Metabolism of arylsulphonates by micro-organisms

1. Species of Pseudomonas capable of degrading arylsulphonates and detergents of the alkylbenzenesulphonate type were isolated from sewage and river water. 2. Benzenesulphinate, benzenesulphonate and toluene-p-sulphonate were rapidly degraded by these organisms with the release of the sulphonate group as sulphite; detergent homologues with a chain length up to 16 carbon atoms (4-n-hexadecyl-benzenesulphonate) also released sulphite. Sulphite oxidation to sulphate in the medium can occur non-enzymically. 3. Growth on benzenesulphonate and toluene-p-sulphonate elicited a catechol 2,3-oxygenase, which effected a ;meta' cleavage of the ring. The metabolic route for benzenesulphonate was determined as: benzenesulphonate-->catechol-->2-hydroxymuconic semialdehyde-->formate and 4-hydroxy-2-oxovalerate-->acetaldehyde and pyruvate; the enzymes catalysing these steps were all inducible. 4. Toluene-p-sulphonate was degraded via 2-hydroxy-5-methylmuconic semialdehyde to formate and 4-hydroxy-2-oxohexanoate and the latter was cleaved to propionaldehyde and pyruvate. Propionaldehyde and propionate were oxidized rapidly by toluene-p-sulphonate-grown cells but slowly by fumarate-grown organisms. 5. The specificity of the catechol 2,3-oxygenase induced by the arylsulphonates, towards catechol and the methylcatechols, varied during the purification and suggested that 3-methylcatechol was probably oxidized by a separate enzyme. Detergents of the alkylbenzenesulphonate type also induced a catechol 2,3-oxygenase in these bacteria. 6. A few isolates, after growth on benzenesulphonate, opened the ring of catechol by an ;ortho' route to form cis-cis-muconate. The enzymes to degrade this intermediate to beta-oxoadipate were also present in induced cells.     

Role of Catechol and the Methylcatechols as Inducers of Aromatic Metabolism in Pseudomonas putida

Pseudomonas putida NCIB 10015 metabolizes phenol and the cresols (methylphenols) by the meta pathway and metabolizes benzoate by the ortho pathway. Growth on catechol, an intermediate in the metabolism of both phenol and benzoate, induces both ortho and meta pathways; growth on 3- or 4-methylcatechols, intermediates in the metabolism of the cresols, induces only the meta pathway to a very limited degree. Addition of catechol at a growth-limiting rate induces virtually no meta pathway enzymes, but high levels of ortho pathway enzymes. The role of catechol and the methylcatechols as inducers is discussed. A method is described for assaying low levels of catechol 1,2-oxygenase in the presence of high levels of catechol 2,3-oxygenase and vice versa.     

Metabolism of 3-chlorobenzoate by a Pseudomonas (diff) spp

Pseudomonas (diff) spp. was isolated from a complex petrochemical sludge, using benzoate as the sole source of carbon. The organism could metabolize 3-chlorobenzoate, releasing approximately 30% of organically bound chloride. 3-Chlorodihydrodihydroxybenzoate and 3-chlorocatechol were confirmed as pathway intermediates by mass spectral and HPLC analysis. About 3-fold higher levels of catechol 1,2-oxygenase were detected in cells grown on 3-chlorobenzoate as compared to that of benzoate. 3-Chlorocatechol inhibited the catechol 1,2-oxygenase activity, when used as assay substrate. A 15-fold purified catechol 1,2-oxygenase had a Km of 0.37 mumole and Vmax of 2.3 with 3-chlorocatechol. Catechol gave Km of 0.2 mumole and Vmax of 40, suggesting that 3-chlorocatechol is not metabolised further and hence blocks the metabolic pathway for 3-chlorobenzoate degradation. In contrast catechol 1,2-oxygenase was not inhibited by 4-chlorocatechol and probably is an intermediate for the total/complete degradation of 3-chlorobenzoate (approx. 30%).     

