Bioinformatic and Biochemical Characterizations of C–S Bond Formation and Cleavage Enzymes in the Fungus <i>Neurospora crassa</i> Ergothioneine Biosynthetic Pathway

Hu, Wentao; Song, Heng; Her, Ampon Sae; Bak, Daniel W.; Naowarojna, Nathchar; Elliott, Sean J.; Qin, Liqiang; Chen, Xiaoping; Liu, Pinghua

doi:10.1021/ol502596z

Cited by 73 publications

(123 citation statements)

References 37 publications

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“…Other bacteria, such as Chloracidobacterium thermophilum and most fungi utilize a cysteine specific type III or IV sulfoxide synthase, in a three-step pathway. [4][5][6][7][8][9] Given the functional importance of Tyr377 we were surprised to find that a large sub-class of EgtB homologs lack this residue. 11 Most of these enzymes are encoded by Proteobacteria but also occur in several species from other phyla (SI Table 1).…”

Section: Introductionmentioning

confidence: 99%

An Alternative Active Site Architecture for O₂ Activation in the Ergothioneine Biosynthetic EgtB from Chloracidobacterium thermophilum

et al. 2019

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Sulfoxide synthases are non-heme iron enzymes that catalyze oxidative carbon-sulfur bond formation between cysteine derivatives and N-a-trimethylhistidine as a key step in the biosynthesis of thiohistidines. The complex catalytic mechanism of this enzyme reaction has emerged as the controversial subject of several biochemical and computational studies. These studies all used the structure of the g-glutamyl cysteine utilizing sulfoxide synthase, MthEgtB from Mycobacterium thermophilum (EC 1.14.99.50), as a structural basis. To provide an alternative model system we have solved the crystal structure of CthEgtB from Chloracidobacterium thermophilum (EC 1.14.99.51) that utilizes cysteine as a sulfur donor. This structure reveals a completely different configuration of active site residues that are involved in oxygen binding and activation. Furthermore, comparison of the two EgtB structures enables a classification of all ergothioneine biosynthetic EgtBs into five sub-types, each characterized by unique active-site features. This active site diversity provides an excellent platform to examine the catalytic mechanism of sulfoxide synthases by comparative enzymology, but also raises the question as to why so many different solutions to the same biosynthetic problem have emerged.

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Section: Introductionmentioning

confidence: 99%

An Alternative Active Site Architecture for O₂ Activation in the Ergothioneine Biosynthetic EgtB from Chloracidobacterium thermophilum

et al. 2019

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“…The biosynthetic pathway in Neurospora crassa was recently reported (Figure 1). 9,10) Egt-1 is a bifunctional enzyme catalyzing successive reactions; the formation of hercynine (HER) with L-His and S-adenosylmethionine (SAM), followed by synthesis of hercynylcysteine-sulfoxide (Cys-HER) with HER, L-cysteine (L-Cys), and O2. Egt-2, a pyridoxal phosphate-dependent C-S lyase, catalyzes ERG formation from Cys-HER with concomitant release of pyruvate and ammonia as side-products.…”

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confidence: 99%

Ergothioneine production with Aspergillus oryzae

Takusagawa

Satoh

Ohtsu

et al. 2019

Bioscience, Biotechnology, and Biochemistry

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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).

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“…The fungal pathway for the biosynthesis of ERG only encompasses two enzymes compared to the five of the bacterial pathway, and eliminates the need for the use of glutamate and energy in the form of ATP. While NcEgt1 has been produced in E. coli for in vitro studies of the enzyme (Hu et al, 2014), to the best of our knowledge, the fungal pathway has to date not been used for ERG production in E. coli . As we have shown S. cerevisiae is able to produce ergothioneine using the fungal pathway, the more energetically efficient biosynthesis pathway of fungi could lead to better product yield.…”

Section: Discussionmentioning

confidence: 99%

Engineering the yeastSaccharomyces cerevisiaefor the production of L-(+)-ergothioneine

Hoek

Darbani

Zugaj

et al. 2019

Preprint

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20 cerevisiae, yeast, nutraceutical 21 22 Abbreviations: ERG L-(+)-ergothioneine, HCO S-(hercyn-2-yl)-L-cysteine S-oxide, PBS 23 phosphate-buffered saline, PI propidium iodide, PLP pyridoxal 5'-phosphate, SAM S-adenosyl-24 L-methionine 25 26Abstract 27 L-(+)-Ergothioneine is an unusual, naturally occurring antioxidant nutraceutical that has been 28 shown to help reduce cellular oxidative damage. Humans do not biosynthesise it, so can acquire 29 it only from their diet; it exploits a specific transporter (SLC22A4) for its uptake. ERG is 30 considered to be a nutraceutical and possible vitamin that is involved in the maintenance of 31 health, and seems to be at too low a concentration in several diseases in vivo. Ergothioneine is 32 thus a potentially useful dietary supplement. Present methods of commercial production rely on 33 extraction from natural sources or on chemical synthesis. Here we describe the engineering of 34 the baker's yeast Saccharomyces cerevisiae to produce ergothioneine by fermentation in defined 35 media. After integrating combinations of ERG biosynthetic pathways from different organisms, 36 we screened yeast strains for their production of ERG. The highest-producing strain was also 37 engineered with known ergothioneine transporters. The effect of amino acid supplementation of 38 the medium was investigated and the nitrogen metabolism of S. cerevisiae was altered by knock-39 out of TOR1 or YIH1. We also optimized the media composition using fractional factorial 40 methods. Our optimal strategy led to a titer of 598 ± 18 mg/L ergothioneine in fed-batch culture 41 in bioreactors. Because S. cerevisiae is a GRAS ('generally recognised as safe') organism that is 42 2 widely used for nutraceutical production, this work provides a promising process for the 43 biosynthetic production of ERG. 44 45

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Bioinformatic and Biochemical Characterizations of C–S Bond Formation and Cleavage Enzymes in the Fungus Neurospora crassa Ergothioneine Biosynthetic Pathway

Cited by 73 publications

References 37 publications

An Alternative Active Site Architecture for O₂ Activation in the Ergothioneine Biosynthetic EgtB from Chloracidobacterium thermophilum

An Alternative Active Site Architecture for O₂ Activation in the Ergothioneine Biosynthetic EgtB from Chloracidobacterium thermophilum

Ergothioneine production with Aspergillus oryzae

Engineering the yeastSaccharomyces cerevisiaefor the production of L-(+)-ergothioneine

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Bioinformatic and Biochemical Characterizations of C–S Bond Formation and Cleavage Enzymes in the Fungus Neurospora crassa Ergothioneine Biosynthetic Pathway

Cited by 73 publications

References 37 publications

An Alternative Active Site Architecture for O2 Activation in the Ergothioneine Biosynthetic EgtB from Chloracidobacterium thermophilum

An Alternative Active Site Architecture for O2 Activation in the Ergothioneine Biosynthetic EgtB from Chloracidobacterium thermophilum

Ergothioneine production with Aspergillus oryzae

Engineering the yeastSaccharomyces cerevisiaefor the production of L-(+)-ergothioneine

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An Alternative Active Site Architecture for O₂ Activation in the Ergothioneine Biosynthetic EgtB from Chloracidobacterium thermophilum

An Alternative Active Site Architecture for O₂ Activation in the Ergothioneine Biosynthetic EgtB from Chloracidobacterium thermophilum