Studies on biosynthetic assembly line of meroterpenoids

• Project Area: Biosynthetic machinery: Deciphering and regulating the system for bioactive metabolite diversification
• Project/Area Number: 22108004
• Research Category: Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)
• Allocation Type: Single-year Grants
• Review Section: Science and Engineering
• Research Institution: The University of Tokyo
• Principal Investigator: ABE IKURO 東京大学, 薬学研究科(研究院), 教授 (40305496)
• Co-Investigator(Kenkyū-buntansha): MATSUDA Yudai 東京大学, 大学院薬学系研究科, 助教 (10720766)
• Co-Investigator(Renkei-kenkyūsha): WAKIMOTO Toshiyuki 東京大学, 大学院薬学系研究科, 准教授 (70363900) ; AWAKAWA Takayoshi 東京大学, 大学院薬学系研究科, 助教 (80609834) ; MORITA Hiroyuki 東京大学, 大学院薬学系研究科, 助教 (20416663)
• Project Period (FY): 2010-04-01 – 2015-03-31
• Project Status: Completed (Fiscal Year 2014)
• Budget Amount: ¥55,640,000 (Direct Cost: ¥42,800,000、Indirect Cost: ¥12,840,000); Fiscal Year 2014: ¥11,570,000 (Direct Cost: ¥8,900,000、Indirect Cost: ¥2,670,000); Fiscal Year 2013: ¥12,220,000 (Direct Cost: ¥9,400,000、Indirect Cost: ¥2,820,000); Fiscal Year 2012: ¥12,740,000 (Direct Cost: ¥9,800,000、Indirect Cost: ¥2,940,000); Fiscal Year 2011: ¥12,610,000 (Direct Cost: ¥9,700,000、Indirect Cost: ¥2,910,000); Fiscal Year 2010: ¥6,500,000 (Direct Cost: ¥5,000,000、Indirect Cost: ¥1,500,000)
• Keywords: メロテルペノイド / 生合成 / 遺伝子クラスター / 多様性創出 / 機能制御 / 二次代謝酵素 / 酵素 / 遺伝子 / 生合成クラスター / 生合成工学

Outline of Final Research Achievements

Meroterpenoids are hybrid natural products of both terpenoid and polyketide origin. We identified several biosynthetic gene clusters of pharmaceutically important fungal meroterpenoids, which includes that is responsible for the production of pyripyropene A, terretonin, anditomin, and andrastin, through reconstituted biosynthesis in a heterologous fungal expression system. Interestingly, the biosynthetic assembly lines are rich sources for interesting enzymes and chemistry, such as previously unknown membrane-bound terpene cyclases and multifunctional oxygenases, for the generation of structurally divergent complex fungal meroterpenoids.

Research Products

• [Journal Article] Uncovering the unusual D-ring construction in terretonin biosynthesis by collaboration of a multifunctional cytochrome P450 and a unique isomerase (2015)
  • Author(s): Matsuda, Y., Iwabuchi, T., Wakimoto, T., Awakawa, T., Abe, I.
  • Journal Title: J. Am. Chem. Soc. Volume: 137 Issue: 9 Pages: 3393-3401
  • DOI: 10.1021/jacs.5b00570
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Complete biosynthetic pathway of anditomin: nature's sophisticated synthetic route to a complex fungal meroterpenoid (2014)
  • Author(s): Matsuda, Y., Wakimoto, T., Mori, T., Awakawa, T., Abe, I.
  • Journal Title: J. Am. Chem. Soc. Volume: 136 Issue: 43 Pages: 15326-15336
  • DOI: 10.1021/ja508127q
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] A methyltransferase initiates terpene cyclization in teleocidin B biosynthesis (2014)
  • Author(s): Awakawa, T., Zhang, L., Wakimoto, T., Hoshino, S., Mori, T., Ito, T., Ishikawa, J., Tanner, M. E., Abe, I.
  • Journal Title: J. Am. Chem. Soc. Volume: 136 Issue: 28 Pages: 9910-9913
  • DOI: 10.1021/ja505224r
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Calyculin biogenesis from a pyrophosphate protoxin produced by a sponge symbiont (2014)
  • Author(s): Wakimoto, T., Egami, Y., Nakashima, Y., Wakimoto,Y., Mori, T., Awakawa,T., Ito, T., Kenmoku, H., Asakawa, Y., Piel, J., Abe, I.
