Patent Grant
8129180
The present invention relates to a protein exhibiting activity of a pyrethrin biosynthetic enzyme, a gene encoding thereof, and vector bearing the gene.
Pyrethrin which is a secondary metabolite contained in pyrethrum exhibits excellent insecticidal activity against insects as well as being an ideal feature as an insecticidal constituent where toxicity against mammals is low, and is widely used for mosquito coils, and insecticide sprays and powders. Recently, demand for pyrethrin has decreased because of the remarkable development of synthetic pyrethroid. However, pyrethrin still has a high utility value as a plant-derived, and environmentally friendly material for insecticides, and further investigation has continued to a point where pyrethrin can be obtained inexpensively and effectively. In particular, the existence value of the above pyrethrin, a secondary metabolite, has been emphasized again, because of increasing oil prices, which is a raw material of synthetic pyrethroid, and the like.
Pyrethrin is mainly extracted from the flower part of pyrethrum. However, the growth duration of pyrethrum until flowering is very long, over three years. It is considered that selection and breeding of high-producing strains of pyrethrum and promotion of pyrethrin biosynthesis in plant cells of the same or different species have a beneficial effect as a means for increasing the efficiency of pyrethrin production.
Pyrethrin has an ester-bonded structure between chrysanthemic acid that is a monoterpene carboxylic acid and rethrolones (alcohols), which is a metabolite of fatty acid oxidation (
Methods for increasing efficiency of the above-described biosynthesis of pyrethrin include use of genes involved in the biosynthesis. In order to implement biosynthesis of pyrethrin, isolation and identification of the relevant gene is crucial.
Meanwhile, various ester compounds produced by plant cells are biosynthesized by catalysis of acyltransferase from CoA thioester of carboxylic acid (acyl-CoA, RCO-S-CoA) and alcohol (R′—OH) as substrates (
As an example of such acyltransferase in the pyrethrin biosynthesis, existence of chrysanthemoyl/pyrethroyl transferase (pyrethrin biosynthetic enzyme) which uses (1R)-trans-chrysanthemoyl-CoA and (S)-pyrethrolone as substrates have been predicted, however, there has been no isolated and specific composition based on an amino acid sequence, and naturally a gene encoding the protein based on such an amino acid sequence is not particularly sought.
Meanwhile, Japanese Patent Application Publication No. H9-504684 discloses an amino acid sequence of chrysanthemyl diphosphate synthase, an enzyme that can catalyze synthesis of chrysanthemyl diphosphate, which is adopted as a raw material for chemical synthesis of pyrethrin, and a sequence of a gene coding a protein based on such an amino acid sequence. However, there has been neither disclosure nor suggestion about the gene encoding the enzyme per se, which can catalyze the above pyrethrin biosynthesis, and the gene coding protein based on such an amino acid sequence. As obvious from the situation in the conventional art, elucidation of the gene encoding the above enzyme protein through identification of the enzyme involved in the pyrethrin biosynthesis, and effective biosynthesis of pyrethrin based on knowledge of genetic engineering have not been achieved.
[Patent Document 1] Japanese patent application publication No. H9-504684
[Non-Patent Document 1] R. Kalscheuer and A. Steinbuchel, A novel bifunctional wax ester synthase/acyl-CoA: diacylglycerol acyltransferase mediates wax ester and triacylglycerol biosynthesis in Acinetobacter calcoaceticus ADP1. J. Biol. Chem. 278:8075-8082 (2003)
[Non-Patent Document 2] J. Luo et al., Convergent evolution in the BAHD family of acyl transferases: identification and characterization of anthocyanin acyl transferases from Arabidopsis thaliana. Plant J. 50:678-695 (2007)
The present invention aims to determine amino acid sequences of the enzyme involved in pyrethrin biosynthesis and a base sequence of the gene thereof, and to construct vectors bearing the gene and transformants, as well as to provide methods for effectively producing pyrethrin by applying such creative techniques to plants with faster growth.
In order to solve the above issues, inventors of the present invention purified a pyrethrin synthesis enzyme protein by using the pyrethrum flower as a raw material and pyrethrin I synthesis activity as an indicator, and by performing crude fractionation of a protein and crude purification by batch method using hydrophobic resin, and then purification with a predetermined combination of chromatography; and finally analyzed an internal amino acid sequence and amino terminal sequence of the relevant protein as is described below. RACE-PCR was performed and a polynucleotide fragment of an unknown part of the sequence was amplified using a cDNA library obtained from a flower part of pyrethrum as temperate and degenerate primers designed based on crude amino acid sequences, which have been clarified through the above analysis.
