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Rational engineering of the TOL meta-cleavage pathway

(1998) Rational engineering of the TOL meta-cleavage pathway. Biotechnology and Bioengineering , 58 (2-3) pp. 240-249. 10.1002/(SICI)1097-0290(19980420)58:2/3<240::AID-BIT17>3.0.CO;2-G.

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Abstract

The meta-cleavage pathway of Pseudomonas putida mt-2 was simulated using a biochemical systems simulation developed by Regan (1996). A non-competitive inhibition term for catechol-2,3-dioxygenase (C230) by 2-OH-pent-2,4-dienoate (K(i) = 150 μM) was incorporated into the model. The simulation predicted steady state accumulation levels in the μM range for metabolites pre-meta- cleavage, and in the μM range for metabolites post-meta-cleavage. The logarithmic gains L[V(i)/-, X(j)] and L[X(i)/-, X(j)] clearly indicated that the pathway was most sensitive to the concentration of the starting substrate, benzoate, and the first enzyme of the pathway, toluate-1,2- dioxygenase (TO). The simulation was validated experimentally; it was found that the amplification of TO increased the steady state flux from 0.024 to 0.091 (mmol/g cell dwt)/h. This resulted in an increased accumulation of a number of the pathway metabolites (intra and extracellularly), especially cis-diol, 4-OH-2-oxovalerate, and 4-oxalocrotonate. Metabolic control analysis indicated that C230 was, in fact, the major controlling enzymic step of the pathway with a scaled control coefficient of 0.83. The amplification of TO resulted in a shift of some of the control away from C230. Catechol- 2,3-dioxygenase, however, remained as the major controlling element of the pathway.

Type: Article
Title: Rational engineering of the TOL meta-cleavage pathway
DOI: 10.1002/(SICI)1097-0290(19980420)58:2/3<240::AID-BIT17>3.0.CO;2-G
UCL classification: UCL > Provost and Vice Provost Offices
UCL > Provost and Vice Provost Offices > UCL BEAMS
UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science
UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science > Dept of Biochemical Engineering
URI: http://discovery.ucl.ac.uk/id/eprint/21956
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