Screening Mutation Site Of MetA, A Critical Enzyme For Methionine Production In E.coli Fermentation

Methionine is one of the sulfur-containing essential amino acids, which plays an essential role on the growth of ruminant animals and livestock. Currently, methionine is mainly produced by chemical synthesis, s ince the development of the biological method is limited by the current technology. Therefore it’s necessary to develop an effective method to improve the efficiency of methionine production and reduce the breeding cost in C hina.The purpose of this study is to optimize the key pathway of the microbial metabolism, and to improve the production efficiency and yield of methionine. O ur research is mainly focus on two points: Firstly, genetically modifying the Methionine met A metabolism in E.coli; Secondly, optimizing the fermentation conditions in the production of methionine.The key reaction of methionine metablism is to generating O-amber acyl homoserine from homoserine, which is catalyzed by homoserine amber acyltransferase(met A, EC 2.3.1.46), controlled by feedback inhibition of methionine and S-adenosine methionine. Here two met A mutants were obtained by using site mutation technique, which were named as met A(Y299C) and met A(T24k). In the met A(Y299C) strain, the 896 th base A in met A was changed into G, thus Tyrosine is replaced by Cysteine. In the met A(T24k) strain, the 71 th base C in met A was changed into A, and the threonine is replaced by Lysine.First of all, protein quantitation examination showed there is no significant difference between two mutanted stains and Wild Type(WT), which proved mutation has no effect on protein expression. In addition, the enzyme activity was downregulated in the met A(T24k) strain, while The modified met A(Y299C) showing a decreased feedback sensitivity to methionine and SAM in comparison to the WT enzyme.In summary, methionine production increased from 6.76 g/L to 10.35 g/L in the met A(Y299C) mutant strain compared to the WT in our study, the above findings demonstrated the importance of met A control points for methionine biosynthesis, which provide invaluable information on developing metabolic engineering strategies for methionine production.