Biotechnology and Bioprocess Engineering 2009, 14: 694-701 DOI/10.1007/s12257-008-0266-2

Characterization of the Phenol Monooxygenase Gene from Chromobacterium

violaceum: Potential Use for Phenol Biodegradation

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Elen Aquino Perpetuo1*, Regina Célia Pereira Marques2, Maria Anita Mendes1, Wanessa Cristina de Lima2, Carlos Frederico Martins Menck2, and

Claudio Augusto Oller do Nascimento1 1CEPEMA, Chemical Engineering Department, University of São Paulo, São Paulo, Brazil

2DNA Repair Laboratory, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil

Abstract In this work, the biodegradation mechanism of phenol and sub products (such as catechol and hydroquinone) in

`ÜêçãçÄ~ÅíÉêáìã=îáçä~ÅÉìã was investigated by cloning and molecular characterization of a phenol monooxygenase

gene in bëÅÜÉêáÅÜá~=Åçäá. This gene (`îãé) is very similar (74 and 59% of similarity and identity, respectively) to the

ortholog from o~äëíçåá~=ÉìíêçéÜ~I bacteria capable of utilizing phenol as the sole carbon source. The phenol biodeg-

radation ability of bK=Åçäá recombinant strains was tested by cell-growth in a minimal medium containing phenol as the

sole source of carbon and release of intermediary metabolites (catechol and hydroquinone). Interestingly, during the

growth of these strains on phenol, catechol, and hydroquinone accumulated transiently in the medium. These me-

tabolites were further analyzed by HPLC. These results indicated that phenol can be initially orto or para hydroxylated

to produce cathecol or hydroquinone, respectively, followed by meta-cleavage of aromatic rings. To verify this infor-

mation, the metabolites obtained from HPLC were submitted to LC/MS to confirm their chemical structure, thereby in-

dicating that the recombinant strains utilize two different routes simultaneously, leading to different ring-fission sub-

strates for the metabolism of phenol. © KSBB

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INTRODUCTION

Phenolic compounds are major environmental pollutants

from industrial processes such as oil refineries, industrial-resin manufacturing, and petroleum-based processing plants. Biodegradation of these compounds by bacteria has been extensively studied and, to date, a large number of phenol-degrading bacteria have been isolated, and their phenol deg-radation pathways have been characterized [1-3]. Bacteria with the ability to degrade phenol are widespread in the envi-ronment and the biodegradation process can be divided in two phases: the upper pathway, involving phenol oxidation to catechol catalyzed by a multi- or a single- component hy-

*Corresponding author

Tel: +55-13-3362-9361 Fax: +55-13-3362-9363

e-mail: elen@cepema.usp.br

droxylase/monooxygenase [4,5], and the lower pathway in-cluding catechol ring fission and subsequent reactions lead-ing to the generation of central metabolism intermediates. Depending on the genetic background of the biodegrading bacteria, the lower pathway may occur via the ortho or the meta-cleavage pathways.

The enzymes involved in phenol degradation are well known and have substrate specificity, which allows several organisms to metabolize phenol. Aerobic degradation of a phenolic compound is known to be initiated by its hydroxy-lation to form corresponding catechol, which is not less toxic than phenol, but could be degraded faster than phenol and it is more biodegradable. Batch studies indicated that the 1,000 mg/L glucose concentration was sufficient to cometabolize and degrade catechol in an aqueous solution up to a concen-tration of 1,000 mg/L in an upflow anaerobic sludge blanket (UASB) reactor [6].

