Systems Metabolic Engineering of <i>Escherichia coli</i> for Efficient <i>de novo</i> Biosynthesis of 2,5-Dimethylpyrazine from Glucose

2,5-Dimethylpyrazine (2,5-DMP) is an important flavor compound in food and a valuable intermediate in pharmaceutical synthesis. Conventional chemical synthesis of 2,5-DMP requires harsh conditions and generates toxic byproducts. Growing demand for natural and sustainable products has made microbial biosynthesis via metabolic engineering an attractive alternative. Here, we achieved <i>de novo</i> biosynthesis of 2,5-DMP in wild-type <i>Escherichia coli</i> W3110 through systems metabolic engineering. An l-threonine overproducing strain, supplying the precursor for 2,5-DMP synthesis, was first constructed. The pathway converting l-threonine to L-2-aminoacetoacetate was then established, enabling 0.41 g/L 2,5-DMP production in shake-flask fermentation. Disruption of competing pathways, introduction of a heterologous aminoacetone oxidase, and deletion of l-threonine exporters further enhanced 2,5-DMP production. A genome-integrated T7 expression system for threonine dehydrogenase eliminated the plasmid dependence. Adaptive laboratory evolution improved 2,5-DMP tolerance and yield, with the evolved strain producing 3.72 g/L in broth and 4.51 g/L after recovery of volatile 2,5-DMP from off-gas─the highest titer reported to date. This study establishes a genetically stable, plasmid-free <i>E. coli</i> platform for efficient and sustainable 2,5-DMP biomanufacturing.