Biphenyl Dioxygenase Research Articles

Black Carbon Impacts on Paraburkholderia xenovorans Strain LB400 Cell Enrichment and Activity: Implications toward Lower-Chlorinated Polychlorinated Biphenyls Biodegradation Potential.

Volatilization of lower-chlorinated polychlorinated biphenyls (LC-PCBs) from sediment poses health threats to nearby communities and ecosystems. Biodegradation combined with black carbon (BC) materials is an emerging bioaugmentation approach to remove PCBs from sediment, but development of aerobic biofilms on BC for long-term, sustained LC-PCBs remediation is poorly understood. This work aimed to characterize the cell enrichment and activity of biphenyl- and benzoate-grown Paraburkholderia xenovorans strain LB400 on various BCs. Biphenyl dioxygenase gene (bphA) abundance on four BC types demonstrated corn kernel biochar hosted at least 4 orders of magnitude more attached cells per gram than other feedstocks, and microscopic imaging revealed the attached live cell fraction was >1.5× more on corn kernel biochar than GAC. BC characteristics (i.e., sorption potential, pore size, pH) appear to contribute to cell attachment differences. Reverse transcription qPCR indicated that BC feedstocks significantly influenced bphA expression in attached cells. The bphA transcript-per-gene ratio of attached cells was >10-fold more than suspended cells, confirmed by transcriptomics. RNA-seq also demonstrated significant upregulation of biphenyl and benzoate degradation pathways on attached cells, as well as revealing biofilm formation potential/cell-cell communication pathways. These novel findings demonstrate aerobic PCB-degrading cell abundance and activity could be tuned by adjusting BC feedstocks/attributes to improve LC-PCBs biodegradation potential.

New PCBs-degrading and hexavalent chromium-resistant bacteria isolated from Lake Mariout, Egypt

Polychlorinated biphenyls (PCBs) are persistent organic pollutants. They were widely used and distributed in the environment causing endocrine disruption, carcinogenicity, and immunotoxicity. The toxic heavy metal, Hexavalent chromium Cr(VI), co-exists with PCBs leading to ecological deterioration and health problems. Thus, bioremediation of the coexisting PCBs and Cr(VI) is essential for the environment and public health. In this study, three bacterial strains identified as Bacillus safensis, Bacillus cereus, and Bacillus vietnamenesis, were isolated. As revealed, various strains belonging to the genus Bacillus degraded PCBs. However, this study can be considered the first report on isolated strains as PCB-degraders that resist Cr(VI) as co-contaminants. All strains had the biphenyl dioxygenase (bphA) gene and grew on PCBs as the sole carbon and energy source and completely remediated 5 mg/l Cr(VI) individually within 24 h. The three isolates showed different response mechanisms to Cr(VI) that enabled Bacillus safensis and Bacillus cereus to grow on PCBs and Cr(VI) as co-contaminants. Bacillus vietnamensis recorded the highest growth on PCBs, but could not grow on PCBs with Cr(VI). Therefore, Bacillus safensis and Bacillus cereus are concluded to be good candidates for bioremediation of PCBs and Cr(VI) as co-contaminants, while Bacillus vietnamensis is more suitable for bioremediation of either PCBs or Cr(VI) individually.

Aerobic Bioaugmentation to Decrease Polychlorinated Biphenyl (PCB) Emissions from Contaminated Sediments to Air.

We conducted experiments to determine whether bioaugmentation with aerobic, polychlorinated biphenyl (PCB)-degrading microorganisms can mitigate polychlorinated biphenyl (PCB) emissions from contaminated sediment to air. Paraburkholderia xenovorans strain LB400 was added to bioreactors containing PCB-contaminated site sediment. PCB mass in both the headspace and aqueous bioreactor compartments was measured using passive samplers over 35 days. Time-series measurements of all 209 PCB congeners revealed a 57% decrease in total PCB mass accumulated in the vapor phase of bioaugmented treatments relative to non-bioaugmented controls, on average. A comparative congener-specific analysis revealed preferential biodegradation of lower-chlorinated PCBs (LC-PCBs) by LB400. Release of the most abundant congener (PCB 4 [2,2′-dichlorobiphenyl]) decreased by over 90%. Simulations with a PCB reactive transport model closely aligned with experimental observations. We also evaluated the effect of the phytogenic biosurfactant, saponin, on PCB bioavailability and biodegradation by LB400. Time-series qPCR measurements of biphenyl dioxygenase (bphA) genes showed that saponin better maintained bphA abundance, compared to the saponin-free treatment. These findings indicate that an active population of bioaugmented, aerobic PCB-degrading microorganisms can effectively lower PCB emissions and may therefore contribute to minimizing PCB inhalation exposure in communities surrounding PCB-contaminated sites.