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Particularly, the meta-cleavage pathway is well estab-lished in bacteria, especially in Pseudomonas sp, where the best characterized genetic information for phenol catabolism via the meta-pathway is that coded by the mega-plasmid pVI150 [7-9]. Some fungi are also able to degrade phenol and on the basis of enzymatic studies, Jones et al. [10] pro-posed two pathways for the metabolism of phenol in Asper-gillus fumigatus. In one route, phenol undergoes ortho-hydroxylation to give catechol, whereas in the other, phenol is hydroxylated in the para-position to produce hydroquinone. Both steps are catalyzed by either a multi- or a mono-component phenol-hydroxylase/monooxygenase [11] (phe-nol-monooxygenase EC 1.14.13.7) and are considered to be the rate-limiting steps in the degrading pathway [12].

Several genes coding for phenol-monooxygenases have been cloned and sequenced from phenol-degrading microor-ganisms [13]. Two types of phenol monooxygenases are known, the single-component type and multi-component type. Among them, multicomponent phenol hydroxylase (mPH) is considered to be the major enzyme in the natural environment. Single and multi-component enzyme systems have been identified among mono- and dioxygenases. All these mPHs are similar in their enzyme structure; they com-prise six subunits, among which the catabolic site exists within the largest subunit (approximately 60 kDa), but some of these enzymes have been found to exhibit different sub-strate specificity for substituted phenols [11].

Chromobacterium violaceum is a Gram-negative β-pro-teobacterium first described at the end of the 19th century, and dominates a variety of ecosystems in tropical and sub-tropical regions. These bacteria have been found to be highly abundant in the water and borders/banks of the Negro river, a major component of the Brazilian Amazon Basin. Its com-plete genome sequence reveals: (A) extensive alternative pathways for energy generation, (B) ~500 ORFs for trans-port-related proteins, (C) complex and extensive systems for stress adaptation and motility, and (D) widespread utilization of quorum sensing for the control of inducible systems, all of which underpin the versatility and adaptability of the organ-ism [14]. In addition, there is a series of previously unknown but important enzymes and secondary metabolites, including paraquat-inducible proteins, drug and heavy-metal-resistance proteins, multiple chitinases, and proteins for the detoxifica-tion of xenobiotics that may have biotechnological applica-tions [15,16]. However, to date no report exists identifying the capacity of C. violaceum strains for phenol degradation.

In this work, we cloned a DNA fragment from C. viola-ceum (strain CVT8), containing the phenol monooxygenase gene (Cvmp) in E. coli. The DNA sequence of the Cvmp was confirmed by previous genome data and compared with those of other phenol monooxygenases genes. The protein encoded by this gene showed high similarity with orthologs from bacteria capable of utilizing phenol as the sole carbon source, such as Ralstonia eutropha [17].

The recombinant clones demonstrated great ability in phe-nol degradation and were able to grow in phenol as the sole carbon source. The release of intermediary metabolites was also analyzed. These results indicated that phenol could be

initially orto or para hydroxylated to produce cathecol or hydroquinone, respectively, followed by meta-cleavage of aromatic rings. This was demonstrated by confirmation of the metabolites chemical structure in LC/MS, indicating that the recombinant strains utilized two different routes simulta-neously, leading to different ring-fission substrates for the metabolism of phenol.

MATERIALS AND METHODS

Bacterial strains, Plasmids, Culture Conditions and

Chemicals

The bacterial strains used in this study were C. violaceum

type strain CVT8 (isolated from Negro River, Brazil) and E. coli DH10b (StratageneTM). Cultures of each bacterial strain were grown in a Luria-Bertani (LB) broth medium, on hori-zontal shakers at 250 rpm and 37ºC. When necessary, am-picilin at 50 μg/mL was added to the medium. The plasmid used was pGEM-T Easy (PromegaTM). All aromatic com-pounds were purchased from Merck, Darmstadt, (Germany) (analytical grade).

DNA Manipulations

The genomic DNA from C. violaceum was isolated using

the Chen and Kuo method [18]. The isolation and construc-tion of plasmids and other DNA manipulations were carried out using standard techniques [19]. All enzymes and mo-lecular biology kits were purchased from InvitrogenTM. Re-striction endonuclease digestion and ligation with T4 DNA ligase were performed according to manufacturer’s instruc-tions. The gel extraction kit was employed for the recovery of DNA fragments from agarose gels. Nucleotide sequencing was carried out by using an automated sequencer (Applied BiosystemsTM), sequence analysis with PC software, and homology by means of BLAST programs.