  • Journal Title: Nature Chem. Biol. Volume: 10 Issue: 8 Pages: 648-655
  • DOI: 10.1038/nchembio.1573
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] An environmental bacterial taxon with a large and distinct metabolic repertoire (2014)
  • Author(s): Wilson, M. C., Mori, T., Ruckert, C., Uria, A. R., Helf, M. J., Takada, K., Gernert, C., Steffens, U. A. E., Heycke, N., Schmitt, S., Rinke, C., Helfrich, E. J. N., Brachmann, A. O., Gurgui, C., Wakimoto. T., Kracht, M., Crusemann, M., Hentschel, U., Abe, I., Matsunaga, S., Kalinowski, J., Takeyama, H., Piel, J.
  • Journal Title: Nature Volume: 506 Issue: 7486 Pages: 58-62
  • DOI: 10.1038/nature12959
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Spiro-ring formation is catalyzed by a multifunctional dioxygenase in austinol biosynthesis (2013)
  • Author(s): Y. Matsuda, T. Awakawa, T. Wakimoto, and I. Abe
  • Journal Title: J. Am. Chem. Soc. Volume: 135 Issue: 30 Pages: 10962-10965
  • DOI: 10.1021/ja405518u
  • Peer Reviewed
• [Journal Article] Reconstituted biosynthesis of fungal meroterpenoid andrastin A (2013)
  • Author(s): Matsuda, Y., Awakawa, T., Abe, I.
  • Journal Title: Tetrahedron Volume: 69 Issue: 38 Pages: 8199-8204
  • DOI: 10.1016/j.tet.2013.07.029
  • Peer Reviewed
• [Journal Article] Cloning and structure-function analyses of quinolone- and acridone-producing novel type III polyketide synthases from Citrus microcarpa (2013)
  • Author(s): Mori, T., Shimokawa, Y., Matsui, T., Kinjo, K., Kato, R., Noguchi, H., Sugio, S., Morita, H., Abe, I.
  • Journal Title: J. Biol. Chem. Volume: 288 Issue: 40 Pages: 28845-28858
  • DOI: 10.1074/jbc.m113.493155
  • Peer Reviewed
• [Journal Article] Multiplexing of combinatorial chemistry in antimycin biosynthesis expands the molecular diversity and utility (2013)
  • Author(s): Yan Y., Chen, J., Zhang, L., Zheng, Q., Han, Y., Zhang, H., Awakawa, T., Abe, I., Liu, W.
  • Journal Title: Angew. Chem. Int. Ed. Volume: 52 Issue: 47 Pages: 12308-12312
  • DOI: 10.1002/anie.201305569
  • Peer Reviewed
• [Journal Article] Identification of a key prenyltransferase involved in biosynthesis of the most abundant fungal meroterpenoids derived from 3,5-dimethylorsellinic acid (2012)
  • Author(s): Itoh, T.
  • Journal Title: ChemBioChem Volume: 13 Issue: 8 Pages: 1132-1135
  • DOI: 10.1002/cbic.201200124
  • Peer Reviewed
• [Journal Article] Terretonin biosynthesis requires methylation as essential step for cyclization. (2012)
  • Author(s): Y. Matsuda, T. Awakawa, T. Itoh, T. Wakimoto, T. Kushiro, I. Fujii, Y. Ebizuka, I. Abe
  • Journal Title: ChemBioChem Volume: 13(12) Issue: 12 Pages: 1738-1741
  • DOI: 10.1002/cbic.201200369
  • Peer Reviewed
• [Journal Article] Biosynthesis: an HR-PKS stereo surprise (2012)
  • Author(s): Abe, I.
  • Journal Title: Nature Chemical Biology Volume: 8 Issue: 4 Pages: 322-323
  • DOI: 10.1038/nchembio.924
  • Peer Reviewed
• [Journal Article] Engineering of plant type III polyketide synthases (2012)
  • Author(s): Wakimoto, I.
