靛藍為一環狀結構化合物，且因為靛藍分子大小使其不易通過細胞膜，以微生物經由代謝途徑生產靛藍容易因為靛藍累積於E.coli內而造成結晶以及毒性問題導致細胞死亡以及產量不佳，且靛藍化合物需要經過破菌等程序才能取得產物，因此我們建構了一體外生產靛藍系統並搭配輔因子再生系統，讓產物不需破菌也能夠取得。
我們採用了surface display方法嘗試解決這個問題，藉由INP(ice-nucleation protein)成功將生產靛藍所需的酵素Corynebacterium glutamicum ATCC13032的flavin-containing monooxygenase(FMO)表現於外細胞膜上，於細胞體外生產靛藍會因為缺乏NADPH而無法生產，為解決此問題我們將建構體外輔因子再生系統，將Candida boidinii的formate dehydrogenase(FDH)也以surface display方法表現，並透過位點突變INP-FDH與formate進行催化反應時能將NADP+還原，藉此系統可將生產靛藍後殘餘的NADP+回收成為NADPH再提供給FMO的催化反應使用。
由於INP-FMO以及INP-FDH之間存在著活性差異，因此我們透過調整兩者的RBS序列以及位點突變INP-FMO，並搭配調整反應菌量，將兩者的反應速率調控為最適生產之條件。以surface display表現FMO與FDH並且成功將兩者以co-culture的方式於體外生產靛藍。接著透過two phase生產系統將基質indole溶於有機相之中，搭配以上測試之最佳生產條件，目前使用有機相diphenylmethane能夠將靛藍的產量分別於水相及有機相提升到410 mg/L及20 mg/L，而靛玉紅產量分別約為10 mg/L及40 mg/L。

Indigo is a kind of cyclic compound. It is hard to pass through cell membranes because of the molecular size of indigo. The production of indigo by microorganisms through metabolic pathways is prone to crystallization and toxicity problems caused by the accumulation of indigo in E.coli, resulting in cell death and poor yield. Moreover, indigo compounds need to undergo procedures such as sterilization to obtain the product. Therefore, we constructed an in vitro production system for indigo and a cofactor regeneration system, so that the product can be obtained without sterilization.
In this study, we adopted the techniques of surface display to address this difficulty. With the aid of INP(ice-nucleation protein), we successfully expressed flavin-containing monooxygenase(FMO) of Corynebacterium glutamicum ATCC13032 onto the outer membrane E.coli BL21 that is an important enzyme to produce indigo. However, in-vitro indigo production needs to add addition NADPH. We provided another way to recycle NADP+ back to NADPH, then we do not necessary keep adding NADPH. Utilize site mutation formate dehydrogenase(FDH) from Candida boidinii with the techniques of surface display to regenerate NADPH can make this indigo in-vitro biosynthesis production system more completely.
Due to the difference in activity between INP-FMO and INP-FDH, it will cause insufficient NADPH cycle efficiency and affect indigo production. Therefore, we adjusted the RBS sequence and site mutation INP-FMO, and adjustment of the amount of reaction bacteria, the reaction rate of the two can be adjusted to the optimal production conditions.
We expressed FMO and FDH with surface display and successfully co-culture to produce indigo in vitro. Then, the substrate indole is dissolved in the organic phase through the two phase extraction system. So far we can use diphenylmethane as the organic phase of the two-phase extraction, which can produce up to about 410 mg/L of indigo and 10 mg/L of indirubin in aqueous phase and 20 mg/L of indigo and 40 mg/L of indirubin in organic phase.

摘要---------i
Abstract-----ii
謝誌---------iii
圖目錄-------vi
表目錄-------vii
第一章、緒論 -1
1.1前言------1
1.2 研究目的及方法----2
第二章、 文獻回顧----3
2.1 靛藍之合成途徑及介紹----3
2.1.1 靛藍簡介----3
2.1.2 生產菌株與靛藍前驅物介紹----4
2.1.3 靛藍於細胞體內代謝途徑之簡介----5
2.2 輔因子還原系統----6
2.2.1 Dehydrogenase簡介----6
2.2.2 Formate dehydrogenase 代謝途徑及簡介----6
2.2.3 突變FDH增加對於NADP+活性----7
2.3 Surface display介紹----8
2.3.1 Surface display簡介與發展----8
2.3.2 不同類型Surface display功能及特性----9
2.3.3 Ice nucleation protein應用與特性----10
2.3 Two phase extraction生產系統----11
2.3.1 Two phase應用於微生物生產系統----11
2.3.2 有機相對於微生物的影響----12
2.3.3 有機相與不同菌種的關係----13
第三章、材料與方法----14
3.1 試劑與化學品----14
3.2 實驗室菌株介紹----14
3.3建構質體----14
3.4培養條件----18
3.4.1 Surface display相關實驗----18
3.5 Immunofluorescence assay測量及實驗方法----18
3.6 Activity assay測量方法----19
3.7 Co-culture實驗方法----20
3.8 Two phase extraction及co-culture實驗方法----20
3.9 酵素FMO於細胞體內之生產條件----21
3.10 顏色產物分析方法----21
第四章、結果與討論----22
4.1 INP成功表現FMO及FDH於外細胞膜----22
4.2 FMO之活性測試與體外生產靛藍----24
4.3 FDH之活性測試----26
4.4 FDH於不同濃度受質之活性測試----27
4.5尋找INP-FMO及INP-FDH最適反應之緩衝液----28
4.6測試最適induce條件----29
4.6.1 比較INP-FMO及INP-FDH不同induce時間之活性表現----29
4.6.2 不同induce溫度對於酵素的影響----30
4.7 以Co-culture建構體外生產靛藍系統----31
4.8 利用RBS對基因表現進行調控----33
4.9 透過位點突變FMO提高催化效率----35
4.10 以two phase 建構co culture 體外生產靛藍系統----36
4.10.1 靛藍產物於有機相分析----36
4.10.2 靛藍產物於水相分析----38
4.11 提升單位菌量以達到更高產量之靛藍產物----39
4.12 於two phase系統長時間進行生產之測試----42
4.12.1 以試管進行微量生產測試----42
4.12.2 以搖瓶進行生產測試----44
4.13 輔因子循環利用之長時間生產測試----46
4.14 FMO之突變位點測試與分析----49
第五章、結論----50
參考文獻----51

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