Es. Miller et Sw. Peretti, Bioconversion of toluene to p-hydroxybenzoate via the construction and characterization of a recombinant Pseudomonas putida, GREEN CHEM, 1(3), 1999, pp. 143-152
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.