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Oxygenases catalyze crucial reactions throughout all domains of life, cleaving molecular oxygen (O 2 ) and inserting one or two of its atoms into organic substrates. Many oxygenases, including those in the cytochrome P450 (P450) and Rieske oxygenase enzyme families, function as multicomponent systems, which require one or more redox partners to transfer electrons to the catalytic center. As the identity of the reductase can change the reactivity of the oxygenase, characterization of the latter with its cognate redox partners is critical. However, the isolation of the native redox partner or partners is often challenging. Here, we report the preparation and characterization of PbdB, the native reductase partner of PbdA, a bacterial P450 enzyme that catalyzes the O-demethylation of para-methoxylated benzoates. Through production in a rhodoccocal host, codon optimization, and anaerobic purification, this procedure overcomes conventional challenges in redox partner production and allows for robust oxygenase characterization with its native redox partner. Key lessons learned here, including the value of production in a related host and rare codon effects are applicable to a broad range of Fe-dependent oxygenases and their components., (Copyright © 2024. Published by Elsevier Inc.)

Enzymes often by far exceed the activity, selectivity, and sustainability achieved with chemical catalysts. One of the main reasons for the lack of biocatalysis in the chemical industry is the poor stability exhibited by many enzymes when exposed to process conditions. This dilemma is exemplified in the usually very temperature-sensitive enzymes catalyzing the Baeyer-Villiger reaction, which display excellent stereo- and regioselectivity and offer a green alternative to the commonly used, explosive peracids. Here we describe a protein engineering approach applied to cyclohexanone monooxygenase from Rhodococcus sp. HI-31, a substrate-promiscuous enzyme that efficiently catalyzes the production of the nylon-6 precursor ε-caprolactone. We used a framework for rapid enzyme stabilization by computational libraries (FRESCO), which predicts protein-stabilizing mutations. From 128 screened point mutants, approximately half had a stabilizing effect, albeit mostly to a small degree. To overcome incompatibility effects observed upon combining the best hits, an easy shuffled library design strategy was devised. The most stable and highly active mutant displayed an increase in unfolding temperature of 13°C and an approximately 33x increase in half-life at 30°C. In contrast to the wild-type enzyme, this thermostable 8x mutant is an attractive biocatalyst for biotechnological applications., (© 2019 The Authors. Biotechnology and Bioengineering Immunity Inflammation Published by Wiley Periodicals, Inc.)

Numerous desulfurizing bacteria from the Rhodococcus genus harbor conserved dsz genes responsible for the degradation of sulfur compounds through 4S pathway. This study describes a newly identified desulfurizing bacterium, Rhodococcus sp. FUM94, which unlike previously identified strains encodes a truncated dsz operon. DNA sequencing revealed a frameshift mutation in the dszA gene, which led to an alteration of 66 amino acids and deletion of other C-terminal 66 amino acids. The resulting DszA polypeptide was shorter than DszA in Rhodococcus sp. IGTS8 reference strain. Despite the truncation, desulfurizing activity of the operon was observed and attributed to the removal of an overlap of dszA and dszB genes, and lack of active site in the altered region. Desulfurization experiments resulted in specific production rate of 6.3 mmol 2-hydroxy biphenyl (kgDCW) -1  h -1 at 2 g l -1 biocatalyst concentration and 68.8% biodesulfurization yield at 20 g l -1 biocatalyst concentration, both at 271 μM dibenzothiophene concentration which is comparable to similar wild-type biocatalysts.

Benzoate 1,2-dioxygenase (BDO) of Rhodococcus opacus 1CP, which carried out the initial attack on benzoate, was earlier shown to be the enzyme with a narrow substrate specificity. A kinetics of interaction between benzoate 1,2-dioxygenase and substituted benzoates was assessed taking into account the enlarged list of the type of inhibition and using whole cells grown on benzoate. The type of inhibition was determined and the constants of a reaction of BDO with benzoate in the presence of 2-chlorobenzoate (2CBA), 3,5-dichlorobenzoate (3,5DCBA), and 3-methylbenzoate (3MBA) were calculated. For 2CBA and 3MBA, the types of inhibition were classified as biparametrically disсoordinated inhibition and transient inhibition (from activation towards inhibition), respectively. The process of not widely recognized pseudoinhibition of a BDO reaction with benzoate by 3,5DCBA was assessed by the vector method for the representation of enzymatic reactions. Ki value was determined for 2CBA, 3MBA, and 3,5DCBA as 337.5, 870.3, and 14.7 μM, respectively.

We describe the development of biocatalysis for producing optically pure straight-chain (S)-epoxyalkanes using styrene monooxygenase of Rhodococcus sp. strain ST-10 (RhSMO). RhSMO was expressed in the organic solvent-tolerant microorganism Kocuria rhizophila DC2201, and the bioconversion reaction was performed in an organic solvent-water biphasic reaction system. The biocatalytic process enantioselectively converted linear terminal alkenes to their corresponding (S)-epoxyalkanes using glucose and molecular oxygen. When 1-heptene and 6-chloro-1-hexene were used as substrates (400 mM) under optimized conditions, 88.3 mM (S)-1,2-epoxyheptane and 246.5 mM (S)-1,2-epoxy-6-chlorohexane, respectively, accumulated in the organic phase with good enantiomeric excess (ee; 84.2 and 95.5%). The biocatalysis showed broad substrate specificity toward various aliphatic alkenes, including functionalized and unfunctionalized alkenes, with good to excellent ee. Here, we demonstrate that this biocatalytic system is environmentally friendly and useful for producing various enantiopure (S)-epoxyalkanes., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Nitro group-containing natural products are rare in nature. There are few examples of N-oxygenases, enzymes that incorporate atmospheric oxygen into primary and secondary amines, characterized in the literature. N-oxygenases have yet to be characterized from the Corynebacterineae, a metabolically diverse group of organisms that includes the genera Rhodococcus, Gordonia, and Mycobacterium. A preliminary in silico search for N-oxygenase AurF gene orthologs revealed multiple protein candidates present in the genome of the Actinomycete Rhodococcus jostii RHAI (RHAI_ro06104). Towards the goal of identifying novel biocatalysts with potential utility for the biosynthesis of nitroaromatics, AurF ortholog RHAI_ro6104 was cloned, expressed and purified in E. coli and amine and nitro containing phenol substrates tested for activity. RHAI-ro06104 showed the highest activity with 4-aminophenol, producing a Vmax of 18.76 μM s(-1) and a Km of 15.29 mM and demonstrated significant activities with 2-aminophenol and 2-amino-5-methylphenol, producing a Vmax of 12.86 and 12.72 μM s(-1) with a Km of 8.34 and 2.81 mM, respectively. These findings are consistent with a substrate range observed in other N-oxygenases, which seem to accommodate substrates that lack halogenated substitutions and side groups directly flanking the amine group. Attempts to identify modulators of RHAI-ro06104 gene activity demonstrated that aromatic amino acids inhibit expression by almost 50%.