  • Journal Title: Methods in Enzymology Volume: 51 Pages: 337-358
  • DOI: 10.1016/b978-0-12-394290-6.00016-1
  • ISBN: 9780123942906
  • Peer Reviewed
• [Journal Article] Prenylation of a nonaromatic carbon of indolylbutenone by a fungal indole prenyltransferase (2012)
  • Author(s): Chen, J.
  • Journal Title: Organic Letters Volume: 14 Issue: 12 Pages: 3080-3083
  • DOI: 10.1021/ol301129x
  • Peer Reviewed
• [Journal Article] Biosynthetic pathway for highly structural diversity of a common dilactone core in antimycin production (2012)
  • Author(s): Yan, Y.
  • Journal Title: Organic Letters Volume: 14 Issue: 16 Pages: 4142-4145
  • DOI: 10.1021/ol301785x
  • Peer Reviewed
• [Journal Article] Novel applications of plant polyketide synthases (2012)
  • Author(s): Abe, I.
  • Journal Title: Current Opinion in Chemical Biology Volume: 16 Pages: 179-185
  • Peer Reviewed
• [Journal Article] Expression, purification, and crystallization of an indole prenyltransferase from Aspergillus fumigatus (2012)
  • Author(s): Chen, J., Morita, H., Kato, R., Noguchi, H., Sugio, S., Abe, I.
  • Journal Title: Acta Crystallogr., Sect.F : Struct.Biol.Cryst.Commun. Volume: 68 Issue: 3 Pages: 355-358
  • DOI: 10.1107/s1744309112004964
  • Peer Reviewed
• [Journal Article] Benzophenone synthase from Garcinia mangostana L.pericarps (2012)
  • Author(s): Natsajee Nualkaew, Hiroyuki Morita, Yoshihiko Shimokawa, Keishi Kinjo, Tetsuo Kushiro, Wanchai De-Eknamkul, Yutaka Ebizuka, Ikuro Abe
  • Journal Title: Phytochem. Volume: 77 Pages: 60-69
  • DOI: 10.1016/j.phytochem.2012.02.002
  • Peer Reviewed
• [Journal Article] Benzalacetone synthase (2012)
  • Author(s): Yoshihiko Shimokawa, Hiroyuki Morita, Ikuro Abe
  • Journal Title: Frontiers in Plant Science Volume: 3 Pages: 57-57
  • DOI: 10.3389/fpls.2012.00057
  • Peer Reviewed
• [Journal Article] Identification of a key prenyltransferase involved in biosynthesis of the most abundant fungal meroterpenoids derived from 3,5-dimethylorsellinic acid (2012)
  • Author(s): Itoh, T., Tokunaga, K., Radhakrishnan, E.K., Fujii, I., Abe, I., Ebizuka, Y., Kushiro, T.
  • Journal Title: ChemBioChem Volume: 13(in press)
  • Peer Reviewed
• [Journal Article] Synthesis of unnatural alkaloid scaffolds by exploiting plant polyketide synthase (2011)
  • Author(s): Morita, H., Yamashita, M., Shi, S.-P., Wakimoto, T., Kondo, S., Kato, R., Sugio, S., Kohno, T., Abe, I.
  • Journal Title: Proc.Natl.Acad.Sci.USA Volume: 108 Issue: 33 Pages: 13504-13509
  • DOI: 10.1073/pnas.1107782108
  • Peer Reviewed
• [Journal Article] Cytotoxic tetramic acid derivative produced by a plant type III polyketide synthase (2011)
  • Author(s): Wakimoto, T., Mori, T., Morita, H., Abe, I.
  • Journal Title: J.Am.Chem.Soc. Volume: 133 Issue: 13 Pages: 4746-4749
  • DOI: 10.1021/ja2006737
  • NAID: 10029237041
  • Peer Reviewed
• [Journal Article] Furan fatty acid as an anti-inflammatory component from green-lipped mussel Perna canaliculus (2011)
  • Author(s): Wakimoto, T., Kondo, H., Nii, H., Kimura, K., Egami, Y., Oka, Y., Yoshida, M., Kida, E., Ye, Y.-p., Akahoshi, S., Asakawa, T., Matsumura, K., Ishida, H., Nukaya, H., Tsuji, K., Kan, T., Abe, I.