An amplified polynucleotide fragment of the base sequence was analyzed with a DNA sequencer. As a result, a full length gene sequence of a pyrethrin biosynthetic enzyme, including a sequence shown in
The present invention adopts the following constitution:
(1) A pyrethrin biosynthetic enzyme, in which raw material of the enzyme is a crude enzyme solution prepared from a pyrethrum flower;
wherein (1R)-trans-chrysanthemoyl-CoA and (S)-pyrethrolone are used as substrates for a test to confirm an active function of the pyrethrum biosynthetic enzyme;
wherein the pyrethrum biosynthetic enzyme is produced by a purification procedure including the sequential steps of:
obtaining precipitate of crude protein fractionation with ammonium sulfate precipitation;
crudely purifying the precipitate by batch method using hydrophobic resin;
purifying the enzyme solution obtained by crude purification, with anion-exchange chromatography;
purifying with hydrophobic chromatography; and
purifying with gel filtration,
and wherein produced enzyme is a protein with a molecular weight of approximately 40,000.
(2) A protein of the following (i) or (ii), corresponding to an enzyme extractable from plant bodies of producing pyrethrin:
(i) a protein consisting of an amino acid sequence shown in Sequence No. 1; or
(ii) a protein consisting of an amino acid sequence including one or more of a substitution, deletion, insertion, and/or addition of an amino acid in the amino acid sequence shown in Sequence No. 1, in which the protein exhibits activity of a pyrethrin biosynthetic enzyme.
(3) A protein consisting of an amino acid sequence shown in Sequence No. 2, or a protein consisting of an amino acid sequence shown in Sequence No. 3, or a protein consisting of an amino acid sequence shown in Sequence No. 4, corresponding to an enzyme extractable from plant bodies producing pyrethrin.
(4) A gene encoding a protein of the following (i) or (ii), corresponding to an enzyme extractable from plant bodies producing pyrethrin:
(i) a protein consisting of an amino acid sequence shown in Sequence No. 1; or
(ii) a protein consisting of an amino acid sequence including one or more of a substitution, deletion, insertion, and/or addition of an amino acid in the amino acid sequence shown in Sequence No. 1, in which the protein exhibits activity of a pyrethrin biosynthetic enzyme,
(5) A gene according to (4), consisting of abase sequence shown in Sequence No. 5; wherein the gene encodes the protein of (i).
(6) A gene according to (5), including a base sequence from 85 to 1179 of the base sequence shown in Sequence No. 5 as an open reading frame.
(7) A gene encoding any of the following proteins: a protein consisting of an amino acid sequence shown in Sequence No. 2, or a protein consisting of an amino acid sequence shown in Sequence No. 3, or a protein consisting of an amino acid sequence shown in Sequence No. 4, corresponding to an enzyme extractable from plant bodies producing pyrethrin.
(8) A vector bearing a gene according to any one of (4) to (7).
As is described in a section of an embodiment of the invention,
Amino acid sequences based on extraction and separation of a pyrethrin biosynthetic enzyme, a process leading to a procedure of base sequence determination of the gene thereof, vectors bearing the gene based on the thus determined base sequence, and transformants introducing such vectors of the present invention are explained as follows. However, the above-described constitution of (1) to (8) is not limited to the following embodiments of the present invention, but embodiments that can be easily substituted and considered from such embodiments are also included.
It is essential to ensure proteins composing a pyrethrin biosynthetic enzyme in advance of the embodiment according to steps in order of the following (a) to (d).
