Co-augmentation of a transport gene mfsT1 in Mycolicibacterium neoaurum with genome engineering to enhance ergothioneine production.

Ergothioneine (EGT) is a rare thiohistidine derivative with exceptional antioxidant properties. The blood level of EGT is considered highly reliable predictors for cardiovascular diseases and mortality, yet animals lack the ability to synthesize this compound. Free plasmids have been previously used to overexpress genes involved in the EGT biosynthetic pathway of Mycolicibacterium neoaurum. Here, we tentatively introduced a putative transporter gene mfsT1 into high-copy plasmids and sharply increased the ratio of extracellular EGT concentration from 18.7% to 44.9%. Subsequently, an additional copy of egtABCDE, hisG, and mfsT1 was inserted into the genome with a site-specific genomic integration tool of M. neoaurum, leading a 2.7 times increase in EGT production. Co-enhancing the S-adenosyl-L-methionine regeneration pathway, or alternatively, the integration of three copies of egtABCDE, hisG and mfsT1 into the genome further increased the total EGT yield by 16.1% (64.6 mg/L) and 21.7% (67.7 mg/L), respectively. After 168-h cultivation, the highest titer reached 85.9 mg/L in the latter strain with three inserted copies. This study provided a solid foundation for genome engineering to increase the production of EGT in M. neoaurum.

Adhesive Ergothioneine Hyaluronate Gel Protects against Radiation Gastroenteritis by Alleviating Apoptosis, Inflammation, and Gut Microbiota Dysbiosis.

Radiation gastroenteritis represents one of the most prevalent and hazardous complications of abdominopelvic radiotherapy, which not only severely reduces patients' life quality but also restricts radiotherapy efficacy. However, there is currently no clinically available oral radioprotector for this threatening disease due to its complex pathogenesis and the harsh gastrointestinal environment. To this end, this study developed a facile but effective oral radioprotector, ergothioneine hyaluronate (EGT@HA) gel, protecting against radiation gastroenteritis by synergistically regulating oxidative stress, inflammation, and gut microbiota. In vitro and cellular experiments verified the chemical stability and free radical scavenging ability of EGT and its favorable cellular radioprotective efficacy by inhibiting intracellular reactive oxidative species (ROS) generation, DNA damage, mitochondrial damage, and apoptosis. At the in vivo level, EGT@HA with prolonged gastrointestinal residence mitigated radiation-induced gastrointestinal tissue injury, apoptosis, neutrophil infiltration, and gut flora dysbiosis. For the first time, this work investigated the protective effects of EGT@HA gel on radiation gastroenteritis, which not only hastens the advancement of the novel gastrointestinal radioprotector but also provides a valuable gastrointestinal radioprotection paradigm by synergistically modulating oxidative stress, inflammation, and gut microbiota disturbance.

[Construction and optimization of ergothioneine-producing Escherichia coli].

Ergothioneine (ERG) is a natural antioxidant that has been widely used in the fields of food, medicine and cosmetics. Compared with traditional plant extraction and chemical synthesis approaches, microbial synthesis of ergothioneine has many advantages, such as the short production cycle and low cost, and thus has attracted intensive attention. In order to engineer an ergothioneine high-yielding Escherichia coli strain, the ergothioneine synthesis gene cluster egtABCDE from Mycobacterium smegmatis and egt1 from Schizosaccharomyces pombe were introduced into E. coli BL21(DE3) to generate a strain E1-A1 harboring the ergothioneine biosynthesis pathway. As a result, (95.58±3.2) mg/L ergothioneine was produced in flask cultures. To further increase ergothioneine yield, the relevant enzymes for biosynthesis of histidine, methionine, and cysteine, the three precursor amino acids of ergothioneine, were overexpressed. Individual overexpression of serAT410STOP and thrA resulted in an ergothioneine titer of (134.83±4.22) mg/L and (130.26±3.34) mg/L, respectively, while co-overexpression of serAT410STOP and thrA increased the production of ergothioneine to (144.97±5.40) mg/L. Eventually, by adopting a fed-batch fermentation strategy in 3 L fermenter, the optimized strain E1-A1-thrA-serA* produced 548.75 mg/L and 710.53 mg/L ergothioneine in glucose inorganic salt medium and rich medium, respectively.

