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PubMed2005

Functional expression system for cytochrome P450 genes using the reductase domain of self-sufficient P450RhF from Rhodococcus sp. NCIMB 9784.

Cytochrome P450RhF from Rhodococcus sp. NCIMB 9784 is a self-sufficient P450 monooxygenase. We report here a simple system for the functional expression of various P450 genes using the reductase domain of this P450RhF, which comprises flavin mononucleotide- and nicotinamide adenine dinucleotide phosphate binding motifs and a [2Fe2S] ferredoxin-like center. Vector pRED was constructed, which carried the T7 promoter, cloning sites for a P450, a linker sequence, and the P450RhF reductase domain, in this order. The known P450 genes, encoding P450cam from Pseudomonas putida (CYP101A) and P450bzo from an environmental metagenome library (CYP203A), were expressed on vector pRED as soluble fusion enzymes with their natural spectral features in Escherichia coli. These E. coli cells expressing the P450cam and P450bzo genes could convert (+)-camphor and 4-hydroxybenzoate into 5-exo-hydroxycamphor and protocatechuate (3,4-dihydroxybenzoate), respectively (the expected products). Using this system, we also succeeded in directly identifying the function of P450 CYP153A as alkane 1-monooxygenase for the first time, i.e., E. coli cells expressing a P450 CYP153A gene named P450balk, which was isolated form Alcanivorax borkumensis SK2, converted octane into 1-octanol with high efficiency (800 mg/l). The system presented here may be applicable to the functional identification of a wide variety of bacterial cytochromes P450.

1-Octanol↗

PubMed2006

Biofilm development of the polyethylene-degrading bacterium Rhodococcus ruber.

We have recently isolated a biofilm-producing strain (C208) of Rhodococcus ruber that degraded polyethylene at a rate of 0.86% per week (r2=0.98). Strain C208 adheres to polyethylene immediately upon exposure to the polyolefin. This initial biofilm differentiates (in a stepwise process that lasts about 20 h) into cell-aggregation-forming microcolonies. Further organization yields "mushroom-like" three-dimensional structures on the mature biofilm. The ratio between the population densities of the biofilm and the planktonic C208 cells after 10 days of incubation was about 60:1, indicating a high preference for the biofilm mode of growth. Analysis of extracellular polymeric substances (EPS) in the biofilm of C208 revealed that the polysaccharides level was up to 2.5 folds higher than that of the protein. The biofilm showed a high viability even after 60 days of incubation, apparently due to polyethylene biodegradation.

Biodegradation, Environmental↗

PubMed2005

The remarkable Rhodococcus erythropolis.

Rhodococcus erythropolis cells contain a large set of enzymes that allow them to carry out an enormous number of bioconversions and degradations. Oxidations, dehydrogenations, epoxidations, hydrolysis, hydroxylations, dehalogenations and desulfurisations have been reported to be performed by R. erythropolis cells or enzymes. This large array of enzymes fully justifies the prospective application of this bacterium in biotechnology.

Biodegradation, Environmental↗

PubMed2005

Transient accumulation of gamma-butyrolactone during degradation of bis(4-chloro-n-butyl) ether by diethylether-grown Rhodococcus sp. strain DTB.

Rhodococcus sp. strain DTB (DSM 44534) grows aerobically on diethylether as sole source of carbon and energy. Dense cell suspension experiments showed that the induced ether-cleaving enzyme system attacks a broad range of ethers like tetrahydrofuran, phenetole and chlorinated alkylethers including Calpha-substituted alkylethers. Identification of metabolites revealed that degradation of the ethers started by an initial attack of the ether bond. Diethylether-grown cells degraded bis(4-chloro-n-butyl) ether via an initial ether scission followed by the transient accumulation of gamma-butyrolactone as intermediate at nearly stoichiometric concentrations.

4-Butyrolactone↗

PubMed2006

Cloning, expression, and characterization of a self-sufficient cytochrome P450 monooxygenase from Rhodococcus ruber DSM 44319.

