| Gaia: | 2,3-Butanediol, acetoin and tetramethylpyrazine have a wide range of applications as important chemicals in the chemical, food and pharmaceutical fields. <i>Bacillus subtilis</i> has a very wide application potential in many industries as a food-safe grade strain and has a good performance as a potential strain for tetramethylpyrazine production. In this study, we constructed a recombinant plasmid with dual promoter to overexpress 2,3-butanediol dehydrogenase and introduced it into <i>Bacillus subtilis BS2</i>. The constructed strain (BS-ppb11) was then optimized for fermentation conditions, and the maximum concentration of 2,3-butanediol produced was 96.5 g/L, which was 36.4% higher than that of the original strain, in which the (R,R)-2,3-butanediol had a chiral purity of 94.7%. Meanwhile, BS-ppb11 produced a maximum concentration of 82.2 g/L acetoin, which was 36.7% higher than that of the original strain. Subsequently, through optimization of metabolic conditions, BS-ppb11 produced 34.8 g/L of tetramethylpyrazine in staged batch replenishment fermentation, which was 95.5% higher than the original strain and was the highest ferritin production reported to date for <i>Bacillus subtilis</i>. In addition, we introduced a photocatalytic coenzyme regeneration system in BS-ppb11 to further improve the metabolic yield of 2,3-butanediol by regulating cofactor homeostasis, which laid the foundation for the subsequent in-depth study of the related mechanism.
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