SIMON N MMG bioremediación dioxinas

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Recent advancements in the bioremediation of

polychlorinated dibenzo-

p

-dioxins

Nicholas Simon

Polychlorinated dibenzo-p-dioxins (PCDDs) have been the subject of research with regard to bioremediation. Species capable of enzymatically degrading PCDDs, such as Sphingomonas wittichii, Pseudomonas veronii, and Cordyceps sinensis, have been identified and characterized. Sphingomonas wittichii has also been success-fully used in the bioremediation of contaminated fly ash. Recombinant Saccharomyces cerevisae and Escherichia coli containing mammalian CYP1A genes have been demonstrated to have great enzymatic activity towards PCDDs, including the known carcinogen 2,3,7,8-tetra-chlorodibenzo-p-dioxin. A mutant P450 complex from Bacillus megaterium has also been shown to have enzymatic activity towards PCDDs, but to a much lesser extent than the mammalian CYP1A complex.

Addresses

Department of Biochemistry and Molecular Biol-ogy, Michigan State University, East Lansing, MI

Corresponding author: simonni1@msu.edu

List of Abbreviations

PCDD polychlorinated dibenzo-p-dioxins DD dibenzo-p-dioxins

CCD chlorinated dibenzo-p-dioxins DCCD dichlorinated dibenzo-p-dioxins MCCD monochlorinated dibenzo-p-dioxins

Introduction

Polychlorinated dibenzo-p-dioxins (PCDDs) are a class of common pollutants that are un-intentional byproducts of pesticide and

her-bicide production and industrial incineration [1]. They are highly toxic, and 2,3,7,8 tetra-chlorodibenzo-p-dioxin (tetra-CCD) has been classified as a human carcinogen, as it is able to bind with a transcriptional regula-tory protein [••2]. While several microor-ganisms are capable of degrading unchlori-nated diobenzo-p-dioxins (DDs), very few are capable of degrading the chlorinated compound, because of the increased stability chlor-ination adds to the compound.

Recently, there have been several efforts to develop a bioremediation strategy for PCDDs. There has been a substantial amount of traditional work using naturally occurring microorganisms, but there have also been attempts to bioengineer recombi-nant organisms to address concerns that the current species that have been characterized will never be a practical solution. Shinkyo

et. al cite the lack of organisms capable of metabolizing 2,3,7,8-tetraCCD and the rates at which they are able to degrade other PCDDs as a major justification for their work recombinant dioxin degrading organ-isms.[3].

Applications of Sphingomonas wittichii RW1

Extensive research has been done on Sphin-gomonas wittichii RW1, which can break-down dibenzofurans and dibenzo-p-di-oxins. This strain is considered to be the best char-acterized strain capable of degrading these compounds [1]. Recently, work was done to establish the ability of this strain to break down PCDDs.

Hong et. al demonstrated the ability of the strain to degrade 2,7-DCDD and 1,2,3,4-MMG445 Basic Biotechnology eJournal, 2006

This review comes from a themed issue based on current advances in the fields of microbiology, biotechnology, and pharmacology. It fulfills in part the assignment of the contributing author in MMG 445, Basic Biotechnology, Department of Microbiology and Molecular Genetics, Fall semester, 2006.

Editors - George M. Garrity and Terry L. Marsh

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Bioremediation of polchloronated dibenzo-p-dioxins 161

tetraCCD. These authors showed that the concentration of 2,7-DCDD in the growth medium dropped approximately 20% over a 120-hour incubation period, and the primary end product was 4-chlorocatechol. In the case of 1,2,3,4-tetraCDD, after 120 hours the concentration of the tetraCDD was low-ered approximately 14%, and was con-verted into two metabolites, 3,4,5,6-tetra-chlorocatechol and 2-methoxy-3,4,5,6-chlo-rophenol. However, it should be noted that the strain was unable to use the two dioxin compounds as a sole carbon source [4].

Nam et. al used S. wittichii RW1 in a biotransformation of 1,2,3,-triCCD and 1,2,3,4,7,8-hexaCCD using a similar proce-dure. Analysis of recovered meta-bolites af-ter the 120 hour incubation period showed that 1,2,3,-triCCD and 1,2,3,4,7,8-hexaCCD were lowered in concentration by approxi-mately 24% and 11%, respectively. 1,2,3-triCCD was de-graded primarily into two end products, trichlorotrihydroxy-diphenyl ether or 3,4,5-trichlorocatechol. 1,2,3,4,7,8-hexaCCD, having a very similar structure to 1,2,3,4-tetraCCD, was broken down into the same major metabolites. An interesting re-sult was that S. wittichii was unable to de-grade 2,3,7-tri and 1,2,3,7,8-pentaCCD, showing the importance of the substitution patterns on bio-degradation of the PCDDs. It was noted that these two compounds had the lowest !Gf of all the compounds tested [5]. This implies that a higher energy of activa-tion is required to react these compounds, which could explain the inability of S. wit-tichii to metabolize them.

