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

Biocatalytic production of p-hydroxybenzoate (HBA) provides improved regios pecificity over Kolbe-Schmitt carboxylation of phenol while achieving signi ficant source reductions in the generation of waste and byproducts. Constru ction of the organism for HBA production was accomplished through two class ical approaches for the engineering of organisms in the production of speci alty chemicals: (1) strain enhancement through chemical mutagenesis to crea te a mutant Pseudomonas putida, EM2839, deficient in HBA degradation, and ( 2) hybrid pathway construction through the recruitment of genes encoding th e toluene-4-monooxygenase (T4MO) (tmoABCDE), p-cresol methylhyroxylase (pch CF), and p-hydroxybenzaldehyde dehydrogenase (phbz) genes from existing pat hways and stably incorporating them into the organism through the use of mi ni-Tn5 transposon systems. Time course measurements of HBA production by resting cells of P. putida EM 2878 in batch cultures revealed that T4MO conversion of toluene to p-creseo l, 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 H BA by P. putida EM2878 showed absolute selectivity for para-hydroxybenzoate production. Maximum HBA concentrations of 35 mg l(-1) were achieved in abo ut 28 hours of operation. However, the rate of HBA production was significa ntly less than that observed during batch studies. The slower rate of HBA p roduction observed in the fed-batch culture was correlated with the degrada tion of specific T4MO polypeptides.