Inventors of the present invention performed fractionation and purification of a pyrethrin biosynthetic enzyme using a crude enzyme solution prepared from a pyrethrum flower as a raw material, (1R)-trans-chrysanthemoyl-CoA and (S)-pyrethrolone as substrates to confirm an active function of the pyrethrin biosynthetic enzyme, and pyrethrin I synthesis activity as an indicator. In other words, as is described in the above (1), fractionation, crude purification and purification were performed in order of crude fractionation by ammonium sulfate precipitation, crude purification by batch method using hydrophobic resin, purification with anion-exchange chromatography, hydrophobic chromatography and gel filtration column, and enzyme reactions to the above-described substrate were performed at each step. An enzyme was obtained by fractionating purified fractions concluded to have pyrethrin biosynthetic enzyme activity. A protein band of a molecular weight 40,000, which is expected to contribute to esterification reaction as an enzyme, was detected by SDS-PAGE of the enzyme (
A produced pyrethrin biosynthetic enzyme protein was successfully secured by transferring the protein obtained by confirming the above band onto a PVDF membrane.
Meanwhile, a specific process through confirmation of the above band is described later in Example 1.
(a) Analysis of Crude Amino Acid Sequences of Enzyme Protein
A purified enzyme secured as is described above was digested with trypsin, a protease, and fragmented into peptides. Then, digested peptide fragments were separated with HPLC, and amino acid residues of the separated peptides were singly disassembled and dissociated from the amino terminal end by the Edman method.
Amino acid residues were determined by analyzing produced phenylthiohydantoin derivatives with HPLC. This series of processes was performed using a peptide sequencer which is a special analytical instrument automated to repeat this reaction and analysis procedures.
As is described above, crude amino acid sequences constituting the pyrethrin biosynthetic enzyme protein were successfully obtained.
(b) Design of Primers and Determination for Gene Sequence of a Full-Length cDNA
Degenerate primers were designed based on base sequences estimated from determined amino acid sequences.
Unknown base sequences between known sequences were determined with PCR (Polymerase Chain Reaction) adopting the above primers (for example, four pairs of degenerate primers).
Next, a polynucleotide serving as an adaptor was previously added to DNA used as substrate of PCR. RACE-PCR was performed simultaneously using primers designed on an adaptor sequence and a known sequence, and DNA fragments containing sequences of both ends were amplified. The amplified DNA fragments were sequenced with DNA sequencer, and a full-length sequence was determined.
The full length sequence of the pyrethrin biosynthetic enzyme determined as is described above is shown in Sequence No. 5 of
(c) Determination for a Full-Length of Amino Acid Sequence
An amino acid sequence coded by the above-described gene corresponding to the above base sequence of Sequence No. 5 was determined.
An amino acid sequence of a pyrethrin biosynthetic enzyme determined in the present invention is shown in Sequence No. 1 of
Even though an amino acid sequence contains one or more of a substitution, deletion, insertion, and/or addition of an amino acid in the amino acid sequence of Sequence No. 1, the protein is included in proteins of a pyrethrin biosynthetic enzyme of the present invention as long as the protein can be extracted from plants containing the pyrethrin biosynthetic enzyme using similar techniques and process with an amino acid sequence of Sequence No. 1, and the protein exhibits a function as a pyrethrin biosynthetic enzyme, because the protein is able to function in the same manner as the enzyme protein shown as Sequence No. 1. Furthermore, as is described in (3), amino acid sequences according to the following sequences also correspond to the protein as a pyrethrin biosynthetic enzyme of the present invention:
As is described later in Example 1, an amino acid sequence shown as Sequence No. 2 is extracted from a pyrethrum flower, therefore it can be naturally purified. Enzymes according to amino acid sequences shown as Sequence Nos. 3 and 4 were obtained by introduction of a vector bearing a sequence encoding the amino acid sequence of Sequence No. 1 into E. coli and expression of the protein in E. coil. However, when an enzyme can be expressed in E. coli as a host, it is highly expected that the enzyme also actually exists as a pyrethrin biosynthetic enzyme in plants, which is able to produce pyrethrin. If the enzyme exists, the enzyme can be obviously extracted and purified from the above plant body using similar techniques and process with the amino acid sequence of Sequence No. 2.
Because protein according to the amino acid sequence of above (2) having activity for pyrethrin biosynthesis exerts the same function as such a protein as well as protein according to the amino acid sequence of Sequence No. 1, and included as a producible protein in the technical concept and the scope of the present invention, the gene of the present invention also consequently includes not only the above gene of (4) coding amino acid sequence shown as Sequence No. 1 but also the genes of above (5) and (6) encoding the above protein of (2) into the technical concept of the present invention. Furthermore, the gene of above (7) encoding a protein of amino acid sequences of the Sequence Nos. 2, 3, and 4 of above (3) is also included in the technical concept of the present invention.