Identification of New Dioxygenases Able to Recognize Polycyclic Aromatic Hydrocarbons with High Aromaticity

Polycyclic aromatic hydrocarbons (PAHs), products from the incomplete combustion of crude oil, are pollutants present in nature. Ring hydroxylating dioxygenase enzymes are able to catalyze polycyclic aromatic hydrocarbons in the biodegradation process with a high degree of stereo-, regio-, and enantiospecificity. In this work, we present the first approximation of the binding modes of 9 PAHs with high aromaticity in the catalytic sites of biphenyl or naphthalene dioxygenases from four microorganisms usually used in bio-remediation processes: Sphingobium yanoikuyae, Rhodococcus jostii RHA1, Pseudomonas sp. C18, and Paraburkholderia xenovorans. Molecular modeling studies of two biphenyl dioxygenases from Sphingobium yanoikuyae and Paraburkholderia xenovorans showed good binding affinity for PAHs with 2–4 benzene rings (fluoranthene, pyrene, and chrysene), and both enzymes had a similar amount of substrate binding. Molecular docking studies using naphthalene dioxygenase from Pseudomonas sp. C18 showed that the enzyme is able to accommodate PAHs with high aromaticity (benzo(a)pyrene, indeno(1,2,3-cd)pyrene), with good docking scores. This study provides important insight into the utility of naphthalene dioxygenases in the degradation of HAPs with high aromaticity.

Augmentation of an Engineered Bacterial Strain Potentially Improves the Cleanup of PCB Water Pollution.

ABSTRACTPolychlorinated biphenyls (PCBs) are recalcitrant organohalide pollutants, consisting of 209 congeners. PCB cleanup in natural landscapes is expected to be achieved by the metabolic activity of microorganisms, but aerobic PCB-degrading bacteria that inhabit sites polluted by PCBs cannot degrade all PCB congeners due to the specificity of their enzymes. In this study, we investigated the degradability of PCBs when a genetically modified PCB-degrading bacterium was compounded with wild-type PCB-degrading bacteria. We used two bacterial strains, Comamonas testosteroni YAZ2 isolated from a PCB-uncontaminated natural landscape and Escherichia coli BL21(DE3) transformed with a biphenyl dioxygenase (BphA) gene from a well-known PCB degrader, Burkholderia xenovorans LB400. The enzymatic specificities of BphA were 2,3-dioxygenation in the YAZ2 and 2,3- and 3,4-dioxygenations in the recombinant E. coli. For the PCB-degrading experiment, a dedicated bioreactor capable of generating oxygen microbubbles was prototyped and used. The combined cells of the recombinant and the wild-type strains with an appropriate composite ratio degraded 40 mg/L of Kaneclor KC-300 to 0.3 ± 0.1 mg/L within 24 h. All of the health-toxic coplanar PCB congeners in KC-300 were degraded. This study suggested that the augmentation of an engineered bacterial strain could improve the cleanup of PCB water pollution. It also revealed the importance of the ratio of the strains with different PCB-degrading profiles to efficient degradation and that the application of oxygen microbubbles could rapidly accelerate the cleanup.IMPORTANCE PCB cleanup technique in a natural environment relies on the use of enzymes from microorganisms, primarily biphenyl dioxygenase and dehalogenase. Herein, we focused on biphenyl dioxygenase and created a recombinant PCB-degrading E. coli strain. Despite the development of environments for the field use of transgenic microbial strains around the world, verification of the applicability of transgenic microbial strains for PCB cleanup in the field has not yet been reported. We tentatively verified the extent to which degradability could be obtained by an augmentation model of a transgenic strain, the enzyme expression of which is easily regulated in rivers and lakes with PCB pollution. Our experiments used a dedicated bioreactor to model the natural landscape and produced results superior to those of bioremediation or biostimulation methods. The application of micro-nano bubbles, which has recently been discussed, to the cleanup of environmental pollution was also found to be useful in this study.