Nam et. al culminated the series of experi-ments by using S. wittichii RW1 to biologi-cally remove DD (Figure 1a) and 5 species of PCDDs from contaminated fly ash, ob-tained from a municipal solid waste incin-erator. Both live and dead cells played a role in PCDD removal, as dead cells were able to remove PCDDs by absorption (Figure 1b). Independent reductions of PCDDs in the biomass culture were 75.5% and 20.2%, for

live and dead cells respectively. Substitution patterns were important in the bioremedia-tion, as highly chlorinated PCDDs such as octochlor-odibenzo-p-dioxin were degraded to a much lower extent than PCDDs with one to three chlorine atoms. The strain was found to have a high initial mortality rate when cultured with PCDDs, with approxi-mately 50% cell death occurring within 24 hours of inoculation. Repeated inoculations and pre-adaptation were found to greatly in-crease the effectiveness of the bioremedia-tion by increasing the amount of viable bac-teria in the culture ••[6]. This study shows that use of S. wittichii RW1 in dioxin biore-mediation of soil may be practical; however, repeated inoculations and pre-adaptation would increase the cost and difficulty of the procedure.

Recently, Aso et. al reported an increase in the dioxin degradation potential of S. wit-tichii through the use of a membrane super-channel. The genes for the superchannel were isolated from the species Sphingo-monas sp. A1, which uses membrane super-channels to take up alginates from the sur-roundings. The genes were inserted into a plasmid, which was then used to transform

S. wittichii cells. The transformed S. wit-tichii cells were able to express the genes and form membrane superchannels, allow-ing them to directly take up DD from the surrounding environ-ment, which resulted in a higher rate of removal of DD than by the wild type ••[7]. No measurements of the ability of the transformed bacteria to take up PCDDs were made; however, if these trans-formed S. wittichii cells can also remove PCDDs at a faster rate, the practicality of us-ing the strain to breakdown dioxin in the field could be greatly improved.

Isolation and characterization of

Pseudomonas veronii PH-03

Hong et. al isolated Pseudomonas veronii

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Figure 1. Removal of dioxins and dioxin metabolites from contaminated fly ash a) The con-centration of dibenzo-p-dioxin (DD), dibenzo-p-furan (DF), and the dioxin metabolites catechol and salicylate is shown with respect to time. The control was a slurry of dead cells and DF. b) The his-togram shows the percent removal of the five polychlorinated dibenzo-p-dioxins used in the ex-periment, which were tetrachlorinated dibenzo-p-dioxin (TCDD), pentachlorinated dibenzo-p-dioxin (PeCDD), hexachlorinated dibenzo-p-dioxin (HxCDD), heptachlorinated dibenzo-p-dioxin (HpCDD), and octochlorinated dibenzo-p-dioxin (OCDD). Adapted from Nam, et. al Water Research 2005, 39:4651-4660.

of dioxins, and was found to be effective (greater than 75% reduction) against DDs and mono- and dichoro-DDs. It was unable to significantly degrade 1,2,3,4 tetracloro-dibenzo-p-dioxin, possibly due to the in-solubility of the compound [8]. To date, no reported attempts have been made to use P. veronii in a soil bioremediation experi-ment. As this strain is further characterized it may become more important as the limits of strains such as S. wittichii are realized.

Cultivation of Cordyceps sinensis

Nakamiya et. al cultivated a fungus, Cordy-ceps sinensis, which is capable of dioxin breakdown. This organism is novel in that it does not degrade dioxins via angular degra-dation like S. wittichii and P. veronii. The exact mechanism has not been determined but a proposed pathway was developed based on the reaction products formed when the organism degraded PCDDs. The pro-posed pathway indicates that the dioxin compounds are degraded at the cyclic ether site, and shortly after a chlorine atom is re-moved. Furthermore, ortho-ring cleavage of the resulting cate-chols occurs, another de-viation from the angular degradation path-way. An exper-iment testing the fungus’ ability to breakdown DD, 2,3,7-triCDD, and

octoCDD showed approximately a 50% re-duction in the initial concentration of each dioxin species after 4 days [••9]. The novel degradative pathway may prove important in breaking down certain compounds more ef-ficiently than bacterial strains like S. wit-tichii and P. veronii, which both use the an-gular degradation pathway.