Molecular Protocols

Molecular protocols were carried out as previously de-

scribed (Sambrook et al., 1989). Briefly, PCR was used to amplify the phenol-monooxygenase gene (1,026 bp) from the C. violaceum genome (protein accession number AAQ-61446). The forward primer 5′-TTGCCAGGTGAAGGTG CTC- 3′ and the reverse primer 5′-TCTTCTCCGACGCC TTCTTCCTGG- 3′ were both obtained from InvitrogenTM. These primers correspond to nucleotide positions 4082123-4082140 and 408175-408199, respectively, of the C. viola-ceum genome sequence. PCR was performed with 1 μg of genomic DNA in 50 μL reaction mixtures by using the con-ditions as follows: initial denaturation at 95ºC for 5 min and then 30 cycles consisting of denaturation at 92ºC for 1 min, followed by annealing at 55ºC for 0.5 min and extension for 3 min at 72ºC. Final elongation was for 10 min. The PCR product was gel purified using the high-pure PCR product purification kit (InvitrogenTM). The purified PCR product

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was cloned by using the pGEM-T Easy Vectors System (PromegaTM). The coding sequence of phenol monooxy-genase was linked to the vector and transferred to E. coli DH10b competent cells by electroporation, using a Gene Pulsar II (Bio-Rad, Tokyo) under the following conditions: voltage 2.5 V, capacitance 25 μF, and resistance 200 Ω.

Induction of the Recombinant Enzymes and Enzyme Activities in Cell Extracts

Cells of E. coli DH10b strains carrying the recombinant

plasmids were grown for 60 min at 37ºC in 10 mL of a SOC medium (20 g, bacto-tryptone; 5 g, bacto-yeast extract; 0.5 g, NaCl; 2.5 mL, 1 M KCl; and 1000 mL, H2O). The cells were plated in a selective medium containing 50 μg of ampicilin/ mL and X-GAL (40 μg/mL), to then be induced with IPTG (1 mM). The clones containing the plasmid with an insert were identified as white colonies (those containing empty plasmids generated blue colonies). The correct construction was confirmed by restriction mapping and DNA sequencing.

Phenol Utilization Test Positive clones were tested for the ability to utilize phenol

as the sole carbon source in a basal salt medium (BS) sup-plemented with phenol (MerckTM) at concentrations of 0.1, 0.2, 0.4, or 0.8 mg/mL in a medium containing 6.8 g of Na2HPO4, 3.0 g of KH2PO4, 0.5 g of NaCl, and 0.5 g of NH4Cl. The pH was adjusted to 6.8. The inoculation was transferred as 1 mL of liquid culture (A600 = 0.8) to 50 mL of BS sup-plied with phenol. Bacterial growth was performed at 37ºC with shaking at 125 rpm for 7 days. Culture media were monitored by absorbance at 600 nm (A600) daily. A600 > 0.3 was considered as positive growth (using phenol). The con-centration of phenol in the medium was determined by ana-lytical methods (HPLC).

Analytical Methods

HPLC was performed on a Shimadzu LC-MS 2010A ana-

lytical system equipped with an on-line degasser auto-sam-pler module, UV detector adjusted to 270 nm, and a Beck-man ODS-C18 column [20]. The analysis was performed using a running 0~60% gradient of solvent B for 20 min at a flow rate of 0.7 mL/min and at room temperature. Solutions of 0.2% acetic acid in water (solvent A) and in acetonitrile (solvent B) were used as elution solvents. Samples of bacte-rial extracts from the phenol test were collected every 2 days and subjected to chromatography. Samples corresponding to zero-time incubation, phenol and medium blanks were ana-lyzed by the same method as controls. A linear calibration curve for phenol quantification by HPLC was constructed. Peaks corresponding to hydrolysis products were collected, each peak then being analyzed by mass spectrometry. Phenol and intermediates were identified and quantified by means of reverse phase high performance liquid chromatography-mass spectrometer (RF-HPLC-MS). The mass spectrometer was equipped with an APCI ionization source.

