Molecular Modification Of L-phenylalanine Metabolic Pathway In Escherichia Coli

In order to keep the food security, a large number of cereal will be stored in China. At thesame time stale rice problem emerges. Deep processing of stale rice to enhance its addedvalue has great economic and social benefits. The main Composition of stale rice is starchwhich can be made into glucose by a simple processing. Using glucose from starch as thecarbon source to produce L-phenylalanine can improve the effectiveness of stale rice well.The aroG and pheA are two important genes of the metabolic pathway of phenylalaninein the aromatic amino acid biosynthetic pathway of E. coli, The aroG gene encodesdeoxy-arabinose-type the heptanone sugar phosphate synthase （DS）, The pheA gene encodesa bifunctional enzyme to catalyse two-step key reaction, That is two functions of branchingmutase （CM） and pre-benzoic acid dehydration （PD）. AroG and PheA are two importantpoints of regulation and the bottleneck of the phenylalanine metabolic pathway. The activityof enzyme are regulated by feedback inhibition, and the expression of enzyme are regulatedby feedback repressor. In this research, we cloned the aroG^fbr, pheAfbrgenes into the pQLinkHvector respectively. At the meantime, we constructed the ppsA, tktA, yddG, aroG^fbrgenes intoa co-expression vector, constructed the PpsA, tktA, yddG, aroG^fbr, pheAfbrgenes into aco-expression vector, constructed the aroG^fbr, pheAfbrgenes into a co-expression vector,constructed the aroG^fbr, pheAfbr, yddG genes into a co-expression vector using geneco-expression technology （LIC）.（the ppsA, tktA, yddG gene cloned by other members of ourresearch group）.In the E. coli aromatic amino acids branch way, the branch acid as a hub material will bedivided into two carbon flow. One branch catalyse by the anthranilic acid synthase to generatetryptophan, Another branch catalyse by the chorismate mutase to generae tyrosine andhenylalanine. In this study, the trpE gene is knocked out in against feedback inhibition ofaromatic amino acids strains ATCC31884useing Red recombination technology, in order toeliminate the competition of the tryptophan branch.In this study, We improved the common pathway and branch pathway, increasedphenylalanine synthesis pathways, broke through the limited-step, eliminated competitioncarbon metabolism flow branch on the basis of increasing the supply of the precursorsubstance and the export of phenylalanine. We let different co-expression plasmid express inATCC31884which removed the tryptophan branch, in order to filter out high-yield strains ofphenylalanine. We laid the foundation for building a more efficient L-phenylalanine engineered strain. At the same time, we used gene cloning technology, gene co-expressiontechnology and gene knockout technology to establish a more comprehensive platform forgenetic manipulation in microorganism, which can provide technical support to improveE.coli efficiency in fermentation.