The Baeyer-Villiger monooxygenases (BVMOs) are microbial enzymes that catalyze the synthetically useful Baeyer-Villiger oxidation reaction. The available BVMO crystal structures all lack a substrate or product bound in a position that would determine the substrate specificity and stereospecificity of the enzyme. Here, we report two crystal structures of cyclohexanone monooxygenase (CHMO) with its product, ε-caprolactone, bound: the CHMO(Tight) and CHMO(Loose) structures. The CHMO(Tight) structure represents the enzyme state in which substrate acceptance and stereospecificity is determined, providing a foundation for engineering BVMOs with altered substrate spectra and/or stereospecificity. The CHMO(Loose) structure is the first structure where the product is solvent accessible. This structure represents the enzyme state upon binding and release of the substrate and product. In addition, the role of the invariant Arg329 in chaperoning the substrate/product during the catalytic cycle is highlighted. Overall, these data provide a structural framework for the engineering of BVMOs with altered substrate spectra and/or stereospecificity.

Bacterial isolates from soils contaminated with (chlorinated) aromatic compounds, which degraded biphenyl/chlorinated biphenyls (CB) and belonged to the genera Rhodococcus and Pseudomonas were studied. Analysis of the 16S rRNA gene sequences was used to determine the phylogenetic position of the isolates. The Rhodococcus cells were found to contain plasmids of high molecular mass (220-680 kbp). PCR screening for the presence of the bphA1 gene, a marker indicating the possibility for induction of 2,3-dioxygenase (biphenyl/toluene dioxygenase subfamily) revealed the presence of the bphAl genes with 99-100% similarity to the homologous genes of bacteria of the relevant species in all pseudomonad and most Rhodococcus isolates. A unique bphA1 gene, which had not been previously reported for the genus, was identified in Rhodococcus sp. G10. The absence of specific amplification of the bphA1 genes in some biphenyl-degrading bacteria (Rhodococcus sp. B7b, B106a, G12a, P2kr, P2(51), and P2m), as well as in an active biphenyl degrader Rhodococcus ruber P25 indicated the absence of the genes encoding the proteins of the biphenyl/toluene dioxygenase subfamily and participation of the enzymes other than this protein family in biphenyl/CB degradation.

Triclosan is an antimicrobial agent, an endocrine disrupting compound, and an emerging contaminant in the environment. This is the first study investigating triclosan biodegradation potential of four oxygenase-expressing bacteria: Rhodococcus jostii RHA1, Mycobacterium vaccae JOB5, Rhodococcus ruber ENV425, and Burkholderia xenovorans LB400. B. xenovorans LB400 and R. ruber ENV425 were unable to degrade triclosan. Propane-grown M. vaccae JOB5 can completely degrade triclosan (5 mg L(-1)). R. jostii RHA1 grown on biphenyl, propane, and LB medium with dicyclopropylketone (DCPK), an alkane monooxygenase inducer, was able to degrade the added triclosan (5 mg L(-1)) to different extents. Incomplete degradation of triclosan by RHA1 is probably due to triclosan product toxicity. The highest triclosan transformation capacity (Tc, defined as the amount of triclosan degraded/the number of cells inactivated; 5.63×10(-3) ng triclosan/16S rRNA gene copies) was observed for biphenyl-grown RHA1 and the lowest Tc (0.20×10(-3) ng-triclosan/16S rRNA gene copies) was observed for propane-grown RHA1. No triclosan degradation metabolites were detected during triclosan degradation by propane- and LB+DCPK-grown RHA1. When using biphenyl-grown RHA1 for degradation, four chlorinated metabolites (2,4-dichlorophenol, monohydroxy-triclosan, dihydroxy-triclosan, and 2-chlorohydroquinone (a new triclosan metabolite)) were detected. Based on the detected metabolites, a meta-cleavage pathway was proposed for triclosan degradation., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Styrene monooxygenase (StyA, SMOA)- and flavin oxidoreductase (StyB, SMOB)-coding genes of styrene-assimilating bacteria Rhodococcus sp. ST-5 and ST-10 were successfully expressed in Escherichia coli. Determined amino acid sequences of StyAs and StyBs of ST-5 and ST-10 showed more similarity with those of Pseudomonas than with self-sufficient styrene monooxygenase (StyA2B) of Rhodococcus. Recombinant enzymes were purified from E. coli cells as functional proteins, and their properties were characterized in detail. StyBs (flavin oxidoreductase) of strains ST-5 and ST-10 have similar enzymatic properties to those of Pseudomonas, but StyB of strain ST-10 exhibited higher temperature stability than that of strain ST-5. StyAs of strains ST-5 and ST-10 catalyzed the epoxidation of vinyl side-chain of styrene and its derivatives and produced (S)-epoxides from styrene derivatives and showed high stereoselectivity. Both StyAs showed higher specific activity on halogenated styrene derivatives than on styrene itself. Additionally, the enzymes could catalyze the epoxidation of short-chain 1-alkenes to the corresponding (S)-epoxides. Aromatic compounds including styrene, 3-chlorostyrene, styrene oxide, and benzene exhibited marked inhibition of SMO reaction, although linear 1-alkene showed no inhibition of SMO activity at any concentration.

Styrene monooxygenases (SMOs) are catalysts for the enantioselective epoxidation of terminal alkenes. Most representatives comprise a reductase and a monooxygenase which are encoded by separate genes (styA, styB). Only six presumed self-sufficient one-component SMOs (styA2B) have previously been submitted to databases, and one has so far been characterized. StyA2B can be supported by another epoxidase (StyA1) encoded by styA1, a gene in direct neighborhood of styA2B. The present report describes the identification of a further styA1/styA2B-like SMO, which was detected in Rhodococcus opacus MR11. Based on the initially available sequences of styA2B-type SMOs, primers directed at conserved sequences were designed and a 7,012-bp genomic fragment from strain MR11 was obtained after PCRs and subsequent genome walking. Six open reading frames (ORFs) were detected and compared to genomic fragments of strains comprising either two- or one-component SMOs. Among the proteins encoded by the ORFs, the monooxygenase StyA1/StyA2B showed the highest divergence on amino acid level when comparing proteins from different sources. That finding, a rare distribution of styA2B genes among bacteria, and the general observation of evolution from simple to complex systems indicate that one-component SMOs evolved from two-component ancestors. Analysis of gene products from styA/styB- and styA1/styA2B-like SMOs revealed that a fusion of styA/styB to styA2B might have happened at least twice among microorganisms. This points to a convergent evolution of one-component SMOs.

The Baeyer-Villiger monooxygenases (BVMOs) are a family of bacterial flavoproteins that catalyze the synthetically useful Baeyer-Villiger oxidation reaction. This involves the conversion of ketones into esters or cyclic ketones into lactones by introducing an oxygen atom adjacent to the carbonyl group. The BVMOs offer exquisite regio- and enantiospecificity while acting on a wide range of substrates. They use only NADPH and oxygen as cosubstrates, and produce only NADP(+) and water as byproducts, making them environmentally attractive for industrial purposes. Here, we report the first crystal structure of a BVMO, cyclohexanone monooxygenase (CHMO) from Rhodococcus sp. HI-31 in complex with its substrate, cyclohexanone, as well as NADP(+) and FAD, to 2.4 Å resolution. This structure shows a drastic rotation of the NADP(+) cofactor in comparison to previously reported NADP(+)-bound structures, as the nicotinamide moiety is no longer positioned above the flavin ring. Instead, the substrate, cyclohexanone, is found at this location, in an appropriate position for the formation of the Criegee intermediate. The rotation of NADP(+) permits the substrate to gain access to the reactive flavin peroxyanion intermediate while preventing it from diffusing out of the active site. The structure thus reveals the conformation of the enzyme during the key catalytic step. CHMO is proposed to undergo a series of conformational changes to gradually move the substrate from the solvent, via binding in a solvent excluded pocket that dictates the enzyme's chemospecificity, to a location above the flavin-peroxide adduct where catalysis occurs.

