Haloperoxidase (HPO, E.C.220.127.116.11) is a metal-containing enzyme oxidizing halonium species, which can be used in the synthesis of halogenated organic compounds, for instance in the production of antimicrobial agents, cosmetics,
, in the presence of halides and H
. To isolate and evaluate a novel non-metal HPO using a culture-independent method, a cassette PCR library was constructed from marine seawater in Japan. We first isolated a novel HPO gene from
ATCC11172 by PCR for constructing the chimeric HPO library (HPO11172). HPO11172 showed each single open-reading frame of 828 base pairs coding for 276 amino acids, respectively, and showed 87% similarity with
IF-3 sequences. Approximately 600 transformants screened for chimeric genes between
ATCC11173 and HPO central fragments were able to identify 113 active clones. Among them, we finally isolated 20 novel HPO genes. Sequence analyses of the obtained 20 clones showed higher homology genes with
strains. Although the HPO A9 clone showed the lowest homology with HPO11172, clones in group B, including CS19, showed a relatively higher homology of 80%, with 70% identy.
cells expressing these HPO chimeric genes were able to successfully bioconvert chlorodimedone with KBr or KCl as substrate.
The oxidization of halonium species such as chloride (Cl
), bromide (Br
), and/or iodide (I
) by enzymatic approaches to synthesize various useful halogenated organic compounds, such as antimicrobial agents and cosmetics, have recently received great attention for selective oxygenation and halogenations of organic substrates
. Since the transformations of such selective oxygenation and halogenations of organic substrates at traditional chemical industry are conducted in very harsh conditions, at high temperatures, and with the use of strong acid or base catalysis, the production process can easily form undesired and toxic by-products, resulting in high costs to obtain the purified, optically active form
Haloperoxidase (HPO, E.C. 18.104.22.168) is a kind of oxygenase catalyzing halonium species and can be classified into four different HPOs: heme-dependant type, heme-flavindependant type, vanadium-dependant type, and non-metaldependant type, according to their coenzyme type. Until now, HPOs have been reported in a wide range of microorganisms, including brown algae (
), red algae (
), gram-negative bacteria (
), and streptomycetes (
), as well as in the marine snail
, the lichen
, and the ascomycetous fungus
. However, to our knowledge, there have been very few reported studies that demonstrates enzyme activity with non-heme-type HPOs, except for
Cassette PCR is a technique developed recently to easily recover useful enzymes from environmental sources without isolating microorganisms whether from culturable or unculturable sources
. This approach might be one of the powerful tools to access diverse enzyme pools than conventional cloning and expression approaches before protein engineering, in order to modify useful catalysis for industrial purposes by using directed evolution that has been proven to be a much more powerful protein engineering tool than conventional rational or random chemical mutagenic approaches. This approach by using cassette PCR and following the directed evolution method sequentially can be applied as a fastest method to creating novel mutants for such purposes as developing thermostability, substrate specificity
, and organic solvent tolerance
In this study, we developed a cassette PCR method for recovering novel genes from environmental sources to search for novel non-metal-type HPOs genes in the marine metagenome.
Materials and Methods
- Cloning and Expression of Non-Metal-Type HPO from Pseudomonas putida
The genes encoding non-metal-type HPOs were amplified directly from four strains of
(ATCC 11172, 12633, 13797, and 17472) genomic DNAs. Two oligonucleotides (forward primer A, 5’-ACAGGAGACCGGACCATGAGCTACGTCAC-3’; reverse primer B, 5’-TCAACTACGAATGAACGCGACCAAATCCGC-3’) were designed to amplify all open reading frame of non-metaltype HPOs on the genome DNAs of
strains. PCR was performed with a KOD plus PCR kit (Toyobo) with the condition of first five cycles of 94℃ for 30 sec, 43℃ for 2 sec, and 74℃ for 1 min and following with 25 cycles of 95℃ for 30 sec, 50℃ for 2 sec, and 74℃ for 1 min. Approximately 0.9 kbp PCR-amplified fragments were purified from 2% agarose gel using the Qiagen Extraction kit (Qiagen) and directly inserted into the
cII sites of the pUC119 cloning vectors (pHPO11172, pHPO12633).
