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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 李昆達 | zh_TW |
| dc.contributor.advisor | Kung-Ta Lee | en |
| dc.contributor.author | 楊子耘 | zh_TW |
| dc.contributor.author | Tzu-Yun Yang | en |
| dc.date.accessioned | 2025-08-21T16:27:43Z | - |
| dc.date.available | 2025-08-22 | - |
| dc.date.copyright | 2025-08-21 | - |
| dc.date.issued | 2025 | - |
| dc.date.submitted | 2025-08-04 | - |
| dc.identifier.citation | Sani, A., Qin, W. Q., Li, J. Y., Liu, Y. F., Zhou, L., Yang, S. Z., & Mu, B. Z. (2024). Structural diversity and applications of lipopeptide biosurfactants as biocontrol agents against phytopathogens: A review. Microbiological Research, 278, 127518. https://doi.org/10.1016/j.micres.2023.127518
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Journal of bacteriology, 196(1), 189–201. https://doi.org/10.1128/JB.00918-13 | - |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/99119 | - |
| dc.description.abstract | 表面活性素（surfactin）與豐原素（fengycin）是由枯草桿菌所產生的脂肽類生物界面活性劑，分別具有抗菌、抗病毒與抗絲狀真菌的活性，應用潛力橫跨生醫、農業與環境工程等多元領域。然而，由於天然生產菌株的產量普遍偏低，限制了其工業化應用的可行性。本研究首先針對枯草桿菌野生型菌株Bacillus subtilis NTU-14進行產能測試與發酵條件優化，測試兩種不同培養基對脂肽產量之影響，結果顯示YGF培養基具有最佳表現（培養48小時後，surfactin與fengycin產量分別為288.39 mg/L與40.41 mg/L）。隨後，本研究建構一個雙宿主穿梭載體系統，使其能同時適用於大腸桿菌與枯草桿菌，用於異源表現導入功能性基因sfp與degQ，分別應用於重建枯草桿菌模式菌株Bacillus subtilis 168的脂肽生合成能力，以及強化NTU-14的脂肽生合成調控效能。本研究確認質體可穩定轉形進入枯草桿菌並成功放大培養，並於後續整合驗證階段，初步比較改造菌株與對照株於不同培養條件下之脂肽產量。儘管異源表現尚未成功展現明確增效，推測可能與誘導條件、表現系統設計或蛋白折疊效率有關，本研究已建立一個具可行性之基因工程與菌株開發平台。未來若結合調控元件優化與培養參數微調，將可建構出具備實用價值的脂肽工業化生產系統。 | zh_TW |
| dc.description.abstract | Surfactin and fengycin are lipopeptide-type biosurfactants produced by Bacillus subtilis, exhibiting antibacterial, antiviral, and antifungal activities against filamentous fungi. These compounds hold significant application potential in diverse fields including biomedicine, agriculture, and environmental engineering. However, the generally low production levels of natural producer strains have limited their feasibility for industrial-scale applications. In this study, we first evaluated the production capacity of the wild-type Bacillus subtilis NTU-14 and optimized its fermentation conditions by testing two different culture medium. The results showed that NTU-14 achieved the highest lipopeptide production when cultivated in YGF medium (288.39 mg/L surfactin and 40.41 mg/L fengycin after 48 hours). Subsequently, a dual-host shuttle vector system compatible with both Escherichia coli and Bacillus subtilis was constructed to enable the heterologous expression of functional genes sfp and degQ. These genes were respectively applied to restore the lipopeptide biosynthetic capacity of the model strain B. subtilis 168 and to enhance the biosynthetic regulation in NTU-14. The plasmids were confirmed to be stably transformed into B. subtilis and successfully maintained during scale-up cultivation. In the final stage, the gene engineering and fermentation strategies were integrated to preliminarily assess the production performance of engineered strains under different cultivation conditions. Although the heterologous