臺灣博碩士論文加值系統

本研究之主旨為利用基因工程對大腸桿菌的代謝途徑進行修改，並配合基因編輯技術強化菌株對碳源之代謝能力及生產目標化學品的代謝途徑，最後使用不同的發酵策略提升目標產物在發酵過程中的產量、產率及轉化率。

In this study, the technology of genetic engineering was applied to modify metabolic pathways of Escherichia coli for overproduction of α-ketoglutarate. The engineered pathways involve sugar metabolism and the synthetic pathway leading to the formation of α-ketoglutarate. Furthermore, various fermentation strategies were empolyed to improve the production yield of and the conversion yiled of α-ketoglutarate.

誌謝 i
摘要 ii
Abstract iii
目錄 iv
圖目錄 vi
表目錄 viii
第一章 緒論 1
1.1 前言 1
1.2 文獻回顧 2
1.2.1 粗甘油 (Crude glycerol) 2
1.2.2 α-酮戊二酸 (Alpha Ketoglutaric acid) 2
1.2.3 生產α-酮戊二酸之菌株 4
1.3 研究動機 5
第二章 實驗方法與材料 6
2.1 菌株儲存與培養 6
2.1.1 菌株儲存 6
2.1.2 菌株培養 7
2.2 DNA(Dexyribonucleic acid)之純化與萃取 8
2.2.1 染色體萃取 8
2.2.2 質體萃取(Plasmid DNA extraction) 9
2.2.3 瓊脂凝膠萃取DNA片段 11
2.3 鏈鎖聚合酶反應 (Polymerase Chain Reaction, PCR) 12
2.4 DNA濃度之量測 13
2.5 基因重組技術 14
2.5.1 凝膠電泳法(Gel electrophoresis) 14
2.5.2　In-Fusion 重組反應 16
2.5.3　λ Red同源重組 (λ Red homologous recombination) 17
2.5.4 位點特異性重組 (Site-specific recombination, SSR) 18
2.5.5 質體插入染色體 (Insertional plasmid) 20
2.6 大腸桿菌勝任細胞(Competent Cell)的製備 22
2.6.1 制備化學法之大腸桿菌勝任細胞 22
2.6.2 製備電擊穿透法之大腸桿菌勝任細胞 23
2.7 大腸桿菌轉化作用(Transformation) 24
2.7.1 化學法製備勝任細胞之轉形 24
2.7.2 電擊穿透法製備勝任細胞之轉形 25
2.8 大腸桿菌P1轉導(Transduction) 26
2.8.1 P1裂解液製備 26
2.8.2 執行P1轉導 27
2.9 菌株馴養 28
2.10 CRISPR / Cas9 剔除質體p15A-MutD5 29
2.11 重組菌株製備 30
2.12 培養基配置 31
2.12.1 M9 salt培養基 31
2.12.2 M9F培養基 31
2.12.3 SMAC salt培養基 32
2.13 醱酵條件 32
2.13.1 搖瓶醱酵條件 32
2.13.2 醱酵槽醱酵條件 1 32
2.13.3 醱酵槽醱酵條件 2 33
2.13.4 醱酵槽醱酵條件 3 33
2.13.5 醱酵溶氧量DO (Dissolved Oxygen) 33
2.14 重組質體建構 34
2.14.1 建構質體pLam-PPS 34
2.14.2 建構質體pUK-NAT-LCI 36
2.14.3 建構質體pTr-CsgBA-LIC 38
2.14.4 建構質體pCL-GFPuv 40
2.14.5 建構質體pND-CsgBA-DocII 42
2.14.6 建構質體pND-CsgBA-CohII 44
第三章 實驗結果與討論 46
3.1 α-酮戊二酸醱酵 46
3.1.1 批次醱酵檢測菌株 AKG-13 48
3.1.2 強化基因ppsA 49
3.1.3 批次醱酵檢測菌株 AKG-14 51
3.1.4 饋料批次醱酵檢測菌株 AKG-14 52
3.1.5 剔除mdhA基因 55
3.1.6 使用高濃度粗甘油進行批次醱酵檢測菌株 AKG-14-157
3.1.7 二階段進料醱酵檢測菌株 AKG-14-1 58
3.1.8 降低進料的氮源濃度檢測菌株 AKG-14-1 59
3.1.9 低溶氧醱酵檢測菌株 AKG-14-1 60
3.1.10 微溶氧醱酵檢測菌株 AKG-14-1 62
3.1.11 剔除kgtP基因 63
3.1.12 醱酵表現整理 65
3.2 細胞固定化 65
3.2.1 觀察LCI蛋白細胞對聚丙烯材質的吸附現象 66
3.2.2 觀察LCI蛋白在Curli蛋白的幫助下細胞對聚丙烯材質的吸附現象 68
3.2.3 觀察CsgB、CsgA蛋白結合 CohII、DocII蛋白在細胞間彼此吸附的現象 71
第四章 結論與未來展望 73
第五章 參考文獻 75
附錄 79
附錄一 研究中所使用到之質體圖 79
附錄二 實驗器材 82
附錄三 數據分析 84
附錄四 引子序列 93

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