以L-苏氨酸为发酵底物的2,5-二甲基吡嗪高产菌株构建

Biosynthesis of 2,5-dimethylpyrazine from L-threonine by a metabolically engineered strain

构建一种以L-苏氨酸为发酵底物的高值化学品2,5-二甲基吡嗪(2,5-dimethylpyrazine,2,5-DMP)生产菌株,为解决L-苏氨酸产能过剩,实现2,5-DMP生物法生产提供可靠思路。通过利用Bacillus subtilis 168(B.subtilis 168)外源表达不同微生物种属来源的L-苏氨酸脱氢酶(L-threonine dehydrogenase,TDH),并比较其利用L-苏氨酸为底物合成2,5-DMP的产量,挑选出2,5-DMP高产菌种,在此基础上进一步外源表达NADH氧化酶(NADH oxidase,NOX),以促进辅因子再生。实验构建了1株高产2,5-DMP的基因工程菌株B.subtilis 168/pMA0911-tdh(E.c)-nox。该菌株以5.83 g/L的L-苏氨酸为底物,发酵24 h后2,5-DMP的产量高达616.04 mg/L,与对照菌株B.subtilis 168/pMA0911相比,产量提高了22.5倍。在TDH过表达的基础上,NOX的参与有利于2,5-DMP产量的提高。该研究首次实现了2,5-DMP高效的生物转化,一方面缓解了L-苏氨酸产能过剩的困境,另一方面有助于实现高值风味化合物2,5-DMP的生物法生产。

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.