Recombinant cells expressing Mammalian CYP1A gene

The CYP1A family of cytochrome P450 complexes is able to degrade PCDDs through the insertion of an oxygen atom to form an epoxide ring as the initial reaction. Multiple hydroxylation reactions and cleav-age of the dioxin ring also appear to be part of the CYP-dependant metabolism [10].

Sakaki et. al used expression plasmids, one containing rat CYP1A1 and the other con-taining rat CYP1A2 with each being coex-pressed with yeast NADPH-P450, and in-serted them into Saccharomyces cerevisiae

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Bioremediation of polchloronated dibenzo-p-dioxins 163

suited to dechlorinating PCDDs than natu-rally occurring microorganisms [10].

An attempt to make a CYP1A enzyme capa-ble of degrading 2,3,7,8-tetraCDD, which is the most toxic PCDD, was made by Shinkyo

et. al. It was assumed that CYP1A could bind to 2,3,7,8-tetraCDD if the binding pocket of the enzyme was enlarged. Site di-rected mutagenesis was used to select-ively transform large amino acid residues into alanine residues. Several mutant forms of the enzyme were created, and cultures were made using recombinant S. cerevisae cells containing the genes for the enzymes. Two mutants, (F240A and F240S) showed activ-ity towards 2,3,7,8-tetraCDD [3]. While these new enzymes certainly show great promise, one has to consider the feasibility of using a lab grown strain of recombinant yeast cells in soil bioremediation. These cells, though more active against PCDDs than naturally occurring microorganisms, would likely have a very high mortality rate in soil.

Neither study addressed the issue of cost of using these cells for bioremediation. In a subsequent paper, Shinkyo et. al cloned the mutant CYP1A gene F240A into plasmids that were inserted into Escherichia coli. Mi-nor changes were made to some of the codons of the gene to optimize it for bacte-rial growth. The F240A expressed in bacte-ria (!F240A) showed catalytic activity to-wards all the same PCDDs as F240A ex-pressed in yeast, but to a lesser degree. The activity of !F240A on 2,3,7,8-tetraCDD was 3 times less than yeast expressed F240A. This could be attributed to the inability of the bacterial system to attach an N-terminus to the protein, or to the differences in the yeast and E. coli systems [••11]. The devel-opment of an E. coli system for expressing CYA1A genes greatly improves the practi-cality of using these recombinants in biore-mediation, as E. coli is easier and cheaper to work with than yeast. However, using a transgenic organism in a bioremediation strategy includes several legal and political factors that must be considered when

weigh-ing the practicality of usweigh-ing a transgenic species in a bio-remediation system.

Use of cytochrome P450 BM-3 isolated from Bacillus megaterium

The ability of mammalian P450 complexes to break down PCDDs led Triarsi et. al to study the ability of bacterial P450 com-plexes to degrade PCDDs. A fatty acid hy-droxylase, P450 BM-3, isolated from Bacil-lus megaterium, was mutated to obtain mu-tant enzymes capable of degrading poly-cyclic aromatic compounds. The activity of the mutant AL4V enzyme and wild type P450 BM-3 against PCDDs was measured using HPLC to measure the metabolites of enzyme mixtures that had been incubated with PCDDs. The mutant activity of the en-zyme towards 1-MCDD, 2,3-DCDD, and 2,3,7-triCDD was approx-imately 12, 2, and 11 times more active than the wild type en-zyme, respectively. Unfortunately this activ-ity was approx-imately 100 times lower than that of the CYP1A1 and ten times lower than CYP1A2 [12]. The use of these mutant enzymes in a bioremediation strategy simply does not seem practical when one considers the extra cost and typical difficulties dis-cussed previously when dealing with a transgenic organism. The levels of dioxin degradation do not appear to be worthwhile, especially when compared to the success found with the mammalian mutant.