Degradation of 2-fluorobenzoate by a pseudomonad

Pseudomonas (spp), isolated from a complex petrochemical sludge, was able to utilize 2-fluorobenzoate as its sole source of carbon and energy. At the end of the growth phase, about 42% of the organically bound fluoride was released. Catechol, 3-fluorocatechol, and 6-fluorodihydrodihydroxybenzoate were confirmed as intermediates by chromatographic and spectral analyses. During 2-fluorobenzoate metabolism, fluoride is eliminated before the aromaticity of the ring is lost. Twofold higher levels of catechol 1,2-oxygenase were detected in 2-fluorobenzoate-grown cells compared with cells grown on benzoate. When used as assay substrates, 3-chlorocatechol showed less catechol 1,2-oxygenase activity than catechol or 4-chlorocatechol. The ability to degrade 4-fluorobenzoate could be transferred toPseudomonas (spp) by the conjugal transfer of plasmid pWR1 fromPseudomonas sp. B13.     

The metabolism of aromatic acids by micro-organisms. Metabolic pathways in the fungi

1. The metabolic pathways of aromatic-ring fission were examined in a range of fungal genera that utilize several compounds related to lignin. 2. Most of the genera, after growth on p-hydroxybenzoate, protocatechuate or compounds that are degraded to the latter (e.g. caffeate, ferulate or vanillate), rapidly oxidized these compounds, but not catechol. 3. Such genera possessed a protocatechuate 3,4-oxygenase and accumulated beta-carboxymuconate as the product of protocatechuate oxidation. This enzyme had a high pH optimum in most organisms; the Rhodotorula enzyme was competitively inhibited by catechol. 4. beta-Carboxymuconate was converted by all competent fungi into beta-carboxymuconolactone, which was isolated and characterized. None of the fungi produced or utilized at significant rates the corresponding bacterial intermediate gamma-carboxymuconolactone. 5. The lactonizing enzymes of Rhodotorula and Neurospora crassa had a pH optimum near 5.5 and approximate molecular weights of 19000 and 190000 respectively. 6. The fungi did not degrade the isomeric (+)-muconolactone, gamma-carboxymethylenebutanolide or beta-oxoadipate enol lactone at significant rates, and thus differ radically from bacteria, where beta-oxoadipate enol lactone is the precursor of beta-oxoadipate in all strains examined. 7. The end product of beta-carboxymuconolactone metabolism by extracts was beta-oxoadipate. 8. Evidence for a coenzyme A derivative of beta-oxoadipate was found during further metabolism of this keto acid. 9. A few anomalous fungi, after growth on p-hydroxybenzoate, had no protocatechuate 3,4-oxygenase, but possessed all the enzymes of the catechol pathway. Catechol was detected in the growth medium in one instance. 10. A strain of Penicillium sp. formed pyruvate but no beta-oxoadipate from protocatechuate, suggesting the existence also of a ;meta' type of ring cleavage among fungi.     

Construction of hybrid xylE genes between the two duplicate homologous genes from TOL plasmid pWW53: comparison of the kinetic properties of the gene products

The two xylE genes for catechol 2,3-oxygenase, encoded by TOL plasmid pWW53, carry a common SalI restriction site within the reading frame. Each gene was cut at the SalI site and the 5' end of each gene spliced to the 3' end of the other to form hybrid genes, from both of which catalytically active catechol 2,3-oxygenase activities were expressed. The kinetic parameters were determined for the gene products of both the hybrid and the wild-type xylE genes with catechol, 3-methylcatechol and 4-methylcatechol as substrates. Comparison of the results suggested firstly, that the C-terminal regions of the enzymes determined both the binding and the catalytic specificity, and, secondly, that the N-terminal region of one of the enzymic gene products contained a secondary binding site which caused inhibition by excess substrate for methylcatechol substrates but not for catechol. One of the wild-type enzymes appeared to have an intrinsically higher activity for all three substrates than the other. This higher activity depended on the presence of both its C- and N-terminal regions, and in both hybrid enzymes, which contained only one of these regions, activity was significantly reduced.     

