Clone And Expression Analysis Of The Aspartokinase Genes From Corynebacterium Pekinense Mutant E31

Methionine is one of the essential amino acids that participate in protein synthesis.It must be gained from food,because it can't be synthetized in body.Methionine provide normal metabolism and growth of sulfur and other important component to body. Methionine also can provide methyl that plays a significant role in our body metabolism and growth.Lacking of methionine will suffocate protein synthesis and damage our body. In Corynebacterium methionine, lysine and threonine were synthetized through aspartate pathway.Aspartokinase is the key enzyme that catalyze the first step reaction of this pathway.however,the activity of the enzyme is inhibited by the end product lysine and threonine. As a result the synthesis of methionine was inhibited.In this study,express plasmid (pET-AK) was constructed by subcloning the structure gene to the down stream of T7 promoter of pET-28a. Then transform this recombinant plasmid into E.coliBL21 (DE3) strain.The recombinant strain was induced by IPTG to express the high active aspartokinase and then inhance the yields of methionine The study contents and results are as follows:1. Aspartokinase genes were amplified by PCR technology from Corynebacterium Pekinense mutant E31 and cloned to plasmid pMD18-T to get the sequence. Sequence analysis of aspartokinase gene revealed:The sequences of aspartokinase genes from C.pekinense mutant E31 shows homologies of 99%,98%, and 97% to those from C.glutamicum, C.flavum and C.crenatum. And the amino acide sequence deduced from ORF shows homologies of 98.8%, 98.8% and 98.5% respectively.2. The recombinant plasmid was transformed into E.coliBL21 (DE3) strain.then induced by IPTG.Through optimizing the induce condition,the optimized condition is Bacteria concentration OD600 for 0.6,the concentration of IPTG for 0.8mM,induction time for 6h.3. By applying response surface methodology (RSM) to optimize the fermentation conditions, The optimized condition:temperature 37℃,pH7.5,inoculum 2%,rotate speed 180r/min.The aspartokinase activity increased to fifty times higher. Methionine yields 2.5 times higher than the origin E.coliBL21.It is concluded that study on the key enzyme in the biosynthetic pathways of methionine is an effective way to enhance the methionine yields.