A new member of class IV of cytochrome P450 monooxygenases was identified in Rhodococcus ruber strain DSM 44319. As the genome of R. ruber has not been sequenced, a P450-like gene fragment was amplified using degenerated primers. The flanking regions of the P450-like DNA fragment were identified by directional genome walking using polymerase chain reaction. The primary protein structure suggests a natural self-sufficient fusion protein consisting of ferredoxin, flavin-containing reductase, and P450 monooxygenase. The only flavin found within the enzyme was riboflavin 5'-monophosphate. The enzyme was successfully expressed in Escherichia coli, purified and characterized. In the presence of NADPH, the P450 monooxygenase showed hydroxylation activity towards polycyclic aromatic hydrocarbons naphthalene, indene, acenaphthene, toluene, fluorene, m-xylene, and ethyl benzene. The conversion of naphthalene, acenaphthene, and fluorene resulted in respective ring monohydroxylated metabolites. Alkyl aromatics like toluene, m-xylene, and ethyl benzene were hydroxylated exclusively at the side chains. The new enzyme's ability to oxidize such compounds makes it a potential candidate for biodegradation of pollutants and an attractive biocatalyst for synthesis.

Amino Acid Sequence↗

PubMed2006

Chloroform degradation by butane-grown cells of Rhodococcus aetherovorans BCP1.

The ability of a Rhodococcus aetherovorans strain, BCP1, to grow on butane and to degrade chloroform in the 0-633 microM range (0-75.5 mg l(-1)) via aerobic cometabolism was investigated by means of resting-cell assays. BCP1 degraded chloroform with a complete mineralization of the organic Cl. The resulting butane and chloroform maximum specific degradation rates were equal to 118 and 22 micromol mg(protein)(-1)day(-1), respectively. Butane inhibition on chloroform degradation was satisfactorily interpreted by means of a model of competitive inhibition, with an inhibition constant equal to 38 % of the estimated butane half-saturation constant, whereas chloroform (at 11 microM) did not inhibit butane utilization. Acetylene (1,720 microM) induced an almost complete inactivation of the degradation of both butane and chloroform, indicating that the studied cometabolic process is mediated by a monooxygenase enzyme. BCP1 proved capable of degrading vinyl chloride and 1,1,2-trichloroethane, but not 1,2-trans-dichloroethylene. BCP1 could grow on the intermediates of the most common butane metabolic pathways and on the aliphatic hydrocarbons from ethane to n-heptane. After growth on n-hexane, it was able to deplete chloroform (13 microM) with a degradation rate higher than that obtained, at the same chloroform concentration, after growth on butane.

Binding, Competitive↗

PubMed2006

Purification, characterization and cloning of aldehyde dehydrogenase from Rhodococcus erythropolis UPV-1.

The enzyme responsible for formaldehyde removal in industrial wastewaters by cells of Rhodococcus erythropolis UPV-1 was identified as a broad-specific aldehyde dehydrogenase (EC 1.2.1.3). The enzyme was purified to electrophoretic homogeneity from ethanol-grown cells with a specific activity of 19.5 U mg-1 protein and an activity recovery of 56%. The enzyme showed an isoelectric point (pI) of 5.3 and was a trimer of 162 kDa consisting of three identical 54-kDa subunits. It was specific for NAD+ and showed hyperbolic kinetics for this coenzyme (Km=90 microM), but sigmoidal kinetics for the aliphatic aldehydes used as substrates. The enzyme affinity for aldehydes increased with their hydrocarbon chain length, ranging from 333 microM for formaldehyde to 85 nM for n-octanal. The corresponding calculated Hill coefficients were in the 1.55-2.77 range. With n-propanal as substrate, the optimum pH and temperature for activity were 9.5-10.0 and 47.5 degrees C, respectively, with an Ea for catalysis of 28.6 kJ mol-1. NAD+ protected the enzyme against thermal inactivation, but aldehydes were ineffective. The activity was severely inhibited by p-hydroxymercuribenzoate, indicating that a thiol was essential for catalysis. The 1,524-bp aldhR gene encoding a 507-amino-acid protein was expressed in cells of Escherichia coli M15 as a hexahistidine-tagged protein.