An amino acid sequence database of known proteins was searched for sequences similar to Sequence No. 1. As a result, such a sequence was not a predicted known acyltransferase, however, some GDSL-motif lipases were similar to the amino acid sequence. Therefore, the above pyrethrin biosynthetic enzyme proteins of (2) and (3) are expected to be proteins similar to GDSL-motif lipase.
In order to confirm biosynthesis activity of the pyrethrin biosynthetic enzyme genes of above (4), (5), (6), and (7), it is appropriate for example, to insert these genes into a vector DNA first, introduce the same to E. coli, express the pyrethrin biosynthetic enzymes as a fusion protein with a histidine-tag in E. coli, purify the expressed pyrethrin biosynthetic enzyme with Ni-NTA affinity chromatography, and then evaluate activity using (1R)-trans-chrysanthemoyl-CoA and (S)-pyrethrolone as a substrate and pyrethrin I synthesis activity as an indicator.
(d) Production of Vector and Transformants Introducing the Vector
The vector of above (8) is produced by inserting any of the genes of above (4), (5), (6) and (7), and exhibits pyrethrin biosynthetic enzyme activity. The above vector can express the inserted gene or gene fragment into hosts such as plants and microorganisms by being introduced into the hosts by well-known transformation methods.
Also, a transformant having the vector of above (8) introduced therein refers to a transformant which introduces a gene or gene fragment related to pyrethrin biosynthesis into a host. A term of “having the above vector introduced” used herein refers to that the gene inserted into the vector is introduced into a host in the manner capable of expressing such a gene using well-known genetic engineering techniques.
Methods for introducing genes include, but are not limited to, a transformation method with Agrobacterium, a particle gun method, a microinjection method and an electroporation method.
When the transformation method with Agrobacterium is used, the relevant gene is inserted into a Ti plasmid vector, the vector is introduced into Agrobacterium, and then the Agrobacterium is infected to the appropriate plant. A tumor (crown gall) is formed at the site of gene introduction. After elimination of Agrobacterium, many plant bodies regenerated from the crown gall are evaluated on activity, and plant bodies that highly express the pyrethrin biosynthetic enzyme can be selected.
Such transformants can express genes related to pyrethrin biosynthesis in their bodies. Therefore, a pyrethrin biosynthetic enzyme may be produced in large quantities by constructing transformants, which have the vector of above (8) bearing a promoter to express such an enzyme in large quantity by using bacterial chromosome and/or chloroplast of plants, blue-green algae, yeasts, or bacteria such as E. coli as a host.
Since the above-described vector contains a gene (or a gene fragment) of a pyrethrin biosynthetic enzyme derived from pyrethrum, plant chromosome and/or chloroplast are preferred as hosts for construction of transformants, in particular, chromosome and/or chloroplast of asteracea plants, which belong to the same family as pyrethrum, are more preferable. Such asteracea plants include, but are not limited to, marigold, African marigold, calendula and zinnia.
Above-described plants also include not only entire plant bodies but also a part of the plant bodies, for example, a leaf, seed, tuber, graft and the like. Furthermore, the above plants also include plant materials (a part of a plant including the flower, stem, fruit, leaf, and root) with growth potential, such as plant tissue, protoplast, cell, callus, organs, plant seed, germ, pollen, ovum, and zygote, derived from genetically-modified plants and progeny thereof transformed previously.
Pyrethrin can be produced by using either one of the protein of above (2) or the above transformants. The present invention provides such a pyrethrin production method. According to the method, pyrethrin can be produced effectively and easily using the above asteracea plants or other plants with obviously faster growth than pyrethrum, and thus, social demand seeking safe and environment-friendly insecticide could be greatly fulfilled by the present invention.
Hereinafter, details of the present invention will be concretely described by referring to Examples as follows.
An enzyme was purified from pyrethrum flowers according to procedures shown in
Detailed explanations regarding the procedures are provided below.
Buffer Compositions Used for Purification
Compositions of buffers used for purification are shown in Tables 1 to 6.
Reaction of Pyrethrin Biosynthetic Enzyme
An enzyme reaction was assayed in each purification step, and enzyme activity of a pyrethrin biosynthetic enzyme in purified fractions was evaluated. A reaction was performed at 25° C. for 1 hour using the reaction composition shown in Table 7 as follows. After the enzyme reaction, 200 μl of hexane was added to the reaction solution. The organic phase was separated and collected therefrom, and 10 μl for each sample was used for HPLC analysis.
