Study On The Combinational Expression Of Key Enzymes In Phenylalanine Synthesis Pathway Of Escherichia Coli

L-phenylalanine,an aromatic alpha amino acid,is one of the eight essential amino acids for human body.L-phenylalanine plays a key role in the medical,feed additives,food processing industries and has been widely applied in many industries.However,it is unable to meet our needs with only rely on the chemical synthesis.So by biological fermentation production of phenylalanine is of great importance.In this article,we modified the metabolic pathway and the key enzymes involved in the biosynthetic pathway of phenylalanine were overexpressed to raise the production of phenylalanine.The main contents of this study were as follows:?1?We knocked out the trpE gene of Escherichia coli try?tyrA to eliminate all the competitive pathways involved in phenylalanine synthesis.In the shake flask fermentation test,we found that the phenylalanine production of Escherichia coli try?tyrA?trpE has improved to 3.5 times compared to original strain Escherichia coli try?tyr A.?2?We overexpressed the aroF and pheA gene,two key enzymes of the phenylalanine synthesis pathway.Furthermore we made an point mutation of pheA,a Thr326 Pro animo replacement was achieved as pheA^fbr to eliminate the feedback inhibition of chorismate mutase P and prephenate dehydratase by phenylalanine.?3?In order to verify how it works on phenylalanine production,four recombinant plasmids were constructed and identified respectively.In the shake flask experiments,we found that the low copy number plasmid pACYC184-aroF and high copy number plasmid pTrc99A-pheA^fbr functioned better in phenylalanine fermentation while it functioned worse when the two plasmids co-expressed in a single host.?4?Moreover,The tandem expression of aroF and pheA^fbr was also applied.In the shake flask fermentation,strain E7—containing the p Trc99 A plasmid constructed with an rrnB terminator and a trc promoter between these two genes,reached the highest yield—1.43g/L,about 2 times of the trpE knockout strain,6 times of the original strain Escherichia coli try.In 5L fermenter test,the phenylalanine yield at 72 h had reached 7.86g/L,5.5 times of shake flask test.