Catechol oxygenases of Pseudomonas putida mutant strains.

Investigation of a mutant strain of Pseudomonas putida NCIB 10015, strain PsU-E1, showed that it had lost the ability to produce catechol 1,2-oxygenase after growth with catechol. Additional mutants of both wild-type and mutant strains PsU-E1 have been isolated that grow on catechol, but not on benzoate, yet still form a catechol 1,2-oxygenase when exposed to benzoate. These findings indicate that either there are separately induced catechol 1,2-oxygenase enzymes, or that there are two separate inducers for the one catechol 1,2-oxygenase enzyme. Comparisons of the physical properties of the catechol 1,2-oxygenases formed in response to the two different inducers show no significant differences, so it is more probable that the two proteins are the product of the same gene. Sufficient enzymes of the ortho-fission pathway are induced in the wild-type strain by the initial substrate benzoate (or an early intermediate) to commit that substrate to metabolism by ortho fission exclusively. A mechanism exists that permits metabolism of catechol by meta fission if the ortho-fission enzymes are unable to prevent its intracellular accumulation.     

Phenol and Benzoate Metabolism by Pseudomonas putida: Regulation of Tangential Pathways

Catechol occurs as an intermediate in the metabolism of both benzoate and phenol by strains of Pseudomonas putida. During growth at the expense of benzoate, catechol is cleaved ortho (1,2-oxygenase) and metabolized via the beta-ketoadipate pathway; during growth at the expense of phenol or cresols, the catechol or substituted catechols formed are metabolized by a separate pathway following meta (2,3-oxygenase) cleavage of the aromatic ring of catechol. It is possible to explain the mutually exclusive occurrence of the meta and ortho pathway enzymes in phenol- and benzoate-grown cells of P. putida on the basis of differences in the mode of regulation of these two pathways. By use of both nonmetabolizable inducers and blocked mutants, gratuitous synthesis of some of the meta pathway enzymes was obtained. All four enzymes of the meta pathway are induced by the primary substrate, cresol or phenol, or its analogue. Three enzymes of the ortho pathway that catalyze the conversion of catechol to beta-ketoadipate enol-lactone are induced by cis,cis-muconate, produced from catechol by 1,2-oxygenase-mediated cleavage. Observations on the differences in specificity of induction and function of the two pathways suggest that they are not really either tangential or redundant. The meta pathway serves as a general mechanism for catabolism of various alkyl derivatives of catechol derived from substituted phenolic compounds. The ortho pathway is more specific and serves primarily in the catabolism of precursors of catechol and catechol itself.     

Microbial degradation of alkylbenzenesulphonates. Metabolism of homologues of short alkyl-chain length by an Alcaligenes sp

1. An organism isolated from sewage and identified as an Alcaligenes sp. utilized benzenesulphonate, toluene-p-sulphonate or phenylethane-p-sulphonate as sole source of carbon and energy for growth. Higher alkylbenzenesulphonate homologues and the hydrocarbons, benzene, toluene, phenylethane and 1-phenyldodecane were not utilized. 2. 2-Phenylpropanesulphonate was metabolized to 4-isopropylcatechol. 3. 1-Phenylpropanesulphonate was metabolized to an ortho-diol, which was tentatively identified, in the absence of an authentic specimen, as 4-n-propylcatechol. 4. In the presence of 4-isopropylcatechol, which inhibited catechol 2,3-dioxygenase, 4-ethylcatechol accumulated in cultures growing on phenylethane-p-sulphonate. 5. Authentic samples of catechol, 3-methylcatechol, 4-methylcatechol, 4-ethylcatechol and 3-isopropylcatechol were oxidized by heat-treated extracts to the corresponding 2-hydroxyalkylmuconic semialdehydes. Ring cleavage occurred between C-2 and C-3. 6. The catechol derived from 1-phenylpropanesulphonate was oxygenated by catechol 2,3-dioxygenase to a compound with all the properties of a 2-hydroxyalkylmuconic semialdehyde, but it was not rigorously identified. 7. The catechol 2,3-dioxygenase induced by growth on benzenesulphonate, toluene-p-sulphonate or phenylethane-p-sulphonate showed a constant ratio of specific activities with catechol, 3-methylcatechol, 4-methylcatechol and 4-ethylcatechol that was independent of the growth substrate. At 60°C, activity towards these substrates declined at an identical first-order rate. 8. Enzymes of the `ortho' pathway of catechol metabolism were present in small amounts in cells grown on benzenesulphonate, toluene-p-sulphonate or phenylethane-p-sulphonate. 9. The catechol 1,2-dioxygenase oxidized the alkylcatechols, but the rates and the total extents of oxidation were less than for catechol itself. The oxidation products of these alkylcatechols were not further metabolized.     

