Study On The Regulation Of Branch Metabolic Pathway In Tryptophan Biosynthesis Of Escherichia Coli

L-tryptophan, as a nessecary amino acid in animals, has a significantfunction both in growth and also metabolism. Recently, L-tryptophan has beendemanded more and more by the international market because its broadapplication in pharmacy, feedstuff and food industry. But because of the longmetabolic pathway and complex regulation mechanism, it is not easy toestablish an engineering bacterium to meet the industrial need. And alsobecause of the expensive cost in both chemical and enzymatic synthesis, theindustrial production of L-tryptophan in China has still not been drived true.This experiment will follow the main idea of establishing an engineeringbacterium which will produce L-tryptophan, concentrate on the branchsynthesis metabolic pathway and try to rebuild a expedite branch pathway forsynthesizing L-tryptophan. The work we have done are:1. We cloned and expressed two genes: trpE and trpD from E.coli trp operon.Because of three negative regulation factors for gene trpE, which includingrepression protein,attenuator and feedback repression, we paid ourconcentration on releasing the negative regulation factors from trpE andreconstructing a feedback-resistant mutant S40FtrpE. And also this mutanteliminates the effects of repression protein and attenuator, so the active ofanthranilate synthetase coded by mutant is 2.9 times than negative comparison.2. In order to cut off the catabolism of tryptophan synthesized by E.coli, we knocked out the gene TnaA from the host bacterium E.coliK12-R and reconstructed a new deficient mutant E.coliK12-RT, which lack of tryptophanase coded by TnaA. The test of tryptophan content from fermentation solution proved that the new reconstructed bacterium E.coliK12-RT can accumulate tryptophan more efficiently.3. We co-expressed different enzymes from the common pathway and t he branch pathway at the same time by two separate strategies. Oneis to clone these two genes in series onto the same plasmid pBV22 0, the other one is to separately clone them onto two different plasmi ds, which can coexist in one host bacterium. Based on this, we const ructed three new recombined plasmids: pBV220-p-trpED-pl-aroG (cis), pBV220-p-trpED-pl-aroG (cis), pBV220-aroG-pl-p-trpED(trans) and alsoa new bi-plasmids system pBV220-S40FtrpED/pZA31-aroG to realize the co-expression. But after the construction, the essay of enzyme ac -tivity and tryptophan content showed inapparent advance.4. We used new recombined bacterium: pBV220-S40FtrpED/E.coliK12-RT to do the elementary fermentation experiment in lab. We compareddifferent culture mediums, cubage, sugar concentration and the altera-tion of PH. And finally picked out the the best condition for furtherexperiments.By research of the branch pathway, the production of tryptophan from our engineering bacterium has improved. But any synthesis metaboiitism in vivo is cooperated by a series of complex regulation mechanisms, including more than one key enzymes and plenty of restrictive steps. So only when all the key enzymes co-expressed and balanced with each other, we can get an efficient genetic engineering bacterium to produce tryptophan with high production.