Enantioselective epoxidation followed by regioselective epoxide opening reaction are the key processes in construction of the polyether skeleton. Recent genetic analysis of ionophore polyether biosynthetic gene clusters suggested that flavin-containing monooxygenases (FMOs) could be involved in the oxidation steps. In vivo and in vitro analyses of Lsd18, an FMO involved in the biosynthesis of polyether lasalocid, using simple olefin or truncated diene of a putative substrate as substrate mimics demonstrated that enantioselective epoxidation affords natural type mono- or bis-epoxide in a stepwise manner. These findings allow us to figure out enzymatic polyether construction in lasalocid biosynthesis., (© 2012 American Chemical Society)

Styrene metabolism genes were isolated from styrene-assimilating bacteria Rhodococcus sp. ST-5 and ST-10. Strain ST-5 had a gene cluster containing four open reading frames which encoded styrene degradation enzymes. The genes showed high similarity to styABCD of Pseudomonas sp. Y2. On the other hand, strain ST-10 had only two genes which encoded styrene monooxygenase and flavin oxidoreductase (styAB). Escherichia coli transformants possessing the sty genes of strains ST-5 and ST-10 produced (S)-styrene oxide from styrene, indicating that these genes function as styrene degradation enzymes. Metabolite analysis by resting-cell reaction with gas chromatography-mass spectrometry revealed that strain ST-5 converts styrene to phenylacetaldehyde via styrene oxide by styrene oxide isomerase (styC) reaction. On the other hand, strain ST-10 lacked this enzyme, and thus accumulated styrene oxide as an intermediate. HPLC analysis showed that styrene oxide was spontaneously isomerized to phenylacetaldehyde by chemical reaction. The produced phenylacetaldehyde was converted to phenylacetic acid (PAA) in strain ST-10 as well as in strain ST-5. Furthermore, phenylacetic acid was converted to phenylacetyl-CoA by the catalysis of phenylacetate-CoA ligase in strains ST-5 and ST-10. This study proposes possible styrene metabolism pathways in Rhodococcus sp. strains ST-5 and ST-10., (Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

4-Nitrophenol (4-NP) is a toxic compound formed in soil by the hydrolysis of organophosphorous pesticides, such as parathion. We previously reported the presence of the 4-NP degradation gene cluster (nphRA1A2) in Rhodococcus sp. strain PN1, which encodes a two-component 4-NP hydroxylase system that oxidizes 4-NP into 4-nitrocatechol. In the current study, another gene cluster (npsC and npsRA2A1B) encoding a similar 4-NP hydroxylase system was cloned from strain PN1. The enzymes from this 4-NP hydroxylase system (NpsA1 and NpsA2) were purified as histidine-tagged (His-) proteins and then characterized. His-NpsA2 showed NADH/FAD oxidoreductase activity, and His-NpsA1 showed 4-NP oxidizing activity in the presence of His-NpsA2. In the 4-NP oxidation using the reconstituted enzyme system (His-NpsA1 and His-NpsA2), hydroquinone (35% of 4-NP disappeared) and hydroxyquinol (59% of 4-NP disappeared) were detected in the presence of ascorbic acid as a reducing reagent, suggesting that, without the reducing reagent, 4-NP was converted into their oxidized forms, 1,4-benzoquinone and 2-hydroxy-1,4-benzoquinone. In addition, in the cell extract of recombinant Escherichia coli expressing npsB, a typical spectral change showing conversion of hydroxyquinol into maleylacetate was observed. These results indicate that this nps gene cluster, in addition to the nph gene cluster, is also involved in 4-NP degradation in strain PN1., (Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Rhodococcus jostii RHA1 is a polychlorinated biphenyl degrader. Multi-component biphenyl 2,3-dioxygenase (BphA) genes of RHA1 encode large and small subunits of oxygenase component and ferredoxin and reductase components. They did not express enzyme activity in Escherichia coli. To obtain BphA activity in E. coli, hybrid BphA gene derivatives were constructed by replacing ferredoxin and/or reductase component genes of RHA1 with those of Pseudomonas pseudoalcaligenes KF707. The results obtained indicate a lack of catalytic activity of the RHA1 ferredoxin component gene, bphAc in E. coli. To determine the cause of inability of RHA1 bphAc to express in E. coli, the bphAc gene was introduced into Rosetta (DE3) pLacI, which has extra tRNA genes for rare codons in E. coli. The resulting strain abundantly produced the bphAc product, and showed activity. These results suggest that codon usage bias is involved in inability of RHA1 bphAc to express its catalytic activity in E. coli.

A meta-cleavage pathway for the aerobic degradation of aromatic hydrocarbons is catalyzed by extradiol dioxygenases via a two-step mechanism: catechol substrate binding and dioxygen incorporation. The binding of substrate triggers the release of water, thereby opening a coordination site for molecular oxygen. The crystal structures of AkbC, a type I extradiol dioxygenase, and the enzyme substrate (3-methylcatechol) complex revealed the substrate binding process of extradiol dioxygenase. AkbC is composed of an N-domain and an active C-domain, which contains iron coordinated by a 2-His-1-carboxylate facial triad motif. The C-domain includes a β-hairpin structure and a C-terminal tail. In substrate-bound AkbC, 3-methylcatechol interacts with the iron via a single hydroxyl group, which represents an intermediate stage in the substrate binding process. Structure-based mutagenesis revealed that the C-terminal tail and β-hairpin form part of the substrate binding pocket that is responsible for substrate specificity by blocking substrate entry. Once a substrate enters the active site, these structural elements also play a role in the correct positioning of the substrate. Based on the results presented here, a putative substrate binding mechanism is proposed.

Two-component flavoprotein monooxygenases are emerging biocatalysts that generally consist of a monooxygenase and a reductase component. Here we show that Rhodococcus opacus 1CP encodes a multifunctional enantioselective flavoprotein monooxygenase system composed of a single styrene monooxygenase (SMO) (StyA1) and another styrene monooxygenase fused to an NADH-flavin oxidoreductase (StyA2B). StyA1 and StyA2B convert styrene and chemical analogues to the corresponding epoxides at the expense of FADH2 provided from StyA2B. The StyA1/StyA2B system presents the highest monooxygenase activity in an equimolar ratio of StyA1 and StyA2B, indicating (transient) protein complex formation. StyA1 is also active when FADH2 is supplied by StyB from Pseudomonas sp. VLB120 or PheA2 from Rhodococcus opacus 1CP. However, in both cases the reductase produces an excess of FADH2, resulting in a high waste of NADH. The epoxidation rate of StyA1 heavily depends on the type of reductase. This supports that the FADH2-induced activation of StyA1 requires interprotein communication. We conclude that the StyA1/StyA2B system represents a novel type of multifunctional flavoprotein monooxygenase. Its unique mechanism of cofactor utilization provides new opportunities for biotechnological applications and is highly relevant from a structural and evolutionary point of view.