Evidence for the induction of catechol-1,2-oxygenase by fulvic acid

Summary The effect of fulvic acid on the growth of a benzoate-metabolizing Arthrobacter sp. has been studied. The Arthrobacter grew exponentially with benzoate and fulvic acid but if fulvic acid was omitted then growth was progressively slower and catechol accumulated. These results indicated that fulvic acid enhanced the synthesis of catechol-1,2-oxygenase. Catechol-1,2-oxygenase activity measurements support this suggestion. re]19750822     

Regulation of ascorbic acid and of xylulose synthesis in rat-liver extracts. The effect of starvation on the enzymes of the glucuronic acid pathway

1. Control of enzyme formation has been examined in the pathways degrading mandelate and p-hydroxymandelate in Pseudomonas fluorescens. 2. The first three enzymes form a group which is common to both pathways and which is co-ordinately induced or repressed. The genes controlling these enzymes are assumed to form a ;regulon'. This group of enzymes is induced by mandelate or p-hydroxymandelate and repressed by benzoate and by p-hydroxybenzoate (the immediate end products resulting from the action of this group of enzymes). 3. Repression is independently exerted by end products of enzymes controlled by succeeding regulons, i.e. by catechol, by protocatechuate and finally by succinate and acetate. 4. The pattern is repeated further along the pathway, so that benzoate oxidase (controlled by the second regulon) is repressed by its immediate end product, catechol, and again by succinate and acetate. 5. Pyrocatechase, an enzyme controlled by the third regulon, is repressed by succinate and acetate. 6. There is a parallel system of multi-sensitive repression mechanisms controlling production of the enzymes that degrade the hydroxy compounds. Again, the enzymes of each regulon are repressed by the immediate end product of their action and by the end products of each succeeding group of enzymes. 7. Repressor activity appears to be exerted by compounds that are likely to occur as such in the external environment or that occur at points of convergence of the degradative pathways of the cell. 8. The net effect of this control system, involving both induction and end-product repression, appears to be that cells will not form inducible degradative enzymes if the end products are already being supplied from without or are being produced by degradation of some alternative source of carbon and energy.     

Control of catechol meta-cleavage pathway in Alcaligenes eutrophus

Alcaligenes eutrophus 335 (ATCC 17697) metabolizes phenol and p-cresol via a catechol meta-cleavage pathway. Studies with mutant strains, each defective in an enzyme of the pathway, showed that the six enzymes assayed are induced by the primary substrate. Studies with a putative polarity mutant defective in the expression of aldehyde dehydrogenase suggested that the structural genes encoding this and subsequent enzymes of the pathway exist in the same operon. From studies with mutant strains that constitutively synthesize catechol 2,3-oxygenase and subsequent enzymes and from the coordination of repression of these enzymes by p-toluate, benzoate, and acetate, it is proposed the catechol 2,3-oxygenase structural gene is situated in this operon (2,3-oxygenase operon). Studies with regulatory mutant strains suggest that the 2,3-oxygenase operon is under negative control.     