  • Journal Title: Proc.Natl.Acad.Sci.USA Volume: 108 Issue: 42 Pages: 17533-17537
  • DOI: 10.1073/pnas.1110577108
  • Peer Reviewed
• [Journal Article] Crystallization and preliminary X-ray analysis of 4-coumarate : CoA ligase from Arabidopsis thaliana (2011)
  • Author(s): Morita, H., Mori, T., Wanibuchi, K., Kato, R., Sugio, S., Abe, I.
  • Journal Title: Acta Crystallogr., Sect.F : Struct.Biol.Cryst.Commun. Volume: 67 Issue: 3 Pages: 409-411
  • DOI: 10.1107/s1744309111002181
  • Peer Reviewed
• [Journal Article] Enzymatic formation of an aromatic dodecaketide by engineered plant polyketide synthase (2011)
  • Author(s): Wanibuchi, K., Morita, H., Noguchi, H., Abe, I.
  • Journal Title: Bioorg.Med.Chem.Lett. Volume: 21 Issue: 7 Pages: 2083-2086
  • DOI: 10.1016/j.bmcl.2011.01.135
  • Peer Reviewed
• [Journal Article] Molecular cloning, modeling and site-directed mutagenesis of type III polyketide synthase from Sargassum binderi (2011)
  • Author(s): Baharum, H., Morita, H., Tomitsuka, A., Lee, F.-C., Ng, K.-Y., Rahim, R.A., Abe, I., Ho, C.-L.
  • Journal Title: Marine Biotech. Volume: 13 Issue: 5 Pages: 845-856
  • DOI: 10.1007/s10126-010-9344-5
  • Peer Reviewed
• [Journal Article] Cytotoxic Tetramic Acid Derivative Produced by a Plant Type-III Polyketide Synthase (2011)
  • Author(s): T.Wakimoto, T.Mori, H.Morita, I.Abe
  • Journal Title: J.Am.Chem.Soc. Volume: 133(In press(掲載確定))
  • NAID: 10029237041
  • Peer Reviewed
• [Journal Article] Structure-Based Engineering of Aloe arborescens Octaketide Synthase (2011)
  • Author(s): K.Wanibuchi, H.Morita, H.Noguchi, I.Abe
  • Journal Title: Bioorg.Med.Chem.Lett. Volume: 21 Pages: 2083-2086
  • Peer Reviewed
• [Journal Article] Structural Basis for The One-pot Formation of The Diarylheptanoid Scaffold by Curcuminoid Synthase from Oryza sativa (2010)
  • Author(s): H.Morita, K.Wanibuchi, H.Nii, R.Kato, S.Sugio, I.Abe
  • Journal Title: Proc.Natl.Acad.Sci.USA Volume: 107 Pages: 19778-19783
  • Peer Reviewed
• [Journal Article] A Structure-Based Mechanism for Benzalacetone Synthase from Rheum palmatum (2010)
  • Author(s): H.Morita, Y.Shimokawa, M.Tanio, R.Kato, H.Noguchi, S.Sugio, T.Kohno, I.Abe
  • Journal Title: Proc.Natl.Acad.Sci.USA Volume: 107 Pages: 669-673
  • Peer Reviewed
• [Journal Article] Structure-Based Engineering of Benzalacetone Synthase (2010)
  • Author(s): H.Morita, K.Wanibuchi, R.Kato, S.Sugio, I.Abe
  • Journal Title: Bioorg.Med.Chem.Lett. Volume: 20 Pages: 5099-5103
  • Peer Reviewed
• [Presentation] 複雑骨格天然物の生合成マシナリーの解明 (2015)
  • Author(s): 阿部郁朗
  • Organizer: 日本化学会 第95春季年会 イブニングセッション
  • Place of Presentation: 千葉県船橋市
  • Year and Date: 2015-03-26
  • Invited
• [Presentation] Engineered Biosynthesis of Medicinal Natural Products (2014)
  • Author(s): 阿部郁朗
  • Organizer: 2014 Fall Annual Convention of Pharmaceutical Society of Korea
  • Place of Presentation: Gyeongju, Korea
  • Year and Date: 2014-10-23 – 2014-10-24
  • Invited
• [Presentation] 天然物の生合成工学 (2014)
  • Author(s): 阿部郁朗
  • Organizer: 第４９回天然物化学談話会
  • Place of Presentation: 岡山県倉敷市
  • Year and Date: 2014-07-02 – 2014-07-04
  • Invited