Rhodococcus sp. JDC-11, capable of utilizing di-n-butyl phthalate (DBP) as the sole source of carbon and energy, was isolated from sewage sludge and confirmed mainly based on 16S rRNA gene sequence analysis. The optimum pH, temperature, and agitation rate for DBP degradation by Rhodococcus sp. JDC-11 was 8.0, 30 degrees C, and 175 rpm, respectively. In addition, the effect of glucose concentration on DBP degradation indicated that low concentration of glucose inhibited the degradation of DBP while high concentrations of glucose increased its degradation. Meanwhile, the substrates utilization test showed that JDC-11 could also utilize other phthalates. Furthermore, the major metabolites of DBP degradation were identified as mono-butyl phthalate and phthalic acid by gas chromatography-mass spectrometry and the metabolic pathway of DBP degradation by Rhodococcus sp. JDC-11 was tentatively speculated. Using a set of new degenerate primer, partial sequence of the 3, 4-phthalate dioxygenase gene was obtained from the strain. Sequence analysis revealed that the phthalate dioxygenase gene of JDC-11 was highly homologous to the large subunit of phthalate dioxygenase from Rhodococcus coprophilus strain G9.

Sequence analysis of a 9-kb genomic fragment of the actinobacterium Rhodococcus opacus 1CP led to identification of an open reading frame encoding a novel fusion protein, StyA2B, with a putative function in styrene metabolism via styrene oxide and phenylacetic acid. Gene cluster analysis indicated that the highly related fusion proteins of Nocardia farcinica IFM10152 and Arthrobacter aurescens TC1 are involved in a similar physiological process. Whereas 413 amino acids of the N terminus of StyA2B are highly similar to those of the oxygenases of two-component styrene monooxygenases (SMOs) from pseudomonads, the residual 160 amino acids of the C terminus show significant homology to the flavin reductases of these systems. Cloning and functional expression of His(10)-StyA2B revealed for the first time that the fusion protein does in fact catalyze two separate reactions. Strictly NADH-dependent reduction of flavins and highly enantioselective oxygenation of styrene to (S)-styrene oxide were shown. Inhibition studies and photometric analysis of recombinant StyA2B indicated the absence of tightly bound heme and flavin cofactors in this self-sufficient monooxygenase. StyA2B oxygenates a spectrum of aromatic compounds similar to those of two-component SMOs. However, the specific activities of the flavin-reducing and styrene-oxidizing functions of StyA2B are one to two orders of magnitude lower than those of StyA/StyB from Pseudomonas sp. strain VLB120.

A novel indigo-producing oxygenase gene, designated ipoA (1,197 bp) was characterized from Rhodococcus sp. strain T104. Three indigo-negative mutations (A58V, P59L, and G251D) were obtained through random mutagenesis using an E. coli mutator strain. Subsequent saturation mutagenesis resulted in the identification of nine and three amino acid substitutions that restore activity in the A58V and P59L mutants, respectively. Activity was not restored in the G251D mutation by any other amino acids. Interestingly, activity in the A58V mutant, where a methyl group is only replaced by an isopropyl side chain, is restored by a variety of amino acids, including polar ones. A molecular modeling study suggests that the residues at positions 58, 59, and 251 of the T104 IpoA enzyme are far from the active site, indicating that the mutations must alter the overall structure of the enzyme.

To understand the response of soil bacteria to the surrounding environment, it is necessary to examine the gene expression profiles of the bacteria in the soil. For this purpose, we developed a new method of extracting RNA from soil reproducibly. Using this new method, we extracted RNA from a field soil, which was sterilized and inoculated with Rhodococcus sp. strain RHA1, a biphenyl degrader isolated from gamma-hexachlorocyclohexane-contaminated soil. Data from agarose gel electrophoresis indicated that the extracted RNA was purified properly. This new method can be applied easily in the preparation of large amounts of RNA. Real-time reverse transcription-polymerase chain reaction (RT-PCR) experiments performed by the TaqMan method suggested that the bphAa gene in this strain, which is involved in the degradation of biphenyl, was induced in the biphenyl amended soil.

The pathways from beta-carotene to astaxanthin are crucial key steps for producing astaxanthin, one of industrially useful carotenoids, in heterologous hosts. Two beta-carotene ketolases (beta-carotene 4,4'-oxygenase), CrtO and CrtW, with different structure are known up to the present. In this paper, we compared the catalytic functions of a CrtO ketolase that was obtained from a marine bacterium Rhodococcus erythropolis strain PR4, CrtO derived from cyanobacterium Synechosistis sp. PCC6803, and CrtW derived from a marine bacterium Brevundimonas sp. SD212, by complementation analysis in Escherichia coli expressing the known crt genes. Results strongly suggested that a CrtO-type ketolase was unable to synthesize astaxanthin from zeaxanthin, i.e., only a CrtW-type ketolase could accept 3-hydroxy-beta-ionone ring as the substrate. Their catalytic efficiency for synthesizing canthaxanthin from beta-carotene was also examined. The results obtained up to the present clearly suggest that the bacterial crtW and crtZ genes are a combination of the most promising gene candidates for developing recombinant hosts that produce astaxanthin as the predominant carotenoid.

Two types of non-homologous beta-carotene ketolases (CrtW and CrtO) were previously described. We report improvement of a CrtO-type of beta-carotene ketolase for canthaxanthin production in a methylotrophic bacterium, Methylomonas sp. 16a, which could use the C1 substrate (methane or methanol) as sole carbon and energy source. The crtO gene from Rhodococcus erythropolis was improved for canthaxanthin production in an E. coli strain engineered to produce high titer carotenoids by error-prone PCR mutagenesis followed by in vitro recombination. The best mutants from protein engineering could produce approximately 90% of total carotenoids as canthaxanthin in the high titer E. coli strain compared to approximately 20% canthaxanthin produced by the starting gene. Canthaxanthin production in Methylomonas was also significantly improved to approximately 50% of total carotenoids by the mutant genes. Further improvement of canthaxanthin production to approximately 93% in Methylomonas was achieved by increased expression of the best mutant gene. Some mutations were found in many of the improved genes, suggesting that these sites, and possibly the regions around these sites, were important for improving the crtO's activity for canthaxanthin production.

Dibenzothiophene (DBT) and its derivatives can be microbially desulfurized by Dsz enzymes. We investigated the expressional characteristics of the dsz operon. The result revealed that the ratio of mRNA quantity of dszA, dszB, and dszC was 11:3.3:1; however, western blot analysis indicated that the expression level of dszB is far lower than that of dszC. Gene analysis revealed that the termination codon of dszA and the initiation codon of dszB overlapped, whereas there was a 13-bp gap between dszB and dszC. In order to get a better, steady expression of DszB, we removed this structure by overlap polymerase chain reaction (PCR) and expressed the redesigned dsz operon in Rhodococcus erythropolis. The desulfurization activity of resting cells prepared from R. erythropolis DR-2, which held the redesigned dsz operon, was about five-fold higher than that of R. erythropolis DR-1, which held the original dsz operon.

