[Identification of key targets for ergothioneine biosynthesis based on random genomic transposition and...

Ergothioneine (ERG), as a natural potent antioxidant, possesses excellent physicochemical properties and diverse physiological activities, thereby exhibiting broad application prospects. However, engineering of existing genetically modified strains is prone to encountering bottlenecks in yield improvement. To identify key regulatory genes involved in ERG biosynthesis and enhance its heterologous production efficiency in Escherichia coli, this study employed E. coli BL21(DE3) as the chassis and constructed a heterologous biosynthetic pathway comprising Trichoderma reesei-derived Tregt1 and Tregt2, yielding a foundational engineered strain P1 with a shake-flask fermentation titer of 650 mg/L. Subsequently, a genome-wide mutant strain library was constructed using a random integration system mediated by Himar1C9 transposase, and combined with a high-throughput screening method based on the mBBr fluorescent probe, four positive mutant strains exhibiting significant phenotypic differences were successfully obtained. Sequencing analysis revealed that the key mutated genes encode phosphoenolpyruvate synthase PpsA, vitamin B₁₂ transporter permease BtuC, DNA helicase RecG, and a bifunctional nicotinamidase/pyrazinamidase PncA. Shake-flask fermentation validation showed that overexpression of recG significantly increased the ERG titer to 797 mg/L, whereas overexpression of the other three genes reduced the yield. This study establishes a systemic strategy combining random transposon integration and high-throughput screening, thereby overcoming the limitations of conventional targeted engineering and laying a foundation for the construction of efficient ERG-producing engineered strains and the advancement of their industrial production.