Modeling of the Biphenyl Dioxygenase α-Subunit Structure of Rhodococcus Strains and Features of the Destruction of Chlorinated and Hydroxylated Biphenyls at Different Temperatures

Modeling of the Biphenyl Dioxygenase α-Subunit Structure of Rhodococcus Strains and Features of the Destruction of Chlorinated and Hydroxylated Biphenyls at Different Temperatures

Predominant Biphenyl Dioxygenase From Legacy Polychlorinated Biphenyl (PCB)-Contaminated Soil Is a Part of Unusual Gene Cluster and Transforms Flavone and Flavanone.

In this study, the diversity of bphA genes was assessed in a 13C-enriched metagenome upon stable isotope probing (SIP) of microbial populations in legacy PCB-contaminated soil with 13C-biphenyl (BP). In total, 13 bphA sequence variants (SVs) were identified in the final amplicon dataset. Of these, one SV comprised 59% of all sequences, and when it was translated into a protein sequence, it exhibited 87, 77.4, and 76.7% identity to its homologs from Pseudomonas furukawaii KF707, Cupriavidus sp. WS, and Pseudomonas alcaliphila B-367, respectively. This same BphA sequence also contained unusual amino acid residues, Alanine, Valine, and Serine in region III, which had been reported to be crucial for the substrate specificity of the corresponding biphenyl dioxygenase (BPDO), and was accordingly designated BphA_AVS. The DNA locus of 18 kbp containing the BphA_AVS-coding sequence retrieved from the metagenome was comprised of 16 ORFs and was most likely borne by Paraburkholderia sp. The BPDO corresponding to bphAE_AVS was cloned and heterologously expressed in E. coli, and its substrate specificity toward PCBs and a spectrum of flavonoids was assessed. Although depleting a rather narrow spectrum of PCB congeners, the efficient transformation of flavone and flavanone was demonstrated through dihydroxylation of the B-ring of the molecules. The homology-based functional assignment of the putative proteins encoded by the rest of ORFs in the AVS region suggests their potential involvement in the transformation of aromatic compounds, such as flavonoids. In conclusion, this study contributes to the body of information on the involvement of soil-borne BPDOs in the metabolism of flavonoid compounds, and our paper provides a more advanced context for understanding the interactions between plants, microbes and anthropogenic compounds in the soil.

Novel clades of soil biphenyl degraders revealed by integrating isotope probing, multi-omics, and single-cell analyses

Most microorganisms in the biosphere remain uncultured and poorly characterized. Although the surge in genome sequences has enabled insights into the genetic and metabolic properties of uncultured microorganisms, their physiology and ecological roles cannot be determined without direct probing of their activities in natural habitats. Here we employed an experimental framework coupling genome reconstruction and activity assays to characterize the largely uncultured microorganisms responsible for aerobic biodegradation of biphenyl as a proxy for a large class of environmental pollutants, polychlorinated biphenyls. We used 13C-labeled biphenyl in contaminated soils and traced the flow of pollutant-derived carbon into active cells using single-cell analyses and protein–stable isotope probing. The detection of 13C-enriched proteins linked biphenyl biodegradation to the uncultured Alphaproteobacteria clade UBA11222, which we found to host a distinctive biphenyl dioxygenase gene widely retrieved from contaminated environments. The same approach indicated the capacity of Azoarcus species to oxidize biphenyl and suggested similar metabolic abilities for species of Rugosibacter. Biphenyl oxidation would thus represent formerly unrecognized ecological functions of both genera. The quantitative role of these microorganisms in pollutant degradation was resolved using single-cell-based uptake measurements. Our strategy advances our understanding of microbially mediated biodegradation processes and has general application potential for elucidating the ecological roles of uncultured microorganisms in their natural habitats.