- Characterization of HPOs from Pseudomonas putida
harboring pHPO11172 was cultivated overnight in 1.6L of LB medium at 30℃ with 0.2 mM IPTG addition (16h).
cells were disrupted by using BugBuster Protein Extraction reagent (Novagen) and centrifuged at 10,000 ×
for 15 min. The obtained cell-free extract was diluted with 50 mM Tris-HCl buffer (pH 8.0) containing 5% (w/v) glycerol and 1 mM phenylmethylsulfonylfluoride and used as the crude enzyme extract for further chromatography. The crude extract (48 ml) was loaded onto an anion-exchange column (DEAE-5PW, 21.5 mm × 15 cm) and washed with the same buffer, and separation was done by a linear gradient of NaCl from 0 to 1 M at 5 ml/min. The active fractions (8 ml) that were concentrated by ultrafiltration using Biomax-10 (Millipore) were then loaded onto a gel-filtration column (TSKgel G3000SW, 21.5 mm × 30 cm) in 10 mM sodium phosphate buffer (pH 7.0), and separation was done at 5 ml/min. The homogeneity of the purified HPO enzyme was evaluated by SDS-PAGE (12.5% acrylamide slab gel, 1 mm thick) by the method of Laemmli
. The protein band was stained with Coomassie Brilliant Blue R-250.
- Recovery of Metagenomic DNA from Seawater
Metagenomic DNA was isolated using seawater (20 L) sampled from Kamaishi, Japan and stored at 4℃ until use. After filtering two times with filter papers (1 m × 47 mm, GV type, Millipore) crude seawater DNA samples were then treated with 10 mM Tris-HCl buffer (pH 8.0) with 200 L of proteinase K (10 mg/ml). After incubating for 30 min at 37℃, 7.5 ml of lysing solution containing 100 mM Tris-HCl buffer (pH 8.0), 0.3 M NaCl, 20 mM EDTA, and 2% sodiumdodecylsulfate was added and extraction was done with a phenol-chloroform solution. The crude metagenomic DNA was ethanol precipitated and dissolved in TE buffer for further study as the marine metagenome template.
- Construction of HPO Cassette PCR Libraries and Screening of Active Genes
Cassette PCR primers used in this study consisted of three parts of DNA primers. We first selected the well-conserved domains of the HPO enzyme family genes in the N-terminal region (FHHGWP) and C-terminal region (DDQIVP) for designing degenerate central oligonucleotide primers for the forward direction (CF) and central oligonucleotide primers for the reverse direction (CR), respectively, in order to amplify various HPO genes from environmental genomic DNA samples. We also prepared four oligonucleotide primers (5F, 5R, 3F, and 3R) for amplifying 5’-arm and 3‘-arm fragments respectively based on the HPO gene in pHPO-11172 from
ATCC11172 as the main template, as shown in
. In order to obtain the full open reading frame of chimeric HPO genes with various central fragments of marine HPOs, the 5R and 3F primers were designed complementary to the 5’-terminal part of CF and CR, respectively. After amplifying central fragments from seawater genomic DNA samples by using the CF and CR primers, cassette oligonucleotides A and B prepared by 5F/3R and 3F/3R were mixed together with 5F and 3R primers of the second PCR for constructing full-length HPO hybrid genes, as shown in
B. The finally obtained full-length PCR product of approximately 0.8 kb was restricted with
dIII restriction enzymes and cloned into pHPO11172 plasmid vector pretreated with the same restriction enzymes. After transforming
JM109 strain with the library, the transformants were cultivated on LB plates containing ampicillin as antibiotics to construct the HPO cassette PCR metagenomic library. In order to select active HPOs from libraries, a high-throughput system was constructed. Colonies harboring the chimeric HPO gene were picked using an automatic colony picker (QARRAY lite, X2601, Genetix), transferred into 2 ml of ampicillin-containing LB medium dispensed in 96-deep-square well plates, and the plates were incubated overnight at 37℃ using a plate orbital shaker (Titramax 1000; Heidolph). Cultures were harvested by centrifugation (2,500 rpm for 10 min) and frozen at -80℃ for 60 min. For the HPO assay, cells were suspended in 1,000 μl of 0.5 mM Na-succinic acid buffer (pH 5.0) containing 20 μM of NaBr, 1 M of phenol red, and 10 M of H
, and incubated at 37℃ for 30 min. The increase in absorbance at 595 nm (purple color) was recorded by a spectrophotometer (SpectraMax Plus; Molecular Devices) to determine the amount of bromophenol blue produced by the HPO activity.
Oligonucleotides used in this study for the cloning and cassette PCR.
Oligonucleotides used in this study for the cloning and cassette PCR.
