| This work is the analysis of metabolic pathway and designing rational genetic modification to optimize cellular properties by using principle of molecular biology. Aromatic metabolites such as tryptophan, phenylalanine, tyrosine which can only be synthesized by plants and microbes are essential amino acids for human and animals. In addition, phenylalanine is used in aspartame production and acts as intermediate and well vector in synthesis of some anti-cancer drugs. So it has increasing commercial demand.Escherichia coli and many other microoganism synthesize aromatic amino acids through the condensation reaction between phosphoenolpyru vate(PEP) and erythrose-4-phosphate(E4P) to form 3-deoxy-D- arabinohep tulosonate 7-phosphate(DAHP). PEP and E4P are limiting substrates for formation of DAHP. In bacterial, many enzymes compete for intracellular PEP, especially the phosphotransferase system which is responsible for glucose transport in E. coli. This system uses PEP as a phosphate donor and converts it to pyruvate, which is less likely to recycle back to PEP. ppsA and tktA are the key genes in central metabolism of aromatic amino acids biosynthesis. ppsA encoding phosphoenolpyrucate synthetase A(PpsA) which catalyzes pyruvate into PEP; tktA encoding transketolase A which plays a major role in erythrose-4-phosphate (E4P) production of pentose pathway. The common biosynthesis pathway of aromatic amino acids includes seven steps from DAHP to chorismate acid. For the common pathway, 3-dehydroquinate(DHQ) synthase(encoded by aroB), 5-enolpyruv- oylshikimate S-phosphate(EPSP) synthase(encoded by aroA), and chorisma- te synthase(encoded by aroC] are rate-limiting enzymes. Chorismate acid is branch point in aromatic amino acids biosynthesis, related to phenylalanine, bifunctional enzymes chorismate mutase/prephenate dehydratase(encoded by pheA) is rate-limiting enzyme. The global regulator CsrA of E. coli is a specific mRNA-binding protein. CsrA negatively regulates several metabolic pathways that are induced post-exponentially, including glycogen biosynthesis, gluconeogenesis, and glycogen catabolism; positively controls gene expression within the glycolytic pathway; and also CsrA modulates the levels ofenzymes that participate directly in PEP metabolism. Several enzymes that indirectly affect PEP are also regulated by CsrA. Thus, a CsrA mutation not only causes a significant elevation in intracellular PEP, but also breaks cellular intrinsic metabolic coordination and channel more carbon flux to phenylalanine biosynthesis. Based on above analysis, this thesis improved genetic engineering bacteria of phenylalanine biosynthesis on the following three respects:1. We amplified ppsA and tktA from E.coli K-12 by PCR and constructed recombinant plasmids of them in pBV220 vector containing PRPL promoter. Because of each gene carrying PL promoter, four productions of ligation were obtained, that are PPT-â… ,PPT- â…¡ ,PTP-â… , and PTP-â…¡. The results of SDS-PAGE demonstrated that the bands at 84kD and 73kD were more intensive than the same ones of the controls. The specific activity of PpsA in crude extracts was increased by 10.8-fold, and TktA, by 3.9-fold.2. When both genes were co-expressed in E.coli, the activity of PpsA varied from 2.1-9.1 fold comparing to control, but the activity of TktA was relatively stable(3.9-4.5 fold) . Whatever the two genes were expressed respectively or cooperatively, both could promote the production of DAHP, the first intermediate of the common aromatic pathway, but co-expression was more effective on forming DAHP and screened PPT-â…¡ and PTP-â… as more effective. The results demonstrate that co-expression of ppsA and tktA can improve the production of DAHP, and what's more, when multigenes co-expressed, the recombinant which has coordinated enzymes activity is optimum.3. We amplified aroA, aroC, aroB of common pathway, CsrA and its flanking sequence of global regulation network from E. colt, and Kan resistant gene from plasmid pET28a.4. Centerpi...
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