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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 林曉青(Hsiao-Ching Lin) | |
| dc.contributor.author | Chien-Ting Chen | en |
| dc.contributor.author | 陳建廷 | zh_TW |
| dc.date.accessioned | 2021-07-11T15:28:18Z | - |
| dc.date.available | 2028-12-31 | |
| dc.date.copyright | 2018-08-24 | |
| dc.date.issued | 2018 | |
| dc.date.submitted | 2018-08-19 | |
| dc.identifier.citation | 1. Geethangili, M., & Tzeng, Y. (2011). Review of Pharmacological Effects ofAntrodia camphorataand Its Bioactive Compounds. Evidence-Based Complementary And Alternative Medicine, 2011, 1-17.
2. Cherng, I., Chiang, H., Cheng, M., & Wang, Y. (1995). Three New Triterpenoids from Antrodia cinnamomea. Journal Of Natural Products, 58(3), 365-371. doi: 10.1021/np50117a004. 3. Liao, P., Kuo, D., Lin, C., Ho, K., Lin, T., & Hwang, S. (2010). Historical spatial range expansion and a very recent bottleneck of Cinnamomum kanehirae Hay. (Lauraceae) in Taiwan inferred from nuclear genes. BMC Evolutionary Biology, 10(1), 124. doi: 10.1186/1471-2148-10-124. 4. Lu, M., El-Shazly, M., Wu, T., Du, Y., Chang, T., & Chen, C. et al. (2013). Recent research and development of Antrodia cinnamomea. Pharmacology & Therapeutics, 139(2), 124-156. 5. Cheng, J., Yang, C., Cheng, C., Wang, Y., Huang, N., & Lu, M. (2005). Characterization and Functional Study ofAntrodia camphorataLipopolysaccharide. Journal Of Agricultural And Food Chemistry, 53(2), 469-474. doi: 10.1021/jf049281a. 6. Peng, C., Chen, K., Peng, R., Chyau, C., Su, C., & Hsieh-Li, H. (2007). Antrodia camphorata extract induces replicative senescence in superficial TCC, and inhibits the absolute migration capability in invasive bladder carcinoma cells. Journal Of Ethnopharmacology, 109(1), 93-103. doi: 10.1016/j.jep.2006.07.009. 7. Peng, C., Chen, K., Peng, R., Su, C., & Hsieh-Li, H. (2006). Human urinary bladder cancer T24 cells are susceptible to the Antrodia camphorata extracts. Cancer Letters, 243(1), 109-119. doi: 10.1016/j.canlet.2005.11.021. 8. Hsu, Y., Kuo, Y., Kuo, P., Ng, L., Kuo, Y., & Lin, C. (2005). Apoptotic effects of extract from Antrodia camphorata fruiting bodies in human hepatocellular carcinoma cell lines. Cancer Letters, 221(1), 77-89. doi: 10.1016/j.canlet.2004.08.012. 9. Yang, H., Chen, C., Chang, W., Lu, F., Lai, Y., & Chen, C. et al. (2006). Growth inhibition and induction of apoptosis in MCF-7 breast cancer cells by Antrodia camphorata. Cancer Letters, 231(2), 215-227. doi: 10.1016/j.canlet.2005.02.004. 10. Chen, Y., Chiu, H., Chao, C., Lin, W., Chao, L., Huang, G., & Kuo, Y. (2013). New Anti-Inflammatory Aromatic Components from Antrodia camphorata. International Journal Of Molecular Sciences, 14(3), 4629-4639. doi: 10.3390/ijms14034629. 11. Geethangili, M., & Tzeng, Y. (2011). Review of Pharmacological Effects ofAntrodia camphorataand Its Bioactive Compounds. Evidence-Based Complementary And Alternative Medicine, 2011, 1-17. doi: 10.1093/ecam/nep108. 12. Chen, C., Chyau, C., & Hseu, T. (2007). Production of a COX-2 inhibitor, 2,4,5-trimethoxybenzaldehyde, with submerged cultured Antrodia camphorata. Letters In Applied Microbiology, 44(4), 387-392. doi: 10.1111/j.1472-765x.2006.02087.x. 13. Tsai, W., Rao, Y., Lin, S., Chou, M., Shen, Y., & Wu, C. et al. (2010). Methylantcinate A induces tumor specific growth inhibition in oral cancer cells via Bax-mediated mitochondrial apoptotic pathway. Bioorganic & Medicinal Chemistry Letters, 20(20), 6145-6148. doi: 10.1016/j.bmcl.2010.08.006. 