The genes for benzene catabolism in Pseudomonas putida ML2 are flanked by two copies of the insertion element IS1489, forming a class-I-type catabolic transposon, Tn5542

Two directly repeated sequences of the IS elements IS1489v1 and IS1489v2 flank the benzene dioxygenase (bedC1C2BA) and the cis-benzene dihydrodiol dehydrogenase (bedD) genes on the catabolic plasmid pHMT112 in Pseudomonas putida ML2, forming a Class-I-type composite transposon, Tn5542. Both IS1489v1 and IS1489v2 contain an identical 1371-bp open reading frame, tnpA, that is preceded by a possible ribosome binding site. The tnpA gene of IS1489v1 is bound by a pair of 40-bp imperfect inverted repeats while that of IS1489v2 is flanked only by the left inverted repeat. The tnpA gene codes for a putative 53-kDa polypeptide of 456 amino acids bearing similarity to transposases encoded on IS elements of P. alcaligenes, P. aeruginosa, P. stutzeri, and Serratia marcescens. The basic nature of the putative TnpA protein with a deduced pI of 8.93 is typical of IS-encoded transposases. Similar to other IS elements, an outward facing promoter was detected at the right end of IS1489v1. Experiments involving the suicide vector, pKNG101, failed to show transposition of Tn5542.     

Trichloroethylene degradation by Escherichia coli containing the cloned Pseudomonas putida F1 toluene dioxygenase genes

Toluene dioxygenase from Pseudomonas putida F1 has been implicated as an enzyme capable of degrading trichloroethylene. This has now been confirmed with Escherichia coli JM109(pDTG601) that contains the structural genes (todC1C2BA) of toluene dioxygenase under the control of the tac promoter. The extent of trichloroethylene degradation by the recombinant organism depended on the cell concentration and the concentration of trichloroethylene. A linear rate of trichloroethylene degradation was observed with the E. coli recombinant strain. In contrast, P. putida F39/D, a mutant strain of P. putida F1 that does not contain cis-toluene dihydrodiol dehydrogenase, showed a much faster initial rate of trichloroethylene degradation which decreased over time.     

Trichloroethylene degradation by Escherichia coli containing the cloned Pseudomonas putida F1 toluene dioxygenase genes.

Toluene dioxygenase from Pseudomonas putida F1 has been implicated as an enzyme capable of degrading trichloroethylene. This has now been confirmed with Escherichia coli JM109(pDTG601) that contains the structural genes (todC1C2BA) of toluene dioxygenase under the control of the tac promoter. The extent of trichloroethylene degradation by the recombinant organism depended on the cell concentration and the concentration of trichloroethylene. A linear rate of trichloroethylene degradation was observed with the E. coli recombinant strain. In contrast, P. putida F39/D, a mutant strain of P. putida F1 that does not contain cis-toluene dihydrodiol dehydrogenase, showed a much faster initial rate of trichloroethylene degradation which decreased over time.     

Initial reactions in the oxidation of naphthalene by Pseudomonas putida.

A strain of Pseudomonas putida that can utilize naphthalene as its sole source of carbon and energy was isolated from soil. A mutant strain of this organism, P. putida 119, when grown on glucose in the presence of naphthalene, accumulates optically pure (+)-cis-1(R),2(S)-dihydroxy-1,2-dihydronaphthalene in the culture medium. The cis relative stereochemistry in this molecule was established by nuclear magnetic resonance spectrometry. Radiochemical trapping experiments established that this cis dihydrodiol is an intermediate in the metabolism of naphthalene by P. Fluorescens (formerly ATCC, 17483), P. putida (ATCC, 17484), and a Pseudomonas species (NCIB 9816), as well as the parent strain of P. putida described in this report. Formation of the cis dihydrodiol is catalyzed by a dioxygenase which requires either NADH or NADPH as an electron donor. A double label procedure is described for determining the origin of oxygen in the cis dihydrodiol under conditions where this metabolite would not normally accumulate. Several aromatic hydrocarbons are oxidized by cell extracts prepared from naphthalene-grown cells of P. putida. The cis dihydrodiol is converted to 1,2-dihydroxynaphthalene by an NAD+-dependent dehydrogenase. This enzyme is specific for the (+) isomer of the dihydrodiol and shows a primary isotope effect when the dihydrodiol is substituted at C-2 with deuterium.     

