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Rhodococcus erythropolis PR4 is able to degrade diesel oil, normal-, iso- and cycloparaffins and aromatic compounds. The complete DNA content of the strain was previously sequenced and numerous oxygenase genes were identified. In order to identify the key elements participating in biodegradation of various hydrocarbons, we performed a comparative whole transcriptome analysis of cells grown on hexadecane, diesel oil and acetate. The transcriptomic data for the most prominent genes were validated by RT-qPCR. The expression of two genes coding for alkane-1-monooxygenase enzymes was highly upregulated in the presence of hydrocarbon substrates. The transcription of eight phylogenetically diverse cytochrome P450 (cyp) genes was upregulated in the presence of diesel oil. The transcript levels of various oxygenase genes were determined in cells grown in an artificial mixture, containing hexadecane, cycloparaffin and aromatic compounds and six cyp genes were induced by this hydrocarbon mixture. Five of them were not upregulated by linear and branched hydrocarbons. The expression of fatty acid synthase I genes was downregulated by hydrocarbon substrates, indicating the utilization of external alkanes for fatty acid synthesis. Moreover, the transcription of genes involved in siderophore synthesis, iron transport and exopolysaccharide biosynthesis was also upregulated, indicating their important role in hydrocarbon metabolism. Based on the results, complex metabolic response profiles were established for cells grown on various hydrocarbons. Our results represent a functional annotation of a rhodococcal genome, provide deeper insight into molecular events in diesel/hydrocarbon utilization and suggest novel target genes for environmental monitoring projects.

Petroleum hydrocarbons are a stubborn pollutant that is difficult to degrade globally, and plant-microbial degradation is the main way to solve this type of pollutant. In this study, the physiological and ecological responses of alfalfa to petroleum hydrocarbons in different concentrations of petroleum hydrocarbon-contaminated soil with KB1 ( Rhodococcus erythropolis ) were analyzed and determined by laboratory potting techniques. The growth of alfalfa (CK) and alfalfa with KB1 (JZ) in different concentrations of petroleum hydrocarbons contaminated soil was compared and analyzed. The results of the CK group showed that petroleum hydrocarbons could significantly affect the activity of alfalfa antioxidant enzyme system, inhibit the development of alfalfa roots and the normal growth of plants, especially in the high-concentration group. KB1 strain had the ability to produce IAA, form biofilm, fix nitrogen, produce betaine and ACC deaminase, and the addition of KB1 could improve the growth traits of alfalfa in the soil contaminated with different concentrations of petroleum hydrocarbons, the content of soluble sugars in roots, and the stress resistance and antioxidant enzyme activities of alfalfa. In addition, the degradation kinetics of the strain showed that the degradation rate of petroleum could reach 75.2% after soaking with KB1. Furthermore, KB1 can efficiently degrade petroleum hydrocarbons in advance and significantly alleviate the damage of high concentration of petroleum hydrocarbons to plant roots. The results showed that KB1 strains and alfalfa plants could effectively enhance the degradation of petroleum hydrocarbons, which provided new ideas for improving bioremediation strategies.

Rhodococcus erythropolis WZ010 was capable of producing optically pure (2S,3S)-2,3-butanediol in alcoholic fermentation. The gene encoding an acetoin(diacetyl) reductase from R. erythropolis WZ010 (ReADR) was cloned, overexpressed in Escherichia coli, and subsequently purified by Ni-affinity chromatography. ReADR in the native form appeared to be a homodimer with a calculated subunit size of 26,864, belonging to the family of the short-chain dehydrogenase/reductases. The enzyme accepted a broad range of substrates including aliphatic and aryl alcohols, aldehydes, and ketones. It exhibited remarkable tolerance to dimethyl sulfoxide (DMSO) and retained 53.6 % of the initial activity after 4 h incubation with 30 % (v/v) DMSO. The enzyme displayed absolute stereospecificity in the reduction of diacetyl to (2S,3S)-2,3-butanediol via (S)-acetoin. The optimal pH and temperature for diacetyl reduction were pH 7.0 and 30 °C, whereas those for (2S,3S)-2,3-butanediol oxidation were pH 9.5 and 25 °C. Under the optimized conditions, the activity of diacetyl reduction was 11.9-fold higher than that of (2S,3S)-2,3-butanediol oxidation. Kinetic parameters of the enzyme showed lower K ₘ values and higher catalytic efficiency for diacetyl and NADH in comparison to those for (2S,3S)-2,3-butanediol and NAD⁺, suggesting its physiological role in favor of (2S,3S)-2,3-butanediol formation. Interestingly, the enzyme showed higher catalytic efficiency for (S)-1-phenylethanol oxidation than that for acetophenone reduction. ReADR-catalyzed asymmetric reduction of diacetyl was coupled with stereoselective oxidation of 1-phenylethanol, which simultaneously formed both (2S,3S)-2,3-butanediol and (R)-1-phenylethanol in great conversions and enantiomeric excess values.