Bioconversion of Guaiacylglycerol-β-guaiacyl ether into vanillin by Burkholderia sp. ISTR5: A genomic and proteomic approach

Long-term research on biofuel production from lignocellulose did not progress much due to strong inter-unit linkages in lignin structure. Lignin contains β-O-4 linkage as a major bond (50–60%) between aromatic compounds to form a complex 3D-structure. The present study aims for the characterization of a novel strain Burkholderia sp. ISTR5 (R5) for β-O-4 linkage disintegration. Guaiacylglycerol-β-guaiacyl ether (GGE) was taken as a representative compound for β-O-4 linkage analysis. We showed that the strain R5 completely degraded GGE in 72 h. Further, gas chromatography-mass spectrometry (GC-MS) profile showed that vanillin is one of the highest produced chemicals during the degradation of GGE with small fractions of other metabolites in the reaction mixture. Nuclear Magnetic Resonance (NMR) analysis demonstrated the fragmentation of β-O-4 bonds and the advent of new chemical bonds. In addition to it, the whole genome analysis showed that R5 contains a superior enzyme chassis for the degradation of aromatic compounds. Enzymes such as glutathione-s-transferase, biphenyl dioxygenase, catalase/peroxidase, hydrolases, and dehydrogenases dominated the genome of R5. Proteomics analysis showed 2 glutathione lyase, 1 glutathione-s-transferase, 1 GMC oxidoreductase, 2 FAD binding oxidoreductase and several hydrolases, peroxidases, and transferases were upregulated on GGE. This study showed the capability of R5 to degrade and bioconvert the β-O-4 linkage lignin model compound GGE into vanillin.

The engineered biphenyl dioxygenases enhanced the metabolism of dibenzofuran

As a model compound of dioxin, dibenzofuran is a persistent environmental pollutant. Several investigations have provided evidence that biphenyl dioxygenase (BPDO) from Burkholderia xenovorans LB400 could be engineered to further enhance the metabolite profile of biphenyl and polychlorinated biphenyl through lateral oxygenation. In this context, we examined the ability of the evolved BphAES283M, BphAEp4-S283M and BphAERR41-S283M to transform dibenzofuran with features of co-planar and ortho-substituted biphenyls. For BphAES283M, BphAEp4-S283M and BphAERR41-S283M, the kcat/Km value toward dibenzofuran was 4.5 times, 3 times and 2.5 times higher than that of the wild-type enzyme, respectively. Meanwhile, biochemical experiments determined that the substitution Ser283Met affected the regiospecificity of product formation, and the primary metabolite produced by BphAES283M was identified as 3,4-dihydro-3,4-dihydroxy-dibenzofuran. The structural analysis revealed residue Met283 as critical to generate a flexible catalytic cavity and a productive orientation of dibenzofuran during the catalytic reaction. Collectively, this study provides the theoretical basis and technical support for the significant development of better promising biocatalysts to effectively degrade dibenzofuran and other aromatic pollutants in the environment.

Microbial and enzymatic degradation of PCBs from e-waste-contaminated sites: a review.

Electronic waste is termed as e-waste and on recycling it produces environmental pollution. Among these e-waste pollutants, polychlorinated biphenyls (PCBs) are significantly important due to ubiquitous, organic in nature and serious health and environmental hazards. PCBs are used in different electrical equipment such as in transformers and capacitors for the purposes of exchange of heat and hydraulic fluids. Bioremediation is a reassuring technology for the elimination of the PCBs from the environment. In spite of their chemical stability, there are several microbes which can bio-transform or mineralize the PCBs aerobically or anaerobically. In this review paper, our objective was to summarize the information regarding PCB-degrading enzymes and microbes. The review suggested that the most proficient PCB degraders during anaerobic condition are Dehalobacter, Dehalococcoides, and Desulfitobacterium and in aerobic condition are Burkholderia, Achromobacter, Comamonas, Ralstonia, Pseudomonas, Bacillus, and Alcaligenes etc., showing the broadest substrate among bacterial strains. Enzymes found in soil such as dehydrogenases and fluorescein diacetate (FDA) esterases have the capability to breakdown PCBs. Biphenyl upper pathway involves four enzymes: dehydrogenase (bphB), multicomponent dioxygenase (bphA, E, F, and G), second dioxygenase (bphC), hydrolase, and (bphD). Biphenyl dioxygenase is considered as the foremost enzyme used for aerobic degradation of PCBs in metabolic pathway. It has been proved that several micro-organisms are responsible for the PCB metabolization. The review provides novel strategies for e-waste-contaminated soil management.