• [Presentation] Biosynthesis of Fungal Meroterpenoids (2014)
  • Author(s): 阿部郁朗
  • Organizer: 97th Canadian Chemistry Conference and Exhibition
  • Place of Presentation: Vancouver, British Columbia, Canada
  • Year and Date: 2014-06-01 – 2014-06-05
  • Invited
• [Presentation] Engineered Biosynthesis of Medicinal Natural Products (2014)
  • Author(s): 阿部郁朗
  • Organizer: 9th International Symposium on Chromatography of Natural Products
  • Place of Presentation: Medical University of Lublin, Lublin, POLAND
  • Year and Date: 2014-05-26 – 2014-05-29
  • Invited
• [Presentation] メロテルペノイド生合成マシナリーの解明と制御 (2014)
  • Author(s): 阿部郁朗
  • Organizer: 日本薬学会 第134回年会（熊本）シンポジウム 「天然物化学の新潮流と将来への展望」
  • Place of Presentation: 熊本県熊本市
  • Invited
• [Presentation] メロテルペノイド生合成マシナリーの解明と制御 (2014)
  • Author(s): 阿部郁朗
  • Organizer: 日本化学会第94春期年会特別企画「バイオインフォマティクスを駆使した生物活性天然物の供給への挑戦」
  • Place of Presentation: 愛知県名古屋市
  • Invited
• [Presentation] Engineered Biosynthesis of Plant Polyphenols (2013)
  • Author(s): Abe, I.
  • Organizer: Banbury Conference on Evolution of Plant Metabolic Diversity
  • Place of Presentation: Cold Spring Harbor, USA
  • Year and Date: 2013-03-06
  • Invited
• [Presentation] Biosynthesis of fungal meroterpenoids (2013)
  • Author(s): Abe, I.
  • Organizer: TERPNET2013, Biosynthesis, Function and Biotechnology of Isoprenoids in Terrestrial and Marine Organisms
  • Place of Presentation: Kolymvari, Crete, Greece
• [Presentation] Expanding the catalytic repertoires of biosynthetic enzymes (2013)
  • Author(s): Abe, I.
  • Organizer: Enzyme Engineering XXII: Emerging topics in Enzyme Engineering
  • Place of Presentation: 富山県富山市
  • Invited
• [Presentation] Engineered biosynthesis of medicinal natural products (2013)
  • Author(s): Abe, I.
  • Organizer: 8. International Conference on Medicinal Chemistry and Timmerman Award 2013
  • Place of Presentation: Jakarta, Indonesia
  • Invited
• [Presentation] Engineered Biosynthesis of Plant Polyphenols (2012)
  • Author(s): Abe, I.
  • Organizer: International Seminar on Natural Product Medicines
  • Place of Presentation: Bandung, Indonesia
  • Year and Date: 2012-11-22
  • Invited
• [Presentation] Expanding the Catalytic Repertoires of Biosynthetic Enzymes (2012)
  • Author(s): Abe, I.
  • Organizer: Directing Biosynthesis III
  • Place of Presentation: Nottingham, UK
  • Year and Date: 2012-09-20
  • Invited
• [Presentation] Engineering Plant Polyketide Synthases (2012)
  • Author(s): Abe, I.
  • Organizer: The International Conference of Natural Product Biosynthesis (8th US-Japan Seminar)
  • Place of Presentation: Awajishima, Japan
  • Year and Date: 2012-06-18
  • Invited
• [Presentation] 二次代謝酵素の機能制御 (2012)
  • Author(s): 阿部郁朗
  • Organizer: 第132回日本薬学会年会シンポジウム「酵素は何をしているのか?最新科学が解き明かす新機構」
  • Place of Presentation: 札幌
  • Year and Date: 2012-03-30
• [Presentation] 二次代謝酵素の潜在的触媒能力と機能制御 (2011)
  • Author(s): 阿部郁朗
  • Organizer: 文部科学省科学研究費補助金「新学術領域研究」生合成マシナリー第1回公開シンポジウム
  • Place of Presentation: 東京
  • Year and Date: 2011-01-08
• [Presentation] Engineering Plant Polykedite Synthases (2011)
  • Author(s): Abe, I.
