This study aimed to explore the impact of Bacillus subtilis E20 threonine dehydrogenase gene on the production of Maotai-flavor by soybean fermentation, the tdh gene was knocked out through homologous recombination, and the mutant strain E20-Δtdh was successfully constructed. The mutant strains and wild strains were inoculated into the soybeans that after high temperature and high-pressure sterilization, and the simulated Maotai flavored fermentation was performed. The sensory evaluation showed that the Maotai-flavor of the fermented product of the mutant strain was not prominent, and a burnt smell was produced. The results of GC-MS analysis showed that the relative content of pyrazine in the fermentation product of the mutant strain was 16.089% lower than that of the wild strain, and 2,5-dimethylpyrazine was not detected. The evaluation results of characteristic aroma components showed that the pyrazines that had a direct impact on the flavor of Maotai were 2,5-dimethylpyrazine rather than tetramethylpyrazine. The above results confirmed that the tdh gene of Bacillus subtilis E20 was closely related to the production of 2,5-dimethylpyrazine, and could significantly affect the formation of Maotai-flavor.
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