Catechol ring-cleavage in Pseudomonas cepacia: the simultaneous induction of ortho and meta pathways

This work demonstrates the ring-cleavage pathways of catechol on Pseudomonas cepacia ATCC 29351, formed upon its growth on salicylate and benzoate, each as a sole carbon source. When grown on salicylate, P. cepacia induces only the catechol ortho pathway by its induction of catechol 1,2-dioxygenase. However, interestingly, benzoate-grown cells induce the ortho and meta pathways for the biodegradation of catechol, by inducing simultaneously catechol 1,2-dioxygenase and 2,3-dioxygenase, respectively, in the ratio of 7:1. The results indicate that P. cepacia ATCC 29351 possesses the genetic capacity for enzymes of both the ortho- and meta-cleavage pathways of benzoate degradation, although the phenotypic expression for the ortho pathway is higher. The simultaneous induction of catechol 1,2- and 2,3-dioxygenase is not detected in salicylate degradation. Although catechol is the metabolic intermediate for both salicylate and benzoate, catechol did not induce either pathway when used as a sole carbon source.     

Expression and substrate specificity of the toluene dioxygenase of Pseudomonas putida NCIMB 11767

 Pseudomonas putida NCIMB 11767 oxidized phenol, monochlorophenols, several dichlorophenols and a range of alkylbenzenes (C₁–C₆) via an inducible toluene dioxygenase enzyme system. Biphenyl and naphthalene were also oxidized by this enzyme. Growth on toluene and phenol induced the meta-ring-fission enzyme, catechol 2,3-oxygenase, whereas growth on benzoate, which did not require expression of toluene dioxygenase, induced the ortho-ringcleavage enzyme, catechol 1,2-oxygenase. Monochlorobenzoate isomers and 2,3,5-trichlorophenol were gratuitous inducers of toluene dioxygenase, whereas 3,4-dichlorophenol was a fortuitous oxidation substrate of the enzyme. The organism also grew on 2,4- and 2,5-dichloro isomers of both phenol and benzoate, on 2,3,4-trichlorophenol and on 1-phenylheptane. During growth on toluene in nitrogen-limited chemostat culture, expression of both toluene dioxygenase and catechol 2,3-oxygenase was positively correlated with increase in specific growth rate (0.11–0.74 h^-1), whereas the biomass yield coefficient decreased. At optimal dilution rates, the predicted performance of a 1-m³ bioreactor supplied with 1 g nitrogen l^-1 for removal of toluene was 57 g day^-1 and for removal of trichloroethylene was 3.4 g day^-1. The work highlights the oxidative versatility of this bacterium with respect to substituted hydrocarbons and shows how growth rate influences the production of competent cells for potential use as bioremediation catalysts. Received: 26 June 1995 / Received revision: 4 September 1995 / Accepted: 20 September 1995     

Localization and functional analysis of transposon mutations in regulatory genes of the TOL catabolic pathway.

Mutant derivatives of the TOL plasmid pWW0-161, containing Tn5 insertions in the xylS and xylR regulatory genes of the catabolic pathway, have been identified and characterized. The two genes are located together on a 1.5- to 3.0-kilobase segment of TOL, just downstream of genes of the enzymes of the meta-cleavage pathway. As predicted by a current model for regulation of the TOL catabolic pathway, benzyl alcohol dehydrogenase, a representative enzyme of the upper (hydrocarbon leads to carboxylic acid) pathway, was induced by m-methylbenzyl alcohol in xylS mutant bacteria but not in a xylR mutant, whereas catechol 2,3-oxygenase, a representative enzyme of the lower (meta-cleavage) pathway, was induced by m-toluate in a xylR mutant but not in the xylS mutants. Unexpectedly, however, catechol 2,3-oxygenase was not induced by m-methylbenzyl alcohol in xylS mutants but was induced by benzyl alcohol and benzoate. These results indicate that expression of the TOL plasmid-encoded catabolic pathway is regulated by at least three control elements, two of which (the products of the xylS and xylR genes) interact in the induction of the lower pathway by methylated hydrocarbons and alcohols and one of which responds only to nonmethylated substrates.     

