Systems Metabolic Engineering of Escherichia coli for Efficient de novo 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 de novo biosynthesis of 2,5-DMP in wild-type Escherichia coli 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 E. coli platform for efficient and sustainable 2,5-DMP biomanufacturing.