expression did not result in significant enhancement of lipopeptide yields, possibly due to induction conditions, expression system limitations, or protein folding inefficiency, this study has established a feasible platform for genetic engineering and strain development. With further improvements in regulatory element design and fermentation parameter refinement, this work may facilitate the development of a practically applicable and scalable lipopeptide production system for industrial use. | en |
| dc.description.provenance | Submitted by admin ntu (admin@lib.ntu.edu.tw) on 2025-08-21T16:27:43Z No. of bitstreams: 0 | en |
| dc.description.provenance | Made available in DSpace on 2025-08-21T16:27:43Z (GMT). No. of bitstreams: 0 | en |
| dc.description.tableofcontents | 口試委員會審定書 i
謝辭 ii 中文摘要 iii Abstract iv 目次 vi 圖表目次 ix 壹、 緒論 1 1.1 脂肽 1 1.1.1 脂肽之簡介 1 1.1.2 脂肽之市場規模 2 1.1.3 非核糖體肽合成酶 3 1.1.4 促進脂肽生合成之條件 4 1.2 表面活性素 5 1.2.1 表面活性素之簡介 5 1.2.2 表面活性素之生合成途徑 6 1.2.3 表面活性素之應用 8 1.3 豐原素 9 1.3.1 豐原素之簡介 9 1.3.2 豐原素之生合成途徑 10 1.3.3 豐原素之應用 13 1.4 枯草桿菌 13 1.4.1 枯草桿菌之簡介 13 1.4.2 枯草桿菌模式菌株168之簡介 15 1.5 以枯草桿菌生產脂肽之策略 15 1.5.1 在枯草桿菌中進行異源表現 15 1.5.1.1 sfp基因之簡介 16 1.5.1.2 degQ基因之簡介 17 1.5.2 穿梭載體之建構策略 18 1.6 研究目的 19 1.7 研究架構 20 貳、 材料與方法 22 2.1 菌株來源 22 2.2 脂肽萃取與乾重測定 24 2.3 高效液相層析分析 26 2.3.1 高效液相層析條件 26 2.3.2 純度與產量計算 26 2.4 質體建構 27 2.4.1 PCR產物之純化 30 2.4.2 DNA定量 30 2.4.3 Gibson組裝 30 2.4.4 抽取質體 30 2.4.5 抽取基因組DNA 31 2.4.6 Zero-Blunt克隆 31 2.5 轉形方法 34 2.5.1 大腸桿菌轉形 34 2.5.2 枯草桿菌轉形 36 2.5.2.1 製備枯草桿菌勝任細胞 36 2.5.2.2 使用化學方法轉形 36 2.4 IPTG誘導與螢光顯微鏡觀察 36 參、 結果 38 3.1 發酵條件優化 38 3.1.1 B. subtilis NTU-14脂肽產能基礎確認 38 3.1.2 培養基條件優化 41 3.2 穿梭載體建構與轉形 45 3.2.1 枯草桿菌轉形試驗與條件優化 45 3.2.2 表現質體構建與確認 47 3.3 基因改造與培養條件整合效益驗證 58 肆、 討論 60 4.1 異源蛋白質無法成功表現之可能原因 60 4.1.1 基因表現匣 60 4.1.2 質體建構 62 伍、 結論 63 陸、 未來展望 64 參考文獻 65 附錄 75 | - |
| dc.language.iso | zh_TW | - |
| dc.subject | 枯草桿菌 | zh_TW |
| dc.subject | 表面活性素 | zh_TW |
| dc.subject | 豐原素 | zh_TW |
| dc.subject | 雙宿主穿梭載體系統 | zh_TW |
| dc.subject | surfactin | en |
| dc.subject | fengycin | en |
| dc.subject | Bacillus subtilis | en |
| dc.subject | dual-host shuttle vector system | en |
| dc.title | 開發以穿梭載體系統應用於枯草桿菌中的脂肽生產工程 | zh_TW |
| dc.title | Development of a Shuttle Vector System to Enhance Lipopeptide Biosynthesis in Bacillus subtilis | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 113-2 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 劉啟德;蘇敬岳;蔡黛華 | zh_TW |
| dc.contributor.oralexamcommittee | Chi-Te Liu;Ching-Yueh Su;Dai-Hua Tsai | en |
| dc.subject.keyword | 枯草桿菌,表面活性素,豐原素,雙宿主穿梭載體系統, | zh_TW |
| dc.subject.keyword | Bacillus subtilis,surfactin,fengycin,dual-host shuttle vector system, | en |
| dc.relation.page | 75 | - |
| dc.identifier.doi | 10.6342/NTU202503761 | - |
| dc.rights.note | 同意授權(限校園內公開) | - |
| dc.date.accepted | 2025-08-08 | - |
| dc.contributor.author-college | 生命科學院 | - |
| dc.contributor.author-dept | 生化科技學系 | - |
| dc.date.embargo-lift | 2025-08-22 | - |
| 顯示於系所單位： | 生化科技學系 | |
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|---|---|---|---|
| ntu-113-2.pdf 授權僅限NTU校內IP使用（校園外請利用VPN校外連線服務） | 11.53 MB | Adobe PDF |
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