Ergothioneine (EGT) is a sulfur-containing, anti-oxidative amino acid derived from histidine. EGT is synthesized in bacteria and fungi but not in animals and plants, and is now recognized as important for human health. Its cost-effective fermentative production has not been elucidated due to the lack of information for productive microorganisms. In this study, we doubled the gene copy for EGT synthesis and deleted the histidine ammonia-lyase gene in a potent EGT-producing methylotrophic bacterium Methylobacterium aquaticum strain 22A, and optimized its culture conditions, resulting in increased EGT production of 7.0 mg EGT/g dry cell weight and 100 mu g EGT/5 mL/7 days. In addition, through screening we found EGT-producing eukaryotic strains of Aureobasidium pullulans and Rhodotorula mucilaginosa, which can produce 1.0 and 3.2 mg EGT/g dry cell weight, 70 and 120 mu g EGT/5 mL/7 days, respectively. This study proposes practical uses of potent EGT-producing recombinant Methylobacterium species and non-recombinant yeast and fungal strains. (C) 2018, The Society for Biotechnology, Japan. All rights reserved.