  • Organizer: Enzyme Engineering XXI
  • Place of Presentation: Veil, Colorado, USA
• [Presentation] Engineering Plant Polykedite Synthases (2011)
  • Author(s): Abe, I.
  • Organizer: Phytochemical Society of North America 50^<th> Anniversary Symposium
  • Place of Presentation: Kohala Coast, Hawaii, USA
• [Presentation] 天然薬物の生合成工学 (2010)
  • Author(s): 阿部郁朗
  • Organizer: 徳島文理大学文部科学省戦略的研究基盤形成支援事業研究報告会
  • Place of Presentation: 徳島
  • Year and Date: 2010-12-22
• [Presentation] Engineering of Plant Polyketide Synthases (2010)
  • Author(s): I.Abe
  • Organizer: International Chemical Congress of Pacific Basin Societies (PACIFICHEM 2010)
  • Place of Presentation: Hawaii, USA
  • Year and Date: 2010-12-15
• [Presentation] Engineering Natural Products Biosynthesis (2010)
  • Author(s): I.Abe
  • Organizer: 9th NRCT-JSPS Joint Seminar
  • Place of Presentation: Bangkok, Thailand
  • Year and Date: 2010-12-09
• [Presentation] 植物二次代謝酵素の潜在的触媒能力と機能拡張 (2010)
  • Author(s): 阿部郁朗
  • Organizer: 第47回 植物化学シンポジウム
  • Place of Presentation: 静岡
  • Year and Date: 2010-11-18
• [Presentation] Engineering Natural Products Biosynthesis (2010)
  • Author(s): I.Abe
  • Organizer: 25th Symposium on Natural Products
  • Place of Presentation: Kaoshung, Taiwan
  • Year and Date: 2010-11-05
• [Presentation] 植物ポリケタイド合成酵素の酵素工学 (2010)
  • Author(s): 阿部郁朗
  • Organizer: 第14回 生体触媒化学シンポジウム「酵素工学と生体触媒化学」
  • Place of Presentation: 静岡
  • Year and Date: 2010-09-23
• [Presentation] Engineering of Plant Polyketide Synthases (2010)
  • Author(s): I.Abe
  • Organizer: Directing Biosynthesis 2010 : Discovery, Evolution, Function
  • Place of Presentation: Durham, United Kingdom
  • Year and Date: 2010-09-15
• [Presentation] Engineering of Plant Polyketide Synthases (2010)
  • Author(s): I.Abe
  • Organizer: 50th Anniversary symposium of the Korean Society for Applied Biological Chemistry
  • Place of Presentation: Gyeongju, Korea
  • Year and Date: 2010-08-25
• [Book] Natural Products: Discourse, Diversity and Design (分担執筆) (2014)
  • Author(s): Abe, I.
  • Total Pages: 300
  • Publisher: Wiley
• [Book] Biosynthesis and Molecular Genetics of Fungal Secondary Metabolites (分担執筆) (2014)
  • Author(s): Abe, I., Matsuda, Y.
  • Total Pages: 300
  • Publisher: Springer
• [Remarks] 東京大学大学院薬学系研究科天然物化学教室ホームページ
  • URL: http://www.f.u-tokyo.ac.jp/~tennen/head.htm
• [Remarks] 東京大学大学院薬学系研究科天然物化学教室
  • URL: http://www.f.u-tokyo.ac.jp/~tennen/head.htm
• [Remarks] 東京大学大学院薬学系研究科天然物化学教室
  • URL: http://www.f.u-tokyo.ac.jp/~tennen/head.htm
• [Remarks]
  • URL: http://www.f.u-tokyo.ac.jp/~tennen/head.htm
• [Remarks]
  • URL: http://www.f.u-tokyo.ac.jp/~tennen/head.htm
• [Patent(Industrial Property Rights)] 植物ポリケタイド合成酵素を用いた3環性非天然型アルカロイドの生産 (2010)
  • Inventor(s): 阿部郁朗、脇本敏幸、森田洋行、山下誠
  • Industrial Property Rights Holder: 東京大学
  • Industrial Property Number: 2010-212208
  • Filing Date: 2010-09-22