Rapid TLC-based preliminary screening of ergothioneine-producing fungi and analysis of conserved EgtB domains.

Ergothioneine (EGT) is a thiol-histidine compound with antioxidant properties and an important dietary component of edible mushrooms. Rapid methods for preliminary screening of EGT-producing fungi remain limited. Here, we developed a thin-layer chromatography (TLC) method using iodine vapour visualization for screening and densitometric estimation of EGT-associated signals. Forty-three fungal isolates collected from edible mushrooms and wild fruiting bodies in Maynooth, Ireland, were screened, identifying eight putative EGT-positive isolates representing Pleurotus, Hypsizygus, Agaricus, Rhodocollybia, and Coprinopsis. Pleurotus pulmonarius and Rhodocollybia butyracea showed the highest signals. TLC could discriminate between EGT and glutathione, and showed a concentration-dependent response. Co-chromatography with an authentic EGT standard supported the assignment of EGT-associated signals. EgtB-related sequence conservation was identified among the analysed isolates showing EGT-associated signals, providing supportive evidence for their potential EGT biosynthetic capacity. Overall, the workflow provides a rapid and cost-effective approach for preliminary screening of fungi for EGT-associated TLC signals.