Microbiome changes and oxidative capability of an anaerobic PCB dechlorinating enrichment culture after oxygen exposure

Marine sediments may represent a sink of persistent organic pollutants including polychlorinated biphenyls (PCBs), toxic compounds prone to reductive or oxidative biodegradation pathways depending on the degree of chlorination and the positions of the chlorine atoms on the biphenyl rings. Superficial marine sediments can be subjected to episodic sediment resuspension by boat traffic and wind action causing the exposure of the underlying anaerobic layer to oxygen. Under these dynamic conditions, a deeper knowledge of the adaptation capability of the autochthonous microbial communities towards severe changes of the reaction environment is required. Insights into the metabolic potential of sediment community members may contribute greatly to the definition of efficient and reliable in situ bioremediation strategies. In this study, an anaerobic PCB-dechlorinating microbial consortium, developed from the chronically polluted marine sediment of Mar Piccolo (Taranto, Italy), was used to evaluate the response of the sediment microbiome to the imposition of aerobic conditions after prolonged anaerobic incubation. Compared to the anaerobic control, a dramatic change in microbiome composition, with a marked increase of Alphaproteobacteria of up to 39.2 % of total operational taxonomic units (OTUs) was revealed by high-throughput 16S rRNA gene sequencing. Accordingly, a decrement of low chlorinated PCBs (up to 58.3 ± 7.5 % for PCB 18) and the concomitant appearance of genes coding for PCB-degrading biphenyl dioxygenase (bph) were observed at the end of the aerobic incubation, suggesting the occurrence of oxidative PCB biodegradation processes.

Degradation of BDE-47 in mangrove sediments under alternating anaerobic-aerobic conditions

Polybrominated diphenyl ethers (PBDEs) resistant to degradation have significant environmental impacts. Anaerobic reductive debromination and aerobic oxidation of PBDEs by microorganisms are main removal mechanisms during natural attenuation, but previous studies often focused on the process under either aerobic or anaerobic condition leading to unsatisfactory removal. The present study aims to remove PBDEs by employing alternating anaerobic-aerobic condition, which is common in inter-tidal mangrove sediments, and elucidate the degradation pathways. During 40-week experiment, BDE-47 reduced with an accumulation of tri-BDEs and di-BDEs as debromination products in all sediments. However, the removal percentages of BDE-47 and the concentrations of debromination congeners varied among flushing regimes. Sediments under less frequent flushing regime (longer duration of aerobic period) had significantly lower concentration and proportion of debromination products, especially BDE-17, than that under more frequent regime (longer anaerobic period). BDE-17 then went through aerobic degradation pathway, as evidenced by the accumulation of its hydroxylation form. Microbial analyses further revealed that less frequent regime favored accumulation of biphenyl dioxygenase gene for aerobic degradation, while more frequent tidal regime promoted growth of dehalogenating bacteria for reductive debromination. This study first time demonstrated that PBDEs in contaminated sediments could be removed under alternating anaerobic-aerobic conditions.

Analysis of Affinity Energy Between Biphenyl Dioxygenase and Polychlorinated Biphenyls Using Molecular Docking

Molecular docking was used to calculate the affinity energy between biphenyl dioxygenases(BphA), i ncluding 1ULJ, 1WQL, 2YFJ, 2YFL, 2GBX, 2XSH, 2E4P, 3GZX, and 3GZY(selected from the Protein Data Bank) and 209 polychlorinated biphenyl(PCB) congeners. The relationships between the calculated affinity energy and the persistent organic pollutant characteristics(migration, octanol-air partition coefficients, lgKOA; persistence, half-life, lgt1/2; toxicity, half-maximal inhibitory concentration, lgIC50; bioaccumulation, bioconcentration factor, lgBCF) of the PCBs were studied to understand the BphA mediated degradation of PCBs. The effect of substituent characteristics on the affinity energy was explored through full factorial experimental design. The affinities of nine kinds of BphA pr oteins on PCBs ranked as follows: 2GBX>2YFJ>2YFL>3GZX>2XSH>3GZY>2E4P>1WQL>1ULJ. The relationships between the calculated affinity energy and the molecular weight, lgKOA, lgBCF, and lgt1/2 of the PCBs were statistically significant(p 0.05). PCBs were more difficult to degrade following an increase in the free energy of binding. Correlation analysis showed that the average affinity energy values of PCBs gradually increased as the number of chlorine atoms increased, r egardless of the substituent position. The substituents at the ortho-positions interacted mainly through a second-order interaction, whereas those at the para-positions did not participate via a second-order interaction.