Cloning and characterization of a novel cis-naphthalene dihydrodiol dehydrogenase gene (narB) from Rhodococcus sp. NCIMB12038

Rhodococcus sp. NCIMB112038 can utilize naphthalene as its sole carbon and energy source. The gene encoding cis-naphthalene dihydrodiol dehydrogenase (narB) of this strain has been cloned and sequenced. Expression of NCIMB12038 cis-naphthalene dihydrodiol dehydrogenase was demonstrated in Escherichia coli cells. narB encodes a putative protein of 271 amino acids and shares 39% amino acid identity with the cis-naphthalene dihydrodiol dehydrogenase from Pseudomonas putida G7. Comparison of NarB with some putative cis-dihydrodiol dehydrogenases from Rhodococcus species revealed significant differences between these proteins. NarB together with two other proteins forms a new group of cis-dihydrodiol dehydrogenases.     

Expression of benzene dioxygenase from Pseudomonas putida ML2 in cis-1,2-cyclohexanediol-degrading pseudomonads

Benzene dioxygenase (BDO; EC 1.14.12.3) from Pseudomonas putida ML2 dihydroxylates benzene to produce cis-1,2-dihydroxy-cyclohexa-3,5-diene. As well as oxidising benzene and toluene, cell-free extracts of Escherichia coli JM109 expressing recombinant BDO oxidised cyclohexene, 1-methylcyclohexene and 3-methylcyclohexene. In an attempt to construct a novel metabolic pathway for the degradation of cyclohexene (via an initial BDO-mediated dihydroxylation of cyclohexene), cis-1,2-cyclohexanediol-degrading bacteria were isolated by enrichment culture. The bedC1C2BA genes encoding BDO (under the control of the tac promoter) were sub-cloned into pLAFR5, successfully conjugated into seven of the Gram-negative cis-1,2-cyclo-hexanediol-degrading isolates and stably maintained and expressed in three of them. However, despite their ability to grow on cis-1,2-cyclohexanediol as sole carbon source, express an active BDO and oxidise cyclohexene, none of the three strains was able to grow on cyclohexene as sole carbon source. Analysis revealed that BDO oxidised cyclohexene to a mixture of two products, a monohydroxylated (2-cyclohexen-1-ol) product and a dihydroxylated (cis-1,2-cyclohexanediol) product; and failure to grow on cyclohexene was attributed to the toxicity of metabolic intermediates accumulating from the 2-cyclohexen-1-ol metabolism.     

Bacterial cis-dihydrodiol dehydrogenases: comparison of physicochemical and immunological protperties.

Cells of Pseudomonas putida NP, Pseudomonas species (NCIB 9816), and a Nocardia species, after growth on naphthalene as sole source of carbon and energy, contain a nicotinamide adenine dinucleotide (NAD+)-dependent enzyme that oxidizes cis-dihydrodiols of mono- and polycyclic aromatic compounds. Similarly, cells of a strain of P. putida biotype A, when grown either on toluene or benzene vapors, were found to contain a dehydrogenase that oxidized dihydrodiols of aromatic hydrocarbons with cis stereochemistry and required NAD+ as an electron acceptor. In all these cases, no enzymatic activity was detected when trans-naphthalene dihydrodiol was used as a substrate. Purified cis-naphthalene dihydrodiol dehydrogenase was injected into rabbits to obtain antibodies. Physiocochemical and immunological properties of cis-dihydrodiol:NAD+ oxidoreductases from four different organisms were examined. Kinetic analysis showed that, in all the cases, enzymes exhibited higher affinity for cis-dihydrodiols than for NAD+ and had pH optima between 8.8 and 9.0. except in the case of the enzyme from Nocarida sp., which showed maximum activity at pH 8.4. Molecular-weight determination of the dehydrogenases from the four different organisms by gel filtration on a Sephadex G-200 column gave values ranging from 92,000 for the enzyme from Nocardia sp. to 160,000 for that from P. putida biotype A. All the dehydrogenases, except the one from Nocardia sp., exhibited immunological cross-reaction with the antibodies prepared against the enzyme purified from P. putida NP.     