14. Rao, Y., Wu, A., Geethangili, M., Huang, M., Chao, W., & Wu, C. et al. (2011). Identification of Antrocin from Antrodia camphorata as a Selective and Novel Class of Small Molecule Inhibitor of Akt/mTOR Signaling in Metastatic Breast Cancer MDA-MB-231 Cells. Chemical Research In Toxicology, 24(2), 238-245. doi: 10.1021/tx100318m. 15. Lin, Y., Ma, L., Lee, Y., Shaw, J., Wang, S., & Chu, F. (2017). Differential Gene Expression Network in Terpenoid Synthesis of Antrodia cinnamomea in Mycelia and Fruiting Bodies. Journal Of Agricultural And Food Chemistry, 65(9), 1874-1886. doi: 10.1021/acs.jafc.6b05386. 16. FURUKAWA, K. (2006). Oxygenases and Dehalogenases: Molecular Approaches to Efficient Degradation of Chlorinated Environmental Pollutants. Bioscience, Biotechnology, And Biochemistry, 70(10), 2335-2348. doi: 10.1271/bbb.60218. 17. Wang, Y., Feng, Y., Cao, X., Liu, Y., & Xue, S. (2018). Insights into the molecular mechanism of dehalogenation catalyzed by D-2-haloacid dehalogenase from crystal structures. Scientific Reports, 8(1). doi: 10.1038/s41598-017-19050-x. 18. Katja E. Hill, A. (2018). Investigation of Two Evolutionarily Unrelated Halocarboxylic Acid Dehalogenase Gene Families. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC93952/. 19. Shinohara, Y., Takahashi, S., Osada, H., & Koyama, Y. (2016). Identification of a novel sesquiterpene biosynthetic machinery involved in astellolide biosynthesis. Scientific Reports, 6(1). doi: 10.1038/srep32865. 20. Göhl, M., & Seifert, K. (2014). Synthesis of the Sesquiterpenes Albicanol, Drimanol, and Drimanic Acid, and the Marine Sesquiterpene Hydroquinone Deoxyspongiaquinol. European Journal Of Organic Chemistry, 2014(31), 6975-6982. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/78907 | - |
| dc.description.abstract | 牛樟芝為台灣特有的藥用菇類，許多研究報導牛樟芝具有廣泛的生物活性，例如: 抗癌症、抗發炎、抗氧化以及抑制腫瘤的活姓。Antrocin為倍半萜 (sesquiteropenoid)之化合物，擁有草椎烷(drimane)以及內酯環(lactone ring)骨架，已知由牛樟芝的子實體分離而得，其對於許多癌細胞具有細胞毒性，能促使癌細胞凋亡或抑制生長。然而，antrocin化合物的取得主要來自於牛樟芝子實體的化學分離以及純化，供應產量有限，因此使antrocin的藥物發展受到限制。目前已有antrocin全合成方法的報導，然而，antrocin在牛樟芝的生合成路徑依然未知。在本研究中，我們利用異源表達與化學實驗鑑定出具有合成antrocin結構骨架功能的萜烯環化酶AncC (terpene cyclase)，此外，我們也發現在此基因簇中有三個氧化酶參與antrocin生合成路徑，分別為AncB (P450 monooxygenase)、AncD (P450 monooxygenase)與AncE (dehydrogenase)，並在異源表達系統重建並分離出四個化合物(2~5)，且均為新化合物。最後我們成功鑑定出在牛樟芝基因組中參與antrocin生合成的基因以及生合成路徑，中間產物之結構經由質譜與核磁共振鑑定，本研究成果未來將可能促進antrocin的藥物發展。 | zh_TW |
| dc.description.abstract | Antrodia cinnamomea is a unique mushroom and indigenous to Taiwan. Many studies have reported a diverse range of biological activities of A. cinnamomea including anticancer, anti-inflammatory, antioxidative, antitumor activity. Antrocin is a drimane-type sesquiterpenoid containing a lactone scaffold isolated from A. cinnamomea. It exhibits potent cytotoxic activity against several cancer cell lines. The supply of antrocin was mainly isolated from the fruiting body of 2-3 years-old A. cinnamomeam, which may limit the development of antrocin as a potential medicine. While the total chemical synthestic strategies were developed to synthesize antrocin molecule, the genetic and enzymatic basis of antrocin remains unknown.Here we reported the identification and characterization of a terpene synthase (AncC) from A. cinnamomea that synthesizes the antrocin skeleton with heterologous reconstitution and biochemical characterization. Besides, we have identified three oxygenases (AncB, AncD and AncE) that catalyzes oxidative modifications to afford novel compounds in the antrocin biosynthetic pathway with heterologous expression and in vivo feeding experiments. The structure of these compounds were characterized with MS and NMR spectroscopic methods. Our study unveiled the mystery of antrocin biosynthesis in the medicinal fungus and may facilitate the development of antrocin as a potential therapeutic agent. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-11T15:28:18Z (GMT). No. of bitstreams: 1 ntu-107-R05b46022-1.pdf: 4506629 bytes, checksum: ef5d74d9e678b8d49e7b91cfe26aacd6 (MD5) Previous issue date: 2018 | en |