Rhodococcus sp. RHA1 induces two biphenyl dioxygenases, the BphA and EtbA/EbdA dioxygenases, during growth on biphenyl. Their subunit genes were expressed in R. erythropolis IAM1399 to investigate the involvement of each subunit gene in their activity and their substrate preferences. The recombinant expressing ebdA1A2A3etbA4 and that expressing bphA1A2A3A4 exhibited 4-chlorobiphenyl (4-CB) transformation activity, suggesting that these gene sets are responsible for the EtbA/EbdA and BphA dioxygenases respectively. When bphA4 and etbA4 were swapped to construct the recombinants expressing ebdA1A2A3bphA4 and bphA1A2A3etbA4 respectively, compatibility between BphA4 and EtbA4 was suggested by their 4-CB transformation activities. When bphA3 and ebdA3 were swapped, incompatibility between BphA3 and EbdA3 was suggested. BphA and EtbA/EbdA dioxygenases exhibited the highest transformation activity toward biphenyl and naphthalene respectively, and also attacked dibenzofuran and dibenzo-p-dioxin. The wide substrate preference of EtbA/EbdA dioxygenase suggested that it plays a more important role in polychlorinated biphenyl (PCB) degradation than does BphA dioxygenase.

In order to construct an expression system for the particulate methane mono-oxygenase (pMMO) gene (pmo), the structural gene cluster pmoCAB amplified from Methylosinus trichosporium OB3b was inserted into a shuttle vector pBS305 under the control of a dsz promoter and transformed into Rhodococcus erythropolis LSSE8-1. A stable transformant was successfully obtained using ethane as the sole carbon source. Fluorescence in situ hybridization results showed that the dsz promoter allowed the pmo genes to be transcribed in the recombinant strain. The effects of Cu2+ and Zn2+ concentrations on cell growth and pMMO activity in ethane-containing medium were examined. It was discovered that 7.5 microM Cu2+ and 1.8 microM Zn2+ were suitable to achieve high cell concentration and pMMO activity, but the amount of methanol accumulated during methane oxidation by the recombinant strain was still low.

Clusters of genes which include determinants for the catalytic subunits of naphthalene dioxygenase (narAa and narAb) were analyzed in naphthalene-degrading Rhodococcus strains. We demonstrated (i) that in the region analyzed homologous gene clusters are separated from each other by nonhomologous DNA, (ii) that there are various degrees of homology between related genes, and (iii) that nar genes are located on plasmids in strains NCIMB12038 and P400 and on a chromosome in P200. These observations suggest that genetic exchange and reshuffling of genetic modules, as well as vertical descent of the genetic information, were the main routes in the evolution of naphthalene degradation in Rhodococcus. These conclusions were supported by studies of transcription patterns in the region analyzed. It was found that the nar region is not organized into a single operon but there are several transcription units which differ in the strains investigated. The narA and narB genes were found to be transcribed as a single unit in all strains analyzed, and their transcription was induced by naphthalene. The putative aldolase gene (narC) was found on the same transcript only in strains P200 and P400. In NCIMB12038 transcription of two more gene clusters was induced by growth on naphthalene. Transcription start sites for narA and narB were found to be different in all of the strains studied. Putative regulatory genes (narR1 and narR2) were transcribed as a single mRNA in naphthalene-induced cells. At the same time, a number of the genes known to be essential for naphthalene catabolism in gram-negative bacteria were not found in the region analyzed.

We reported previously that the Rhodococcus erythropolis strain AN12 synthesizes the monocyclic carotenoids 4-keto gamma-carotene and gamma-carotene. We also identified a novel lycopene beta-monocyclase in this strain. Here we report the identification of the rest of the carotenoid synthesis genes in AN12. Two of these showed apparent homology to putative phytoene dehydrogenases. Analysis of Rhodococcus knockout mutants suggested that one of them ( crtI) encodes a phytoene dehydrogenase, whereas the other ( crtO) encodes a beta-carotene ketolase. Expression of the beta-carotene ketolase gene in an Escherichia coli strain which accumulates beta-carotene resulted in the production of canthaxanthin. In vitro assays using a crude extract of the E. coli strain expressing the crtO gene confirmed its ketolase activity. A crtO homologue (DR0093) from Deinococcus radiodurans R1 was also shown to encode a beta-carotene ketolase, despite its sequence homology to phytoene dehydrogenases. The Rhodococcus and Deinococcus CrtO ketolases both catalyze the symmetric addition of two keto groups to beta-carotene to produce canthaxanthin. Even though this activity is similar to the CrtW-type of ketolase activity, the CrtO ketolases show no significant sequence homology to CrtW-type ketolases. The presence of six conserved regions may be a signature for the CrtO-type of beta-carotene ketolases.

Biphenyl dioxygenase is the enzyme that catalyzes the stereospecific dioxygenation of the aromatic ring. This enzyme has attracted the attention of researchers due to its ability to oxidize polychlorinated biphenyls, which is one of the serious environmental contaminants. We determined the crystal structure of the terminal oxygenase component of the biphenyl dioxygenase (BphA1A2) derived from Rhodococcus strain sp. RHA1 in substrate-free and complex forms. These crystal structures revealed that the substrate-binding pocket makes significant conformational changes upon substrate binding to accommodate the substrate into the pocket. Our analysis of the crystal structures suggested that the residues in the substrate-binding pocket can be classified into three groups, which, respectively, seem to be responsible for the catalytic reaction, the orientation/conformation of the substrate, and the conformational changes of the substrate-binding pocket. The cooperative actions of residues in the three groups seem to determine the substrate specificity of the enzyme.

Rhodococcus sp. I24 can oxygenate indene via at least three independent enzyme activities: (i) a naphthalene inducible monooxygenase (ii) a naphthalene inducible dioxygenase, and (iii) a toluene inducible dioxygenase (TID). Pulsed field gel analysis revealed that the I24 strain harbors two megaplasmids of approximately 340 and approximately 50 kb. Rhodococcus sp. KY1, a derivative of the I24 strain, lacks the approximately 340 kb element as well as the TID activity. Southern blotting and sequence analysis of an indigogenic, I24-derived cosmid suggested that an operon encoding a TID resides on the approximately 340 kb element. Expression of the tid operon was induced by toluene but not by naphthalene. In contrast, naphthalene did induce expression of the nid operon, encoding the naphthalene dioxygenase in I24. Cell free protein extracts of Escherichia coli cells expressing tidABCD were used in HPLC-based enzyme assays to characterize the indene bioconversion of TID in vitro. In addition to 1-indenol, indene was transformed to cis-indandiol with an enantiomeric excess of 45.2% of cis-(1S,2R)-indandiol over cis-(1R,2S)-indandiol, as revealed by chiral HPLC analysis. The Km of TID for indene was 380 microM. The enzyme also dioxygenated naphthalene to cis-dihydronaphthalenediol with an activity of 78% compared to the formation of cis-indandiol from indene. The Km of TID for naphthalene was 28 microM. TID converted only trace amounts of toluene to 1,2-dihydro-3-methylcatechol after prolonged incubation time. The results indicate the role of the tid operon in the bioconversion of indene to 1-indenol and cis-(1S,2R)-indandiol by Rhodococcus sp. I24.