A major concern among the environmental agencies includes the emission of sulfurous gas into the environment. Consequently, the oil agencies are in constant search of alternative processes aiming the reduction of sulfur content in fuels. One of the technologies commonly used is the hydrodesulfurization (HDS), but this is a high-cost process that also requires high temperature and pressure. A complementary alternative to HDS is biodesulfurization (BDS) involving the use of specific microorganisms to the removal of sulfur present in the carbon chain, using the oxidation pathway “4S”, in which there is cleavage of carbon–sulfur bond, and maintaining the calorific value of the organic molecule. The BDS is a low-cost technique when compared with HDS. For this process to occur, activation of specific enzymes is needed, which is controlled by dsz ABC genes. Therefore, strategies to optimize this process have been of great importance to the oil refineries. For decades, attempts to try to implement BDS in the industry have been made, but difficulties in obtaining satisfactory results led the researchers to seek new knowledge about this bioprocess. The need of more studies concerning implementation on an industrial scale of this process is evident, since this biotechnology is a promising alternative to refineries in the near future.

It was investigated the potential of Rhodococcus erythropolis AW3 as a biosorbent for the removal of crystal violet (CV) dye from natural water and real effluents. The biosorbent was characterized by flow cytometry, Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), energy X-ray dispersive spectroscopy (EDS), and point of zero charge (pHZPC). Batch biosorption experiments were performed to optimize different parameters involved in the biosorption process. The equilibrium was reached at 90 min at the optimum biosorbent dose of 0.50 g L−1 and pH of 9.0. Results indicated that Langmuir isotherm model was the most suitable to represent the experimental data, and the highest biosorption capacity was 289.8 mg g−1. Kinetic data were well fitted with the pseudo-second-order model. The thermodynamic study showed that the process was favorable, exothermic, and associated with an increase of entropy. Finally, it was demonstrated that the biosorption process using Rhodococcus erythropolis AW3 could be successfully applied to remove CV from natural water and effluents derived from clinical and industrial activities.

Rhodococcus sp. has a broad catabolic diversity and unique enzymatic capabilities, and it is able to adapt under extreme conditions. Thereby, the production of this remarkable bacterium has a great biotechnological and industrial importance. In this sense, we sought to improve the R. erythropolis ATCC 4277 growth through a central composite design, by varying the components of nutrient medium (glucose, malt extract, yeast extract, CaCO 3 ), temperature, and agitation. It was found that the concentrations of glucose and malt extract are not statistically significant, being reduced of 4.0 and 10.0 g L -1 to 2.0 and 5.0 g L −1 , respectively. The CaCO 3 concentration and temperature were also diminished of 2.0 to 1.16 g L −1 and 28 to 23.7 °C, respectively. Optimal growth conditions provided a 240% increase in final biomass concentration, an increment in specific growth rate, and a growth yield coefficient about five times greater. Application of the optimal conditions in biodesulfurization and biodenitrogenation processes showed that desulfurization capability is not associated with optimal growth conditions; however, it was achieved a 47% of nitrogen removal in the assay containing 10% ( w / w ) of heavy gas oil. Graphical Abstract ᅟ

Rhodococcus erythropolis N9T-4, isolated from stored crude oil, shows extremely oligotrophic features and can grow on a basal medium without any additional carbon, nitrogen, sulfur, and energy sources, but requires CO 2 for its oligotrophic growth. Transmission electron microscopic observation showed that a relatively large and spherical compartment was observed in a N9T-4 cell grown under oligotrophic conditions. In most cases, only one compartment was observed per cell, but in some cases, it was localized at each pole of the cell, suggesting that it divides at cell division. We termed this unique bacterial compartment an oligobody. The oligobody was not observed or very rarely observed in small sizes under nutrient rich conditions, whereas additional carbon sources did not affect oligobody formation. Energy dispersive X-ray spectroscopy analysis revealed remarkable peaks corresponding to phosphorus and potassium in the oligobody. The oligobodies in N9T-4 cells could be stained by Toluidine blue, suggesting that the oligobody is composed of inorganic polyphosphate and is a type of acidocalcisome. Two genes-encoding polyphosphate kinases, ppk1 and ppk2 , were found in the N9T-4 genome: ppk1 disruption caused a negative effect on the formation of the oligobody. Although it was suggested that the oligobody plays an important role for the oligotrophic growth, both ppk -deleted mutants showed the same level of oligotrophic growth as the wild-type strain.

