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KMID : 0545120000100060789
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Journal of Microbiology and Biotechnology
2000 Volume.10 No. 6 p.789 ~ p.796
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Effects of tktA, aroFFBR,and aroL Expression in the Tryptophan-Producing Escherichia coli
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Kim, Tae Hyun
Kwak, Joon Hyeok/Lee, Se Yong/Lee, Heung Shick/Goong, Suk Nam
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Abstract
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In order to analyze the effects of tktA, aroF^FBR, and aroL expression in a tryptophan-producing Escherichia coli, a series of plasmids carrying the genes were constructed. Introduction of tktA, aroF^FBR, and aroL into the E. coli strain resulted in approximately 10-20 fold increase in the activities of transketolase, the feedback inhibition-resistant 3-deoxyD-arabinoheptulsonate-7-phosphate synthase, and shikimate kinase. Expression of aroF^FBR in the aroB mutant strain of E. coli resulted in the accumulation of 10mM of 3-deoxy-Darabinoheptulsonate-7-phosphate (DAHP) in the medium. Simultaneous expression of tktA and aroF^FBR in the strain further increased the amount of excreted DAHP to 20mM. In contrast, the mutant strain which has no gene introduced accumulated 0.5mM of DAHP. However, the expression of tktA and aroF^FBR in a tryptophan-producing E. coli strain did not lead to the increased production of tryptophan, but instead, a significant amount of shikimate, which is an intermediate in the tryptophan biosynthetic pathway, was excreted to the growth medium. Despite the fact that additional expression of shikimate kinase in the strain could possibly remove 90% of excreted shikimate to 0.1mM, the amount of tryptophan produced was still unchanged. Removing shikimate using a cloned aroL gene caused the excretion of glutamate, which suggests disturbed central carbon metabolism. However, when cultivated in a complex medium, the strain expressing tktA, aroF^FBR, and aroL produced more tryptophan than the parental strain. These data indicate that additional rate-limiting steps are present in the tryptophan biosynthetic pathway, and the carbon flow to the terminal pathway is strictly regulated. Expressing tktA in E. coli cells appeared to impose a great metabolic burden to the cells as evidenced by retarded cell growth in the defined medium. Recombinant E. coli strains harboring plasmids which carry the tktA gene showed a tendency to segregate their plasmids almost completely within 24h.
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