| 2,5-dimethylpyrazine is a high value-added product widely used in the food and pharma-ceutical fields as an intermediate in the synthesis of anti-hypertensive or hypoglycemic drugs such as glipizide and acipimox,and as a food additive.Currently,the most commonly used method for the preparation of 2,5-dimethylpyrazine is chemical synthesis,but this method often suffers from the problems of difficult product separation and toxic by-product generation,which poses a great limitation to the application of 2,5-dimethylpyrazine in the food industry.In contrast,the biosynthetic process for the preparation of 2,5-dimethylpyrazine is mild and less environmentally polluting,and is more suitable for food production.However,the biosynthetic efficiency of 2,5-dimethylpyrazine is very low and cannot meet the demand of industrial production.To solve this problem,this study successfully constructed a microbial cell factory for the efficient synthesis of 2,5-dimethylpyrazine by rearranging the intracellular metabolic flow of Escherichia coli by means of gene editing,which laid the theoretical and experimental foundation for the industrial production and application of 2,5-dimethylpyrazine.The main research contents include:（1）Screening of different sources of L-threonine-3-dehydrogenase（TDH）.Since TDH is the only key enzyme in the biosynthetic pathway of 2,5-dimethylpyrazine,we selected TDH from different sources for exogenous expression in E.coli BL21 and screened for the highest TDH enzyme activity by SDS-PAGE analysis,enzyme activity analysis,and the yield analysis of whole-cell catalytic synthesis of 2,5-dimethylpyrazine.The highest TDH catalytic activity of E.coli K-12 source was found,and the recombinant strain E.coli BL21/pET28a-Ectdh was successfully constructed as the basic strain for the subsequent study,because it had the highest TDH enzyme activity(132.0 m U·mg-1)and the yield of whole-cell catalytic synthesis of2,5-dimethylpyrazine was 603.0 mg·L-1,which was much higher than the other five recombi-nant strains;（2）Exogenous expression of aminoacetone oxidase.In order to improve the efficiency of the non-enzymatic synthesis of 2,5-dimethylpyrazine from the intermediate product aminoac-etone,we constructed a recombinant strain E.coli BL21/pET28a-Ectdh-soaao by introducing an exogenous aminoacetone oxidase（So AAO）,and the enzymatic activity of So AAO was determined to be 83.6 m U·mg-1.The whole-cell catalytic synthesis of 2,5-dimethylpyrazine was 1006.0 mg·L-1,which was significantly increased compared to the TDH single-expression recombinant strain E.coli BL21/pET28a-Ectdh.This confirmed that So AAO had a facilitative effect on the synthesis of 2,5-dimethylpyrazine;（3）Knockout of the kbl gene blocks the branching carbon flow pathway.2-amino-3-keto-butyric acid CoA ligase（KBL）catalyzes the production of glycine from L-threonine and affects the efficiency of 2,5-dimethylpyrazine synthesis.Therefore,we constructed a recomb-inant strain E.coli BL21Δkbl/pET28a-Ectdh-soaao by knocking out the kbl gene via CRISPR-cas9 and co-overexpressing TDH and So AAO.It showed a significant increase in yield and carbon atom yield for the whole-cell catalytic synthesis of 2,5-dimethylpyrazine,the yield was 1528.0 mg·L-1and the carbon atom yield was 28.2%.This confirmed the inhibitory effect of KBL on the synthesis of 2,5-dimethylpyrazine;（4）Optimization of whole-cell catalytic reaction conditions and system scale-upexperi-ments.The reaction conditions were optimized for the whole-cell-catalyzed synthesis of2,5-dimethylpyrazine by recombinant strain E.coli BL21Δkbl/pET28a-Ectdh-soaao.The opti-mized conditions were:40℃,pH 9.0,substrate concentration 10 g·L-1,and a 5 L fermenter scale-upexperiment was conducted under these conditions.The whole-cell catalytic yield of2,5-dimethylpyrazine reached 1682.0 mg·L-1for 24 h,with a carbon yield of 30.2%,which was the highest level reported so far,and a significant increase in carbon yield compared to the highest level reported so far（17.0%）. |
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