麦角硫因(ergothioneine,EGT)是一种含硫组氨酸衍生物,广泛应用于食品、化妆品及医药等领域。然而,当前国内外针对EGT生物合成的研究以合成途径明确的分岐杆菌与粗糙脉孢菌为主,而作为EGT主要天然来源的食药用真菌,其合成酶研究相对较少。该研究通过生物信息学分析结合LC-MS检测,鉴定出PTR-Egt1和PTR-Egt2为食药用真菌虎奶菇中催化EGT合成的2个关键酶。为实现EGT的高效合成,成功在大肠杆菌中共表达这2种关键酶,并通过优化蛋白标签组合,筛选出携带SUMO标签的PTR-Egt1与携带MBP标签的PTR-Egt2为最佳重组方案。在优化条件下,发酵120 h发酵液中EGT产量达到74.20 mg/L。进一步研究表明,添加前体物质L-组氨酸显著提升了EGT产量,当组氨酸添加量为1 g/L、发酵时间为120 h时,EGT产量提高至124.03 mg/L,生产效率达到1.03 mg/(L·h)。该研究系统地探索了虎奶菇来源EGT合成酶的功能及其异源表达优化策略,为食药用真菌中EGT的高效生物合成提供了重要的理论依据和实践指导。
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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