Oxidation of substituted phenols by Pseudomonas putida F1 and Pseudomonas sp. strain JS6.

The biodegradation of benzene, toluene, and chlorobenzenes by Pseudomonas putida involves the initial conversion of the parent molecules to cis-dihydrodiols by dioxygenase enzyme systems. The cis-dihydrodiols are then converted to the corresponding catechols by dihydrodiol dehydrogenase enzymes. Pseudomonas sp. strain JS6 uses a similar system for growth on toluene or dichlorobenzenes. We tested the wild-type organisms and a series of mutants for their ability to transform substituted phenols after induction with toluene. When grown on toluene, both wild-type organisms converted methyl-, chloro-, and nitro-substituted phenols to the corresponding catechols. Mutant strains deficient in dihydrodiol dehydrogenase or catechol oxygenase activities also transformed the phenols. Oxidation of phenols was closely correlated with the induction and activity of the toluene dioxygenase enzyme system.     

Oxidation of substituted phenols by Pseudomonas putida F1 and Pseudomonas sp. strain JS6

The biodegradation of benzene, toluene, and chlorobenzenes by Pseudomonas putida involves the initial conversion of the parent molecules to cis-dihydrodiols by dioxygenase enzyme systems. The cis-dihydrodiols are then converted to the corresponding catechols by dihydrodiol dehydrogenase enzymes. Pseudomonas sp. strain JS6 uses a similar system for growth on toluene or dichlorobenzenes. We tested the wild-type organisms and a series of mutants for their ability to transform substituted phenols after induction with toluene. When grown on toluene, both wild-type organisms converted methyl-, chloro-, and nitro-substituted phenols to the corresponding catechols. Mutant strains deficient in dihydrodiol dehydrogenase or catechol oxygenase activities also transformed the phenols. Oxidation of phenols was closely correlated with the induction and activity of the toluene dioxygenase enzyme system.     

Cloning and overexpression of the 3-hydroxyisobutyrate dehydrogenase gene from pseudomonas putida E23

The structural gene for NAD+-dependent 3-hydroxyisobutyrate dehydrogenase (EC 1.1.1.31) from Pseudomonas putida E23 was cloned in Escherichia coli cells to obtain a large amount of the enzyme and its nucleotides were sequenced to study its structural relationship with other proteins. The gene encoded a polypeptide containing 295 amino acid residues and was in a cluster with the gene for methylmalonate semialdehyde dehydrogenase. Transformed E. coli cells overproduced 3-hydroxyisobutyrate dehydrogenase, and the recombinant enzyme was purified to homogeneity with a high yield. Lysine and asparagine residues, which are important in catalysis of the 3-hydroxyacid dehydrogenase family, are conserved in this enzyme.     

Efficient degradation of trichloroethylene by a hybrid aromatic ring dioxygenase.

Engineering of hybrid gene clusters between the toluene metabolic tod operon and the biphenyl metabolic bph operon greatly enhanced the rate of biodegradation of trichloroethylene. Escherichia coli cells carrying a hybrid gene cluster composed of todC1 (the gene encoding the large subunit of toluene terminal dioxygenase in Pseudomonas putida F1), bphA2 (the gene encoding the small subunit of biphenyl terminal dioxygenase in Pseudomonas pseudoalcaligenes KF707), bphA3 (the gene encoding ferredoxin in KF707), and bphA4 (the gene encoding ferredoxin reductase in KF707) degraded trichloroethylene much faster than E. coli cells carrying the original toluene dioxygenase genes (todC1C2BA) or the original biphenyl dioxygenase genes (bphA1A2A3A4).     

