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BACKGROUND: Ergothioneine (EGT) is a high‐value food functional factor that cannot be synthesized by humans and other vertebrates, and the low yield limits its application. RESULTS: In this study, the optimal fermentation temperature, fermentation time, initial pH, inoculum age, and inoculation ratio on EGT biosynthesis of Rhodotorula mucilaginosa DL‐X01 were optimized. In addition, the effects of three key precursor substances – histidine, methionine, and cysteine – on fungal EGT synthesis were verified. The optimal conditions were further obtained by response surface optimization. The EGT yield of R. mucilaginosa DL‐X01 under optimal fermentation conditions reached 64.48 ± 2.30 mg L−1 at shake flask fermentation level. Finally, the yield was increased to 339.08 ± 3.31 mg L−1 (intracellular) by fed‐batch fermentation in a 5 L bioreactor. CONCLUSION: To the best of our knowledge, this is the highest EGT yield ever reported in non‐recombinant strains. The fermentation strategy described in this study will promote the efficient biosynthesis of EGT in red yeast and its sustainable production in the food industry. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

[Display omitted] • Untreated molasses could be utilized by R. mucilaginosa DL-X01 as carbon source. • Fish bone meal enzymatic hydrolysate was used to replace yeast extract and peptone. • The amount of molasses and fish bone meal added were optimized. • Feedstock cost of producing EGT is reduced by 331 % compared to using YPD broth. Ergothioneine (EGT) is a high-value natural antioxidant that cannot be synthesized by the human body. This study showed that Rhodotorula mucilaginosa DL-X01 can use untreated molasses and fish bone meal enzymatic hydrolysate as the substrates to synthesize EGT. By optimizing the growth conditions, the EGT yield reached 29.39 mg/L when molasses and fish bone meal (FBM) were added at 60 g/L and 400 g/L respectively. Finally, the EGT yield was increased to 216.25 mg/L by fed-batch fermentation in a 5 L bioreactor. Compared with the fermentation by yeast extract peptone dextrose medium, the feedstock cost of EGT production was reduced by 330.91 % by using molasses and FBM as substrates. These results showed that R. mucilaginosa DL-X01 can produce high-value EGT using two cheap processing by-products, molasses and FBM, which is of great significance for environmental protection and sustainable development. [ABSTRACT FROM AUTHOR]

Ergothioneine (EGT) is a natural histidine-derived compound with strong antioxidant activity. This research evaluated the potential of EGT to protect against liver injury induced by carbon tetrachloride (CCl 4) both in vivo and in vitro for the first time. Animal results showed that daily supplement of EGT (continuous gavage for 21 days) significantly reduced the levels of elevated serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST), regulated inflammatory factor levels, and improved antioxidant enzyme activity in mice after CCl 4 treatment. In addition, cell results showed that exposure to CCl 4 causes significant oxidative stress in LO2 cells, which was mitigated after EGT pretreatment. Furthermore, EGT treatment reduced reactive oxygen species production and improved mitochondrial function compared with the model group, which was potentially mediated by the activation of the Nrf2/ARE pathway. These findings provided a scientific theoretical basis for the application of EGT in prevention of liver injury. • EGT can be well-absorbed in Caco2 cells monolayers. • EGT supplement can reduce ROS and lipid peroxidation levels in the liver. • EGT pretreatment can alleviate the oxidative damage and apoptosis induced by CCl 4. • EGT alleviates oxidative damage by regulating the Nrf2/ARE signaling pathway. [ABSTRACT FROM AUTHOR]

Ergothioneine (EGT) is a natural sulfur-containing amino acid with potent antioxidant and cytoprotective activities. In this study, the oxidation tolerance of 23 different Rhodotorula strains were screened with hydrogen peroxide (H 2 O 2) as the inhibitory condition. By measuring their carotenoid and EGT production and correlation analysis with H 2 O 2 tolerance, the results showed that the carotenoid synthesis capacity has no effect on H 2 O 2 tolerance, in contrast to a strong positive correlation between EGT yield and H 2 O 2 tolerance of Rhodotorula species. In addition, a high EGT-producing strain R. mucilaginosa DL-X01 was screened with the yield of 38.48 ± 2.71 mg/L in a shaking flask fermentation. To the best of our knowledge, this is the highest EGT yield ever reported in non-recombinant strains. Growth kinetic data showed that DL-X01 had a longer lag phase in the presence of exogenous oxidant, but a shorter generation time and higher specific growth rate after entering the exponential phase. The results of this study provided a potential simple and rapid method for screening high EGT-producing strains, which could promote the efficient production of EGT by microorganisms and further development of the red yeast in the food and biotechnology industries. • Carotenoid content had no effect on H 2 O 2 tolerance of Rhodotorula species. • EGT yield of Rhodotorula species positively correlated with H 2 O 2 tolerance. • Rhodotorula mucilaginosa DL-X01 has excellent ergothioneine production capacity. [ABSTRACT FROM AUTHOR]