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.     

Degradation of diphenylether by Pseudomonas cepacia Et4: enzymatic release of phenol from 2,3-dihydroxydiphenylether

2,3-Dihydroxybiphenyl dioxygenase from Pseudomonas cepacia Et 4 was found to catalyze the ring fission of 2,3-dihydroxydiphenylether in the course of diphenylether degradation. The enzyme was purified and characterized. It had a molecular mass of 240 kDa and is dissociated by SDS into eight subunits of equal mass (31 kDa). The purified enzyme was found to be most active with 2,3-dihydroxybiphenyl as substrate and showed moderate activity with 2,3-dihydroxydiphenylether, catechol and some 3-substituted catechols. The K _m-value of 1 M for 2,3-dihydroxydiphenylether indicated a high affinity of the enzyme towards this substrate. The cleavage of 2,3-dihydroxydiphenylether by 2,3-dihydroxybiphenyl dioxygenase lead to the formation of phenol and 2-pyrone-6-carboxylate as products of ring fission and ether cleavage without participation of free intermediates. Isotope labeling experiments carried out with ¹⁸O₂ and H₂ ¹⁸O indicated the incorporation of ¹⁸O from the atmosphere into the carboxyl residue as well as into the carbonyl oxygen of the lactone moiety of 2-pyrone-6-carboxylate. Based on these experimental findings the reaction mechanism for the formation of phenol and 2-pyrone-6-carboxylate is proposed in accordance with the mechanism suggested by Kersten et al. (1982).Non-standard abbreviations DPE diphenylether - 2,3-dihydroxy-DPE 2,3-dihydroxydiphenylether - PCA 2-pyrone-6-carboxylic acid - 2,3-dihydroxy-BP dioxygenase 2,3-dihydroxybiphenyl dioxygenase - GC gas chromatography     

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.     

Growth rate-dependent expression of phenol-assimilation pathways in Alcaligenes eutrophus JMP 134–the influence of formate as an auxiliary energy source on phenol conversion characteristics

 Alcaligenes eutrophus JMP 134 was continuously (carbon-source-limited) grown on phenol to determine the maximum growth rates (μ_max) as a function of the phenol assimilation pathways expressed. During growth on phenol as the sole source of carbon and energy, an almost exclusive expression of the ortho cleavage pathway (catechol 1,2-dioxygenase) was observed at initially low growth rates. This allowed a μ_max of 0.28 h^-1. The induction of the meta cleavage pathway (catechol 2,3-dioxygenase), which appeared at around 0.25 h^-1, resulted in a further increase in the growth rate to 0.40 h^-1 after the enzyme activities of this pathway had been correspondingly expressed. Hence, two maximum growth rates, one for the ortho and one for the meta cleavage pathway, exist for the growth of A. eutrophus JMP 134 on phenol. Growth on phenol was stimulated by formate, which served as an auxiliary energy source in this strain. The simultaneous utilization of phenol and formate at a molar ratio of 1:5.2 resulted in an increase of the yield coefficient from about 0.75 g dry mass/g phenol to 1.25 g/g. Furthermore, formate exerted a pronounced effect on the growth rate. At a molar ratio of phenol to formate of 1:4.2, the growth rate was increased to 0.42 h^-1, despite the exclusive induction of the ortho cleavage pathway. The meta cleavage pathway was expressed during growth on this substrate mixture at about 0.4 h^-1. However, this did not enable a significant increase of the growth rate beyond 0.4 h^-1. This is attributed to an exhaustion of the capacity for formate oxidation at this rate. The results are discussed with respect to energy production capabilities when phenol is assimilated as an energy-deficient heterotrophic substrate. Received: 13 November 1995/Received revision: 15 April 1996/Accepted: 22 April 1996     

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.     