| dc.description.tableofcontents | TABLE OF CONTENTS
摘要 I ABSTRACT II TABLE OF CONTENTS III LIST OF FIGURES V LIST OF TABLE VII 1 Introduction 1 1-1 Introduction of Antrodia cinnamomea and its bioactivity 1 1-2 Introduction of Antrocin bioactivity and limition of development 3 1-3 Introduction of HAD-like enzyme 4 1-4 The purpose of this study. 5 2 Materials and Methods 6 2-1 Strains and Culture Conditions 6 2-2 Chemicals and Chemical Analysis 6 2-3 General Molecular Biology Experiments. 7 2-4 Construction of plasmid for AncA, AncB, AncC and AncD expression in S. cerevisiae. 7 2-5 Construction of pAncB-AncC-xw55 for coexpression in S. cerevisiae. 8 2-6 Biotransformation of S. cerevisiae expressing AncA or AncC 8 2-7 Biotransformation of S. cerevisiae expressing AncB and AncC 8 2-8 Biotransformation of S. cerevisiae expressing AncC and AncE. 9 2-9 Biotransformation of S. cerevisiae expressing AncC and AncD. 9 2-10 Biotransformation of S. cerevisiae expressing AncC, AncB and AncE 9 2-11 Biotransformation of S. cerevisiae expressing AncC, AncB, AncD and AncE… 9 2-12 Construction of plasmid for AncA or AncC expression in Pichia pastoris 9 2-13 Overexpression and Purification of AncA or AncC from Pichia pastoris. 10 2-14 In vitro assay of AncA or AncC 11 2-15 Isolation and Purification of Albicanol (1). 11 2-16 Isolation and Purification of m/z 239 (2) and m/z 237 (3). 12 2-17 Isolation and Purification of m/z 219 (4). 14 2-18 Isolation and Purification of m/z 253 (5). 15 3 Results and discussion 18 3-1 Deduced functions of genes within the anc cluster. 18 3-2 Heterologous expression of AncA in S. cerevisiae. 19 3-3 Verification of the function of AncA. 20 3-4 Heterologous expression of AncC in S. cerevisiae 21 3-5 Verification of the function of AncC. 22 3-6 Amino acid comparison of the AncA and AncC enzymes 24 3-7 AncC Coexpression of AncC with AncB, AncD and AncE in S. cerevisiae. …………………………………………………………………………….24 3-8 Feeding experiments of AncB, AncD and AncE in S. cerevisiae. 29 3-9 Structure elucidation of compound(1) 33 3-10 Elucidate structure of compound m/z 239 (2) 35 3-11 Structural elucidation of compound m/z 237 (3a and 3b) 38 3-12 Elucidate structure of compound m/z 219 (4) 43 3-13 Elucidate structure of compound m/z 253 (5) 46 Reference 49 Appendix 53 LIST OF FIGURES Figure 1. Chemical structures of antroquinonol and its derivatives10. Antroquinonol (1), antroquinonol B (2), 4-acetylantroquinonol B (3). 2 Figure 2. The structure of antrocin. 3 Figure 3. Drim-8-ene-11-ol biosynthesis pathway in previous study19 4 Figure 4. Putative biosynthesis pathway of Antrocin in our study. 5 Figure 5. Verification of the function of AncA. LCMS analysis. Extracted ion chromatograom of (A) S. cerevisiae BJ5464-NpgA expressing AncA; (B) S. cerevisiae BJ5464-NpgA wild type. 19 Figure 6. Functional characterization of AncA. 20 Figure 7. Verification of the function of AncC. The LCMS analysis (extracted ion chromatograom) of (A) S. cerevisiae BJ5464-NpgA expressing AncC; (B) S. cerevisiae BJ5464-NpgA wild type. 21 Figure 8. The SDS-PAGE of purified AncC (58.2 KDa). 22 Figure 9. The GC-MS profiles (TIC) of AncC from the vitro reactions. 23 Figure 10. LC-MS profiles of metabolites extracted from the culture of S. cerevisiae strains expressing target cluster genes. 25 Figure 11. LC-MS profiles of metabolites extracted from the culture of S. cerevisiae expressing target cluster genes. 27 Figure 13. The biosynthetic pathway of antrocin mapped using heterologous expression and feeding experiments in this work. 28 Figure 12. Structure of compounds 1 ~ 5. 