, J.E. Functional benefits of ergothioneine and

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Med., 2012, 54, S4–S8.

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(7) Kalaras, M.D.; Richie J.P.; Calcagnotto, A.; Beelman, R.B. Mushrooms: A rich source of the

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antioxidants ergothioneine and glutathione. Food Chem., 2017, 233, 429–433

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(8) Genghof, D.S.; Van Damme, O. Biosynthesis of ergothioneine and hercynine by mycobacteria.

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J. Bacteriol. 1964, 87, 852–862.

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(9) Seebeck, F.P. In vitro reconstitution of Mycobacterial ergothioneine biosynthesis. J. Am.

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Chem. Soc., 2010, 132, 6632–6633.

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(10) Kawano, Y.; Onishi, F.; Shiroyama, M.; Miura, M.; Tanaka, N.; Oshiro, S.; Nonaka, G.;

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Nakanishi, T.; Ohtsu, I. Improved fermentative L-cysteine overproduction by enhancing a newly

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identified thiosulfate assimilation pathway in Escherichia coli. Appl. Microbiol. Biotechnol., 2017,

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(11) Jones, G.W.; Doyle, S.; Fitzpatrick, D.A. The evolutionary history of the genes involved in the

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biosynthesis of the antioxidant ergothioneine. Gene, 2014, 549, 161–170.



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(12) Watanabe, K.; Yamano, Y.; Murata, K.; Kimura, A. The nucleotide sequence of the gene for

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γ-glutamylcysteine synthetase of Escherichia coli. Nucleic Acids Res., 1986, 14, 4393–4400.

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(13) Vit, A.; Misson, L.; Blankenfeldt, W.; Seebeck, F.P. Ergothioneine biosynthetic

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methyltransferase EgtD reveals the structural basis of aromatic amino acid betaine biosynthesis.

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ChemBioChem, 2015, 16, 119–125.

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(14) Kari, C.; Nagy, Z.; Kovács, P.; Hernádi, F. Mechanism of the growth inhibitory effect of

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cysteine on Escherichia coli. J. Gen. Microbiol., 1971, 68, 349–356.

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FIGURE CAPTIONS

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Figure 1. ERG biosynthetic pathway.

309

Figure 2. Plasmids for ERG production.

310

Figure 3. LC-ESI-MS analysis of EgtC reaction products.

311

(a) Traces at 250 nm of reaction products. The reaction (40 µL) was carried out by adding

312

purified recombinant EgtC (3.9 µM) (i) or boiled EgtC (ii) to the EgtB reaction solution at 25°C

313

for 17 h.

314

(b) MS spectrum of EgtC reaction product (ESI positive mode).


Page 20 of 41

Page 21 of 41


315

Figure 4. LC-ESI-MS analysis of EgtE reaction products.

316

(a) Traces at 250 nm of ERG standard (i) and reaction products (ii). The reaction (40 µL) was

317

carried out by adding purified recombinant EgtE (2.5 µM) and EgtC (3.9 µM) (ii) to boiled

318

supernatant of EgtB reaction mixture containing 2 mM dithiothreitol at 25°C for 17 h.

319

(b) MS spectra of ERG standard (i) and EgtE reaction product (ii) (ESI positive mode).

320

Figure 5. Culture profiles of strain ET1. E. coli BW25113 harboring pCF1s-MsD (strain ET1)

321

was cultured in M9Y medium supplemented with L-His and L-Met. After 3 h cultivation, 0.5 mM

322

of IPTG was added into the medium. Data are presented as mean values with standard errors

323

from three independent experiments.

324

Figure 6. Culture profiles of strain ET2. E. coli BW25113 harboring pCF1s-MsD and

325

pQE1a-mMsB (strain ET2) was cultured in M9Y medium supplemented with L-His and L-Met.

326

After 3 h cultivation, 0.5 mM of IPTG was added into the medium. Data are presented as mean

327

values with standard errors from three independent experiments.

328

Figure 7. Culture profiles of strain ET3. E. coli BW25113 harboring pCF1s-MsD,

329

pQE1a-mMsB, and pAC1c-hMsC/hMsE (strain ET3) was cultured in M9Y medium

330

supplemented with L-His and L-Met. After 3 h cultivation, 0.5 mM of IPTG was added into the



331

medium. Data are presented as mean values with standard errors from three independent

332

experiments.

333


Page 22 of 41

Page 23 of 41


334

Table 1. Bacterial strains used in this study.