Isolation and characterization of four novel Gram-positive bacteria associated with the rhizosphere of two endemorelict plants capable of degrading a broad range of aromatic substrates

Four new Gram-positive, phenol-degrading strains were isolated from the rhizospheres of endemorelict plants Ramonda serbica and Ramonda nathaliae known to exude high amounts of phenolics in the soil. Isolates were designated Bacillus sp. PS1, Bacillus sp. PS11, Streptomyces sp. PS12, and Streptomyces sp. PN1 based on 16S rDNA sequence and biochemical analysis. In addition to their ability to tolerate and utilize high amounts of phenol of either up to 800 or up to 1,400 mg l⁻¹ without apparent inhibition in growth, all four strains were also able to degrade a broad range of aromatic substrates including benzene, toluene, ethylbenzene, xylenes, styrene, halogenated benzenes, and naphthalene. Isolates were able to grow in pure culture and in defined mixed culture on phenol and on the mixture of BTEX (benzene, toluene, ethylbenzene, and xylenes) compounds as a sole source of carbon and energy. Pure culture of Bacillus sp. PS11 yielded 1.5-fold higher biomass amounts in comparison to mixed culture, under all conditions. Strains successfully degraded phenol in the soil model system (2 g kg⁻¹) within 6 days. Activities of phenol hydroxylase, catechol 1,2-dioxygenase, and catechol 2,3-dioxygenase were detected and analyzed from the crude cell extract of the isolates. While all four strains use ortho degradation pathway, enzyme indicative of meta degradation pathway (catechol 2,3-dioxygenase) was also detected in Bacillus sp. PS11 and Streptomyces sp. PN1. Phenol degradation activities were induced 2 h after supplementation by phenol, but not by catechol. Catechol slightly inhibited activity of catechol 2,3-dioxygenase in strains PS11 and PN1.     

Changes in structure,activity and metabolism of aerobic granules as a microbial response to high phenol loading

Four column-type sequential aerobic sludge blanket reactors were fed with phenol as the sole carbon and energy source and operated at loading rates of 1.0, 1.5, 2.0 and 2.5 kg phenol m^–3 day^–1. The results indicated that phenol loading exerted a profound influence on the structure, activity and metabolism of the aerobic granules. Compact granules with good settling ability were maintained at loadings up to 2.0 kg phenol m^–3 day^–1, and structurally weakened granules with enhanced production of extracellular polymers and proteins and significantly lower hydrophobicities were observed at the highest loading of 2.5 kg phenol m^–3 day^–1. Specific oxygen uptake rate, catechol 2,3-dioxygenase (C23O) and catechol 1,2-dioxygenase (C12O) activities peaked at a loading of 2.0 kg phenol m^–3 day^–1, and declined thereafter. Granules degraded phenol completely in all four reactors, mainly through the meta cleavage pathway as C23O activities were significantly higher than C12O activities. At the highest loading applied, the anabolism and catabolism of microorganisms were regulated such that phenol degradation proceeded exclusively via the meta pathway, apparently to produce more energy for overstimulation of protein production against phenol toxicity. This work contributes to a better understanding of the ability of aerobic granules to handle high-strength industrial wastewaters containing chemicals that are normally inhibitory to microbial growth.     

Isolation and Properties of Phenol Utilizing Microorganisms with Special Reference to Catechol 1,2-Oxygenase

Phenol utilizing yeasts were isolated from soil. The relationship were examined between distribution of phenol uptake rate using intact cells and distribution of the activities of catechol 1,2-oxygenase which is one of the key enzymes in phenol metabolism. Two of the isolates showed catechol 1,2-oxygenase activity even when grown in glucose medium, though the enzyme activity was about 1% of the full activity induced by phenol. Partially constitutive mutants for catechol 1,2-oxygenase were obtained by mutagenesis of an inducible strain. The level of mutant enzyme activity was close to that of the isolated constitutive strain. One isolate, Trichosporon cutaneum, preferentially utilized phenol to glucose in medium containing both phenol (200 ppm) and glucose (0.1%), until the concentration of phenol decreased to 10–20 ppm.     

Monohydroxylation of phenol and 2,5-dichlorophenol by toluene dioxygenase in Pseudomonas putida F1.

Pseudomonas putida F1 contains a multicomponent enzyme system, toluene dioxygenase, that converts toluene and a variety of substituted benzenes to cis-dihydrodiols by the addition of one molecule of molecular oxygen. Toluene-grown cells of P. putida F1 also catalyze the monohydroxylation of phenols to the corresponding catechols by an unknown mechanism. Respirometric studies with washed cells revealed similar enzyme induction patterns in cells grown on toluene or phenol. Induction of toluene dioxygenase and subsequent enzymes for catechol oxidation allowed growth on phenol. Tests with specific mutants of P. putida F1 indicated that the ability to hydroxylate phenols was only expressed in cells that contained an active toluene dioxygenase enzyme system. 18O2 experiments indicated that the overall reaction involved the incorporation of only one atom of oxygen in the catechol, which suggests either a monooxygenase mechanism or a dioxygenase reaction with subsequent specific elimination of water.     