Aldehyde Dehydrogenase↗

PubMed2006

Cloning, sequencing, and expression of a novel epoxide hydrolase gene from Rhodococcus opacus in Escherichia coli and characterization of enzyme.

An epoxide hydrolase gene of about 0.8 kb was cloned from Rhodococcus opacus ML-0004, and the open reading frame (ORF) sequence predicted a protein of 253 amino acids with a molecular mass of about 28 kDa. An expression plasmid carrying the gene under the control of the tac promotor was introduced into Escherichia coli, and the epoxide hydrolase gene was successfully expressed in the recombinant strains. Some characteristics of purified recombinant epoxide hydrolase were also studied. Epoxide hydrolase showed a high stereospecificity for L: (+)-tartaric acid, but not for D: (+)-tartaric acid. The epoxide hydrolase activity could be assayed at the pH ranging from 3.5 to 10.0, and its maximum activity was obtained between pH 7.0 and 7.5. The enzyme was sensitive to heat, decreasing slowly between 30 degrees C and 40 degrees C, and significantly at 45 degrees C. The enzyme activity was activated by Ca(2+) and Fe(2+), while strongly inhibited by Ag(+) and Hg(+), and slightly inhibited by Cu(2+), Zn(2+), Ba(2+), Ni(+), EDTA-Na(2) and fumarate.

Amino Acid Sequence↗

PubMed2007

Bioconversion of butyronitrile to butyramide using whole cells of Rhodococcus rhodochrous PA-34.

Butyramide is an important chemical commodity, which is used for the synthesis of hydroxamic acids and electrorheological fluids and for the preparation of beta-amodoorganotin compounds. The nitrile hydratase (Nhase) of Rhodococcus rhodochrous PA-34 catalyzed the conversion of butyronitrile to butyramide. The maximum Nhase activity [18 U/mg dry cell weight (dcw)] of whole cells of R. rhodochrous PA-34 was observed at pH 7.0 with 10% (v/v) butyronitrile and 1 mg cells (dcw)/ml reaction mixture at 10 degrees C. The cells of R. rhodochrous PA-34 retained almost 50% activity when incubated for 1 h in the presence of 85% (v/v) butyronitrile. A yield of 597 g of butyramide (6.8 M) was obtained using 60% (v/v) butyronitrile, 1 g cells (dry weight) in a 1-l batch reaction at 10 degrees C for 6 h.

Amides↗

PubMed2006

Utilization of hydrophobic bacterium Rhodococcus opacus B-4 as whole-cell catalyst in anhydrous organic solvents.

Rhodococcus opacus strain B-4, which has recently been isolated as an organic solvent-tolerant bacterium, has a high hydrophobicity and exhibits a high affinity for hydrocarbons. This bacterium was able to survive for at least 5 days in organic solvents, including n-tetradecane, oleyl alcohol, and bis(2-ethylhexyl) phthalate (BEHP), which contained water less than 1% (w/v). The biocatalytic ability of R. opacus B-4 was demonstrated in the essentially nonaqueous BEHP using indigo production from indole as a model conversion. By the catabolism of oleic acid for NADH regeneration, indigo production increased up to 71.6 microg ml(-1) by 24 h.

Alkanes↗

PubMed2001

Long-term repeated biodesulfurization by immobilized Rhodococcus erythropolis KA2-5-1 cells.

In this study, biodesulfurization (BDS) was carried out using immobilized Rhodococcus erythropolis KA2-5-1 in n-tetradecane containing dibenzothiophene (DBT) as a model oil (n-tetradecane/immobilized cell biphasic system). The cells were immobilized by entrapping them with calcium alginate, agar, photo-crosslinkable resin prepolymers (ENT-4000 and ENTP-4000), and urethane prepolymers (PU-3 and PU-6); and it was found that ENT-4000-immobilized cells had the highest DBT desulfurization activity in the model oil system without leakage of cells from the support. Furthermore, ENT4000-immobilized cells could catalyze BDS repeatedly in this system for more than 900 h with reactivation; and recovery of both the biocatalyst and the desulfurized model oil was easy. This study would give a solution to the problems in BDS, such as the troublesome process of recovering desulfurized oil and the short life of BDS biocatalysts.