Conclusions

With recent advancements, bioremediation strategies for dioxin removal are becoming increasingly possible. Currently, the S. wit-tichii RW1 shows great promise, evidenced by the successful dioxin removal from fly ash and new recombinant technologies which may improve the ability of the strain to uptake dioxin. Recombinant microorgan-isms containing mammalian CYP1A genes also show great promise, but this research is still in its infancy. The metabolic rates re-ported are impressive, but all tests done so far have used recombinant yeast and E. coli

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a hardier strain capable of surviving in soil, it seems hypocritical for CYP1A researchers to question the practicality of using naturally occurring organisms. Certainly more re-search is needed in this area, as it seems to be a promising method with regard to the breakdown of 2,3,7,8-tetraCCD.

Acknowledgments

I would like to thank all of the colleagues and classmates who provided help and guidance while writing this paper, as well as Dr. Garrity and Dr. Marsh for providing a venue in which to publish it.

References and recommended reading

Papers of special significance that have been published within the period of review are highlighted as follows:

• of significance •• of special significance

1. Nojiri H, Omori T: Molecular bases of aerobic bacterial degradation of dioxins: Involvement of angular dioxygenation. Bioscience Biotech-nology and Biochemistry 2002, 66:2001-2016.

••2. Mandal PK: Dioxin: a review of its environmental effects and its aryl hydrocarbon receptor biology.

Journal of Comparative Physiology B-Biochemical Systemic and Environ-mental Physiology 2005, 175:221-230. This is an excellent review outlining the health risks caused by PCDDs, particularly 2,3,7,8-tetraCDD.

3. Shinkyo R, Sakaki T, Takita T, Ohta M, Inouye K: Generation of 2,3,7,8-TCDD-metabolizing enzyme by modifying rat CYP1A1 through site directed mutagenesis. Biochemical and Bio-physical Research Communications 2003, 308:511-517.

4. Hong H-B, Chang Y-S, Nam I-H, Fortnagel P, Schmidt S: Biotrans-formation of 2,7-Dichloro- and 1,2,3,4-Tetrachloro-p-Dioxin by Sphingo-monas wittichii RW1. Applied and Environ-mental Microbiology 2002, 68:2584-2588.

5. Nam I-H, Kim YM, Schmidt S, Chang Y-S:

Biotransformation of 1,2,3-Tri- and 1,2,3,4,7,8-Hexachloro-p-Dioxin by

Sphingomonas wittichii Strain RW1.

Applied and Environmental Microbiology 2005, 72:112-116.

••6. Nam I-H, Hong H-B, Kim Y-M, Kim B-H, Murugesan K, Chang Y-S: Biological removal of polychlorinated dibenzo-p-dioxins from incinerator fly ash by

Sphingomonas wittichii RW1. Water Research 2005, 39:4651-4660.

This study showed the effectiveness of S. wittichii RW1 in the bioremediation of contaminated fly ash obtained from a municipal incinerator.

••7. Aso Y, Miyamoto Y, Harada KM, Momma K, Kawai S, Hashimoto W, Mikami B, Murata K: Engineered membrane superchannel improves bioremediation potential of dioxin-degrading bacteria. Nature Biotech-nology 2006, 24:188-189.

In this study, genes for a membrane channel were inserted into S. wittichii RW1, allowing for increased uptake of dioxin molecules, increasing dioxin removal rate.

8. Hong H-B, Nam I-H, Kumarasamy M, Kim YM, Chang Y-S: Biodegradation of dibenzo-p-dioxin, dibenzofuran, and chlorodibenzo-p-dioxins by Pseudo-monas veronii PH-03. Biodegradation 2004, 15:303-313.

••9. Nakamiya K, Hashimoto S, Ito H, Edmonds JS, Yasuhara A, Morita M:

Degradation of dioxins by cyclic ether degrading fungus, Cordyceps sinensis. Fems Microbiology Letters 2005, 248:17-22.

In this study a fungus, Cordyceps sinensis, was isolated and found to have a novel dioxin metabolism pathway.

10. Sakaki T, Shinkyo R, Takita T, Ohta M, Inouye K: Biodegradation of poly-chlorinated dibenzo-p-dioxins by recombinant yeast expressing rat CYP1A subfamily. Archives of Biochemistry and Biophysics 2002, 401:91-98.

••11. Shinkyo R, Kamakura M, Ikushiro S, Inouye K, Sakaki T: Biodegradation of dioxins by recombinant Escherichia coli expressing rat CYP1A1 or its mutant. Applied Microbiology and Biotechnology 2006, 72:584-590.

In this study, mutant rat CYP1A1 genes were successfully expressed in E. coli cells and used in the breakdown of PCDDs.

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