Summary: Researchers were able to produce 0.6 grams of ergothioneine, a natural amino acid that has promising effects on Alzheimer’s disease in animal studies, per liter of yeast broth via a small scale fermentation process.
Source: Technical University of Denmark
Ergothioneine is a natural amino acid with antioxidative properties. It prevents cellular stress, which can lead to brain diseases, neurological damage and cancer. In rats and roundworms, research shows that ergothioneine has promising effects in preventing neurodegenerative diseases such as dementia and Alzheimer’s. Also, it has been reported that patients suffering from neurodegenerative diseases have significantly lower blood levels of ergothioneine than others. These findings suggest that ergothioneine might have great potential as a vitamin to prevent or delay the onset of those diseases.
Currently, it is both complicated and expensive to produce ergothioneine with chemical synthesis. However, by engineering and optimising baker’s yeast, scientists from The Novo Nordisk Foundation Center for Biosustainability (DTU Biosustain) has for the first time exploited the potential of making ergothioneine in yeast in a bio-based fashion.
In a study published in Frontiers in Bioengineering and Biotechnology Journal, the researchers were able to produce 0.6 gram of ergothioneine pr. liter yeast broth in a small-scale fermentation process.
Too expensive for consumers
Because of today’s expensive chemical production routes, current market prices of ergothioneine are very high compared to vitamins such as vitamin C and vitamin D that also prevent certain diseases. Thus, one of the main goals for the scientists is to further optimise the production of ergothioneine to reach a higher yield, so it can be sold to the consumer at a much cheaper price in the future.
One of the main reasons for ergothioneine being so expensive at the moment is that the chemical process is costly and the yields fairly low. Furthermore, it has not been tested for its efficacy for the prevention or treatment of neurodegenerative diseases in humans yet. But since the safety assessment of ergothioneine has already been done, it is ‘simply’ a question of being able to produce enough.
Before the scientists were able to produce ergothioneine in a biobased fashion, some explored the possibility of simply extracting ergothioneine from mushrooms. But again, this would be extremely expensive and require mushroom farms taking up areas of potential farming land.
“By making this important antioxidant in a biobased fashion, you avoid using chemicals or farmland. Yeast is far better at producing ergothioneine than humans or mushrooms could ever be,” says the first author Steven van der Hoek.
Enzymes are the key
In nature, ergothioneine is produced by bacteria and fungi, but the enzymes bacteria and fungi use for making ergothioneine make up slightly different pathways.
In the study, the scientists chose to screen enzymes from different fungi and from the bacterium Mycobacterium smegmatis in various combinations to identify the clones with the highest ergothioneine production. As production host they used yeast, and they discovered that two specific enzymes NcEgt1 and CpEgt2, both fungal enzymes, made the best combination.
Furthermore, they also investigated potential ergothioneine transporters to increase the yield from their yeast strain. Unfortunately, this did not have any effect.
One thing that worked was to add amino acids that operate as building blocks of ergothioneine to the medium. By doing this, they were able to increase the production of ergothioneine significantly.
Thus, optimisation of the medium was one of the important steps to increase the production to 0.6 g/L in 84 hours, which compares well with the current best-reported production in E. coli that gets 1.3 g/L in 216 hours.
“The bacterial pathway in E. coli uses a lot of energy while the fungal pathway in yeast doesn’t. That could lead to a production benefit. Also, yeast is a safe and well-known production host for food supplements” says Steven van der Hoek.
Currently, the scientists are trying to increase productivity by engineering the strain further to make a commercially viable product.
The authors of this study also stress that the positive effects of ergothioneine have so far only been reported in animal models, and, hence, it is too soon to say if this will work in humans. Regardless, ergothioneine production in a larger scale than today could be important to get access to a beneficial dietary supplement.
Technical University of Denmark
Anders Østerby Mønsted – Technical University of Denmark
The image is credited to Technical University of Denmark.
Original Research: Open access
“Engineering the Yeast Saccharomyces cerevisiae for the Production of L-(+)-Ergothioneine”. Steven van der Hoek et al.
Frontiers in Bioengineering and Biotechnology doi:10.3389/fbioe.2019.00262.
Engineering the Yeast Saccharomyces cerevisiae for the Production of L-(+)-Ergothioneine
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.