Agar↗

PubMed2001

High cell density cultivation of Rhodococcus opacus for lipid production at a pilot-plant scale.

The triacylglycerol (TAG)-accumulating bacterium Rhodococcus opacus strain PD630 was investigated with respect to the fermentative production of TAGs consisting of an unusually high fraction of fatty acids with an odd-number of carbon atoms and unsaturated monoenic fatty acids from sugar beet molasses and sucrose. Fed-batch fermentations were optimized at the 30-1 scale in a stirred tank bioreactor at 30 degrees C using a mineral salts medium, which contained sugar beet molasses and sucrose as sole carbon sources. Approximately 37.5 g cell dry matter (CDM) per liter was the highest cell density that was obtained at that scale with a TAG content in the cells of 52%. This fermentative process was also applied to a 500-1 pilot-plant scale. Cell densities as high as 18.4 g CDM per liter were obtained, and 42% of the sucrose present in the medium was converted into cell mass which consisted of 38.4% TAGs.

Biotechnology↗

PubMed1996

Simultaneous utilization of pyridine and fructose by Rhodococcus opacus UFZ B 408 without an external nitrogen source.

A bacterium classified as Rhodococcus opacus, which is able to use pyridine (a potentially growth-inhibiting substrate) as its sole source of carbon, energy and nitrogen, was isolated. In a carbon-limited chemostat culture, the kinetics was determined for growth on both pyridine and a mixture of pyridine and fructose (9 mM/22.15 mM). With growth on pyridine, stable steady states were achieved up to dilution rates of about 0.1 h-1. A further increase in the dilution rate resulted in the progressive accumulation of pyridine in the culture liquid and the cells were washed out. The maximum specific growth rate (mu max = 0.23 h-1) and the Ks value (0.22 mM) for growth on pyridine were determined from the residual pyridine concentrations measured within the range of stable steady states. With growth on the substrate mixture, the specific pyridine consumption rates and the residual pyridine concentrations were lower at similar dilution rates than with growth on pyridine alone, and stable steady states were established at dilution rates of up to 0.13 h-1. The maximum pyridine degradation rate was enhanced to 270 mg pyridine l-1 h-1 compared to 210 mg pyridine l-1 h-1 with growth on pyridine as a single substrate. An external nitrogen source did not need to be added in the case of growth on the substrate mixture. Fructose was assimilated by means of ammonium released from pyridine. Analysis of the nitrogen balance furnished proof that pyridine is an energy-deficient substrate; pyridine was assimilated and dissimilated at a ratio of 1 mol/0.67 mol respectively. The resulting yield coefficient was about 0.55 g dry weight/g pyridine. Moreover, it was demonstrated that, in regard to the biologically usable energy, 1 mol pyridine corresponds to 0.43 mol fructose.

Biodegradation, Environmental↗

PubMed1997

Cell-linked and extracellular cholesterol oxidase activities from Rhodococcus erythropolis. Isolation and physiological characterization.

Rhodococcus erythropolis cells growing in a cholesterol-free glycerol-containing mineral medium displayed very low levels of a cell-wall-bound cholesterol oxidase activity. Addition of cholesterol induced a marked increase in the synthesis of this enzyme, which reached a maximum within 6 days and was subsequently followed by the appearance of extracellular cholesterol oxidase in the culture broth. Significant levels of induction were only achieved when cholesterol emulsified with Tween 80. The presence of chloramphenicol at the time of induction completely prevented the emergence of both enzymatic forms, suggesting the requirement of de novo protein synthesis. Upon transfer of cholesterol-growing cultures to fresh medium lacking cholesterol, the extracellular cholesterol oxidase was quickly erased, while the activity of the particulate enzyme decreased sharply. The electrophoretic pattern on native Western blotting as well as on sodium dodecyl sulphate/polyacrylamide gels, together with kinetic data, strongly support the idea that the particulate and extracellular cholesterol oxidases are two different forms of the same enzyme with an estimated molecular mass of 55 kDa.

Chloramphenicol↗

PubMed1999

Degradation of alicyclic molecules by Rhodococcus ruber CD4.