Proteome analysis of Pseudomonas sp. K82 biodegradation pathways

Pseudomonas sp. K82 is a soil bacterium that can degrade and use monocyclic aromatic compounds including aniline, 3-methylaniline, 4-methylaniline, benzoate and p-hydroxybenzoate as its sole carbon and energy sources. In order to understand the impact of these aromatic compounds on metabolic pathways in Pseudomonas sp. K82, proteomes obtained from cultures exposed to different substrates were displayed by two-dimensional gel electrophoresis and were compared to search for differentially induced metabolic enzymes. Column separations of active fractions were performed to identify major biodegradation enzymes. More than thirty proteins involved in biodegradation and other types of metabolism were identified by electrospray ionization-quadrupole time of flight mass spectrometry. The proteome analysis suggested that Pseudomonas sp. K82 has three main metabolic pathways to degrade these aromatic compounds and induces specific metabolic pathways for each compound. The catechol 2,3-dioxygenase (CD2,3) pathway was the major pathway and the catechol 1,2-dioxygenase (beta-ketoadipate) pathway was the secondary pathway induced by aniline (aniline analogues) exposure. On the other hand, the catechol 1,2-dioxygenase pathway was the major pathway induced by benzoate exposure. For the degradation of p-hydroxybenzoate, the protocatechuate 4,5-dioxygenase pathway was the major degradation pathway induced. The nuclear magnetic resonance analysis of substrates demonstrated that Pseudomonas sp. K82 metabolizes some aromatic compounds more rapidly than others (benzoate > p-hydroxybenzoate > aniline) and that when combined, p-hydroxybenzoate metabolism is repressed by the presence of benzoate or aniline. These results suggest that proteome analysis can be useful in the high throughput study of bacterial metabolic pathways, including that of biodegradation, and that inter-relationships exist with respect to the metabolic pathways of aromatic compounds in Pseudomonas sp. K82.     

Biodegradation of phenanthrene by<Emphasis Type="Italic"> Pseudomonas</Emphasis> sp. strain PP2: novel metabolic pathway,role of biosurfactant and cell surface hydrophobicity in hydrocarbon assimilation

Pseudomonas sp. strain PP2 isolated in our laboratory efficiently metabolizes phenanthrene at 0.3% concentration as the sole source of carbon and energy. The metabolic pathways for the degradation of phenanthrene, benzoate and p-hydroxybenzoate were elucidated by identifying metabolites, biotransformation studies, oxygen uptake by whole cells on probable metabolic intermediates, and monitoring enzyme activities in cell-free extracts. The results obtained suggest that phenanthrene degradation is initiated by double hydroxylation resulting in the formation of 3,4-dihydroxyphenanthrene. The diol was finally oxidized to 2-hydroxymuconic semialdehyde. Detection of 1-hydroxy-2-naphthoic acid, alpha-naphthol, 1,2-dihydroxy naphthalene, and salicylate in the spent medium by thin layer chromatography; the presence of 1,2-dihydroxynaphthalene dioxygenase, salicylaldehyde dehydrogenase and catechol-2,3-dioxygenase activity in the extract; O(2) uptake by cells on alpha-naphthol, 1,2-dihydroxynaphthalene, salicylaldehyde, salicylate and catechol; and no O(2) uptake on o-phthalate and 3,4-dihydroxybenzoate supports the novel route of metabolism of phenanthrene via 1-hydroxy-2-naphthoic acid --> [alpha-naphthol] --> 1,2-dihydroxy naphthalene --> salicylate --> catechol. The strain degrades benzoate via catechol and cis,cis-muconic acid, and p-hydroxybenzoate via 3,4-dihydroxybenzoate and 3-carboxy- cis,cis-muconic acid. Interestingly, the culture failed to grow on naphthalene. When grown on either hydrocarbon or dextrose, the culture showed good extracellular biosurfactant production. Growth-dependent changes in the cell surface hydrophobicity, and emulsification activity experiments suggest that: (1) production of biosurfactant was constitutive and growth-associated, (2) production was higher when cells were grown on phenanthrene as compared to dextrose and benzoate, (3) hydrocarbon-grown cells were more hydrophobic and showed higher affinity towards both aromatic and aliphatic hydrocarbons compared to dextrose-grown cells, and (4) mid-log-phase cells were significantly (2-fold) more hydrophobic than stationary phase cells. Based on these results, we hypothesize that growth-associated extracellular biosurfactant production and modulation of cell surface hydrophobicity plays an important role in hydrocarbon assimilation/uptake in Pseudomonas sp. strain PP2.     

