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

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.

Keywords: Saccharomyces cerevisiae; ergothioneine; medium optimization; metabolic engineering; nutraceutical; yeast.

Figure 1

Pathways toward ergothioneine biosynthesis in bacteria and fungi.

Figure 2

Ergothioneine production in strains with different ERG pathway combinations on three variations of synthetic complete medium: (A) AA: 20 g/l glucose and 1 g/l of each L-His, L-Met, and L-Cys, (B) Glu: 40 g/l glucose, (C) FiT: 60 g/l EnPump substrate for slow glucose release to simulate fed-batch conditions.

Figure 3

Ergothioneine production by yeast strains expressing human ERG transporter SLC22A4X or a putative ERG transporter from M. smegmatis on three variations of synthetic complete medium: (A) AA: 20 g/l glucose and 1 g/l of each L-His, L-Met, and L-Cys, (B) Glu: 40 g/l glucose, (C) FiT: 60 g/l EnPump substrate for slow glucose release to simulate fed-batch conditions, (D) Microscope images of yeast cells with ergothioneine transporters linked to GFP. To the left are Brightfield images and to the right GFP images. Top panels; ST8461, ergothioneine producing strain without transporter. Middle-top panels, ST8921, ergothioneine producing strain with a putative transporter from Mycobacterium smegmatis linked to GFP at the N-terminus. Middle panels, ST8922, ergothioneine producing strain with a putative transporter from Mycobacterium smegmatis linked to GFP at the C-terminus. Middle-bottom panels, ST8923, ergothioneine producing strain with a transporter from Homo sapiens linked to GFP at the C-terminus. Bottom panels, ST8924, ergothioneine producing strain with a transporter from Homo sapiens linked to GFP at the N-terminus.

Figure 4

The effect of gene knock-outs linked to nitrogen metabolism on the production of ergothioneine in the production strain with the MsErgT transporter in different media. Genomic alterations are shown as well. (A) Glu: SC + 40 g/l glucose (B) FiT: SC + 60 g/l EnPump substrate, 0.6% reagent A.

Figure 5

Production of ergothioneine over time in the production strain with or without transporter in different media compositions. (A,D) Glu: SC + 40 g/l glucose, (B,E) AA: SC + 40 g/l glucoe + 1 g/l His/Met/Cys, (C,F) AA2: SC + 40 g/l glucose + 2 g/l His/Cys/Met.

Figure 6

Median concentration of ergothionine produced after 24 and 48 h. The values are additive. Open bars represent values obtained after 24 h while the closed bars represent values obtained after 48 h.

Figure 7

The effect of the integration of a second copy of Egt enzymes on the production of ergothioneine in the high producing strain in different media. Genomic alterations are shown as well. (A) Glu: SC + 40 g/l glucose (B) FiT: SC + 60 g/l EnPump substrate, 0.6% reagent A.

Figure 8

Fed-batch cultivation of ERG-producing strain ST8927. The cultivations were performed in duplicate, the average values are shown. The error bars show standard deviations. The additions of minerals, trace metals, and/or vitamins are indicated by black arrows. At 60.5 h, we added 2 g (NH₄)₂SO₄, 0.5 g MgSO₄, 4 ml trace metals solution, and 2 ml vitamin solution, at 73.5 h we added 0.5 g MgSO₄, 4 ml trace metals solution 2 ml vitamin solution and at 75.5 h we added 2 g (NH₄)₂SO₄. At 60.5 and 75.5 h, 2 g (NH₄)₂SO₄ was added as a sterile 100 g/l solution. At 60.5 and 73.5 h, 0.5 g MgSO₄ was added as a sterile 50 g/l solution, while 4 ml sterile trace metals solution and 2 ml sterile vitamin solution were added.