Activity Measurement of Pyrethrin Biosynthesis Enzyme with HPLC
HPLC analysis was performed using SCL-10A VP (programming unit), DGU-14A (deaeration unit), LC-6AD (pump), CTO-10AS VP (sample injection and column oven unit), and SPD-10AV VP (optical detector) produced by SHIMADZU, and data were processed with CLASS-VP software from SHIMADZU. A Cadenza C-18 column from IMTAKT (0.46 cm I.D.×10 cm L.) was used, and absorption at 230 nm was measured at 40° C., at a flow rate of 1 ml/min. Acetonitrile:H2O (65:35) was used as mobile phase.
HPLC analysis results are exemplified in
Preparation of Crude Enzyme
A crude enzyme was prepared from 500 g of pyrethrum flower buds according to the following procedures. Ice cold 1.5 L of Buffer A and polyvinyl pyrrolidone (1/10 volume (w/v) of Buffer A) were added to the buds, and the buds were homogenized using a blender. A homogenate was filtered with a four-layered cheesecloth, and filtrate was centrifuged at 8,000×g for 20 min at 4° C. Collected supernatant was mixed with 100 mL of DOWEX (1×4, 100-200 Cl FORM) (Muromachi technos CO., LTD), stirred with a stirrer for 10 min, and centrifuged at 8,000×g for 20 min at 4° C. Supernatant was collected as a crude enzyme solution, and provided for further purification.
Fractionation by Ammonium Sulfate Precipitation
Ammonium sulfate was ground using a pestle and a mortar in advance, and resolved and mixed using a stirrer by portions into the crude enzyme solution obtained by the above-described preparation to a concentration of 30% saturation of ammonium sulfate. After allowing it to stand for 30 min, the solution was centrifuged at 8,000×g for 20 min at 4° C. (g represents gravitational acceleration). Supernatant was collected, and ammonium sulfate was added so that a concentration of ammonium sulfate becomes 80% saturation. After allowing it to stand for one night, the solution was centrifuged at 8,000×g for 20 min at 4° C. and the enzyme fraction was obtained as precipitate.
Crude Purification by Batch Method Using Hydrophobic Resin
The precipitate obtained by the above-described fractionation was suspended into Buffer F, and Phenyl Sepharose (GE Healthcare) was added to the solution. After mixing using a stirrer for 30 min, the mixture was separated using a Buchner funnel. Phenyl Sepharose remaining in the Buchner funnel was washed with 500 mL of Buffer F, and then protein absorbed by the resin was eluted with 500 mL of Buffer B. Ammonium sulfate was added into the collected elute to 1 M of concentration, and then 20 mL of Phenyl Sepharose (GE Healthcare) was added to the solution. After mixing using a stirrer for 30 min, the mixture was transferred and settled in Econo-Column (Bio-Rad), protein absorbed by the resin was eluted with 50 mL of Buffer B. Eluate was transferred into a cellophane dialysis tubing, dialyzed in 2 L of desalting buffer for 2 hours by stirring the buffer using a stirrer, and followed by another desalting after buffer change for 3 hours. The desalted enzyme solution was further purified by column chromatography using an AKTA explorer (GE Healthcare) system.
Purification with Anion-Exchange Chromatography
The enzyme solution obtained by the above-described crude purification with the batch method was further purified by anion-exchange chromatography using a Q Sepharose column according to the following conditions.
Purification with Hydrophobic Chromatography
The enzyme solution obtained by the above-described anion-exchange chromatography was further purified by hydrophobic chromatography using a Phenyl Superose column according to the following conditions.
Purification with Gel Filtration
The enzyme solution obtained by the above-described hydrophobic chromatography was further purified by gel filtration using a Superdex 75 column according to the following conditions.
An enzyme purified by the above-described methods was separated with SDS-PAGE, and the degree of purification was confirmed with silver staining. The result of silver staining is shown in
Procedures from analysis of the above-described crude amino acid sequence (a) or (b) to determination of a full length amino acid sequence of the pyrethrin biosynthetic enzyme obtained in Example 1, further, preparation of transformant introducing a vector bearing a gene, which codes protein of the above-described amino acid sequence, were performed as follows.
