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Applied and Environmental Microbiology, September 2003, p. 5120-5127, Vol. 69, No. 9
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.9.5120-5127.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Genetic Engineering of a Highly Solvent-Tolerant Pseudomonas putida Strain for Biotransformation of Toluene to p-Hydroxybenzoate

María-Isabel Ramos-González,1* Arie Ben-Bassat,2 María-Jesús Campos,1 and Juan L. Ramos1

Department of Biochemistry and Molecular and Cellular Biology of Plants, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, 18008 Granada, Spain,1 Central Research and Development, DuPont Experimental Station, Wilmington, Delaware 19880-03282

Received 6 February 2003/ Accepted 18 June 2003

The solvent-tolerant strain Pseudomonas putida DOT-T1E has been engineered for biotransformation of toluene into 4-hydroxybenzoate (4-HBA). P. putida DOT-T1E transforms toluene into 3-methylcatechol in a reaction catalyzed by toluene dioxygenase. The todC1C2 genes encode the {alpha} and ß subunits of the multicomponent enzyme toluene dioxygenase, which catalyzes the first step in the Tod pathway of toluene catabolism. A DOT-T1E{Delta}todC mutant strain was constructed by homologous recombination and was shown to be unable to use toluene as a sole carbon source. The P. putida pobA gene, whose product is responsible for the hydroxylation of 4-HBA into 3,4-hydroxybenzoate, was cloned by complementation of a Pseudomonas mendocina pobA1 pobA2 double mutant. This pobA gene was knocked out in vitro and used to generate a double mutant, DOT-T1E{Delta}todCpobA, that was unable to use either toluene or 4-HBA as a carbon source. The tmo and pcu genes from P. mendocina KR1, which catalyze the transformation of toluene into 4-HBA through a combination of the toluene 4-monoxygenase pathway and oxidation of p-cresol into the hydroxylated carboxylic acid, were subcloned in mini-Tn5Tc and stably recruited in the chromosome of DOT-T1E{Delta}todCpobA. Expression of the tmo and pcu genes took place in a DOT-T1E background due to cross-activation of the tmo promoter by the two-component signal transduction system TodST. Several independent isolates that accumulated 4-HBA in the supernatant from toluene were analyzed. Differences were observed in these clones in the time required for detection of 4-HBA and in the amount of this compound accumulated in the supernatant. The fastest and most noticeable accumulation of 4-HBA (12 mM) was found with a clone designated DOT-T1E-24.

* Corresponding author. Mailing address: Department of Biochemistry and Molecular and Cellular Biology of Plants, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Profesor Albareda, 1, 18008 Granada, Spain. Phone: 34-958-181600. Fax: 34-958-129600. E-mail: maribel.ramos{at}eez.csic.es.

Applied and Environmental Microbiology, September 2003, p. 5120-5127, Vol. 69, No. 9
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.9.5120-5127.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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