This study is aiming to develop a reliable approach to solve the overcapacity of L-threonine and produce 2,5-dimethylpyrazine (2,5-DMP), a high-value chemical. A metabolically engineered strain for 2,5-DMP production was constructed, using L-threonine as substrate. L-Threonine dehydrogenase (TDH) derived from different microbial species was separately overexpressed in Bacillus subtilis 168 and L-threonine was used as the substrate to synthesize 2,5-DMP. The high 2,5-DMP- yield strain was selected, and the NADH oxidase (NOX) was further exogenously expressed in the strain to promote cofactor regeneration. Finally, a genetic engineered strain B. subtilis 168/pMA0911-tdh (E. c)-nox with high 2,5-DMP-yield was obtained. After 24 h of fermentation, the yield of 2,5-DMP reached 616.04 mg/L using 5.83 g/L of L-threonine as the substrate. Comparing with the control strain B. subtilis 168/pMA0911, the yield was increased by 22.5 times. On the basis of TDH overexpression in B. subtilis 168, overexpression of NOX promoted the production of 2,5-DMP. In conclusion, this was the first study to achieve efficient biotransformation of 2,5-DMP. Also, it has the potential to alleviate the overcapacity of L-threonine. Therefore, this strain is able to promote the biological production of the high-value flavor compound 2,5-DMP.
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