335

Strains

336

M. smegmatis JCM6386 ERG producer

337

E. coli

Descriptions

Source JCMa

338

XL1-Blue

hsdR17, recA1, endA1, gyrA96, thi-1, supE44, relA1, lac[F’, proAB, lacIqZ∆M15, Tn10(TcR)] Nippon Gene

339

BL21(DE3)

F−, ompT hsdSB(rB− mB−) gal dcm (DE3)

Merck

340

BW25113

rrnB3 ∆lacZ4787 hsdR514 ∆(araBAD)567 ∆(rhaBAD)568 rph-1

NIGb

341

ET1

BW25113 harboring pCF1s-MsD

This study

342

ET2

BW25113 harboring pCF1s-MsD, pQE1a-mMsB

This study

343

ET3

BW25113 harboring pCF1s-MsD, pQE1a-mMsB, pAC1c-hMsC/hMsE

This study

344

ET4

BW25113 harboring pCF1s-MsD, pQE1a-mMsB/EcA, pAC1c-hMsC/hMsE

This study

345

a

346

b

Japan Collection of Microorganisms, Riken BioResource Center. National Institute of Genetics

347 348



Page 24 of 41

349

Table 2. Plasmids used in this study.

350

Plasmids

Description

Source

351

pQE1a-Red

Protein production plasmid, tac promoter, ColE1 ori, ApR

Lab stock

352

pCF1s-Red

Protein production plasmid, tac promoter, CDF ori, SmR

Lab stock

353

pET-21a

Protein production plasmid, T7 promoter, pBR322 ori, ApR

Merck

354

pACYCDuet-1

Protein production plasmid, T7 promoter, p15A ori, CmR

Merck

355

pQE1a-MsB

pQE1a-Red derivative, production of EgtB

This study

356

pQE1a-mMsB

pQE1a-MsB derivative, production of MBP-fused EgtB

This study

357

pQE1a-mMsB/EcA

pQE1a-mMsB derivative, coproduction of MBP-fused EgtB and GshA

This study

358

pQE1a-MsC

pQE1a-Red derivative, production of EgtC

This study

359

pQE1a-hMsC

pQE1a-MsC derivative, production of 6×His-tagged EgtC

This study

360

pQE1a-MsE

pQE1a-Red derivative, production of EgtE

This study

361

pQE1a-hMsE

pQE1a-Red derivative, production of 6×His-tagged EgtE

This study

362

pAC1c-hMsC/hMsE

pACYCDuet-1 derivative, coproduction of 6×His-tagged EgtC and EgtE

This study

363

pCF1s-MsD

pCF1s-Red derivative, production of EgtD

This study

364 365


Page 25 of 41


366

Table 3. Culture profiles of ET3 and ET4 strains.

367

Strains

368

OD

HER

γGC-HER

Cys-HER

ERG

[–]

[mg/L]

[mg/L]

[mg/L]

[mg/L]

ND

10 ± 2

19 ± 2

1±1

9±1

17 ± 1

ND

9±0

24 ± 4

369

ET3a

10.1 ± 0.5

73 ± 15

370

ET4a

8.7 ± 0.2

121 ± 12

371

ET3b

11.1 ± 0.5

48 ± 17

372 373

ND, not detected.

374

Data after 72 h cultivation are presented as mean values with standard error from three independent experiments.

375

a

ET3 and ET4 were cultured in M9Y media supplemented with L-His and L-Met.

376

b

ET3 was cultured in M9Y media supplemented with L-His, L-Met, and thiosulfate.

377



N

NH L -His

H 2N

3 x L -Met + 3 x ATP

COOH

3 x SAM

HER production

EgtD 3 x SAH N

NH

HER N

EgtA/ + L-Glu GshA + ATP

L-Cys

COO

γGC + O 2

EgtB

COOH

H N

H 2N

γGC-HER production

COOH O

O

S

γGC-HER N

NH

N COO

EgtC

L -Glu H 2N

COOH

ERG production

O S N

NH

Cys-HER

N COO

EgtE

Pyruvate + NH 3, [O] S HN

NH

ERG N

378

COO

379 380

Figure 1. ERG biosynthetic pathway.

381


Page 26 of 41

Page 27 of 41


382 383 384

Figure 2. Plasmids for ERG production.

385



386 387 388

Figure 3. LC-ESI-MS analysis of EgtC reaction products.

389

(a) Traces at 250 nm of reaction products. The reaction (40 µL) was carried out by adding

390

purified recombinant EgtC (3.9 µM) (i) or boiled EgtC (ii) to the EgtB reaction solution at 25°C

391

for 17 h.

392

(b) MS spectrum of EgtC reaction product (ESI positive mode).

393 394


Page 28 of 41

Page 29 of 41


395 396 397

Figure 4. LC-ESI-MS analysis of EgtE reaction products.

398

(a) Traces at 250 nm of ERG standard (i) and reaction products (ii). The reaction (40 µL) was

399

carried out by adding purified recombinant EgtE (2.5 µM) and EgtC (3.9 µM) (ii) to boiled

400

supernatant of EgtB reaction mixture containing 2 mM dithiothreitol at 25°C for 17 h.

