l‐Threonine Catabolism Driven Flavor Formation: A Key Pathway for Soy Sauce‐Like Aroma in Bacillus subtilis Fermentation

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 (BJ3‐2Δ tdh and BJ3‐2Δ kbl ) and a double mutant (BJ3‐2Δ tdh ‐ 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.