The Regulation Of Phenylalanine Biosynthesis In Escherichia Coli

Objectives1 To construct the strains which are suitable for industrial production of phenylalanine at low cost, which in turn reduces the expense of aspartame synthesis.2 To establish the technical platform of E. coli anabolic gene regulation in order to set up the basis for transforming metabolic pathway of aromatic amino acid in E. coli in the future. Not only it can be used to enhance the production capacity and rate of aromatic amino acid, such as phenylalanine, tryptophan, and tyrosine, but also can be used to import the foreign genes which could be used to synthesize other aromatic compounds, such as indigo, indole acetic acid, catechol, quinic acid and its derivatives.Materials and MethodsThe phenylalanine synthetic pathway was modified in E. coli strain MG1655 by taking pZE12 as the vector.1 The gene tyrA encoding key enzyme in tyrosine metabolic pathways was knocked out. The gene pheL which has the inhibitory effect on the function of gene pheA was also knocked out. For better gene regulation and less background noise, the genes pheA and aroF were also knocked out.2 DAHP synthase gene aroF, branch mutase/Pre-benzoic acid dehydrase gene pheA were cloned from E. coli, The two genes were connected in series and cloned in the pZE12 plasmid. The recombined plasmids were transformed and expressed in the gene knockout strains.3 3-dehydroquinate synthase gene aroB and shikimate kinase gene aroL were cloned from E. coli, and they were connected in series and were cloned into the expressive vector pET22b. The aroL-aroB genes were digested and re-connected into pZE12 pheA-aroF. The recombined plasmids were transformed and expressed in the gene knockout bacterial strains.4 Phosphoenol pyruvate synthase A gene ppsA and Transketolase A gene tktA were cloned from E.coli, and connected in series. The ppsA- tktA then was cloned in the expressive vector pET22b, then ppsA-tktA genes were digested and connected into pZE12 pheA-aroF. The recombined plasmids were transformed and expressed in the gene knockout bacterial strains.Results1 The results of PCR identification:pheL, pheA, tyrA and aroF genes were successfully knocked out from E.coli MG1655.2 The results of PCR and restriction enzyme digestion showed that the recombinant plasmids of pZE12 AF, pZE12 AFLB and pZE12 AFPT were successfully constructed. The gene pairs of pheA & aroF, ppsA & tktA and aroL & aroB were hooked up and cloned into pZE12.3 The results of SDS-PAGE showed that the expression of pheA and aroF encoded proteins were high, while the expressions of aroL, aroB, ppsA and tktA encoded proteins were low.4 Comparing with the wild-type E.coli MG1655, the yields of phenylalanine were increased 13,10.3 and 5.1 folds in the genetic reconstructed bacterial strains containing pZE12 AF, pZE12 AFPT and pZE12 AFLB respectively. ConclusionThe overexpression of genes pheA, aroF, aroL, aroB, ppsA and tktA related to the phenylalanine metabolic pathway of E. coli can significantly increase the yield of phenylalanine. The highest yield was found in stains with pheA and aroF genes. The high expression of enzyme is the key factor of enhancing the phenylalanine yield via the genetic re-constructed bacterial strains. The well balanced expression levels among the key enzyme genes were important to achieve the most efficient use of carbon flow to improve the yield of phenylalanine.