Co-metabolism of methyl- and chloro-substituted catechols by an Achromobacter sp. possessing a new meta-cleaving oxygenase

Co-metabolism of 3-methylcatechol, 4-chlorocatechol and 3,5-dichlorocatechol by an Achromobacter sp. was shown to result in the accumulation of 2-hydroxy-3-methylmuconic semialdehyde, 4-chloro-2-hydroxymuconic semialdehyde and 3,5-dichloro-2-hydroxymuconic semialdehyde respectively. Formation of these products indicated that cleavage of the aromatic nucleus of the substituted catechols was accomplished by a new meta-cleaving enzyme, catechol 1,6-oxygenase. This enzyme was equally active on both chloro- and methyl-substituted catechols.     

Metabolism of toluene and xylenes by Pseudomonas (putida (arvilla) mt-2: evidence for a new function of the TOL plasmid.

Pseudomonas putida (arvilla) mt-2 carries genes for the catabolism of toluene, m-xylene, and p-xylene on a transmissible plasmid, TOL. These compounds are degraded by oxidation of one of the methyl substituents via the corresponding alcohols and aldehydes to benzoate and m- and p-toluates, respectively, which are then further metabolised by the meta pathway, also coded for by the TOL plasmid. The specificities of the benzyl alcohol dehydrogenase and the benzaldehyde dehydrogenase for their three respective substrates are independent of the carbon source used for growth, suggesting that a single set of nonspecific enzymes is responsible for the dissimilation of the breakdown products of toluene and m- and p-xylene. Benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase are coincidently and possible coordinately induced by toluene and the xylenes, and by the corresponding alcohols and aldehydes. They are not induced in cells grown on m-toluate but catechol 2,3-oxygenase can be induced by m-xylene.     

The Production of Catechols from Benzene and Toluene by Pseudomonas Putida in Glucose Fed-Batch Culture

Catechol and 3-methylcatechol were produced from benzene and toluene respectively using different mutants of Pseudomonas putida. P. putida 2313 lacked the extradiol cleavage enzyme, catechol 2,3-oxygenase, allowing overproduction of 3-methylcatechol from toluene to a level of 11.5 mM (1.27 g·1^-1) in glucose fed-batch culture. P. putida 6(12), a mutant of P. putida 2313, lacked both catechol-oxygenase and catechol 1,2-oxygenase, and accumulated catechol from benzene to a level of 27.5mM(3g·1^-1).

In both biotransformations product formation ceased within 10 hours of feeding the aromatic substrate, and this was due to product inhibition by the catechols. The primary site of catechol toxicity was inhibition of the aromatic dioxygenase. Neither cis-toluene dihydrodiol cis-1,2-dihydroxy-3-methylcyclohexa-3,5-diene), nor cis-benzene dihydrodiol (cis-l,2-dihydroxy-3-methylcyclohexa-3,5-diene) dehydrogenase was significantly inhibited by catechol overproduction whereas both ring activating dioxygenases were inhibited within 4-6 hours of the maximum product concentration being attained.

3-Methylcatechol overproduction from toluene was also studied using a continuous product removal system. Granular activated charcoal removed 3-methylcatechol efficiently and was easily regenerated by washing with ethyl acetate. Using P. putida 2313, it was shown that the final product concentration increased approximately fourfold. Additional products were formed and the significance of these are discussed.