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研究生: 賴逸宸
Yi-Chen Lai
論文名稱: 利用解脂耶氏酵母生產大量脂肪酸及高價值二羧酸
Massive Production of Fatty Acids and High Value Dicarboxylic Acids in Yarrowia lipolytica
指導教授: 蔡伸隆
Shen-Long Tsai
口試委員: 李振綱
Cheng-Kang Lee
王勝仕
Sheng-Shih Wang
葉怡均
Yi-Chun Yeh
蔡伸隆
Shen-Long Tsai
學位類別: 碩士
Master
系所名稱: 工程學院 - 化學工程系
Department of Chemical Engineering
論文出版年: 2020
畢業學年度: 108
語文別: 中文
論文頁數: 134
中文關鍵詞: 解脂耶氏酵母脂肪酸二羧酸癸二酸酰基輔酶A氧化酶硫酯酶細胞色素P450NADPH-細胞色素P450還原酶脂肪醇氧化酶同源置換β氧化ω氧化
外文關鍵詞: Yarrowia lipolytica, Fatty acids, Dicarboxylic acids, Sebacic acid, Acyl-CoA oxidase, Thioesterase, Cytochrome P450, NADPH-cytochrome P450 reductase, Fatty-alcohol oxidase, Homologous recombination, β-oxidation, ω-oxidation
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  • 碩士學位論文指導教授推薦書 II 碩士學位考試委員審定書 III 摘要 IV ABSTRACT V 致謝 VI 目錄 VII 圖目錄 XI 表目錄 XIV 壹、 緒論 1 1. 1 前言 1 1. 2 研究動機與目的 2 1. 3 研究內容 3 貳、 文獻回顧 5 2. 1 解脂耶氏酵母(Yarrowia lipolytica)的介紹與近期研究 5 2. 2 解脂耶氏酵母的代謝路徑 7 2. 2. 1 葡萄糖的代謝路徑及硫酯酶對脂肪酸碳鏈長度之影響 7 2. 2. 2 烷烴類的代謝路徑 9 2. 2. 3 β氧化 (β-Oxidation) 11 2. 2. 4 ω氧化 (ω-Oxidation) 13 2. 3 癸二酸的應用 16 參、 實驗材料與方法 17 3. 1 實驗摘要 17 3. 2 材料與儀器 19 3. 2. 1 藥品 19 3. 2. 2 實驗用之生物製劑及套組 23 3. 2. 3 儀器 25 3. 2. 4 菌株 27 3. 2. 5 質體 27 3. 2. 6 引子(Primer) 28 3. 3 實驗方法 32 3. 3. 1 大腸桿菌之培養 32 3. 3. 2 酵母菌之培養 32 3. 3. 3 小量質體純化法(Miniprep) 33 3. 3. 4 小量質體純化法(ZR Plasmid Miniprep – Classic Kit) 35 3. 3. 5 小量質體純化法(Tools Plasmid Mini Kit) 36 3. 3. 6 提取酵母菌基因組(YeaStar Genomic DNA Kit) 37 3. 3. 7 聚合酶鏈鎖反應(Polymerase chain reaction, PCR) 38 3. 3. 8 瓊脂糖凝膠電泳分析(Agarose gel electrophoresis) 40 3. 3. 9 瓊脂糖凝膠回收DNA (DNA recovery) 42 3. 3. 10 酶切反應(Digestion) 43 3. 3. 11 核酸接合反應(DNA ligation) 45 3. 3. 12 Gibson assembly 46 3. 3. 13 大腸桿菌勝任細胞(Ultra competent cell)的製備 48 3. 3. 14 大腸桿菌之轉型作用(Transformation) 50 3. 3. 15 大腸桿菌電脈衝穿孔術之勝任細胞(Electrocompetent cell)的製備 51 3. 3. 16 大腸桿菌之電脈衝穿孔術(Electroporation) 52 3. 3. 17 線性化(Linearization) 53 3. 3. 18 解脂耶氏酵母的轉型作用 54 3. 3. 19 菌株保存 56 3. 3. 20 解脂耶氏酵母的發酵測試 57 3. 3. 21 發酵液的分析前處理 59 3. 3. 22 氣相層析質譜儀(GC-MS)的操作 60 3. 3. 23 繪製癸二酸二(三甲基矽基)酯的檢量線 63 肆、 結果與討論 64 4. 1 pYAL1質體的建構 64 4. 2 pYAL1::FAO1(NTC)質體的建構 65 4. 3 pYAL1::FAO1-CPR1(NTC)質體的建構 68 4. 4 pYAL1::FAO1-ALK1(NTC)質體的建構 70 4. 5 C10 Yarrowia lipolytica Po1g integrated pYAL1::FAO1(NTC)菌株的建構 72 4. 6 C10 Yarrowia lipolytica Po1g integrated pYAL1::FAO1-CPR1(NTC)菌株的建構 74 4. 7 C10 Yarrowia lipolytica Po1g integrated pYAL1::FAO1-ALK1(NTC)菌株的建構 76 4. 8 繪製癸二酸二(三甲基矽基)酯的檢量線 79 4. 9 菌株的發酵實驗 82 4. 9. 1. 菌株生長情況 82 4. 9. 2. 發酵液內所含之癸二酸濃度 86 4. 9. 3. 菌株發酵液內不同碳鏈長度之二羧酸的比例關係 90 伍、 結論 93 5. 1 總結 93 5. 2 未來與展望 95 陸、 參考文獻 96 柒、 附錄 103 附錄一、 質體建構流程圖 103 附錄二、 質譜圖 114

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