The present work describes investigations on the bacterial degradation of the alicyclic molecule cyclododecane. It represents a structure where the initial degradative steps have to be similar to a "subterminal" attack as there is no "terminal" part of the molecule. We were able to show that the gram-positive bacterium Rhodococcus ruber CD4 DSM 44394 oxidizes cyclododecane to the corresponding alcohol and ketone, the latter being subject to ring fission by a Baeyer-Villiger oxygenase. This key enzyme is an NADPH- and O2-dependent flavoprotein with a substrate specificity for bigger rings. The further metabolism of the resulting lactone gives rise to an omega-hydroxyalkanoic acid that is susceptible to common beta-oxidation. Due to its alicyclic character and its ring size, cyclododecane is comparable to aliphatic bridge components that are an important element in the coal texture. They contribute to the three-dimensional coal structure and thus could serve as a valuable target for the oxidative abilities of R. ruber CD4 to reduce the molecular mass of coal.

Biodegradation, Environmental↗

PubMed2000

Evidence for diverse oxidations in the catabolism of toluene by Rhodococcus rhodochrous strain OFS.

Rhodococcus rhodochrous strain OFS grew on toluene as a sole source of carbon and energy with a maximum growth rate of 0.011 h(-1). Initial reaction products were extracted, derivatized and identified by GC-MS. Oxygen consumption studies indicated that OFS grown on an aliphatic substrate required an induction period before oxidizing toluene. OFS grown on toluene transformed an array of aromatic ground water pollutants including styrene, ethylbenzene and chlorobenzene. Products of these transformations were identified. The sole product of chlorobenzene biotransformation was 4-chlorophenol. Products from toluene oxidation included 3- and 4-methylcatechol as well as benzyl alcohol, p-cresol and cis-toluene dihydrodiol. The identification of these and the products of other aromatic substrate conversions affirm that oxidation occurred on the functional group as well as directly on the aromatic nucleus.

Biodegradation, Environmental↗

PubMed2001

Biological production of optically active muconolactones by Rhodococcus rhodochrous.

Optically active (-)-3-methylmuconolactone was biologically produced using a mutant strain of Rhodococcus rhodochrous N75 that is capable of metabolizing 4-methylcatechol via a modified ortho-cleavage pathway. The mutant strain (CJ30) was prepared by mutagenesis using N-methyl-N'-nitro-N-nitrosoguanidine and found to be blocked in the degradation of 3-methyl-muconolactone. Cells of the mutant CJ30, which had been previously grown on yeast extract and induced with p-toluate, transformed p-toluate (11.5 mM) to optically active (-)-3-methylmuconolactone with a yield of 53%. The structure of 3-methylmuconolactone was confirmed by NMR spectroscopy and mass spectrometry. Cell-free extracts of R. rhodochrous N75 also transformed a range of 4-alkylcatechols, such as 4-ethylcatechol, 4-iso-propylcatechol, and 4-tert-butylcatechol, to the corresponding 4-alkyl-substituted muconolactones.

Culture Media↗

PubMed2006

Isolation and characterization of a carbendazim-degrading Rhodococcus sp. djl-6.

Bacterium djl-6, capable of degrading carbendazim, was isolated by continuous enrichment culture originating from carbendazim-treated soil. The isolate was identified as Rhodococcus sp. according to its phenotypic features, physiologic and biochemical characteristics, and phylogenetic analysis. The strain could use carbendazim as sole carbon or nitrogen source. It showed a high average degradation rate of 55.56 mg . L(-1) . d(-1) in M9 medium amended with carbendazim. High-pressure liquid chromatography-mass spectrometry (HPLC-MS) analysis showed the presence of 2-aminobenzimidazole, benzimidazole, and an unknown metabolite with molecular ions (M(+)) of m/z 104.8 and 118.5. The degradation in the isolate djl-6 seems to be initiated with the cleavage of the methyl carbemate side chain, resulting in the formation of 2-aminobenzimidazole and benzimidazole. This is the first report of the intermediates benzimidazole and 2-aminobenzimidazole found together in the culture filtrate of pure bacterium.

Benzimidazoles↗