The crystal structure of the 4-chlorocatechol 1,2-dioxygenase from the Gram-positive bacterium Rhodococcus opacus (erythropolis) 1CP, a Fe(III) ion-containing enzyme involved in the aerobic biodegradation of chloroaromatic compounds, has been solved by multiple wavelength anomalous dispersion using the weak anomalous signal of the two catalytic irons (1 Fe/257 amino acids) and refined at a 2.5 A resolution (R(free) 28.7%; R factor 21.4%). The analysis of the structure and its comparison with the structure of catechol 1,2-dioxygenase from Acinetobacter calcoaceticus ADP1 (Ac 1,2-CTD) highlight significant differences between these enzymes. The general topology of the present enzyme comprises two catalytic domains (one for each subunit) related by a noncrystallographic 2-fold axis and separated by a common alpha-helical zipper motif consisting of five N-terminal helices from each subunit; furthermore the C-terminal tail is shortened significantly with respect to the known Ac 1,2-CTD. The presence of two phospholipids binding in a hydrophobic tunnel along the dimer axis is shown here to be a common feature for this class of enzyme. The active site cavity presents several dissimilarities with respect to the known catechol-cleaving enzyme. The catalytic nonheme iron(III) ion is bound to the side chains of Tyr-134, Tyr-169, His-194, and His-196, and a cocrystallized benzoate ion, bound to the metal center, reveals details on a novel mode of binding of bidentate inhibitors and a distinctive hydrogen bond network with the surrounding ligands. Among the amino acid residues expected to interact with substrates, several are different from the corresponding analogs of Ac 1,2-CTD: Asp-52, Ala-53, Gly-76, Phe-78, and Cys-224; in addition, regions of largely conserved amino acid residues in the catalytic cleft show different shapes resulting from several substantial backbone and side chain shifts. The present structure is the first of intradiol dioxygenases that specifically catalyze the cleavage of chlorocatechols, key intermediates in the aerobic catabolism of toxic chloroaromatics.

Rhodococcus sp. strain T104 is able to utilize both limonene and biphenyl as growth substrates. Furthermore, T104 possesses separate pathways for the degradation of limonene and biphenyl. Previously, we found that a gene(s) involved in limonene degradation was also related to indigo-producing ability. To further corroborate this observation, we have cloned and sequenced a 8,842-bp genomic DNA region with four open reading frames, including one for indole oxygenase, which converts indole to indigo (a blue pigment), The reverse transcription PCR data demonstrated that the identified indole oxygenase gene is specifically induced by limonene, thereby implicating this gene in the degradation of limonene by T104.

Based on structural, biochemical, and genetic data, the soluble diiron monooxygenases can be divided into four groups: the soluble methane monooxygenases, the Amo alkene monooxygenase of Rhodococcus corallinus B-276, the phenol hydroxylases, and the four-component alkene/aromatic monooxygenases. The limited phylogenetic distribution of these enzymes among bacteria, together with available genetic evidence, indicates that they have been spread largely through horizontal gene transfer. Phylogenetic analyses reveal that the alpha- and beta-oxygenase subunits are paralogous proteins and were derived from an ancient gene duplication of a carboxylate-bridged diiron protein, with subsequent divergence yielding a catalytic alpha-oxygenase subunit and a structural beta-oxygenase subunit. The oxidoreductase and ferredoxin components of these enzymes are likely to have been acquired by horizontal transfer from ancestors common to unrelated diiron and Rieske center oxygenases and other enzymes. The cumulative results of phylogenetic reconstructions suggest that the alkene/aromatic monooxygenases diverged first from the last common ancestor for these enzymes, followed by the phenol hydroxylases, Amo alkene monooxygenase, and methane monooxygenases.

Rhodococcus sp. strain MS11 was isolated from a mixed culture. It displays a diverse range of metabolic capabilities. During growth on 1,2,4-trichlorobenzene, 1,2,4,5-tetrachlorobenzene (1,2,4,5-TeCB) and 3-chlorobenzoate stoichiometric amounts of chloride were released. It also utilized all three isomeric dichlorobenzenes and 1,2,3-trichlorobenzene as the sole carbon and energy source. Furthermore, the bacterium grew well on a great number of n-alkanes ranging from n-heptane to n-triacontane and on the branched alkane 2,6,10,14-tetramethylpentadecane (pristane) and slowly on n-hexane and n-pentatriacontane. It was able to grow at temperatures from 5 to 30 degrees C, with optimal growth at 20 degrees C, and could tolerate 6 % NaCl in mineral salts medium. Genes encoding the initial chlorobenzene dioxygenase were detected by using a primer pair that was designed against the alpha-subunit (TecA1) of the chlorobenzene dioxygenase of Ralstonia (formerly Burkholderia) sp. strain PS12. The amino acid sequence of the amplified part of the alpha-subunit of the chlorobenzene dioxygenase of Rhodococcus sp. strain MS11 showed >99 % identity to the alpha-subunit of the chlorobenzene dioxygenase from Ralstonia sp. strain PS12 and the parts of both alpha-subunits responsible for substrate specificity were identical. The subsequent enzymes dihydrodiol dehydrogenase and chlorocatechol 1,2-dioxygenase were induced in cells grown on 1,2,4,5-TeCB. During cultivation on medium-chain-length n-alkanes ranging from n-decane to n-heptadecane, including 1-hexadecene, and on the branched alkane pristane, strain MS11 produced biosurfactants lowering the surface tension of the cultures from 72 to

The terminal oxygenase component of the biphenyl dioxygenase (BphA1A2 complex) was over-expressed with a novel over expression system in recombinant Rhodococcus strain and purified. The purified enzyme has been crystallized by the hanging drop vapor diffusion method and subjected to X-ray diffraction analysis. The crystals belong to the tetragonal system in the space group P4(1)2(1)2 or P4(3)2(1)2 and diffract to better than 2.2A resolution.

The dsz desulfurization gene cluster from Rhodococcus erythropolis strain KA2-5-1 was transferred into R. erythropolis strain MC1109, unable to desulfurize light gas oil (LGO), using a transposon-transposase complex. As a result, two recombinant strains, named MC0203 and MC0122, were isolated. Resting cells of strain MC0203 decreased the sulfur concentration of LGO from 120 mg l(-1) to 70 mg l(-1) in 2 h. The LGO-desulfurization activity of strain MC0203 was about twice that of strain MC0122 and KA2-5-1. The 10-methyl fatty acids of strain MC0203 were about 28%-41% that of strain MC1109. It is likely that strain MC0203 had a mutation involving alkylenation or methylation of delta9-unsaturated fatty acids caused by the transposon inserted in the chromosome, which increased the fluidity of cell membranes and enhanced the desulfurization activity.

Rhodococcus erythropolis IGTS8 that possesses dibenzothiophene sulfone monooxygenase mutated at residue 345 (Q345A), can degrade octyl sulfide on which the wild strain cannot grow. Residue 345 and the neighbouring residues were changed by site-directed mutagenesis. Only DszA changed at residue 345 gave an altered C-S bond cleavage pattern of 3-methyl DBT sulfone. This residue is therefore involved in C-S bond cleavage specifically for alkylated DBT sulfone.

