Redesigning Metabolic Routes: Manipulation of TOL Plasmid Pathway for Catabolism of Alkylbenzoates

Abstract

Increasing quantities of man-made organic chemicals are released each year into the biosphere. Some of these compounds are both toxic and relatively resistant to physical, chemical, or biological degradation, and they thus constitute an environmental burden of considerable magnitude. Genetic manipulation of microbial catabolic pathways offers a powerful means by which to accelerate evolution of biodegradative routes through which such compounds might be eliminated from the environment. In the experiments described here, a catabolic pathway for alkylbenzoates specified by the TOL plasmid of Pseudomonas was restructured to produce a pathway capable of processing a new substrate, 4-ethylbenzoate. Analysis of critical steps in the TOL pathway that prevent metabolism of 4-ethylbenzoate revealed that this compound fails to induce synthesis of the catabolic enzymes and that one of its metabolic intermediates inactivates catechol 2,3-dioxygenase (C23O), the enzyme that cleaves the aromatic ring. Consequently, the pathway was sequentially modified by recruitment of genes from mutant bacteria selected for their production of either an altered pathway operon regulator that is activated by 4-ethylbenzoate or an altered C23O that is less sensitive to metabolite inactivation. The redesigned pathway was stably expressed and enabled host bacteria to degrade 4-ethylbenzoate in addition to the normal substrates of the TOL pathway.

3- and 4-alkylphenol degradation pathway in Pseudomonas sp. strain KL28: genetic organization of the lap gene cluster and substrate specificities of phenol hydroxylase and catechol 2,3-dioxygenase

“…The specific activity of LapB was determined using a cell extract of E. coli DH5a(pJJC23O) in the same phosphate buffer by measuring the rate of formation of meta-cleavage products from (substituted) catechols. The wavelengths (absorption coefficients in mM 21 cm 21 ) used to monitor the formation of the metacleavage products of catechol, 3-methylcatechol, 4-methylcatechol, 4-ethylcatechol and 2,3-dihydroxybiphenyl were 376 (40), 389 (11?9), 382 (24?5), 381 (36?0) and 434 (19?8) nm, respectively (Bayly et al, 1966;Duggleby & Williams, 1986;Ramos et al, 1987;Seah et al, 1998). Protein concentrations were determined using the BCA protein assay (Pierce) with BSA as the standard.…”

“…Cell extracts were obtained as previously described (Cho et al, 2000) and used to determine if the three C23Os could form the same product from a given catechol substrate. Assay mixtures (total 1 ml) contained potassium phosphate buffer (0?1 M, pH 7?5), catechol substrate (final concentration 0?1 mM) and cell extract (75 mg) and incubated (Bayly et al, 1966;Duggleby & Williams, 1986;Ramos et al, 1987;Seah et al, 1998). Protein concentrations were determined using the BCA protein assay (Pierce) with BSA as the standard.…”

“…The site of ring cleavage of the C23O of strain KL28 (LapB) was determined by comparing the visible spectra of the ring fission products with those formed by TodE and XylE. The latter two enzymes are found in the degradation pathways of toluene and xylenes and are known to catalyse the dioxygenation of 3-and 4-methylcatechol by (2,3) proximal cleavage (Cerdan et al, 1994;Cho et al, 2000;Klecka & Gibson, 1981;Ramos et al, 1987). In addition, XylE and TodE are known to catalyse the dioxygenation of 4-ethylcatechol and 2,3-dihydroxybiphenyl, respectively, via the same mode of ring cleavage (Cerdan et al, 1994;Cho et al, 2000;Ramos et al, 1987).…”

Tracking genetically engineered bacteria: monoclonal antibodies against surface determinants of the soil bacterium Pseudomonas putida 2440

“…MAbs against surface determinants of P. putida 2440, the host bacterium for recombinant TOL plasmid and other recombinant plasmids (4,9,35), were sought with a screening strategy that involved an anti-antibody coupled to FITC and intact microorganisms as the antigen source. To select highly specific MAbs, a large battery of eubacteria was used in the screening.…”

“…P. putida 2440 has been used as the host for recombinant TOL plasmids able to eliminate aromatic hydrocarbons that cannot be degraded by non-pWWO-modified pathways (1,35). Furthermore, P. putida 2440 has been shown to be an excellent recipient for recombinant plasmids through conjugation and transformation (4), and it has been used in several biotransformation processes, e.g., production of benzyl alcohols (9).…”