The globalization of food culture has driven the growth of the flavor market, with Bacillus subtilis-fermented soybean products widely used as traditional Asian condiments due to their distinctive soy sauce-like aroma. However, the molecular mechanism underlying aroma formation remains incompletely understood. Building on previous systematic multi-omics studies, this study identifies a key role for l-threonine catabolism in regulating pyrazine biosynthesis. Using homologous recombination, we performed targeted knockouts of the tdh and kbl genes to generate single mutants (BJ3-2Δtdh and BJ3-2Δkbl) and a double mutant (BJ3-2Δtdh-kbl). Headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry (HS-SPME-GC-MS), along with sensory evaluation, revealed that the tdh knockout drastically reduced 2,5-dimethylpyrazine (2,5-DMP) levels by 96.79%. The double knockout achieved a 97.73% reduction and completely eliminated 3-ethyl-2,5-dimethylpyrazine (EDMP). Interestingly, the kbl knockout alone enhanced 2,5-DMP production by 65.34%, and the odor activity value of EDMP increased to 10.50. Exogenous supplementation experiments confirmed that 2,5-DMP is a critical contributor to nutty, sweet, and buttery aromas, while also intensifying smoky undertones. Overall, this study elucidates the regulatory function of l-threonine catabolism in pyrazine biosynthesis, linking microbial metabolism to food flavor and offering a metabolic engineering strategy for targeted aroma modulation in fermented foods. PRACTICAL APPLICATIONS: This research provides valuable insights into the mechanism underlying the formation of soy sauce-like aroma, laying the foundation for optimizing 2,5-dimethylpyrazine (2,5-DMP) concentrations to fine-tune aroma profiles. These findings may support the environmentally friendly production of high-quality soy sauce-like aroma foods, potentially reducing reliance on artificial additives.