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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 蘇南維(Nan-Wei Su) | |
| dc.contributor.author | I-Shu Wang | en |
| dc.contributor.author | 王奕舒 | zh_TW |
| dc.date.accessioned | 2021-07-10T21:40:22Z | - |
| dc.date.available | 2021-07-10T21:40:22Z | - |
| dc.date.copyright | 2020-09-14 | |
| dc.date.issued | 2020 | |
| dc.date.submitted | 2020-08-14 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76916 | - |
| dc.description.abstract | 本研究室先前已知利用源自於Bacillus subtilis BCRC80517的類黃酮磷酸酯合成酶可將quercetin、kaempferol及fisetin轉化生成相對應的磷酸酯衍生物。然而，利用微生物本身直接對上述三種化合物進行生物轉化時，卻僅有fisetin可得到與in vitro酵素轉化時相同的磷酸酯衍生物。由化學結構及轉化產物分析轉化基質的結果，推測原因為quercetin、kaempferol在結構上共同具有5號碳之羥基，相較於類黃酮磷酸酯合成酶，與B. subtilis中的quercetin 2,3-dioxygenase親和性較高，而quercetin 2,3-dioxygenase會在化合物的2號及3號碳進行氧化反應，導致其開環，無法轉化生成磷酸酯衍生物，fisetin無5號碳之羥基，則能以磷酸化途徑進行代謝。 本研究利用磷酸酯合成酶轉化fisetin，產物純化後再經由NMR及LC-MS/MS鑑定，確認其為磷酸酯衍生物，磷酸酯鍵分別位於3’及4’號碳上。由於fisetin在B. subtilis BCRC80517中能以磷酸化途徑進行轉化，推測將quercetin 2,3-dioxygenase活性抑制，則5號碳上具羥基之黃酮醇類同樣能以此途徑進行代謝。本研究第二部分建立同源重組及CRISPR-Cas9系統，以基因剔除方式去除quercetin 2,3-dioxygenase。B. subtilis 168為模式菌株，以其作為基因編輯之對象。首先，確認B. subtilis 168具有類黃酮磷酸酯合成酶及quercetin 2,3-dioxygenase後，再建構同源重組及CRISPR-Cas9系統之載體，並配合電穿孔進行突變，最後分別建立篩選模式挑選突變菌株。同源重組以抗生素進行篩選，CRISPR-Cas9系統則以quercetin作為指標，未經過基因編輯之菌株於含quercetin的固態培養基上會產生透明圈，且突變後主要的代謝途徑被剔除，推測會導致菌株生長速度較慢，因此挑選生長慢且未產生透明圈之菌株，再進行定序。 | zh_TW |
| dc.description.abstract | Flavonols are a group of secondary metabolites in plants and possess versatile physiological activities for human health. However, most of them are water insoluble, resulting in the limitation of oral administration applications. Our previous studies revealed Bacillus subtilis BCRC80517 was capable of converting several types of flavonoids into highly water-soluble phosphate conjugates. Nevertheless, flavonols with C-5-hydroxyl group in structure, such as quercetin and kaempferol, could not be transformed into the corresponding phosphate conjugates by the bacterium, but ring-opened in the C-ring of structure by bacterial constitutive quercetin 2,3-dioxygenase (qdoI) instead. Interestingly, when B. subtilis BCRC80517 cultivated with flavonols without C-5-hydroxyl group in structure, such as fisetin, the main transformation products would be the phosphate conjugates rather than the ring-opened products. In this work, we isolated the bioconversion products and identified two novel