Characterization and engineering control of the effects of reactive oxygen species on the conversion of sterols to steroid synthons in Mycobacterium neoaurum.

The conversion of sterols to steroid synthons by engineered mycobacteria comprises one of the basic ways for the production of steroid medications in the pharmaceutical industry. Here, we revealed that high amounts of reactive oxygen species (ROS) generate during the conversion process of sterols, which impairs the cell viability of mycobacterial cells and thus hinders the conversion of sterols to steroid synthons. Accordingly, the endogenous antioxidants for detoxifying ROS in mycobacteria, ROS scavenging enzymes and low molecular weight thiols, were examined. The results revealed that three antioxidants, catalase (CAT), mycothiol (MSH), and ergothioneine (EGT), demonstrated efficacy toward neutralizing the excessive ROS produced during sterol metabolism. CAT overexpression or MSH or EGT augmentation enhanced the conversion of phytosterols to 22-hydroxy-23,24-bisnorchol-4-ene-3-one (4-HBC) by 18.9%, 23.8%, and 32.1%, respectively, and also enhanced the cell viability, indicating the benefits of these antioxidants in reducing ROS-induced stress. Further combinatorial augmentation of CAT, MSH, and EGT demonstrated enhanced effects toward intracellular ROS scavenging, resulting in 54.2% greater cell viability and 47.5% enhancement in 4-HBC production. These findings indicated that the excessive ROS induces cell stress, in turn limiting the conversion of sterols, whereas neutralization of the excessive ROS by combined control of CAT, MSH, and EGT serves as an effective strategy to boost the conversion productivity of sterols to steroid synthons.

Ergothioneine production with Aspergillus oryzae.

To establish a reliable and practical ergothioneine (ERG) supply, we employed fermentative ERG production using Aspergillus oryzae, a fungus used for food production. We heterologously overexpressed the egt-1 and -2 genes of Neurospora crassa in A. oryzae and succeeded in producing ERG (231.0 mg/kg of media, which was 20 times higher than the wild type). Abbreviations: ERG: ergothioneine; HER: hercynine; Cys-HER: hercynylcysteine-sulfoxide; SAM: S-adenosylmethionine; SAH: S-adenosylhomocysteine; l-His: l-histidine; l-Cys: l-cysteine; LC-ESI-MS: liquid chromatography-electrospray ionization-mass spectrometry.

Generation and characterization of thiol-deficient Mycobacterium tuberculosis mutants.

Mycothiol (MSH) and ergothioneine (ERG) are thiols able to compensate for each other to protect mycobacteria against oxidative stress. Gamma-glutamylcysteine (GGC), another thiol and an intermediate in ERG biosynthesis has detoxification abilities. Five enzymes are involved in ERG biosynthesis, namely EgtA, EgtB, EgtC, EgtD and EgtE. The role of these enzymes in the production of ERG had been unclear. On the other hand, the enzyme MshA is known to be essential for MSH biosynthesis. In this manuscript, we describe the raw data of the generation and characterization of Mycobacterium tuberculosis (M.tb) mutants harbouring a deletion of the gene coding for each of these enzymes, and the raw data of the phenotypic characterization of the obtained thiol-deficient M.tb mutants. High throughput screening (HTS) of off-patent drugs and natural compounds revealed few compounds that displayed a higher activity against the thiol-deficient mutants relative to the wild-type strain. The mode of action of these drugs was further investigated. Raw data displaying these results are described here.

The functional interplay of low molecular weight thiols in Mycobacterium tuberculosis.