Fig. 1. Cell growth of `K=îáçä~ÅÉìã strain CVT8 on phenol con-

taining media. The strain was incubated aerobically in a

minimal medium containing different concentrations of

phenol. Cell growth was determined by absorbance (A600

nm).

RESULTS AND DISCUSSION

Identification of a Phenol-monooxygenase Activity in=`K=îáçä~ÅÉìã under Different Growth Conditions

C. violaceum, strain CVT8 was grown on a BS medium

with phenol as the source of carbon under aerobiosis (Fig. 1). Although slow, there was a clear growth in the medium con-taining up to 0.2 mg/mL of phenol. This indicates that these cells are able to degrade phenol, probably due to constitutive phenol-monooxygenase activity, as the genome of C. violaceum has a potential ortholog of this protein.

Protein Sequence of the `îéã Gene The predicted amino acid sequence of Cvpm showed sig-

nificant similarities with those of several hydroxylases be-longing to the monooxygenase family. As shown in Fig. 2, the deduced Cvpm amino acid sequence encoded 324 resi-dues and comparison with others available from GenBank revealed a high similarity (74%) with the ortholog from R. eutropha, also belonging to the β-Proteobacteria group [17]. The Cvpm was aligned with other sequences from related phenol-degrading bacteria and a phylogenetic tree con-structed by using the neighbor joining method. The phyloge-netic tree (Fig. 3) showed that monooxygenases have a high level of variability among phenol-degrading bacteria and can be separated into three distinct groups. Cvpm is clustered together with Nitrosomonas europae, a bacterium that is known to transform aromatic compounds [21], and is close to other β-Proteobacteria orthologs. It is interesting to ob-serve that the distribution of these proteins in the phyloge-netic tree is highly variable, with several orthologs from un-related bacteria branching together. Moreover, in several bacteria, this gene is carried by plasmids. These features

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Remp MAYQVTVMPSGHKFEVAADETILGAALRHSIGLPYGCKNGACGSCKGRVLEGTIVQGDHA 60 Cvmp MTCQVKVLPSGHTFGVEAHETILEAALRQGVGLPYGCRDGACGACKGKVLEGEVSQDGFQ 60 Remp PAALTAQEKTEGRALFCCANAASDVTIECREVHGAGDIPIKKVPCRVTAIERLADDVIAI 120 Cvmp EKALSAAERAQGMALFCCSRPKGDVSIEAREVTGVGDIQIKTLPCRVEKIDKI-HDVAVL 119 Remp KLQLPATERMQYLAGQYVEFLLRDGKRRSYSIATPPHEDGPIELHIRHMPGGAFTDYVFG 180 Cvmp KLKLPVSERLQFRAGQYIDILMKDGKKRSFSIANAPHDDAFLELHIRHQPGGSFSEYVFH 179 Remp AREGQPAMKERDILRFEGPLGSFFLREESEAPIILLASGTGFAPIKAIVEHAAYTGIQRP 240 Cvmp Q------MKEREIMRFKGPMGSFFLREESDKPIVLIASGTGFAPVKGIIEHAIHHGITRP 233 Remp MTLYWGGRRPKDLYMHALCEQWARELPNFRYVPVISDALPEDNWQGRTGFVHQAVIADHP 300 Cvmp MQFYWGARTKADLYMSELAEGWAAAHPNIRYIPVLSEALPEDGWTGRTGFVHQAVLEDFA 293 Remp DLSGHEVYACGAPVMINAARGDFTRQCKLHEDAFFADSFTSEADM 345 Cvmp DLSGHQVYACGAPVMVEAAHGTFIRERGLPEDEFFSDAFFLAKDM 338

Fig. 2. Alignment of the protein sequence of deduced phenol monooxygenase from `K=îáçä~ÅÉìã (Cvpm) and oK=ÉìíêçéÜ~ (Remp),

showing 59% of identity and 74% of similarity between them. Amino acids in bold letters are identical.

indicate that this monooxygenase may be prone to lateral gene transfer among different bacterial species.