- Bioconversion of Chlorodimedone
Transformants showing HPO conversion activity in the HPO chimeric library screening were selected and used to inoculate 5 ml of ampicillin-containing LB medium dispensed in 24-deepsquare well plates. The plates were incubated, and cells grown in the plates were collected and suspended in 0.5 mM Na-succinic acid buffer (pH 5.0) as described above. After centrifugation, the cells were washed with cold 0.85% (w/v) NaCl solution and frozen at -80℃ for 1 h. For the synthesis reaction, the cells were mixed with 2.5 ml of substrate solution comprising glycine and 3,4-dihydroxybenzaldehyde; the pH of the substrate solution was adjusted to 6.5 with 1 N HCl. After mixing by vigorous vortexing, the synthesis of
-DOPS was carried out at 37℃ for 5 h. After the reaction, the
cells were discarded by centrifugation (10,000 ×g for 10 min) and the obtained supernatant was analyzed by HPLC.
Results and Discussion
- Cloning and Activity of Non-Metal HPO from Pseudomonas putida
To obtain the sequence information of non-metal HPO genes for further cassette PCR template construction, we selected four strains of
(ATCC 11172, 12633, 13797, and 17472) since
IF-3 currently is already known for possessing a non-metal HPO gene. When PCR was done on the genomic DNAs of these four strains, the PCR product was amplified on the genomic DNA of
ATCC11172 and ATCC 12633 only. Although we tried to changed the PCR conditions, no amplifying products were found from
ATCC 13797 and 17472. When the PCR products were sequenced, the cloned HPO gene from
ATCC11172 and ATCC 12633 showed each single open-reading frame of 828 base pairs coding for 276 amino acids, respectively, and comparison of these two genes with
IF-3 sequences showed 87% similarities. Almost identical similarity between the two obtained genes was shown, as 97%. The comparison of the deduced amino acids of these two genes from
ATCC11172 and ATCC 12633 with
IF-3 sequences showed 93% and 98% similarities, respectively. From the analysis of these sequences, the genes from
ATCC11172 and ATCC 12633 contained significantly conserved glycine serine motifs that are typically shown in the bacterial haloperoxygenase family (
, Gly95-X-Ser-X-Gly99). Moreover, we could identify Ser 97, Asp227, and His256, which are usually found in the non-metal haloperoxygenase active site. The recombinant
JM109, transformed with pHPO-11172 under the control of the tac promoter, was induced with 0.2 mM IPTG for 9 h to overproduce soluble active recombinant HPO (
, lane 1). The recombinant HPO was purified as a single band, as shown in
lane 4, through two-step chromatography as described above. The molecular mass estimated by SDS-PAGE of the purified HPO was 30 kDa, as expected. When the purified enzyme was examined for HPO activity as shown described Materials and methods, bromophenol blue was produced from phenol red as the substrate by HPO activity (
Analysis of the purified HPO enzyme. (A) SDS-PAGE of purified HPO enzyme expressed in E. coli harboring pHPO11172 obtained from P. putida ATCC11172. The purified HPO enzyme from E. coli harboring pHPO11172 was separated by 12.5% polyacrylamide gel and analyzed by Coomassie brilliant blue staining solution. Lane M, molecular mass standard; lane 1, whole-cell extract of the recombinant E. coli harboring pHPO11172; lane 2, crude enzyme after anion-exchange chromatography; lane 3, crude enzyme after gel filtration; lane 4, the purified enzyme. (B) Reactions catalyzed by the purified HPO enzyme. 1, phenol red; 2, phenol red blue; 3, HPO enzyme reaction.