Aromatic-ring-hydroxylating dioxygenases (ARHDOs) are key enzymes in the aerobic bacterial metabolism of aromatic compounds. They are of biotechnological importance as they function as biocatalysts in the stereospecific synthesis of chiral synthons and the degradation of aromatic pollutants. This report describes the development and validation of a system for the rapid isolation and characterization of specific ARHDO activities. The system is based on the identification of ARHDO gene segments that encode the enzymes' major functional determinants, on consensus primers for the direct amplification of such partial genes and on a 'recipient' ARHDO gene cluster for the insertion of the amplified segments. Previously, it has been shown that neither the N- nor the C-terminal portions but only the core region of the large or alpha-subunit of a class II ARHDO significantly influence substrate and product spectra. On the basis of these observations, consensus primers were designed for the amplification of the gene segment encoding the catalytic core of the large subunit. These primers were tested on 11 bacterial isolates known to metabolize aromatic compounds. In 10 cases, a gene fragment of expected length was amplified. DNA sequencing confirmed similarity to ARHDO alpha-subunit gene cores. The heterologously well-expressible bphA gene cluster of Burkholderia sp. strain LB400 was modified to facilitate the in-frame insertion of amplified segments. It was used successfully to express the resulting hybrid gene clusters and to form catalytically active chimaeric ARHDOs. The metabolic properties of these enzymes differed significantly from each other and from the parental ARHDO of strain LB400. These results indicate that the system described here can be used to rapidly isolate and functionally characterize ARHDO activities, starting from isolated strains, mixtures of organisms or samples of nucleic acids. Applications of the system range from the recruitment of novel ARHDO activities to an improved characterization of natural ARHDO diversity.

To clone novel type 1 Baeyer-Villiger monooxygenase (BVMO) genes, we isolated or collected 25 bacterial strains able to grow on alicyclic compounds. Twelve of the bacterial strains yielded polymerase chain reaction (PCR) fragments with highly degenerate primers based on the sequences of known and putative BVMOs. All these fragments were found to encode peptides homologous to published BVMO sequences. The complete BVMO genes and flanking DNA were cloned from a Comamonas, a Xanthobacter and a Rhodococcus strain using the PCR fragments as probes. BVMO genes cloned from the first two strains could be expressed to high levels in Escherichia coli using standard expression vectors, and the recombinants converted cyclopentanone and cyclohexanone to the corresponding lactones. The Rhodococcus BVMO, a putative steroid monooxygenase, could be expressed after modification of the N-terminal sequence. However, recombinants expressing this protein did not show activity towards progesterone. An esterase homologue located directly upstream of the Xanthobacter BVMO gene and a dehydrogenase homologue encoded directly downstream of the Comamonas sp. NCIMB 9872 BVMO gene were also expressed in E. coli and shown to specify lactone hydrolase and cyclohexanol dehydrogenase activity respectively.

mRNA differential display has been used to identify cyclohexanone oxidation genes in a mixed microbial community derived from a wastewater bioreactor. Thirteen DNA fragments randomly amplified from the total RNA of an enrichment subculture exposed to cyclohexanone corresponded to genes predicted to be involved in the degradation of cyclohexanone. Nine of these DNA fragments are part of genes encoding three distinct Baeyer-Villiger cyclohexanone monooxygenases from three different bacterial species present in the enrichment culture. In Arthrobacter sp. strain BP2 and Rhodococcus sp. strain Phi2, the monooxygenase is part of a gene cluster that includes all the genes required for the degradation of cyclohexanone, while in Rhodococcus sp. strain Phi1 the genes surrounding the monooxygenase are not predicted to be involved in this degradation pathway but rather seem to belong to a biosynthetic pathway. Furthermore, in the case of Arthrobacter strain BP2, three other genes flanking the monooxygenase were identified by differential display, demonstrating that the repeated sampling of bacterial operons shown earlier for a pure culture (D. M. Walters, R. Russ, H. Knackmuss, and P. E. Rouvière, Gene 273:305-315, 2001) is also possible for microbial communities. The activity of the three cyclohexanone monooxygenases was confirmed and characterized following their expression in Escherichia coli.

Two mutants of the dibenzothiophene-desulfurizing Rhodococcus erythropolis KA2-5-1, strains MS51 and MS316, which express a high level of desulfurizing activity in the presence of sulfate, were isolated using the transposome technique. The level of dibenzothiophene-desulfurization by cell-free extracts prepared from mutants MS51 and MS316 grown on sulfate was about five-fold higher than that by cell-free extracts of the wild-type. This result was consistent with results of Western-blot analysis using antisera specific for DszA, DszB and DszC, the enzymes involved in the desulfurization of dibenzothiophene. Gene analysis of the mutants revealed that the same gene was disrupted in mutants MS51 and MS316 and that the transposon-inserted gene in these strains was the gene for cystathionine beta-synthase, cbs. The cbs mutants also expressed high levels of Dsz enzymes when methionine was used as the sole source of sulfur.

Protein engineering by segment exchange was used to distinguish between regions of major and minor influence on the structure of the substrate-binding pocket of a biphenyl dioxygenase (BDO). Eight chimaeric enzyme systems were generated that each consisted of a hybrid hydroxylase alpha subunit (BphA1) containing segments from Burkholderia sp. strain LB400 and Rhodococcus globerulus P6, and of a hydroxylase beta subunit (BphA2), a ferredoxin (BphA3) and a ferredoxin reductase (BphA4) from strain LB400. All hybrid bphA1 genes were expressed at high levels. Seven of the resulting fusion subunits functionally interacted with the other polypeptides of the dioxygenase system to yield catalytically active enzymes. Changes in the regiospecificity of substrate attack, monitored by the formation of seventeen different dioxygenation products obtained from seven chlorobiphenyls, were used to monitor effects of segment exchanges on the structure of the BDO substrate-binding site. Exchanges of neither the beta subunit nor the N- and C-terminal regions of the alpha subunit exerted significant influences. All BDO regions that showed major effects on the substrate-binding pocket were located between approximately positions 165 and 395 of the alpha subunit. Within this part of the enzyme, in addition to segments identified previously, a subregion which is involved in ligation of the mononuclear iron significantly influenced the regiospecificity of substrate dioxygenation. Moreover, the results indicate that the construction of appropriate hybrid genes may be used as a general strategy to overcome problems in obtaining heterologous BDO activities in Escherichia coli or other host organisms.

Sixteen actinomycetes capable of utilizing dibenzofuran as a sole source of carbon and energy were isolated, including Rhodococcus, Microbacterium, and Terrabacter genera. Heretofore, no dibenzofuran-utilizing strain belonging to the genus Microbacterium has been reported. Five extradiol dioxygenase genes (dfdB, and edil to 4) of the strain Rhodococcus sp. YK2 were cloned and analyzed. The nucleotide sequence of dfdB gene was almost identical to the bphC1 gene of Terrabacter sp. DPO360, which was involved in dibenzofuran metabolism in this strain. Southern and Northern hybridization analyses using these extradiol dioxygenase genes as probes suggest that the dfdB gene in YK2 was conserved in diverse dibenzofuran-utilizing actinomycetes; also, the dfdB gene was the only expressed gene among five extradiol dioxygenase genes in the medium containing DF as a sole carbon source. These results suggest that the dfdB gene is important for dibenzofuran metabolism not only in the strain YK2, but also in diverse dibenzofuran-degrading actinomycetes.