Cloning of the `K=îáçä~ÅÉìã Phenol-monooxygenase Gene

The sequence of the Cvpm gene of C. violaceum was used

to design primers to amplify the complete gene from the CVT8 strain by polymerase chain reaction (PCR). The gene was inserted into the plasmid pGEM, where it can be ex-pressed under the control of the beta-galactosidase promoter. The gene was confirmed by DNA sequencing. E. coli DH10b bearing the gene Cvpm was grown on a LB medium with ampicilin and cell-free extracts were prepared to meas-ure phenol-monooxygenase activity associated with the cloned genes, by using the phenol as a substrate. A cell-free extract from E. coli DH10b bearing an empty pGEM plas-mid was used as control for phenol-monooxygenase activity. No significant activity was observed.

Identification of Phenol-utilizing bK=Åçäá Clones

E. coli clones containing the Cvmp gene were cultured in either a solid or a liquid BS medium supplemented with phenol as the sole carbon source at concentrations of 0.2, 0.4, or 0.8 mg/mL (Fig. 4). Cell growth was analyzed for 7 days and compared with the C. violaceum wild type. These clones were able to grow up to 0.8 mg/mL in phenol, whereas C.

violaceum grew only up to 0.2 mg/mL (Fig. 1). E. coli con-taining an empty plasmid did not grow in a medium with phenol, confirming the need of the Cvmp gene for this growth.

Since phenol was the sole source of carbon for the recom-binant E. coli, this was also measured and it was noted that its degradation is inversely associated with the growth of the degrading strain (Fig. 5). Indeed, we observed similar kinet-

ics trends between phenol-limited growth and phenol degra-dation. These results further confirmed that recombinant bacteria effectively metabolize phenol for cell growth.

Determination of Phenol-monooxygenase Activity

Cultures were grown for 72 h and then tested for phenol

degradation. Fig. 6A shows the chromatograms upon LC/MS analysis for 72 h cultures in phenol degradation tests. The residual amount of phenol in the culture of the recombinant strain was much smaller than that of the host strain harboring the empty vector (data not shown). Small peaks were de-tected at approximately 11 and 13 min of elution (Fig. 6B), of which the mass spectra were in a full agreement with those of authentic catechol and hydroquinone (Fig. 6C); this suggesting that phenol was converted to these metabolites. These results show that the gene Cvpm encoded an enzyme with monooxygenase activity, as suggested by in silico com-parison. The average phenol degradation rate (0.05 mmol/ L/h) of this monooxygenase enzyme was calculated through dividing the total amount of substrate consumed by the time required for consumption.

Various hydroxylated compounds are formed in the proc-ess of phenol degradation by bacteria. Catechol and hydro-quinone are examples of metabolites released during phenol degradationl by monooxygenase enzymes. Metabolites ex-tracted from culture media of phenol-degrading bacteria in-dicated hydroxylation of phenol, this pathway being well characterized in Pseudomonas sp. [22,23]. Although its ge-netic organization was different from that of the phenol monooxygenases found in Pseudomonas sp. [24], normally multi-component, the Cvpm gene showed considerable iden-tity to the gene corresponding to single-component phenol monooxygenase in R. eutropha E2 [17] and one component (P5) of the phenol monooxygenase in Pseudomonas sp CF 600 [11].

