Ergothioneine (EGT), a sulfur-containing histidine derivative, is widely used in the fields of food, cosmetics and pharmaceutical industries.While much of the current research on EGT biosynthesis focuses on well-characterized pathways in Mycolicibacterium smegmatis and Neurospora crassa, limited attention has been given to EGT synthases in edible and medicinal fungi, which are the primary natural sources of EGT.In this study, bioinformatics analysis and LC-MS were utilized to identify two key enzymes, PTR-Egt1 and PTR-Egt2, involved in EGT biosynthesis in the edible macrofungus Pleurotus tuber-regium.These enzymes were then co-expressed in Escherichia coli for efficient EGT production.Optimization of protein tags led to the selection of SUMO-tagged PTR-Egt1 and MBP-tagged PTR-Egt2 as the optimal recombinant configuration.Under 120 h fermentation conditions, EGT production reached 74.20 mg/L.Further optimization revealed that the addition of L-histidine significantly boosted EGT yield, achieving a maximum of 124.03 mg/L and a production efficiency of 1.03 mg/(L·h) after 120 hours with 1 g/L histidine supplementation.This study provides a systematic investigation into the function of EGT synthesis enzymes from P.tuber-regium and offers optimization strategies for heterologous expression, laying a solid foundation for the efficient biosynthesis of EGT in edible and medicinal fungi.
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