Site-directed Mutagenesis And Expression Of Key Enzyme Gene (aroG, TrpBA) Of Tryptophan Pathways Synthesis In The E.coli

L-tryptophan is the essential amino acid for protein biosynthesis. Many hormones andphysiologically active substances,5-hydroxy-tryptamine, indole acetic acid, nicotinic acid,pigments,come from tryptophan in vivo departure. Therefore, building bacteria ofL-tryptophan-producing is important for the production of human life. In this study, the keyenzyme gene of DAHP synthase aroG in the metabolic pathways of Escherichia coli ismutatied, and co-expressed with another key enzyme gene trpBA. The purpose of this study isto obtain the gene aroG^fbrof lifting of phenylalanine feedback inhibition, and build a newengineered strain of L-tryptophan-producing for research.We analysis the tertiary structure of DAHP synthase by bioinformatics and a feedback in-hibition resistant aroG^fbr（C632T） gene was cloned by SOE-PCR. The mutation of S211Fleads the binding hydrophobic pocket region space of sub-phenylalanine to become small andstables hydrophobic pocket area.So it can weaken the binding ability of phenylalanine. Theexperiments, in the presence of1mM phenylalanine, showed that the specific enzyme activityof wild-type DAHP synthase was0.5（±0.12） U/mg. The specific enzyme activity of mutantedDAHP synthase was4.25（±0.11） U/mg. It was8.5-folds to the enzyme activity of the wild.In this study, engineered strain of E.coli JM109/pEtac-aroG^fbr-tac-trpBA and controlstrain of E.coli JM109/pEtac-aroG-tac-trpBA were constructed. Different recombinants wereinduced. The SDS-PAGE result and specific enzyme activities of different recombinants wereanalyzed indicated that: the target proteins were expressed successfully. The preliminaryfermentation of different recombinants was researched. The results showed that thetryptophan production of mutant strains was11.47times for starting strain E.coli JM109/pEtac. It was1.4times for the wild-type strain E.coli JM109/pEtac-aroG-tac-trpBA.Meanwile，in the presence of1mM phenylalanine, the DAHP synthase’s specific enzymeactivity of wild recombinant bacteria E.coli JM109/pEtac-aroG-tac-trpBA was0.4（±0.10）U/mg. DAHP synthase’s specific enzyme activity was3.6（±0.10） U/mg of mutantrecombinant bacteria E.coli JM109/pEtac-aroG^fbr-tac-trpBA. The specific enzyme activity ofmutant was increased significantly. So the mutation weakens the inhibition of phenylalanineto DAHP synthesis enzyme, effectively. It was further proof that weakening the inhibition ofphenylalanine to DAHP synthesis resultes in the DAHP synthase’s enzyme activity improved.It enhances carbon flow to synthesis of tryptophan.The tryptophan fermentation conditions of recombinant bacteria E.coli JM109/pEtac-aroG^fbr-tac-trpBA was optimizated. The optimum reaction conditions were as follows:15g/Lglucose,0.06M KH2PO3,1.5mM IPTG induction concentration,10%inoculum size, shakingspeed200and250r/min,36h of fermentation. After initial optimization, the tryptophanproduction of E.coli JM109/pEtac-aroG^fbr-tac-trpBA was up to36.3mg/L. The tryptophanproduction increased1.2times with the wild-type strain E.coli JM109/pEtac-aroG-tac-trpBAand increased1.86times comparison with before optimization.