Biodesulfurization (BDS) is considered a complementary technology to the traditional hydrodesulfurization treatment for the removal of recalcitrant sulfur compounds from petroleum products. BDS was investigated in a bubble column bioreactor using two-phase media. The effects of various process parameters, such as biocatalyst age and concentration, organic fraction percentage (OFP), and type of sulfur compound—namely, dibenzothiophene (DBT), 4-methyldibenzothiophene (4-MDBT), 4,6-dimethyldibenzothiophene (4,6-DMDBT), and 4,6-diethyldibenzothiophene (4,6-DEDBT)—were evaluated, using resting cells of Rhodococcus erythropolis IGTS8. Cells derived from the beginning of the exponential growth phase of the bacterium exhibited the highest biodesulfurization efficiency and rate. The biocatalyst performed better in an OFP of 50% v/v. The extent of DBT desulfurization was dependent on cell concentration, with the desulfurization rate reaching its maximum at intermediate cell concentrations. A new semi-empirical model for the biphasic BDS was developed, based on the overall Michaelis-Menten kinetics and taking into consideration the deactivation of the biocatalyst over time, as well as the underlying mass transfer phenomena. The model fitted experimental data on DBT consumption and 2-hydroxibyphenyl (2-HBP) accumulation in the organic phase for various initial DBT concentrations and different organosulfur compounds. For constant OFP and biocatalyst concentration, the most important parameter that affects BDS efficiency seems to be biocatalyst deactivation, while the phenomenon is controlled by the affinities of biodesulfurizing enzymes for the different organosulfur compounds. Thus, desulfurization efficiency decreased with increasing initial DBT concentration, and in inverse proportion to increases in the carbon number of alkyl substituent groups.

Rhodococcus erythropolis N9T-4 shows extremely oligotrophic growth requiring atmospheric CO₂ and forms its colonies on an inorganic basal medium (BM) without any additional carbon source. Screening of a random mutation library constructed by a unique genome deletion method that we established indicated that the aceA, aceB, and pckG genes encoding isocitrate lyase, malate synthase, and phosphoenolpyruvate carboxykinase, respectively, were requisite for survival on BM plates. The aceA- and aceB deletion mutants and the pckG deletion mutant grew well on BM plates containing L-malate and D-glucose, respectively, suggesting that the glyoxylate (GO) shunt and gluconeogenesis are essential for the oligotrophic growth of N9T-4. Interestingly, most of the enzyme activities in the TCA cycle were observed in the cell-free extract of N9T-4, with perhaps the most important exception being α-ketoglutarate dehydrogenase (KGDH) activity. Instead of the KGDH activity, we detected a remarkable level of α-ketoglutarate decarboxylase (KGD) activity, which is the activity exhibited by the E1 component of the KGDH complex in Mycobacterium tuberculosis. The recombinant KGD of N9T-4 catalyzed the decarboxylation of α-ketoglutarate to form succinic semialdehyde (SSA) in a time-dependent manner. Since N9T-4 also showed a detectable SSA dehydrogenase activity, we concluded that N9T-4 possesses a variant TCA cycle, which uses SSA rather than succinyl-CoA. These results suggest that oligotrophic N9T-4 cells utilize the GO shunt to avoid the loss of carbons as CO₂ and to conserve CoA units in the TCA cycle.

The gene encoding a (2R,3R)-2,3-butanediol dehydrogenase from Rhodococcus erythropolis WZ010 (ReBDH) was over-expressed in Escherichia coli and the resulting recombinant ReBDH was successfully purified by Ni-affinity chromatography. The purified ReBDH in the native form was found to exist as a monomer with a calculated subunit size of 37180, belonging to the family of the zinc-containing alcohol dehydrogenases. The enzyme was NAD(H)-specific and its optimal activity for acetoin reduction was observed at pH 6.5 and 55 °C. The optimal pH and temperature for 2,3-butanediol oxidation were pH 10 and 45 °C, respectively. The enzyme activity was inhibited by ethylenediaminetetraacetic acid (EDTA) or metal ions Al3+, Zn2+, Fe2+, Cu2+ and Ag+, while the addition of 10% (v/v) dimethyl sulfoxide (DMSO) in the reaction mixture increased the activity by 161.2%. Kinetic parameters of the enzyme showed lower Km values and higher catalytic efficiency for diacetyl and NADH in comparison to those for (2R,3R)-2,3-butanediol and NAD+. The activity of acetoin reduction was 7.7 times higher than that of (2R,3R)-2,3-butanediol oxidation when ReBDH was assayed at pH 7.0, suggesting that ReBDH-catalyzed reaction in vivo might favor (2R,3R)-2,3-butanediol formation rather than (2R,3R)-2,3-butanediol oxidation. The enzyme displayed absolute stereospecificity in the reduction of diacetyl to (2R,3R)-2,3-butanediol via (R)-acetoin, demonstrating its potential application on the synthesis of (R)-chiral alcohols.