derivatives, fisetin 4’-O-phosphate and fisetin 3’-O-phosphate, by LC-MS/MS and 1H, 13C and 31P NMR spectral data. For flavonols with C-5-hydroxyl group, knocking out the qdoI gene might be a crucial approach to produce phosphorylated conjugates. B. subtilis 168-type strain was used as the host for eliminating the qdoI gene through homologous recombination and CRISPR-Cas9. For homologous recombination, neomycin resistance was used to positively select transformed colonies. For CRISPR-Cas9 system, the clear zone formed by quercetin degradation was used as negative selection criteria to screen the mutant strains. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-10T21:40:22Z (GMT). No. of bitstreams: 1 U0001-1008202010212200.pdf: 6918477 bytes, checksum: d9f004ec67fb8e4758f3ea803163b14c (MD5) Previous issue date: 2020 | en |
| dc.description.tableofcontents | 口試委員審定書 I 誌謝 II 中文摘要 IV Abstract V 縮寫對照表 VI 目錄 VIII 圖目錄 XIII 表目錄 XVI 第一章 前言 1 第二章 文獻回顧 2 第一節 類黃酮 2 1. 類黃酮簡介 2 2. 類黃酮之生理活性 2 3. 類黃酮之生物可利用率 4 3-1. BCS分類系統 4 3-2. 黃酮醇於人體之代謝與吸收 4 3-3. 黃酮醇類於人體之口服生物可利用率 6 第二節 類黃酮之微生物轉換 13 第三節 磷酸化酵素 14 1. 磷酸化酵素簡介 14 2. 磷酸苯酯合成酶(phenylphosphate synthase, PPS) 15 第四節 槲皮素雙氧化酶 19 1. 槲皮素雙氧化酶 19 2. LmrA/YxaF regulon 20 第五節 基因剔除法 22 1. 同源重組 22 2. CRISPR-Cas9系統 24 第三章 材料與方法 26 第一節 實驗架構 26 第二節 實驗材料 27 1. 菌株 27 1-1 微生物轉換菌株 27 1-2 磷酸化酵素表現菌株 27 1-3 建構質體保存菌株 27 2. 培養基 28 2-1 Bacillus subtilis 168及Bacillus subtilis BCRC80517之培養基 28 2-2 Escherichia coli BPE∷pET47b(+)/BL21(DE3)之培養基 28 2-3 Escherichia coli DH5α∷pUC57-qdoI-NEO、Escherichia coli DH5α∷pGEMT-sgRNA184及Escherichia coli DH5α∷pEMT-sgRNA700之培養基 29 2-4勝任細胞之培養基 30 2-5同源重組篩菌之培養基 31 2-6 CRISPR-Cas9系統篩菌之培養基 32 3. 緩衝液 32 3-1 Enzyme buffer 32 3-2 Cell lysis buffer 32 4. 試藥與溶劑 33 5. 儀器設備 35 第三節 實驗方法 37 1. FPS酵素轉換黃酮醇類 37 1-1菌株活化與種菌培養 37 1-2 以IPTG誘導重組蛋白基因大量表現 37 1-3 BPE粗酵素液之製備 37 1-4 蛋白質定量方法 37 1-5 Fisetin酵素轉換 38 1-6分析級高效液相層析儀分析條件 38 2. 酵素轉換fisetin 產物之結構分析 39 2-1 以半製備級高效液相層析儀純化類黃酮磷酸酯衍生物 39 2-2 半製備級高效液相層析儀分離類黃酮磷酸酯衍生物之條件 39 2-3 高效液相層析串聯式質譜儀分析類黃酮衍生物之條件 39 2-4 核磁共振光譜分析類黃酮衍生物之條件 40 3. B. subtilis 168轉換genistein 40 3-1 種菌之培養 40 3-2 微生物轉換genistein 40 3-3 分析級高效液相層析儀分析條件 40 4. B. subtilis 168轉換quercetin 41 4-1 種菌之培養 41 4-2 微生物轉換quercetin 41 4-3 分析級高效液相層析儀分析條件 41 5. 