Biological oxidation of cyclic ketones normally results in formation of the corresponding dicarboxylic acids, which are further metabolized in the cell. Rhodococcus ruber strain SC1 was isolated from an industrial wastewater bioreactor that was able to utilize cyclododecanone as the sole carbon source. A reverse genetic approach was used to isolate a 10-kb gene cluster containing all genes required for oxidative conversion of cyclododecanone to 1,12-dodecanedioic acid (DDDA). The genes required for cyclododecanone oxidation were only marginally similar to the analogous genes for cyclohexanone oxidation. The biochemical function of the enzymes encoded on the 10-kb gene cluster, the flavin monooxygenase, the lactone hydrolase, the alcohol dehydrogenase, and the aldehyde dehydrogenase, was determined in Escherichia coli based on the ability to convert cyclododecanone. Recombinant E. coli strains grown in the presence of cyclododecanone accumulated lauryl lactone, 12-hydroxylauric acid, and/or DDDA depending on the genes cloned. The cyclododecanone monooxygenase is a type 1 Baeyer-Villiger flavin monooxygenase (FAD as cofactor) and exhibited substrate specificity towards long-chain cyclic ketones (C11 to C15), which is different from the specificity of cyclohexanone monooxygenase favoring short-chain cyclic compounds (C5 to C7).

To address the multiplicity of aromatic ring hydroxylation dioxygenases, we used PCR amplification and denaturing gradient gel electrophoresis (DGGE). The amplified DNA fragments separated into five bands, A to E. Southern hybridization analysis of RHA1 total DNA using the probes for each band showed that band C originated from a couple of homologous genes. The nucleotide sequences of the bands showed that bands A, C, and E would be parts of new dioxygenase genes in RHA1. That of band B agreed with the bphA1 gene, which was characterized previously. That of band D did not correspond to any known gene sequences. The regions including the entire open reading frames (ORFs) were cloned and sequenced. The nucleotide sequences of ORFs suggested that the genes of bands A, C, and E may respectively encode benzoate, biphenyl, and polyhydrocarbon dioxygenases. Northern hybridization indicated the induction of the gene of band A by benzoate and biphenyl, and that of the gene of band C by biphenyl and ethylbenzene, supporting the above notions. The gene of band E was not induced by any of these substrates. Thus the combination of DGGE and Southern hybridization enable us to address the multiplicity of the ring hydroxylation dioxygenase genes and to isolate some of them.

The bopXYZ genes from the gram-positive bacterium Rhodococcus sp. strain 19070 encode a broad-substrate-specific benzoate dioxygenase. Expression of the BopXY terminal oxygenase enabled Escherichia coli to convert benzoate or anthranilate (2-aminobenzoate) to a nonaromatic cis-diol or catechol, respectively. This expression system also rapidly transformed m-toluate (3-methylbenzoate) to an unidentified product. In contrast, 2-chlorobenzoate was not a good substrate. The BopXYZ dioxygenase was homologous to the chromosomally encoded benzoate dioxygenase (BenABC) and the plasmid-encoded toluate dioxygenase (XylXYZ) of gram-negative acinetobacters and pseudomonads. Pulsed-field gel electrophoresis failed to identify any plasmid in Rhodococcus sp. strain 19070. Catechol 1,2- and 2,3-dioxygenase activity indicated that strain 19070 possesses both meta- and ortho-cleavage degradative pathways, which are associated in pseudomonads with the xyl and ben genes, respectively. Open reading frames downstream of bopXYZ, designated bopL and bopK, resembled genes encoding cis-diol dehydrogenases and benzoate transporters, respectively. The bop genes were in the same order as the chromosomal ben genes of P. putida PRS2000. The deduced sequences of BopXY were 50 to 60% identical to the corresponding proteins of benzoate and toluate dioxygenases. The reductase components of these latter dioxygenases, BenC and XylZ, are 201 residues shorter than the deduced BopZ sequence. As predicted from the sequence, expression of BopZ in E. coli yielded an approximately 60-kDa protein whose presence corresponded to increased cytochrome c reductase activity. While the N-terminal region of BopZ was approximately 50% identical in sequence to the entire BenC or XylZ reductases, the C terminus was unlike other known protein sequences.

Alpha subunit genes of initial polyaromatic hydrocarbon (PAH) dioxygenases were used as targets for the PCR detection of PAH-degrading strains of the genera Pseudomonas, Comamonas and Rhodococcus which were obtained from activated sludge or soil samples. Sequence analysis of PCR products from several Pseudomonas strains showed that alpha subunits (nahAc allele) of this genus are highly conserved. PCR primers for the specific detection of alpha subunit genes of initial PAH dioxygenases from Pseudomonas strains were not suitable for detecting the corresponding genes from the genera Comamonas and Rhodococcus. Southern analysis using a heterologous gene probe derived from the P. putida OUS82 PAH dioxygenase alpha subunit identified segments of the PAH-degradation gene cluster from C. testosteroni strain H. Parts of this gene cluster containing three subunits of the initial PAH dioxygenase were isolated. These three subunits [ferredoxin (pahAb), alpha (pahAc) and beta (pahAd) subunit] were amplified by PCR as one fragment and expressed in Escherichia coli DH5alpha, resulting in an active initial dioxygenase with the ability to transform indole and phenanthrene. The DNA sequence alignment of alpha subunits from C. testosteroni H and various PAH-degrading bacteria permitted the design of new primers and oligonucleotide probes which are useful for the detection of the initial PAH dioxygenases from strains of Pseudomonas, Comamonas and Rhodococcus.

Analysis of the bacterial population of soil surface samples from a creosote-contaminated site showed that up to 50% of the culturable micro-organisms detected were able to utilise a mixture of cresols. From fifty different microbial isolates fourteen that could utilise more than one cresol isomer were selected and identified by 16S rRNA analysis. Eight isolates were Rhodococcus strains and six were Pseudomonas strains. In general, the Rhodococcus strains exhibited a broader growth substrate range than the Pseudomonas strains. The distribution of various extradiol dioxygenase (edo) genes, previously associated with aromatic compound degradation in rhodococci, was determined for the Rhodococcus strains by PCR detection and Southern-blot hybridization. One strain, Rhodococcus sp. II exhibited the broadest growth substrate range and possessed five different edo genes. Gene disruption experiments indicated that two genes (edoC and edoD) were associated with isopropylbenzene and naphthalene catabolism respectively. The other Rhodococcus strains also possessed some of the edo genes and one (edoB) was present in all of the Rhodococcus strains analysed. None of the rhodococcal edo genes analysed were present in the Pseudomonas strains isolated from the site. It was concluded that individual strains of Rhodococcus possess a wide degradative ability and may be very important in the degradation of complex mixtures of substrates found in creosote.