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Metabolic engineering of a toluene degradation pathway

Jackson, Graham Andrew; (1996) Metabolic engineering of a toluene degradation pathway. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

A mathematical model simulating metabolic flux through the TOL plasmid pWWO meta-cleavage pathway of Pseudomonas putida mt-2 (Regan et al., 1991) has been experimentally investigated. Two of the predicted flux controlling steps in the pathway, the xylXYZ encoded benzoate dioxygenase (plasmid pQR189) and the xylI encoded oxalocrotonate decarboxylase (plasmid pQR194) have been individually co-expressed with the entire meta-cleavage pathway encoded on plasmid pQR150 in the same E.coli JM107 cell. Co-expressing pQR189 and pQR150 in the same E.coli cell during benzoate metabolism has suggested a possible role of benzoate dioxygenase in the reactivation of catechol 2,3-dioxygenase. Upon co-expression of pQR194 with pQR150, pathway metabolism was apparently shut-down in the presence of benzoate. It was predicted that this was due to an initial over-accumulation of the product of xylI, 2-hydroxypent-2, 4-dienoate. Kinetic analysis has revealed that 2-hydroxypent-2, 4-dienoate acts as a noncompetitive inhibitor of catechol 2, 3-dioxygenase, with a Ki of 1.49*10-4 M. This, together with other findings, suggests that the physical coupling of oxalocrotonate decarboxylase and the xylJ encoded 2-oxopent-4-enoate hydratase exists to prevent the accumulation of this toxic intermediate. Hybrid dioxygenases xylX:bphA1A2A3A4 (pQR191) and bphA1:xylYZ, (pQR192) have been shown to be non-functional, thus indicating that enzyme subunits displaying an amino acid sequence identity of only 27% can not be interchanged. The substrate range of the TOL plasmid meta-cleavage pathway has been broadened as a result of the co-expression of the nahA encoded naphthalene dioxygenase from P.putida G7 with the meta-cleavage pathway of the TOL plasmid. Such a dual-dioxygenase system is capable of the metabolism of naphthalene, biphenyl and benzoate as far as ring-cleavage intermediates.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Metabolic engineering of a toluene degradation pathway
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Applied sciences
URI: https://discovery.ucl.ac.uk/id/eprint/10098284
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