28 Figure 14. LC-MS profile of feeding experiment to verify the fuction of AncB. 30 Figure 15. LC-MS profile of feeding experiment to verify the fuction of AncD. 31 Figure 16. LC-MS profile of feeding experiment to verify the function of AncE. 32 Figure 17. 1H-1H COSY, HMBC and NOESY correlations of 2. 36 Figure 18. 1H-1H COSY, HMBC and NOESY correlations of 3a 40 Figure 19. 1H-1H COSY, HMBC and NOESY correlations of 3b. 40 Figure 20. 1H-1H COSY, HMBC and NOESY correlations of 4. 44 Figure 21. 1H-1H COSY, HMBC and NOESY correlations of 5. 47 Figure 22. 1H NMR spectrum of 1, m/z 205. 54 Figure 23. 13C NMR spectra of 1, m/z 205. 55 Figure 24. 1H NMR spectrum of compound 2, mz239 56 Figure 25. 13C NMR spectrum of compound 2, mz239 57 Figure 26. COSY spectrum of 2, mz 239. 58 Figure 27. HMBC spectrum of 2, mz 239. 59 Figure 28. HSQC spectrum of 2, mz 239. 60 Figure 29. NOESY spectrum of 2, mz 239. 61 Figure 30. NOESY spectrum of 2, mz 239 (expended). 62 Figure 31. NOESY spectrum of 2, mz 239 (expended). 63 Figure 32. 1H NMR spectrum of compound 3, mz237. 64 Figure 33. 13C NMR spectrum of compound 3, mz237. 65 Figure 34. COSY spectrum of compound 3, mz237. 66 Figure 35. COSY spectrum of compound 3, mz237 (expended). 67 Figure 36. HSQC spectrum of compound 3, mz237. 68 Figure 37. HMBC spectrum of compound 3, mz237. 69 Figure 38. NOESY spectrum of compound 3, mz237. 70 Figure 39. NOESY spectrum of compound 3, mz237 (expended). 71 Figure 40. 1H NMR spectrum of 4, m/z 219. 72 Figure 41. 13C NMR spectrum of 4, m/z 219. 73 Figure 42. COSY spectrum of compound 4, mz219. 74 Figure 43. COSY spectrum of compound 4, mz219 (expended). 75 Figure 44. HSQC spectrum of compound 4, mz219. 76 Figure 45. HSQC spectrum of compound 4, mz219 (expended). 77 Figure 46. HMBC spectrum of compound 4, mz219. 78 Figure 47. HMBC spectrum of compound 4, mz219 (expended). 79 Figure 48. NOESY spectrum of compound 4, mz219 . 80 Figure 49. 1H NMR spectrum of 5, m/z 253. 81 Figure 50. 13C NMR spectrum of 5, m/z 253. 82 Figure 51. COSY spectrum of compound 5, mz253. 83 Figure 52. COSY spectrum of compound 5, mz253 (expended). 83 Figure 53. HSQC spectrum of compound 5, mz253. 83 Figure 54. HMBC spectrum of compound 5, mz253. 83 Figure 55. NOESY spectrum of compound 5, mz253. 83 LIST OF TABLE Table 1. Primers used in this study. 16 Table 2. Deduced functions of genes within the Anc cluster. 18 Table 3. 1H (500 MHz) and 13C (125 MHz) NMR data of albicanol (1) 34 Table 4. 1H NMR (500 MHz) and 13C NMR (125 MHz) spectroscopic data of m/z 239(2) in acetone-d6. 37 Table 5. 1H NMR (500 MHz) and 13C NMR (125 MHz) spectroscopic data of 3a in acetone-d6. 41 Table 6. 1H NMR (500 MHz) and 13C NMR (125 MHz) spectroscopic data of 3b in acetone-d6. 42 Table 7. 1H NMR (500 MHz) and 13C NMR (125 MHz) spectroscopic data of m/z 219 (4) in acetone-d6. 45 Table 8. 1H NMR (500 MHz) and 13C NMR (125 MHz) spectroscopic data of m/z 253 (5) in acetone-d6. 47 | |
| dc.language.iso | zh-TW | |
| dc.title | 牛樟芝之倍半萜Antrocin的生合成途徑解析與異源表達 | zh_TW |
| dc.title | Identification and Heterologous Reconstitution of the Antrocin Biosynthetic Pathway from Antrodia cinnamomea | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 106-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 吳世雄(Shih-Hsiung Wu),梁博煌(Po-Huang Liang),陳榮傑(Rong-Jie Chein) | |
| dc.subject.keyword | 牛樟芝,antrocin,?烯環化?,?,倍半?, | zh_TW |
| dc.subject.keyword | Antrodia cinnamomea,antrocin,terpene synthase,terpene,sesquiterpenoid, | en |
| dc.relation.page | 87 | |
| dc.identifier.doi | 10.6342/NTU201803984 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2018-08-20 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科學研究所 | zh_TW |
| dc.date.embargo-lift | 2028-12-31 | - |
| 顯示於系所單位： | 生化科學研究所 | |
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