Journal of Microbiology and Biotechnology

  • 제10권6호
  • /
  • Pages.789-796
  • /
  • 2000
  • /
  • 1017-7825(pISSN)
  • /
  • 1738-8872(eISSN)
한국미생물·생명공학회 (The Korean Society for Microbiology and Biotechnology)
권호 표지

Effects of tktA, $aroF^{FBR}$and aroL Expression in the Tryptophan-Producing Escherichia coli

  • 발행 : 2000.12.01
PDF 다운로드
⟨ 이전 논문 다음 논문 ⟩

초록

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-deoxy-D-arabinoheptulsonate-7-phosphate synthase, and shikimate kinase. Expression of $aroF^{FBR}$ in the aroB mutant strain of E. coli resulted in the accumulation of 10 mM of 3-deoxy-D-arabinoheptulsonate-7-phosphate (DAHP) in the medium. Simultaneous expression of tktA and $aroF^{FBR}$ in the strain further increased the amount of excreted DAHP to 20 mM. In contrast, the mutant strain which has no gene introduced accumulated 0.5 mM 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 0.1 mM, 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.

키워드

참고문헌

  1. Escherichia coli and Salmonella typhimurium: Cellular and molecular biology, (1st ed.) Derivations and genotypes of some mutant derivatives of Escherichia coli K-12 Bachmann, B. J.;F. C. Neidhrdt(eds);J. L. Ingraham(eds);K. B. Low(eds);B. Magasanik(eds)M. Schaechter(eds);H. E. Umbarger(eds)
  2. J. Chromatogr. v.329 High-performance liquid chromatography of shikimate pathway intermediates Moldave, M. M.;J. R. Coggins
  3. J. Bacteriol. v.165 Nucleotide sequence of the transcription unit containing the aroL and aroM genes from Escherichia coli K-12 DeFeyter, R. C.;B. E. Davidson;J. Pittard
  4. J. Bacteriol. v.165 Genetic and molecular analysis of aroL, the gene for shikimate kinase II in Escherichia coli K-12 DeFeyter, R. C.;J. Pittard
  5. J. Am. Chem. Soc. v.114 Biocatalytic synthesis of aromatics from D-glucose: The role of transketolase Draths, K.;D. Pompliano;D. Conley;J. Frost;A. Berry;G. Disbrow;R. Staveskey;J. Lievense
  6. Methods Enzymol. v.152 Assay for 3-deoxy-D-arabinoheptulosonic acid-7-phosphate synthase Gollub, E.;H. Zalkin;D. Sprin
  7. Annu. Rev. Microbiol. v.49 Biocatalytic synthesis of aromatics from D-glcose: Renewable microbial sources of aromatic compound Frost, J.;K. Drath
  8. Genetic manipulation of Escherichia coli for improvement of tryptophan production Hong, K. W.
  9. J. Bacteriol. v.175 Identification and characterization of the tktB gene encoding a second transketolase in Escherichia coli K-12 Iida, A.;S. Teshiba;K. Mizobuchi
  10. Appl. Environ. Microbiol. v.65 Hyperproduction of tryptophan by Corynebacterium glutamicum with the modified pentose phosphate pathway Ikeda, M.;R. Katsumata
  11. Bio/Technology v.11 Hyperproduction of tryptophan by Corynebacterium glutamicum by pathway engineering Katsumata, R.;M. Ikeda
  12. J. Microbiol. Biotechnol. v.7 Gene amplification of aceA and aceB in lysine-producing Corynebacterium glutamicum ssp. lactofermentum ATCC21799 Kim, H.-J.;Y. Kim;H.-S. Lee
  13. J. Microbiol. Biotechnol. v.8 Strain improvement of yeast for ethanol production using a combined treatment of electric field and chemical mutagen N-mehty-N-nitro-N-nitrosoguanidine Kim, K.;J.-Y. Lee
  14. J. Mocrobiol. Biotechnol. v.6 Site-specific disruption of glyoxylate bypass and its effect in lysine-producing Corynebacterium lactofermentum strain Kim, Y.;H.-S. Lee
  15. Molecular cloning: A laboratory manual Maniatis, T.;J. Sambrook;E. Fritsch
  16. The overexpression of tktA gene encoding transketolase results in the accumulation of 3-deoxy-D-arabinoheptulsonate-7-phosphate and shikimic acid in Escherichia coli Namgoog, S.
  17. Proc. Natl. Acad. Sci. USA v.79 Biosynthesis of aromatic compounds: C NMR spectroscopy of whole Escherichia coli cells Ogiono, T.;C. Garner;J. L. Markley;K. M. Herrmann
  18. J. Bacteriol. v.174 Stimulationof glucose catabolism in Escherichia coli by a potential futile cycle Patnaik, R.;W. D. Roof;R. F. Young;J. C. Liao
  19. Biotechnol. Bioeng. v.46 Pathway engineering for production of aromatics in Escherichia coli:Confimation of stoichiometric analysis by independent modulationof aroG, tktA, and activities Patnaik, R.;R. Spitzner;J. C. Liao
  20. Appl. Environ. Microbiol. v.60 Engineering of Escherichia coli central metabolosm for aromatic metabolite production with near theoretocal yield Patnaik, R.;J. C. Liao
  21. J. Microbiol. Biotechnol. v.9 Isolation of a high-yield mutant strain for L-proline production, and its fermentation conditions Ryu, W.-S.;H.-W. Jang;K.-H. Cho;S.-J. Chang;Y.-W. Ryu;Y.-H. Park
  22. J. Am. Chem. Soc. v.118 Synthetic modification of the Escherichia coli chromosome: Enhancing the biocatalytic conversion of glucose into aromatic chemicals Snell, K.;K. Draths;J. Frost
  23. Amino acids biosynthesis and genetic regulation Tryptophan: Biosynthesis, regulation, and large-scale production Somerville, R. L.;Herrman, K(eds.);R. Senerville(eds.)
  24. Biochim. Biophys. Acta v.1216 Nucleotide sequence of the Escherichia coli K-12 transkerolase (tkt) gene Sprenger, G.A.
  25. J. Microbiol. Biotechnol. v.9 Metabolic analysis of poly(3-hydroxybutyrate) production by recombinant Escherichia coli Wong, H. H.;R. J. van Wegen;J.-I. Choi;S. Y. Lee;A. P. J. Middelberg
  26. J. Microbiol. Biotechnol. v.9 Cloning, nucleotide sequencing, and characterization of the ptsG gene encoding glucose-specific enzyme II of the phosphotransferase system from Brevibacterium lactofermentum Yoon, K.-H.;K.-N. Lee;J.-K. Lee;S. C. Park
  27. J.Microbiol. Biotechnol. v.8 Cloning expression, and nucleotide sequencing of the gene encoding glucose permease of phosphotransferase systen from Brevibacterium ammoniagenes Yoon, K.-H.;H. Yim;K.-H. Jung