Cloning And Expression Of Trp A-E And Trp Operon Gene Of Escherichia Coli

Amino acid is the fundamental unit of protein,with extremely widespread uses in medicine, food, chemical industry, animal husbandry and agriculture . L-tryptophan (L-Trp) as one of the eight essential amino acids is the indispensible component in amino acid transfusion and other preparation. Because of the rapid development of the usage of L-tryptophan in feedingstuff and medical industry, and the low producing capacity all over the world, to building a way to increase the production of L-tryptophan is most significative. These years, as the rapid development of gene engineering, we try to find an effective way to improve the production of L-tryptophan by producing bacterium which can achieve high production.The enzymes in E.coli which synthesize L-tryptophan are coded by five continuous genes(trpE,trpD,trpC,trpB,trpA).These structure genes are regulated by upstream promoter, operator and trpR gene. TrpR gene is negative regulator to the following structure genes, but it is far away from the P- o -trpEDCBA. So we respectively got two L-tryptophan operon with and without promoter by PCR, cloned them to T carrier separately, then transfered them to expression carriers pBV220 and pET22b(+) , induced the expression of aim genes and tested the activity of AS and tryptophan synthase.We found that the structure genes on pET22b(+) with its own promoter have got the most activity compared with others, so we chose that to be the one be altered. The next step, we changed the two neighboring tryptophan codons in attenuator by site mutant, after which the expression and activity of enzymes coded by tryptophan operon have got improvement.This experiment is mainly to improve the enzyme activity and finally improve the production of L- tryptophan by cloning the whole operon and increasing gene copies. Besides, we also analyzed the self-promoter's effect in expressing and compared the influence of different carriers with diverse intensity promoters to the expreesion and activity. Finally, we have chosen the best situation for site mutant in attenuator and realized the improvement of expression and enzyme activity.There are lots of difficulties in building high production tryptophan engineering bacterium by metabolism engineering. All key enzymes in metabolic network should be considered. It is proved that too high expression of enzyme protein is harmful for synthesis of L-tryptophan, because the high expression easilly lead to inclusion body. Therefore, how to regulate each kind of enzyme's expression in the network becomes the cruces of this experiment. This experiment have made the fundation of further alteration of tryptophan operon and might be the first step in building a high production tryptophan engineering bacterium.