BACKGROUND: Three low molecular weight thiols are synthesized by Mycobacterium tuberculosis (M.tb), namely ergothioneine (ERG), mycothiol (MSH) and gamma-glutamylcysteine (GGC). They are able to counteract reactive oxygen species (ROS) and/or reactive nitrogen species (RNS). In addition, the production of ERG is elevated in the MSH-deficient M.tb mutant, while the production of MSH is elevated in the ERG-deficient mutants. Furthermore, the production of GGC is elevated in the MSH-deficient mutant and the ERG-deficient mutants. The propensity of one thiol to be elevated in the absence of the other prompted further investigations into their interplay in M.tb. METHODS: To achieve that, we generated two M.tb mutants that are unable to produce ERG nor MSH but are able to produce a moderate (ΔegtD-mshA) or significantly high (ΔegtB-mshA) amount of GGC relative to the wild-type strain. In addition, we generated an M.tb mutant that is unable to produce GGC nor MSH but is able to produce a significantly low level of ERG (ΔegtA-mshA) relative to the wild-type strain. The susceptibilities of these mutants to various in vitro and ex vivo stress conditions were investigated and compared. RESULTS: The ΔegtA-mshA mutant was the most susceptible to cellular stress relative to its parent single mutant strains (ΔegtA and ∆mshA) and the other double mutants. In addition, it displayed a growth-defect in vitro, in mouse and human macrophages suggesting; that the complete inhibition of ERG, MSH and GGC biosynthesis is deleterious for the growth of M.tb. CONCLUSIONS: This study indicates that ERG, MSH and GGC are able to compensate for each other to maximize the protection and ensure the fitness of M.tb. This study therefore suggests that the most effective strategy to target thiol biosynthesis for anti-tuberculosis drug development would be the simultaneous inhibition of the biosynthesis of ERG, MSH and GGC.

Gamma-glutamylcysteine protects ergothioneine-deficient Mycobacterium tuberculosis mutants against oxidative and nitrosative stress.

Mycobacterium tuberculosis (M.tb.), the causative agent of tuberculosis (TB), cannot synthesize GSH, but synthesizes two major low molecular weight thiols namely mycothiol (MSH) and ergothioneine (ERG). Gamma-glutamylcysteine (GGC), an intermediate in GSH synthesis, has been implicated in the protection of lactic acid bacteria from oxidative stress in the absence of GSH. In mycobacteria, GGC is an intermediate in ERG biosynthesis, and its formation is catalysed by EgtA (GshA). GGC is subsequently used by EgtB in the formation of hercynine-sulphoxide-GGC. In this study, M.tb. mutants harbouring unmarked, in-frame deletions in each of the fives genes involved in ERG biosynthesis (egtA, egtB, egtC, egtD and egtE) or a marked deletion of the mshA gene (required for MSH biosynthesis) were generated. Liquid chromatography tandem mass spectrometry analyses (LC-MS) revealed that the production of GGC was elevated in the MSH-deficient and the ERG-deficient mutants. The ERG-deficient ΔegtB mutant which accumulated GGC was more resistant to oxidative and nitrosative stress than the ERG-deficient, GGC-deficient ΔegtA mutant. This implicates GGC in the detoxification of reactive oxygen and nitrogen species in M.tb.

Organic cation transporter-mediated ergothioneine uptake in mouse neural progenitor cells suppresses proliferation and promotes differentiation into neurons.