同源重組之載體及CRISPR-Cas9系統建構 42 5-1 B. subtilis 168基因體DNA抽取 42 5-2 聚合酶連鎖反應(Polymerase Chain Reaction, PCR) 42 5-3 PCR產物純化及定序 43 5-4 B. subtilis 168對neomycin濃度抗性之測試 43 5-5 建立進行同源重組突變之載體pUC57-qdoI-NEO 43 5-6 選殖菌株之轉型作用(Transformation) 44 5-7 篩選轉型成功之菌株 44 5-8 qdoI序列之pam site分析 44 5-9 建立體外轉錄之質體pGEMT-sgRNA184及pGEMT-sgRNA700 44 5-10 選殖菌株之轉型作用 (Transformation) 47 5-11 篩選pGEMT-sgRNA184及pGEMT-sgRNA700組合成功之載體 47 5-12限制酶剪切pGEMT-sgRNA184及pGEMT-sgRNA700載體 48 5-13 sgRNA之DNA序列轉錄至RNA序列 49 5-14組裝Cas9/sgRNA複合物 49 6. B.subtilis 168之勝任細胞的建構及電轉型之作用 50 6-1 B.subtilis 168之勝任細胞的建構 50 6-2 B.subtilis 168電轉型之條件 50 6-3 同源重組之選殖菌株篩選 50 6-4 CRISPR-Cas9系統之選殖菌株篩選 51 第四章 結果與討論 52 第一節 BsFPS蛋白質對黃酮醇類化合物之轉換 52 1. FPS之製備 53 2. FPS對fisetin之轉換 53 3. 結構鑑定酵素轉換fisetin之產物 54 3-1 半製備級高效液相層析儀純化漆黃素fisetin轉換之產物 54 3-2 質譜及NMR結構鑒定結果 56 第二節 微生物B. subtilis 168轉換quercetin及genistein 60 1. B. subtilis 168之微生物轉換 60 1-1 微生物轉換quercetin 60 1-2 微生物轉換genistein 62 第三節 建構同源重組載體及CRISPR-Cas9 系統 64 1. 建構同源重組之載體 65 1-1 B. subtilis 168 qdoI定序 65 1-2 B. subtilis 168抗生素抗性測試 65 1-3 建構同源重組質體之載體 66 1-4. 篩選成功組合之質體 68 2. 建構CRISPR-Cas9之系統 69 2-1 B. subtilis 168 qdoI基因之pam site分析 69 2-2 sgRNA載體設計 69 2-3 篩選成功組合之質體 69 2-4利用限制酶剪切載體 72 2-5 sgRNA之轉錄及Cas9/sgRNA複合物建構 72 3. 同源重組及CRISPR-Cas9系統之篩菌方法建立 74 3-1 同源重組之篩菌方法建立 74 3-1 CRISPR-Cas9系統之篩菌方法建立 74 第五章 結論 76 第六章 參考文獻 77 | |
| dc.language.iso | zh-TW | |
| dc.subject | CRISPR-Cas9 | zh_TW |
| dc.subject | 枯草菌 | zh_TW |
| dc.subject | 槲皮素雙氧化酶 | zh_TW |
| dc.subject | 磷酸化 | zh_TW |
| dc.subject | 黃酮醇 | zh_TW |
| dc.subject | 同源重組 | zh_TW |
| dc.subject | quercetin 2 | en |
| dc.subject | CRISPR-Cas9 | en |
| dc.subject | homologous recombination | en |
| dc.subject | 3-dioxygenase | en |
| dc.subject | Bacillus subtilis | en |
| dc.subject | flavonol | en |
| dc.subject | phosphorylation | en |
| dc.title | qdoI基因對枯草菌BCRC80517進行黃酮醇化合物磷酸化之影響 | zh_TW |
| dc.title | The effect of qdoI on the flavon-3-ol phosphorylation by Bacillus subtilis BCRC80517 | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 108-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 李敏雄(Min-Hsiung Lee),陳錦樹(Chin-Shuh Chen),賴進此(Jinn-Tsyy Lai),胡紹揚(Shao-Yang Hu) | |
| dc.subject.keyword | 枯草菌,黃酮醇,磷酸化,槲皮素雙氧化酶,同源重組,CRISPR-Cas9, | zh_TW |
| dc.subject.keyword | Bacillus subtilis,flavonol,phosphorylation,quercetin 2,3-dioxygenase,homologous recombination,CRISPR-Cas9, | en |
| dc.relation.page | 83 | |
| dc.identifier.doi | 10.6342/NTU202002767 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2020-08-16 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科技學系 | zh_TW |
| 顯示於系所單位： | 生化科技學系 | |
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