Metabolic Regulation Of The Escherichia Coli Aromatic Amino Acid Biosynthetic Pathway

Aromatic amino acids are important fine chemical products and intermediates of drug synthesis,which can be numerously used in drug synthesis,food and chemistry industry.In this study,the metabolic engineering was applied for genetic engineering bacteria of tryptophan.After constructing high throughput method for aromatic amino acid determination, this work modified the tryptophan biosynthesis pathway in E.Coli by means of molecular biology methods.The aim was to redirect more carbon flow into the aromatic amino acids biosynthesis branch especially to tryptophan biosynthesis,all the work was as the follows:1.The central metabolism pathway of E.Coli MG1655was modified by co-expressing ppsA and tktA for more PEP and E4P supply.The P_LlacO-1-ppsA-T1 fragment and P_LlacO-1-tktA-T1 fragment which were amplified from pZE12-ppsA and pZE12-tktA respectively were co-expressed in pZA31,leading to the increase of ppsA activity and tktA activity about 3 folds and 3.5 folds respectively.2.The common and branch pathway of aromatic amino acid biosynthesis pathway were further modified:The trpR gene was knocked out by RED recombination system to eliminate the repression control of trpR gene product on the key genes for tryptophan biosynthesis and transport in MG1655 genome,the tryptophan degradation was blocked by tnaA gene knockout.Colony PCR result indicated these two genes were removed from the genome.The lacR-tetR-SP^r was transferred from BWRI genome to the engineered bacterial-MG1655ΔR and MG1655ΔRT for the induction of P_LlacO-1and P_LtetO-1further,the new strains were named MG1655ΔR-RI and MG1655ΔRT-RI.The rate-limited steps in aromatic amino acid biosynthesis pathway were modified by co-expression of P_LlacO-1-aroG^fbr-T1 and P_LlacO-1-trpED^fbr-T1 in pZE12 which were amplified form pZE12-aroG^fbrand pZE12-trpED^fbr.The activity of DAHP synthase and anthranilate synthase encoded by aroG^fbrand trpED^fbrwas increased about 4 folds and 7 folds respectively.3.The effects of glucose content,IPTG concentration,liquid level on the tryptophan production of the engineered strain pZA31-ppsA-tktA&pZE12-aroG^fbr-trpED^fbr/MG1655ΔRT -RI were studied by the orthogonal design experiment.The optimum fermentation condition was found out by orthogonal test analysis with the value:liquid level 100ml in 500ml triangle bottle,1%glucose,0.5mM IPTG.The tryptophan production was about 1.3g·L^-1under the optimum condition.4.The expression of aroG^fbrand trpED^fbrwas tuned by engineered E.Coli small RNAs, and its effect on mRNA levels,enzyme activity and tryptophan production was examined.The P_LlacO-1-cr-sodB1-aroG^fbr-T1 and P_LlacO-1-cr-sodB2-trpED^fbr-T1 were co-expressed in pZE12.The rhyB mutant rhyB1 which was expressed from P_LtetO-1in the pZA31,can pair with the cr-sodB1 and cr-sodB2 which lies ahead of aroG and trpED,leading to the degradation of the two genes at different rate.Although the mRNA levels of trpED^fbrand aroG^fbr,the activity of DAHP synthase and anthranilate synthase were decreased by the engineered small RNAs,the tryptophan production of the the engineered strain. pZA31-rhyB1&pZE12-cr-sodB1-aroG^fbr-cr-sodB2-trpED^fbr/MG1655ΔR-RI was increased about 56%.In summary,the tryptophan biosynthesis pathway in E.Coli was modified by means of molecular biology method.The gene ppsA,tktA,aroG^fbrand trpED^fbrwere expressed,at the same time,the tnaA and trpR were knockedout by RED recombination system.The effects of glucose content,IPTG concentration,liquid level on the tryptophan production of the engineered strain were studied.The tryptophan production reached about 1.3 g·L^-1under optimum condition.The effect of tuning the expression of multiple genes on tryptophan production was also examined,it was shown that the tryptophan production was improved by 56%due to the modulation by the engineered small RNAs.This work settles the ground work for biosynthesis of other amino acid and provides a reference for genetic engineering applied to metabolic engineering.