Bioconversion of toluene to p-hydroxybenzoate . via the construction and characterization of a recombinant Pseudomonas putida

Abstract

Biocatalytic production of p-hydroxybenzoate (HBA) provides improved regiospecificity over Kolbe–Schmitt carboxylation of phenol while achieving significant source reductions in the generation of waste and byproducts. Construction of the organism for HBA production was accomplished through two classical approaches for the engineering of organisms in the production of specialty chemicals: (1) strain enhancement through chemical mutagenesis to create a mutant Pseudomonas putida, EM2839, deficient in HBA degradation, and (2) hybrid pathway construction through the recruitment of genes encoding the toluene-4-monooxygenase (T4MO) (tmoABCDE), p-cresol methylhyroxylase (pchCF), and p-hydroxybenzaldehyde dehydrogenase (phbz) genes from existing pathways and stably incorporating them into the organism through the use of mini-Tn5 transposon systems.

Time course measurements of HBA production by resting cells of P. putida EM2878 in batch cultures revealed that T4MO conversion of toluene to p-creseol, the first step in the pathway, significantly constrained the carbon flux in the pathway, yielding a maximum rate of HBA production of 1.61 ± 0.15 nmol min^–1 mg protein^–1. In fed-batch culture, toluene conversion to HBA by P. putida EM2878 showed absolute selectivity for para-hydroxybenzoate production. Maximum HBA concentrations of 35 mg l^–1 were achieved in about 28 hours of operation. However, the rate of HBA production was significantly less than that observed during batch studies. The slower rate of HBA production observed in the fed-batch culture was correlated with the degradation of specific T4MO polypeptides.

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