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Fig. 3. Neighbor-joining tree based on the amino acid se-

quences of monooxygenases showing the relationship

between `K=îáçä~ÅÉìã and known phenol-degrading bac-

teria. Sequence corresponding to ORF CV3784 (`îãé

gene) from `K=îáçä~ÅÉìã was used for calculation.

The data indicated significant growth of the recombinant

E.coli harboring Cvmp in the presence of high concentrations of phenol, and its utilization as a source of carbon. The deg-radation of phenol was also analyzed by the extraction and identification of metabolites in HPLC. The recombinant clone transformed phenol into catechol and hydroquinone, most likely due to the action of the monooxygenase enzyme. Normally, the pathway used by bacteria to promote phenol hydroxylation is the orto-pathway to give catechol. In this case, two different routes operating simultaneously and lead-ing to different ring-fission substrates are proposed for the metabolism of phenol. In one route, phenol undergoes ortho-hydroxylation to give catechol and in the other, it is hy-

Fig. 4. Cell growth of recombinant bK= Åçäá=harboring the `îéã

gene (CV3784) incubated aerobically in a minimal me-

dium containing different concentrations of phenol. Cell

growth was determined by absorbance (A600 nm).

Fig. 5. Biodegradation of phenol by recombinant bK=Åçäá=harbor-

ing the `îéã gene (CV3784). The clone was incubated

aerobically in a minimal medium containing initially 0.4

mg/mL (4.25 mM) of phenol and starting inoculum of 3

mg/flask (wet weight). The amount of phenol in the me-

dium was defined (open symbols). Cell growth was de-

termined by absorbance (closed symbols).

droxylated in the para-position to produce hydroquinone (Fig. 7). This fact was also observed by Jones et al. [10] in A. fu-migatus. However, the appearance of hydroquinone in the medium during growth on phenol suggested the unusual situation that a second pathway might be operating simulta-neously for phenol dissimilation. There was the possibility that hydroquinone was a dead-end product, formed as a side reaction due to the lack of regiospecificity in phenol monooxygenase. Monooxygenase purified from the yeast Trichosporum cutaneum was shown to be active with other phenolic substrates such as hydroquinone or catechol [25,26].

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A

B

C

Fig. 6. Biodegradation of phenol (0.4 mg/mL) by recombinant bK=Åçäá=harboring the `îéã gene (CV3784) and metabolite formation

(catechol and hydroquinone). (A) Profile of the phenol solution (0.4 mg/mL), (B) metabolites from phenol biodegradation (cate-

chol and hydroquinone), with 80% reduction of the initial phenol concentration after 3 days of incubation, and (C) the profile of

the mass spectra of phenol, catechol, and hydroquinone appears.

Fig. 7. Proposed pathways for the metabolism of phenol in re-

combinant bK= Åçäá harboring the `îãé=gene. The com-

pounds indicated are I, phenol; IIA, catechol; and IIB, hy-

droquinone.

CONCLUSION

In this paper, the gene Cvmp from C. violaceum was func-

tionally characterized. Recombinant bacteria were able to support higher concentrations of phenol than conventional ones (wild or acclimated). These results demonstrate the functionality and laboratory testing of a recombinant bacteria, and point to a potential use in synthetic wastewater treatment. Furthermore, an understanding of the crucial information emerging from this genome expression profiling, as well as, detailed investigation of the biological role of candidate genes as targets of phenol or derivatives toxicity or as deter-minants of resistance in C. violaceum, will instigate the de-velopment of more rational strategies for developing bacteria with greater solvent tolerance. This may have an impact on bioremediation and whole-cell biotransformation in media with organic solvents.

Acknowledgments We thank Prof. Dra. Lucymara F.

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Agnez-Lima (Universidade Federal do Rio Grande do Norte, Natal, RN, Brazil) for the C. violaceum strains. CNPq (Brasília, DF, Brazil) and FAPESP (São Paulo, SP, Brazil) provided financial support for this work.

Received November 8, 2008; accepted May 16, 2009

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