- Construction and Isolation of Novel HPO Clones from Seawater Metagenomic Chimera Library
Since the cloned HPO (pHPO-11172) from
ATCC11172 was confirmed for the desirabed activity of HPO, we designed and constructed a cassette PCR library in combination with high-throughput screening by using seawater in order to find more novel HPO genes directly without strain screening procedure, as described in Materials and Methods. We first designed degenerated primers, CF and CR, by comparison of the deduced amino acid sequences from HPO sequences from various sources registered in DDBJ/GenBank. Multi-alignment for comparison of the HPO enzyme family genes revealed well-conserved amino acid sequences in the N-terminal region (FHHGWP) and C-terminal region (DDQIVP). By using these degenerate primers, we were able to successfully amplify HPO genes from seawater metagenomic DNA pools. The obtained PCR products were digested with
HI endonucleases for direct cloning and expression and recovered from a 12% agarose gel. The purified
RI-BamHI restricted PCR products were cloned into the pHPO11172 plasmid vector and the resulting plasmid was transformed into
. The obtained clones were grown on LB plates containing ampicillin. Approximately 600 transformants were screened for increase in the absorbance at 595 nm (purple color) as increasing bromophenol blue produced by the HPO activity, as described above, identifying 113 active clones. Although most of the clones showed a decreased or equal HPO activity compared with clone pHPO11172, 20 mutants (named clones A2, A4, A6, A9, A12, A15, A18, A21, A23, A27, B1, B2, B5, B6, B7, B8, B10, B27, B28, and B29) obtained in the first screening round displayed enhanced HPO activities compared with the wild-type HPOs (
). As shown in
, we sequenced the obtained 20 clones and their phylogenetic relationship was examined, revealing seawater HPOs were able to group into three groups; A, B as the major group and C as the minor group. Major cluster A and B contained 7 or 6 chimera genes each, showing higher homology genes with
strains. The lowest homology through all groups with
ATCC11173 was the HPO A9 clone, showing almost 66% identity and homology with 78%. The B27 HPO clone included in group A showed almost similar identity of 97% and 98% homology with the HPO gene from
ATCC11173. However, clones in group B, including CS19 HPO clone, showed relatively higher homology of approximately 80% with 70% identity as shown in
Construction scheme of the functionally active chimeric HPO library from the seawater metagenome based on pHPO11172 derived from P. putida ATCC11172. (A) Preparation of the metagenomic DNA pool from the seawater environmental sample. (B) First step for cassette PCR. In order to get the 5’-arm and 3’-arm, CF and CR were designed based on the amino acid sequences of HPO gene-cloned pHPO11172 derived from P. putida ATCC11172. (C) Second step for cassette PCR. The three fragments including the 5’-arm, 3’-arm, and central fragments, from the seawater environmental sample was used for functional assembly of chimeric HPO. (D) Chimeric HPO library construction and high-throughput screening by using a 96-well plate.
Multiple alignment of the obtained chimeric HPOs.
HPO activity of the obtained chimeric HPOs. The obtained transformants were screened by increased absorbance of bromophenol blue at 595 nm (purple color), which was produced by the obtained chimeric HPOs from A2 to B29. Crude extract of E. coli harboring pHPO11172 from P. putida ATCC11172 used as control (P).
Phylogenetic tree of chimeric HPOs. A phylogenetic tree of the amino acid sequences of chimeric HPOs obtained from cassette PCR was analyzed by using the ClustalW web program (http://www.ddbj.nig.ac.jp/search/clustalw-e.html).
- Bioconversion of Chlorodimedone Using Novel HPOs
The time course of bioconversion of chlorodimedone (2-chloro-5,5-dimethylcyclohexhexan-1,3-dione) that is extremely reactive to electrophilic substitution by HPO enzyme was examined to confirm the hybrid HPO genes. This method can quantify HPO’s ability to oxidize halonium such as KBr or KCl as substrate, but does not guarantee its efficacy in brominating inactivated aromatics. The whole cells of
without the HPO gene, as negative control, did not convert chlorodimedone constantly for 200 min, showing no HPO activity as expected (data not shown). Moreover, wild-type HPO from
ATCC11173 showed relatively slow bioconversion activity (as shown in
) on the substrate halonium KBr or KCl during 200 min. Compared with wild-type HPO,
containing clone A12 HPO that selected most higher enzyme activity clones, bioconverted most of the substrate within 180 h. This result showed that novel HPOs obtained in this study from environmental sources of marine seawater are more effective than the wild-type HPO for the efficient conversion of substrate halonium KBr or KCl, strongly indicating that the cassette PCR method used in this study is a very useful approach to achieve various useful genes from environmental sources. We are currently trying to enhance the HBO activity by directed evolution by using these HBO genes.
Bioconversion of chlorodimedone using the novel HPO. Symbols: ○, E. coli containing pHPO11172 from P. putida ATCC11172 with halonium KBr; △, E. coli containing pHPO11172 from P. putida ATCC11172 with halonium KCl; ●, E. coli containing clone A12 HPO with halonium KBr; ▲, E. coli containing clone A12 HPO with halonium KCl.
In conclusion, we report that cloning of various novel non-heme-type HPO hybrid enzymes from a seawater metagenome was successfully done. The obtained novel HPO hybrid enzymes showed various diverse gene pools with enhanced HPO activity. These results strongly indicate that the cassette PCR method used in this study is a very useful approach to achieve various useful genes from environmental sources.
This work was supported by the New Energy and Industrial Technology Development Organization (NEDO), and also partially by the Sunchang Country (High Value-Added) Food Technology Development Program, Ministry for Food, Agriculture, Forestry and Fisheries, Korea (number 311036-3).
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