Microbial conversion of indene to indandiol: a key intermediate in the synthesis of CRIXIVAN

Indene is oxidized to mixtures of cis- and trans-indandiols and related metabolites by Pseudomonas putida and Rhodococcus sp. isolates. Indene metabolism is consistent with monooxygenase and dioxygenase activity. P. putida resolves enantiomeric mixtures of cis-1,2-indandiol by further selective oxidation of the 1R, 2S-enantiomer yielding high enantiomeric purity of cis-(1S, 2R)-indandiol, a potential intermediate in the synthesis of indinavir sulfate (CRIXIVAN), a protease inhibitor used in the treatment of AIDS. Molecular cloning of P. putida toluene dioxygenase in Escherichia coli confirmed the requirement for the dihydrodiol dehydrogenase in resolving racemic mixtures of cis-indandiol. Rhodococcus sp. isolates convert indene to cis-(1S, 2R)-indandiol at high initial enantiomeric excess and one isolate also produces trans-(1R, 2R)-indandiol, suggesting the presence of monooxygenase activity. Scale up and optimization of the bioconversions to these key synthons for chiral synthesis of potential intermediates for commercial manufacture of indinavir sulfate are described.     

Cloning and expression in Escherichia coli of the toluene dioxygenase gene from Pseudomonas putida NCIB11767

The genes encoding toluene dioxygenase, toluene cis-glycol dehydrogenase and catechol 2.3-oxygenase from Pseudomonas putida NCIB 11767 were cloned and expressed in Escherichia coli HB101 on a 20 kb fragment. The recombinant strain produced indigo and a variety of other coloured products. Although the enzymes were expressed in the absence of inducers, further induction was observed in the presence of toluene or benzene, implying the presence of regulatory elements on the 20 kb insert.     

Combination of the tod and the tol pathways in redesigning a metabolic route of Pseudomonas putida for the mineralization of a benzene, toluene, and p-xylene mixture.

Construction of a hybrid strain which is capable of mineralizing components of a benzene, toluene, and p-xylene mixture simultaneously was attempted by redesigning the metabolic pathway of Pseudomonas putida. Genetic and biochemical analyses of the tod and the tol pathways revealed that dihydrodiols formed from benzene, toluene, and p-xylene by toluene dioxygenase in the tod pathway could be channeled into the tol pathway by the action of cis-p-toluate-dihydrodiol dehydrogenase, leading to complete mineralization of a benzene, toluene, and p-xylene mixture. Consequently, a hybrid strain was constructed by cloning todC1C2BA genes encoding toluene dioxygenase on RSF1010 and introducing the resulting plasmid into P. putida mt-2. The hybrid strain of P. putida TB105 was found to mineralize a benzene, toluene, and p-xylene mixture without accumulation of any metabolic intermediate.     

Selectivity Studies of the Benzene cis -Dihydrodiol Dehydrogenase Enzyme from Pseudomonas putida ML2 with Vicinal Diol Substrates

The enantiopure (1 S, 2 S )- cis -dihydrodiol metabolites 2B - 5B have been obtained in low yield from the corresponding monosubstituted halobenzene substrates 2A - 5A, using a wild-type strain of Pseudomonas putida (ML2) containing benzene dioxygenase (BDO). Benzene cis -dihydrodiol dehydrogenase (BCD) from P. putida ML2 and naphthalene cis -dihydrodiol dehydrogenase (NCD) from P. putida 8859 were purified and used in a comparative study of the stereoselective biotransformation of cis -dihydrodiol enantiomers 2B - 5B. The BCD and NCD enzymes were found to accept cis -dihydrodiol enantiomers of monosubstituted benzene cis -dihydrodiol substrates 2B - 5B of opposite absolute configuration. The acyclic alkene 1,2-diols 10 - 17 were also found to be acceptable substrates for BCD.     

Toluene degradation by Pseudomonas putida F1. Nucleotide sequence of the todC1C2BADE genes and their expression in Escherichia coli

