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Science 30 January 1987:
Vol. 235. no. 4788, pp. 593 - 596
DOI: 10.1126/science.3468623
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Articles

Science, Vol 235, Issue 4788, 593-596
Copyright © 1987 by American Association for the Advancement of Science

articles

Redesigning metabolic routes: manipulation of TOL plasmid pathway for catabolism of alkylbenzoates

JL Ramos, A Wasserfallen, K Rose, and KN Timmis

Increasing quantities of man-made organic chemicals are released each year into the biosphere. Some of these compounds are both toxic and relatively resistant to physical, chemical, or biological degradation, and they thus constitute an environmental burden of considerable magnitude. Genetic manipulation of microbial catabolic pathways offers a powerful means by which to accelerate evolution of biodegradative routes through which such compounds might be eliminated from the environment. In the experiments described here, a catabolic pathway for alkylbenzoates specified by the TOL plasmid of Pseudomonas was restructured to produce a pathway capable of processing a new substrate, 4-ethylbenzoate. Analysis of critical steps in the TOL pathway that prevent metabolism of 4-ethylbenzoate revealed that this compound fails to induce synthesis of the catabolic enzymes and that one of its metabolic intermediates inactivates catechol 2,3-dioxygenase (C23O), the enzyme that cleaves the aromatic ring. Consequently, the pathway was sequentially modified by recruitment of genes from mutant bacteria selected for their production of either an altered pathway operon regulator that is activated by 4-ethylbenzoate or an altered C23O that is less sensitive to metabolite inactivation. The redesigned pathway was stably expressed and enabled host bacteria to degrade 4-ethylbenzoate in addition to the normal substrates of the TOL pathway.


THIS ARTICLE HAS BEEN CITED BY OTHER ARTICLES:
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Y. R. Sevastsyanovich, R. Krasowiak, L. E. H. Bingle, A. S. Haines, S. L. Sokolov, I. A. Kosheleva, A. A. Leuchuk, M. A. Titok, K. Smalla, and C. M. Thomas (2008)
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Quantitative Structure-Activity Relationship for the Cleavage of C3/C4-Substituted Catechols by a Prototypal Extradiol Catechol Dioxygenase with Broad Substrate Specificity.
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J. Biochem. 135, 721-730
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3- and 4-alkylphenol degradation pathway in Pseudomonas sp. strain KL28: genetic organization of the lap gene cluster and substrate specificities of phenol hydroxylase and catechol 2,3-dioxygenase.
J. J. Jeong, J. H. Kim, C.-K. Kim, I. Hwang, and K. Lee (2003)
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Novel Approach to the Improvement of Biphenyl and Polychlorinated Biphenyl Degradation Activity: Promoter Implantation by Homologous Recombination.
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Catalytic Properties of the 3-Chlorocatechol-Oxidizing 2,3-Dihydroxybiphenyl 1,2-Dioxygenase from Sphingomonas sp. Strain BN6.
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Adenosylcobalamin-Mediated Methyl Transfer by Toluate cis-Dihydrodiol Dehydrogenase of the TOL Plasmid pWW0.
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Pleiotropic Effects of Adaptation to a Single Carbon Source for Growth on Alternative Substrates.
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Combined Physical and Genetic Map of the Pseudomonas putida KT2440 Chromosome.
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Crystal Structure of the Biphenyl-Cleaving Extradiol Dioxygenase from a PCB-Degrading Pseudomonad.
S. Han, L. D. Eltis, K. N. Timmis, S. W. Muchmore, and J. T. Bolin (1995)
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Genetic engineering of bacteria from managed and natural habitats.
S. Lindow, N. Panopoulos, and B. McFarland (1989)
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Assemblage of ortho cleavage route for simultaneous degradation of chloro- and methylaromatics.
F Rojo, D. Pieper, K. Engesser, H. Knackmuss, and K. Timmis (1987)
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