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.     

Development of Catechol 2,3-Dioxygenase-Specific Primers for Monitoring Bioremediation by Competitive Quantitative PCR

Benzene, toluene, xylenes, phenol, naphthalene, and biphenyl are among a group of compounds that have at least one reported pathway for biodegradation involving catechol 2,3-dioxygenase enzymes. Thus, detection of the corresponding catechol 2,3-dioxygenase genes can serve as a basis for identifying and quantifying bacteria that have these catabolic abilities. Primers that can successfully amplify a 238-bp catechol 2,3-dioxygenase gene fragment from eight different bacteria are described. The identities of the amplicons were confirmed by hybridization with a 238-bp catechol 2,3-dioxygenase probe. The detection limit was 10² to 10³ gene copies, which was lowered to 10⁰ to 10¹ gene copies by hybridization. Using the dioxygenase-specific primers, an increase in catechol 2,3-dioxygenase genes was detected in petroleum-amended soils. The dioxygenase genes were enumerated by competitive quantitative PCR with a 163-bp competitor that was amplified using the same primers. Target and competitor sequences had identical amplification kinetics. Potential PCR inhibitors that could coextract with DNA, nonamplifying DNA, soil factors (humics), and soil pollutants (toluene) did not impact enumeration. Therefore, this technique can be used to accurately and reproducibly quantify catechol 2,3-dioxygenase genes in complex environments such as petroleum-contaminated soil. Direct, non-cultivation-based molecular techniques for detecting and enumerating microbial pollutant-biodegrading genes in environmental samples are powerful tools for monitoring bioremediation and developing field evidence in support of natural attenuation.     

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.     

Aromatic metabolism in Rhizobium trifolii-catechol 1,2-dioxygenase

Catechol 1,2-dioxygenase has been purified 46-fold from cells of Rhizobium trifolii TA1 grown on benzoate plus glucose. The dioxygenase had a molecular weight of 107,000 and a sub-unit molecular weight of 59,000. The enzyme had a K _m of 2 M for catechol and also cleaved 4-methylcatechol. The dioxygenase contained 2 g atoms of Fe³⁺ per mole of enzyme which could be removed by treatment with 1,10-phenanthroline, resulting in a complete loss of activity; reactivation of the enzyme occurred specifically with Fe³⁺.     

Production of toluene cis-glycol by Pseudomonas putida in glucose feb-batch culture

Toluene was oxidized by a mutant strain of Pseudomonas putida (strain NG1) to toluene Cis-Glycol (TCG). Product was accumulated in fed-batch cultures to concentrations (18-24 g/L) higher than hitherto achieved. In vitro activities of toluene dioxygenase from P. Putida NG1 were fivefold lower than that from the toluene-grown wild-type organism, whereas comparable activities of both catechol 2,3- and catechol 1,2-oxygenase were obtained; irreversible inhibition of toluene dioxygenase activity by TCG was shown in vitro. Ammonia deprivation during the production phase limited the growth of revertant organisms but had little effect on either the duration (25h) of the process or the final concentration of TCG achieved. The rate of glucose utilization decreased throughout the biotransformation and cell death accompanied the cessation of TCG accumulation in cultures. These changes were a consequence of TCG formation and a cooperative toxic effect was demonstrated for toluene and TCG. Adenylate energy charge values decreased from ca. 0.8 to 0.2 over the course of the biotransformation but were maintained above 0.5 in the absence of TCG. Similarly, cellular AMP levels increased dramatically during biotransformation, presumably as a consequence of RNA degradation, but were maintained at low levels in the absence of TCG. The results suggest that TCG is the mediate of a gradual deterioration in the state of the culture which leads to a loss of both in vivo and in vitro toluence dioxygenase activity and a marked decrease in culture viability.     

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.     