401

(b) MS spectra of ERG standard (i) and EgtE reaction product (ii) (ESI positive mode).

402



200

Page 30 of 41

15 HER

Conc. [mg/L]

10 100

OD [–]

OD

150

5 50

0

0 0

24

48

Times [h]

403 404 405

Figure 5. Culture profiles of strain ET1. E. coli BW25113 harboring pCF1s-MsD (strain ET1)

406

was cultured in M9Y medium supplemented with L-His and L-Met. After 3 h cultivation, 0.5 mM

407

of IPTG was added into the medium. Data are presented as mean values with standard errors

408

from three independent experiments.

409


Page 31 of 41


120

15 HER

100 10 Cys-HER

60

OD

40

OD [–]

Conc. [mg/L]

γGC-HER

80

5

20 0

0 0

410 411

24

48

Times [h]

412

Figure 6. Culture profiles of strain ET2. E. coli BW25113 harboring pCF1s-MsD and

413

pQE1a-mMsB (strain ET2) was cultured in M9Y medium supplemented with L-His and L-Met.

414

After 3 h cultivation, 0.5 mM of IPTG was added into the medium. Data are presented as mean

415

values with standard errors from three independent experiments.

416



Conc. [mg/L]

80

HER

γGC-HER

Cys-HER

ERG

15

OD

10

60 40

OD [–]

100

Page 32 of 41

5 20 0

0 0

24

48

72

Times [h]

417 418 419

Figure 7. Culture profiles of strain ET3. E. coli BW25113 harboring pCF1s-MsD,

420

pQE1a-mMsB, and pAC1c-hMsC/hMsE (strain ET3) was cultured in M9Y medium

421

supplemented with L-His and L-Met. After 3 h cultivation, 0.5 mM of IPTG was added into the

422

medium. Data are presented as mean values with standard errors from three independent

423

experiments.

424


Page 33 of 41

425


TOC Graphic

426 N

NH

COOH

EgtB

H N

H 2N

COOH O

O

N

S

GshA

γ-Glu-Cys

COO

N

NH

Cys + Glu 3 × Met

3 × SAM

N H 2N

EgtD

O

S N H 2N

427

NH

HN

EgtC

S NH

N

EgtE

N COOH

COO

COOH

N

COO

Heterologous and high production of ergothioneine in E. coli COO

NH



Figure 1. ERG biosynthetic pathway. 271x398mm (600 x 600 DPI)

ACS Paragon Plus Environment

Page 34 of 41

Page 35 of 41


egtD

HER production

pCF1s-MsD lacI

CloDF13 ori

MBP

γGC-HER production

egtB

MBP

ColE1 ori

ApR

lacI

ColE1 ori

ApR

tac promoter

T7 terminator

His-tag

rrnB terminator

egtC

egtE

pAC1c-hMsE/hMsC lacI

gshA

egtB

pQE1a-mMsB/EcA

pQE1a-mMsB lacI

ERG production

SmR

p15A ori

CmR


MBP MBP-tag

cspA terminator


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 purified recombinant EgtC (3.9 µM) (i) or boiled EgtC (ii) to the EgtB reaction solution at 25°C for 17 h. (b) MS spectrum of EgtC reaction product (ESI positive mode).

68x21mm (600 x 600 DPI)


Page 36 of 41

Page 37 of 41


Figure 4. LC-ESI-MS analysis of EgtE reaction products. (a) Traces at 250 nm of ERG standard (i) and reaction products (ii). The reaction (40 µL) was carried out by adding purified recombinant EgtE (2.5 µM) and EgtC (3.9 µM) (ii) to boiled supernatant of EgtB reaction mixture containing 2 mM dithiothreitol at 25°C for 17 h. (b) MS spectra of ERG standard (i) and EgtE reaction product (ii) (ESI positive mode). 80x35mm (600 x 600 DPI)


15of 41 Journal of Agricultural and Food Chemistry Page 38 HER OD

150

10 100 5 50 0


0

24

Times [h]

0 48

OD [–]

Conc. [mg/L]

200

Page 120 39 of 41 Journal of Agricultural and Food Chemistry HER

15

γGC-HER

80 60

10

Cys-HER OD

40

5

20 0


0

24

Times [h]

0 48

OD [–]

Conc. [mg/L]

100

Conc. [mg/L]

80

15of 41 Journal of Agricultural and Food Chemistry Page 40 HER γGC-HER Cys-HER

ERG

OD

10

60 40

5

20 0 0


24

48

Times [h]

0 72

OD [–]

100

Page 41 of 41


TOC graphic 48x27mm (600 x 600 DPI)


Heterologous and High Production of ... - ACS Publications

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