Engineering the Yeast Saccharomyces cerevisiae for the Production of L-(+)-Ergothioneine.

Steven A Van Der Hoek,Behrooz Darbani,Irina Borodina,Karolina E Zugaj,Douglas B Kell,Jacqueline B Medina,Milica Randelovic,Bala Krishna Prabhala,Mathias Bernfried Biron

doi:10.3389/fbioe.2019.00262

Abstract

L-(+)-Ergothioneine (ERG) is an unusual, naturally occurring antioxidant nutraceutical that has been shown to help reduce cellular oxidative damage. Humans do not biosynthesise ERG, but acquire it from their diet; it exploits a specific transporter (SLC22A4) for its uptake. ERG is considered to be a nutraceutical and possible vitamin that is involved in the maintenance of health, and seems to be at too low a concentration in several diseases in vivo. Ergothioneine is thus a potentially useful dietary supplement. Present methods of commercial production rely on extraction from natural sources or on chemical synthesis. Here we describe the engineering of the baker's yeast Saccharomyces cerevisiae to produce ergothioneine by fermentation in defined media. After integrating combinations of ERG biosynthetic pathways from different organisms, we screened yeast strains for their production of ERG. The highest-producing strain was also engineered with known ergothioneine transporters. The effect of amino acid supplementation of the medium was investigated and the nitrogen metabolism of S. cerevisiae was altered by knock-out of TOR1 or YIH1. We also optimized the media composition using fractional factorial methods. Our optimal strategy led to a titer of 598 ± 18 mg/L ergothioneine in fed-batch culture in 1 L bioreactors. Because S. cerevisiae is a GRAS (“generally recognized as safe”) organism that is widely used for nutraceutical production, this work provides a promising process for the biosynthetic production of ERG.

Highlights

Ergothioneine (ERG) (2-mercaptohistidine trimethylbetaine, IUPAC name (2S)-3-(2-Thioxo2,3-dihydro-1H-imidazol-4-yl)-2-(trimethylammonio)propanoate) is a naturally occurring antioxidant that can be found universally in plants and mammals (Melville, 1959); it possesses a tautomeric structure, but is mainly present in the thione form at physiological pH (Figure 1)
The 16 yeast strains with different pathway variants were cultivated in three different media and the intra- and extracellular concentrations of ergothioneine were measured (Figure 2)
Ergothioneine is an antioxidant with many potential health benefits (Cheah and Halliwell, 2012; Ames, 2018; Halliwell et al, 2018)

Summary

Introduction

Ergothioneine (ERG) (2-mercaptohistidine trimethylbetaine, IUPAC name (2S)-3-(2-Thioxo2,3-dihydro-1H-imidazol-4-yl)-2-(trimethylammonio)propanoate) is a naturally occurring antioxidant that can be found universally in plants and mammals (Melville, 1959); it possesses a tautomeric structure, but is mainly present in the thione form at physiological pH (Figure 1). Ergothioneine was discovered in 1909 in the ergot fungus Claviceps purpurea (Tanret, 1909), and its structure was determined 2 years later (Barger and Ewins, 1911). Plants are thought to take ergothioneine excreted by fungi up from the soil (Audley and Tan, 1968; Tan and Audley, 1968) or through symbiotic relationships (Park et al, 2010; Guo et al, 2016)

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Conclusion