Aerobic degradation of polychlorinated biphenyls by Alcaligenes sp. JB1: metabolites and enzymes

In contrast to the degradation of penta-and hexachlorobiphenyls in chemostat cultures, the metabolism of PCBs by Alcaligenes sp. JB1 was shown to be restricted to PCBs with up to four chlorine substituents in resting-cell assays. Among these, the PCB congeners containing ortho chlorine substituents on both phenyl rings were found to be least degraded. Monochloro-benzoates and dichlorobenzoates were detected as metabolites. Resting cell assays with chlorobenzoates showed that JB1 could metabolize all three monochlorobenzoates and dichlorobenzoates containing only meta and para chlorine substituents, but not dichlorobenzoates possessing an ortho chlorine substituent. In enzyme activity assays, meta cleaving 2,3-dihydroxybiphenyl 1,2-dioxygenase and catechol 2,3-dioxygenase activities were constitutive, whereas benzoate dioxygenase and ortho cleaving catechol 1,2-dioxygenase activities were induced by their substrates. No activity was found for pyrocatechase II, the enzyme that is specific for chlorocatechols. The data suggest that complete mineralization of PCBs with three or more chlorine substituents by Alcaligenes sp. JB1 is unlikely.Abbreviations PCB polychlorinated biphenyls - CBA chlorobenzoate - D di - Tr tri - Te tetra - Pe penta- - H hexa     

Specificity of monohydric phenol oxidations by meta cleavage pathways in Pseudomonas aeruginosa T1

Summary The oxidation of several mono-hydric phenols by wild type and mutant strains of Pseudomonas aeruginosa T1 has been studied. The data suggest, that a non-specific enzyme sequence of the meta cleavage pathway is induced by all of these phenols, which can catalyze the oxidation of phenol and its analogues to pyruvate, a fatty acid and a carbonyl compound, according to the general scheme of Dagley et al. (1964). Mutants unable to grow on phenol (hydroxylase-negative), have been isolated, and they are also unable to grown on or oxidize the cresols and the xylenols. Revertants of these mutants regain the capacity to grow on all these phenols and are indistinguishable from the wild type. Induced-substrate relationships for the earlier enzymes of the pathway have been determined, e.g., phenol in addition to catechol and the methylcatechols is an inducer for catechol 2,3-oxygenase. Analysis of the enzymic content of cells grown in a variety of steadystate conditions shows (a) that the ratio of the specific activities of the phenol hydroxylase and catechol 2,3-oxygenase is constant for each of their analogous substrates; and (b) that induction and catabolite repression of catechol 2,3-oxygenase and the muconic semialdehyde hydrolyase are coordinate, but that control of the phenol hydroxylase is independent.Howard Hughes Medical Institute Investigator.     

The catechol 2,3-dioxygenase gene and toluene monooxygenase genes from Burkholderia sp. AA1, an isolate capable of degrading aliphatic hydrocarbons and toluene

Burkholderia sp. AA1 isolated from a diesel fuel-contaminated site degraded toluene, as well as a wide range of alkanes from decane (C₈) to pentacosane (C₂₅) as sole carbon and energy sources. This strain also utilized m-toluate, p-toluate, o-toluate, and m-cresol as sole carbon and energy sources. Toluene- and toluate-grown cells showed catechol 2,3-dioxygenase activity and indole oxidation activity that is exhibited by some toluene oxygenation enzymes. The catechol 2,3-dioxygenase gene (catB) was cloned and sequenced. Its deduced amino acid sequence is analogous to the extradiol dioxygenases cloned from a variety of microorganisms. A DNA fragment containing the genes for the indole oxidation activity was cloned and sequenced. A seven-gene cluster designated as tbhABCDEFG was identified. Significant similarities were found with multicomponent monooxygenase systems for toluene, benzene and phenol from different bacterial strains. Journal of Industrial Microbiology & Biotechnology (2000) 25, 127–131. Received 28 July 1999/ Accepted in revised form 28 June 2000     

Catechol oxygenase induction in Pseudomonas aeruginosa

1. The transfer from benzenesulphonate to benzoate as a growth substrate for Pseudomonas aeruginosa strain A resulted in a change in the enzymic route by which catechol was degraded; at intermediate stages it was possible to obtain cells containing the enzymes of both the ;ortho' and ;meta' metabolic pathways. 2. A similar result was effected by the reverse transfer, benzoate to benzenesulphonate. 3. Catechol itself always elicited a catechol 2,3-oxygenase in uninduced cells, but the product of this reaction, 2-hydroxymuconic semialdehyde, and biochemically related compounds such as 4-hydroxy-2-oxovalerate, unexpectedly induced a catechol 1,2-oxygenase. 4. Both types of catechol oxygenase are strongly repressed by the metabolic end products of both the ;ortho' and ;meta' pathways, but there was no inhibition of enzymic activity by these end products.     

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