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

Abstract

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

Engineering Mycolicibacterium neoaurum for the production of antioxidant ergothioneine

“…EGT is a powerful antioxidant with cytoprotective effects (Ames, 2018;Cheah & Halliwell, 2012). The reconstruction of the EGT synthesis pathway in the commonly used heterogenous biological chassis is a promising strategy (Kamide et al, 2020;Tanaka et al, 2019;van der Hoek et al, 2019), whereas integrating numerous exogenous genes into the host chromosome while maintaining the stability of cell growth and production is still a challenge (Li et al, 2017;Manderscheid et al, 2016), especially the integration of several genes by plasmid vectors (Kamide et al, 2020). Hence, in this study, we mined the production potential of the natural EGT-producing M. neoaurum and described an engineering strategy for the gradual enhancement of EGT production.…”

“…In recent years, the continuous upgrading of synthetic biology toolkits has provided promising solutions for the heterologous synthesis of EGT. To date, several representative strains have been engineered for the heterologous biosynthesis of EGT (Kamide et al, 2020;Kim et al, 2022;Takusagawa et al, 2019;Tanaka et al, 2019;van der Hoek et al, 2019van der Hoek et al, , 2021van der Hoek et al, , 2022. The highest production of EGT (2.39 ± 0.08 g/L) was achieved in 160 h in the engineered Saccharomyces cerevisiae in fedbatch fermentation without supplementation of amino acids (van der Hoek et al, 2022).…”

Evaluation of Anti-Aflatoxin M1 effects of heat-killed cells of Saccharomyces cerevisiae in Brazilian commercial yogurts

“…Yeasts treated with heat (76.39%) and ultrasound (74.20%) also showed high percentages of reduction. An important advantage of using S. cerevisiae as the AFM 1 binder in yogurts is the overall acceptance of this yeast without restrictions in the food industry, considering its classification as a GRAS organism ( Van der Hoek et al, 2019). Besides, the low costs of adding S. cerevisiae biomass in yogurts provide a viable alternative to the dairy industry to reduce the AFM 1 contamination in the product during the storage period.…”

“…S. cerevisiae is one of the most important yeasts used in the food industry, also being considered a GRAS ("generally recognized as safe") organism (Van der Hoek et al, 2019). Thus, a biological approach for reducing aflatoxin based on S. cerevisiae strains that are already used in food products is an attractive alternative to reduce the AFM Pires RC et al…”