Recent advances in the bio-remediation of persistent organic pollutants and its effect on environment

Abstract This paper presents a comprehensive review of the current state of research activities on the application of biodegradation/bioremediation for removing persistent organic pollutants (POPs) such as pesticides, PCBs, PAHs and PPCPs from wastewater. Several potential enzymes have been reported from various microorganisms, which breakdown the complex compounds through biodegradation, biostimulation or bioaugmentation process. Various microorganisms, harbouring numerous plasmids and catabolic genes, acclimatize to these environmentally unfavourable conditions by gene duplication, mutational drift, hypermutation, and recombination. Genetic aspects of some major POP catabolic genes such as biphenyl dioxygenase (bph), hydroxylation, phosphotriesterases and oxygenase etc. assist in degradation organic pollutants. However with the advent of technological advancements in genetic engineering is being considered the possibility for the role of genetically modified organisms, metagenomics and metabolomics is being considered to develop low cost, effective and reliable method for detection, determination and removal of ultra-trace concentration of POPs. In addition, development in microbial fuel cell, nano-materials, biofilms and constructed wetlands augments the biodegradation process. Therefore, this review highlights the effect of POPs on the environment, health hazards, microbial degradation and its mechanism along with brief study of advanced oxidation process for wastewater treatment.

Identification of biphenyl-metabolising microbes in activated biosludge using cultivation-independent and -dependent approaches

Microbes have important roles in removing organic pollutants in wastewater treatment plants (WWTPs), especially in mineralising recalcitrant persistent organic pollutants (POPs) such as polychlorinated biphenyls (PCBs). However, the majority of the microorganisms that metabolise these pollutants in situ remain elusive owing to barriers of traditional techniques in unravel yet-to-be-cultivated microbes. In this study, DNA stable-isotope probing (SIP) coupled with high-throughput sequencing was applied to identify the microbes responsible for PCB degradation in the activated biosludge of a WWTP using 13C-labelled biphenyl (BP). Results of time-course SIP revealed different bacteria and archaea involved in BP metabolism, which dominated the BP-degrading community at different time points. BP degradation by the genera Spartobacteria, Alicyclobacillus, Flavobacterium and the order Cenarchaeales has not been reported previously. The abundance of biphenyl dioxygenase (bphA) genes increased over time and a novel bphA gene was identified from the 13C-heavy DNA fraction. In addition, three cultivable BP degraders were isolated, but did not participate in BP degradation in situ or contain the identified bphA genes. Taken together, these data reveal the huge potential and important roles of yet-to-be-cultivated microbes responsible for PCB degradation in activated biosludge, providing fundamental knowledge on WWTP management to remove POPs.

Metabolic and Evolutionary Insights in the Transformation of Diphenylamine by a Pseudomonas putida Strain Unravelled by Genomic, Proteomic, and Transcription Analysis.

Diphenylamine (DPA) is a common soil and water contaminant. A Pseudomonas putida strain, recently isolated from a wastewater disposal site, was efficient in degrading DPA. Thorough knowledge of the metabolic capacity, genetic stability and physiology of bacteria during biodegradation of pollutants is essential for their future industrial exploitation. We employed genomic, proteomic, transcription analyses and plasmid curing to (i) identify the genetic network of P. putida driving the microbial transformation of DPA and explore its evolution and origin and (ii) investigate the physiological response of bacterial cells during degradation of DPA. Genomic analysis identified (i) two operons encoding a biphenyl (bph) and an aniline (tdn) dioxygenase, both flanked by transposases and (ii) two operons and several scattered genes encoding the ortho-cleavage of catechol. Proteomics identified 11 putative catabolic proteins, all but BphA1 up-regulated in DPA- and aniline-growing cells, and showed that the bacterium mobilized cellular mechanisms to cope with oxidative stress, probably induced by DPA and its derivatives. Transcription analysis verified the role of the selected genes/operons in the metabolic pathway: DPA was initially transformed to aniline and catechol by a biphenyl dioxygenase (DPA-dioxygenase); aniline was then transformed to catechol which was further metabolized via the ortho-cleavage pathway. Plasmid curing of P. putida resulted in loss of the DPA and aniline dioxygenase genes and the corresponding degradation capacities. Overall our findings provide novel insights into the evolution of the DPA degradation pathway and suggests that the degradation capacity of P. putida was acquired through recruitment of the bph and tdn operons via horizontal gene transfer.