The nucleotide sequence of the todC1C2BADE genes which encode the first three enzymes in the catabolism of toluene by Pseudomonas putida F1 was determined. The genes encode the three components of the toluene dioxygenase enzyme system: reductaseTOL (todA), ferredoxinTOL (todB), and the two subunits of the terminal dioxygenase (todC1C2); (+)-cis-(1S, 2R)-dihydroxy-3-methylcyclohexa-3,5-diene dehydrogenase (todD); and 3-methylcatechol 2,3-dioxygenase (todE). Knowledge of the nucleotide sequence of the tod genes was used to construct clones of Escherichia coli JM109 that overproduce toluene dioxygenase (JM109(pDT-601]; toluene dioxygenase and (+)-cis-(1S, 2R)-dihydroxy-3-methylcyclohexa-3,5-diene dehydrogenase (JM109(pDTG602]; and toluene dioxygenase, (+)-cis-(1S, 2R)-dihydroxy-3-methylcyclohexa-3,5-diene dehydrogenase, and 3-methylcatechol 2,3-dioxygenase (JM109(pDTG603]. The overexpression of the tod-C1C2BADE gene products was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The three E. coli JM109 strains harboring the plasmids pDTG601, pDTG602, and pDTG603, after induction with isopropyl-beta-D-thiogalactopyranoside, oxidized toluene to (+)-cis-(1S, 2R)-dihydroxy-3-methylcyclohexa-3,5-diene, 3-methylcatechol, and 2-hydroxy-6-oxo-2,4-heptadienoate, respectively. The tod-C1C2BAD genes show significant homology to the reported nucleotide sequence for benzene dioxygenase and cis-1,2-dihydroxycyclohexa-3,5-diene dehydrogenase from P. putida 136R-3 (Irie, S., Doi, S., Yorifuji, T., Takagi, M., and Yano, K. (1987) J. Bacteriol. 169, 5174-5179). In addition, significant homology was observed between the nucleotide sequences for the todDE genes and the sequences reported for cis-1,2-dihydroxy-6-phenylcyclohexa-3,5-diene dehydrogenase and 2,3-dihydroxybiphenyl-1,2-dioxygenase from Pseudomonas pseudoalcaligenes KF707 (Furukawa, K., Arimura, N., and Miyazaki, T. (1987) J. Bacteriol. 169, 427-429).     

Stereospecific hydroxylation of indan by Escherichia coli containing the cloned toluene dioxygenase genes from Pseudomonas putida F1.

Escherichia coli JM109(pDTG601), containing the todC1C2BA genes encoding toluene dioxygenase from Pseudomonas putida F1, oxidizes indan to (-)-(1R)-indanol (83% R) and trans-1,3-indandiol. Under similar conditions, P. putida F39/D oxidizes indan to (-)-(1R)-indanol (96% R), 1-indanone, and trans-1,3-indandiol. The differences in the enantiomeric composition of the 1-indanols formed by the two organisms are due to the presence of a 1-indanol dehydrogenase in P. putida F39/D that preferentially oxidizes (+)-(1S)-indanol.     

Selectivity Studies of the Benzene cis -Dihydrodiol Dehydrogenase Enzyme from Pseudomonas putida ML2 with Vicinal Diol Substrates

The enantiopure (1 S , 2 S )- cis -dihydrodiol metabolites 2B - 5B have been obtained in low yield from the corresponding monosubstituted halobenzene substrates 2A - 5A , using a wild-type strain of Pseudomonas putida (ML2) containing benzene dioxygenase (BDO). Benzene cis -dihydrodiol dehydrogenase (BCD) from P. putida ML2 and naphthalene cis -dihydrodiol dehydrogenase (NCD) from P. putida 8859 were purified and used in a comparative study of the stereoselective biotransformation of cis -dihydrodiol enantiomers 2B - 5B . The BCD and NCD enzymes were found to accept cis -dihydrodiol enantiomers of monosubstituted benzene cis -dihydrodiol substrates 2B - 5B of opposite absolute configuration. The acyclic alkene 1,2-diols 10 - 17 were also found to be acceptable substrates for BCD.     

Stereospecific hydroxylation of indan by Escherichia coli containing the cloned toluene dioxygenase genes from Pseudomonas putida F1.

Escherichia coli JM109(pDTG601), containing the todC1C2BA genes encoding toluene dioxygenase from Pseudomonas putida F1, oxidizes indan to (-)-(1R)-indanol (83% R) and trans-1,3-indandiol. Under similar conditions, P. putida F39/D oxidizes indan to (-)-(1R)-indanol (96% R), 1-indanone, and trans-1,3-indandiol. The differences in the enantiomeric composition of the 1-indanols formed by the two organisms are due to the presence of a 1-indanol dehydrogenase in P. putida F39/D that preferentially oxidizes (+)-(1S)-indanol.