Analysis of Amino Acid Segments Including Pyrethrin Biosynthetic Enzyme Protein
The protein band obtained in Example 1 was excised from a gel, and used as a sample for analysis of an internal amino acid sequence. Also, the band of an SDS-PAGE was transferred to a PVDF membrane, detected with Coomassie Blue staining, and the excised band was used for N-terminal amino acid analysis. This series of manipulations was performed according to well-known methods.
As a result of the above-described amino acid analyses, examples of crude amino acid sequences, Sequence Nos. 6, 7 and 8, including the enzyme protein shown in
Design of Primers and Determination of a Full-Length cDNA Sequence
A full-length cDNA sequence (
Determination for a Full-Length of Amino Acid Sequence
The N-terminal amino acid sequence of the pyrethrin biosynthetic enzyme determined by the above analysis is a sequence without a portion from the N-terminal to serine (S) 27 of the amino acid sequence of Sequence No. 1, as shown in
Meanwhile, a protein with amino acid sequences of Sequence Nos. 3 and 4 is obtained by abundant expression of a gene encoding a protein with amino acid sequence of Sequence No. 1, which are integrated into a vector and introduced into E. coli. It has already been pointed out in an embodiment (c) of the present invention that this protein has a high possibility of having activity of a pyrethrin biosynthetic enzyme and to exist in plants, which is capable of producing pyrethrin.
Production of Vector and Transformant
Construction of vector and transformant bearing the above-described pyrethrin biosynthetic enzyme gene were performed as follows.
Available vector in the present invention includes existing vectors used for transformation of microorganisms, plants, and plant cells. It is substantially predictable based on common knowledge of one skilled in the art that such vectors are able to contain a constitutive or inducible promoter to express known genes; a protein of facilitating solubilization and purification of expressed protein such as a histidine-tag, glutathione S-transferase; fusion protein such as maltose-binding protein; a drug resistance gene facilitating selection of transformants, and replication origins for binary vector system of Agrobacterium in addition to a part of the above full length gene encoding the above pyrethrin biosynthetic enzyme.
Specifically, for example, pET vector (Novagen), pGEX vector (GE Healthcare), and pMAL vector (New England Biolab) can be used for introduction to microorganisms such as E. coli. Vectors appropriate for introduction into plants with Agrobacterium include pBI101 and pBI121. There is no specific limitation on the type of vectors when the vector is introduced into plant cells by electroporation or particle gun method. Also, the above drug resistance genes include resistance genes of ampicillin, kanamycin and hygromycin. As examples of the above promoters, 35S promoter derived from a cauliflower mosaic virus (constitutive promoter) or promoters of heat shock-induced proteins (inducible promoter) can be used. Replication origins include replication origins derived from Ti or Ri plasmids. It is substantially predictable that construction of these transformants is feasible based on common knowledge of one skilled in the art.
When the above-described transformants are constructed with microorganisms such as E. coli and yeast, conversion of a substance using a microbial cell system becomes possible. Furthermore, construction of the above-described transformants using asteracea plants such as African marigold, calendula and zinnia, which are known to produce a small amount of pyrethrin for less than practical use, and improvement in ability of pyrethrin synthesis enable effective production of pyrethrin in plants which grow faster than pyrethrum. Thus, this is useful for production of insecticides.
The present invention discloses amino acid sequences of enzymes related to pyrethrin biosynthesis and a base sequence of a gene thereof, and thus provides perspective that highly useful and safe pyrethrin as a raw material of insecticides can be inexpensively and effectively produced by using fast growing plants. Therefore, this indicates a possibility that the present invention can make a great contribution to the insecticide industry.
Furthermore, the present invention can be used in all industrial areas using pyrethroids for insecticides, in particular, the area of insecticidal instruments and devices using pyrethroids as active ingredients, such as mosquito/fly coils, insecticide sprays, heat and transpiration devices for liquid insecticides, and electrothermal mosquito mats.
[Sequence Listing]
| Number | Date | Country | Kind |
|---|---|---|---|
| 2008-208295 | Aug 2008 | JP | national |
| Number | Date | Country |
|---|---|---|
| A-9-504684 | May 1997 | JP |
| Number | Date | Country | |
|---|---|---|---|
| 20100062510 A1 | Mar 2010 | US |
