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

ABSTRACT

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 α and β subunits of the multicomponent enzyme toluene dioxygenase, which catalyzes the first step in the Tod pathway of toluene catabolism. A DOT-T1EΔtodC 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-T1EΔtodCpobA, 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-T1EΔtodCpobA. 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.