The aim of the present study is to clarify the functional expression and physiological role in neural progenitor cells (NPCs) of carnitine/organic cation transporter OCTN1/SLC22A4, which accepts the naturally occurring food-derived antioxidant ergothioneine (ERGO) as a substrate in vivo. Real-time PCR analysis revealed that mRNA expression of OCTN1 was much higher than that of other organic cation transporters in mouse cultured cortical NPCs. Immunocytochemical analysis showed colocalization of OCTN1 with the NPC marker nestin in cultured NPCs and mouse embryonic carcinoma P19 cells differentiated into neural progenitor-like cells (P19-NPCs). These cells exhibited time-dependent [(3)H]ERGO uptake. These results demonstrate that OCTN1 is functionally expressed in murine NPCs. Cultured NPCs and P19-NPCs formed neurospheres from clusters of proliferating cells in a culture time-dependent manner. Exposure of cultured NPCs to ERGO or other antioxidants (edaravone and ascorbic acid) led to a significant decrease in the area of neurospheres with concomitant elimination of intracellular reactive oxygen species. Transfection of P19-NPCs with small interfering RNA for OCTN1 markedly promoted formation of neurospheres with a concomitant decrease of [(3)H]ERGO uptake. On the other hand, exposure of cultured NPCs to ERGO markedly increased the number of cells immunoreactive for the neuronal marker ßIII-tubulin, but decreased the number immunoreactive for the astroglial marker glial fibrillary acidic protein (GFAP), with concomitant up-regulation of neuronal differentiation activator gene Math1. Interestingly, edaravone and ascorbic acid did not affect such differentiation of NPCs, in contrast to the case of proliferation. Knockdown of OCTN1 increased the number of cells immunoreactive for GFAP, but decreased the number immunoreactive for ßIII-tubulin, with concomitant down-regulation of Math1 in P19-NPCs. Thus, OCTN1-mediated uptake of ERGO in NPCs inhibits cellular proliferation via regulation of oxidative stress, and also promotes cellular differentiation by modulating the expression of basic helix-loop-helix transcription factors via an unidentified mechanism different from antioxidant action.

Ergothioneine is a secreted antioxidant in Mycobacterium smegmatis.

Ergothioneine (ERG) and mycothiol (MSH) are two low-molecular-weight thiols synthesized by mycobacteria. The role of MSH has been extensively investigated in mycobacteria; however, little is known about the role of ERG in mycobacterial physiology. In this study, quantification of ERG at various points in the growth cycle of Mycobacterium smegmatis revealed that a significant portion of ERG is found in the culture media, suggesting that it is actively secreted. A mutant of M. smegmatis lacking egtD (MSMEG_6247) was unable to synthesize ERG, confirming its role in ERG biosynthesis. Deletion of egtD from wild-type M. smegmatis and an MSH-deficient mutant did not affect their susceptibility to antibiotics tested in this study. The ERG- and MSH-deficient double mutant was significantly more sensitive to peroxide than either of the single mutants lacking either ERG or MSH, suggesting that both thiols play a role in protecting M. smegmatis against oxidative stress and that ERG is able to partly compensate for the loss of MSH.

Domain-specific fluorescence resonance energy transfer (FRET) sensors of metallothionein/thionein.

Each of the two domains of mammalian metallothioneins contains a zinc-thiolate cluster. Employing site-directed mutagenesis and chemical modification, fluorescent probes were introduced into human metallothionein (isoform 2) with minimal perturbations of the structures of these clusters. The resulting FRET (fluorescence resonance energy transfer) sensors are specific for each domain. The design and construction of a sensor for the alpha-domain cluster is based on a FRET pair where a C-terminally added tryptophan serves as the donor for a fluorescence acceptor attached to a free cysteine in the linker region between the two domains. Molecular modeling studies and steady-state fluorescence polarization anisotropy measurements suggest unrestricted motion of the tryptophan donor, but limited motion of the AEDANS ([[(amino)ethyl]amino]naphthalene-1-sulfonic acid) acceptor, putting constraints on the use of the alpha-domain sensor with this FRET pair as a spectroscopic ruler. The fluorescent metallothioneins allow distance measurements during binding and removal of metals in the individual domains. The overall dimensions of the apoprotein, thionein, for which no structural information is available, do not seem to be significantly different from those of the holoprotein. The single- and double-labeled fluorescent metallothioneins overcome a longstanding impediment in studies of the function of this protein, namely its lack of intrinsic probe characteristics.