Engineered <i>Bacillus subtilis</i> for the Production of Tetramethylpyrazine,(R,R)-2,3-Butanediol and Acetoin
Lin Shi,
Yuan Lin,
Jiaao Song,
Hongxing Li,
Yinhao Gao,
Yonghong Lin,
Xiaowen Huang,
Wu Meng,
Weishuai Qin
Affiliations
Lin Shi
State Key Laboratory of Biobased Material and Green Papermaking (LBMP), Qilu University of Technology (Shandong Academy of Sciences), Jinan 250353, China
Yuan Lin
State Key Laboratory of Biobased Material and Green Papermaking (LBMP), Qilu University of Technology (Shandong Academy of Sciences), Jinan 250353, China
Jiaao Song
State Key Laboratory of Biobased Material and Green Papermaking (LBMP), Qilu University of Technology (Shandong Academy of Sciences), Jinan 250353, China
Hongxing Li
State Key Laboratory of Biobased Material and Green Papermaking (LBMP), Qilu University of Technology (Shandong Academy of Sciences), Jinan 250353, China
Yinhao Gao
State Key Laboratory of Biobased Material and Green Papermaking (LBMP), Qilu University of Technology (Shandong Academy of Sciences), Jinan 250353, China
Yonghong Lin
State Key Laboratory of Biobased Material and Green Papermaking (LBMP), Qilu University of Technology (Shandong Academy of Sciences), Jinan 250353, China
Xiaowen Huang
State Key Laboratory of Biobased Material and Green Papermaking (LBMP), Qilu University of Technology (Shandong Academy of Sciences), Jinan 250353, China
Wu Meng
State Key Laboratory of Biobased Material and Green Papermaking (LBMP), Qilu University of Technology (Shandong Academy of Sciences), Jinan 250353, China
Weishuai Qin
College of Biology and Brewing Engineering, Taishan University, Taian 271000, China
2,3-Butanediol, acetoin and tetramethylpyrazine have a wide range of applications as important chemicals in the chemical, food and pharmaceutical fields. Bacillus subtilis 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 Bacillus subtilis BS2. 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 Bacillus subtilis. 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.