Relationship between the binding free energy and PCBs\u2019 migration, persistence, toxicity and bioaccumulation using a combination of the molecular docking method and 3D-QSAR

The molecular docking method was used to calculate the binding free energies between biphenyl dioxygenase and 209 polychlorinated biphenyl (PCB) congeners. The relationships between the calculated binding free energies and migration (octanol–air partition coefficients, KOA), persistence (half-life, t1/2), toxicity (half maximal inhibitory concentration, IC50), and bioaccumulation (bioconcentration factor, BCF) values for the PCBs were used to gain insight into the degradation of PCBs in the presence of biphenyl dioxygenase. The relationships between the calculated binding free energies and the molecular weights, KOA, BCF, and t1/2 values for the PCBs were statistically significant (P < 0.01), whereas the relationship between the calculated binding free energies and the IC50 for the PCBs was not statistically significant (P > 0.05). The electrostatic field, derived from three-dimensional quantitative structure–activity relationship studies, was a primary factor governing the binding free energy, which agreed with literature findings for KOA, t1/2, and BCF. Comparative molecular field analysis and comparative molecular similarity indices analysis contour maps showed that the binding free energies, KOA, t1/2, and BCF values for the PCBs decreased simultaneously when substituents with electropositive groups at the 3-position or electronegative groups at the 3′-position were introduced. This indicated the binding free energy was correlated with the persistent organic pollutant characteristics of PCBs. Furthermore, low binding free energies improved the degradation of the PCBs and simultaneously decreased the KOA, t1/2, and BCF values, thereby reducing the persistent organic pollutant characteristics of PCBs in the environment. These results are expected to be beneficial in providing a theoretical foundation for further elucidation of the degradation and molecular modification of PCBs.

Insights into the genome and proteome of Sphingomonas paucimobilis strain 20006FA involved in the regulation of polycyclic aromatic hydrocarbon degradation.

In order to study the mechanisms regulating the phenanthrene degradation pathway and the intermediate-metabolite accumulation in strain S. paucimobilis 20006FA, we sequenced the genome and compared the genome-based predictions to experimental proteomic analyses. Physiological studies indicated that the degradation involved the salicylate and protocatechuate pathways, reaching 56.3% after 15 days. Furthermore, the strain degraded other polycyclic aromatic hydrocarbons (PAH) such as anthracene (13.1%), dibenzothiophene (76.3%), and fluoranthene. The intermediate metabolite 1-hydroxy-2-naphthoic acid (HNA) accumulated during phenanthrene catabolism and inhibited both bacterial growth and phenanthrene degradation, but exogenous-HNA addition did not affect further degradation. Genomic analysis predicted 126 putative genes encoding enzymes for all the steps of phenanthrene degradation, which loci could also participate in the metabolism of other PAH. Proteomic analysis identified enzymes involved in 19 of the 23 steps needed for the transformation of phenanthrene to trichloroacetic-acid intermediates that were upregulated in phenanthrene cultures relative to the levels in glucose cultures. Moreover, the protein-induction pattern was temporal, varying between 24 and 96h during phenanthrene degradation, with most catabolic proteins being overexpressed at 96 h-e. g., the biphenyl dioxygenase and a multispecies (2Fe-2S)-binding protein. These results provided the first clues about regulation of expression of phenanthrene degradative enzymes in strain 20006FA and enabled an elucidation of the metabolic pathway utilized by the bacterium. To our knowledge the present work represents the first investigation of genomic, proteomic, and physiological studies of a PAH-degrading Sphingomonas strain.

5 改変Biphenyl Dioxygenase遺伝子導入細菌による各種芳香環化合物の生物変換 : "BioCombiChem"技術の確立を目指して(口頭発表の部)

5 改変Biphenyl Dioxygenase遺伝子導入細菌による各種芳香環化合物の生物変換 : "BioCombiChem"技術の確立を目指して(口頭発表の部)