目前使用氧化還原平衡為基礎的代謝演化(metabolic evolution)，都是針對可以消耗輔因子(cofactor) NADH之合成代謝途徑，以提高產量所衍生的方法。代謝演化的進行，必須依賴菌體內因修飾發酵代謝途徑後，在無氧培養時，會產生NADH累積，造成菌株幾乎不能生長的情況下，若此菌株同時有額外表現唯一一個會消耗NADH之生產目標物的合成代謝途徑，為了能消耗無氧條件所累積的NADH而產生了驅動力，讓碳通量(carbon flux)往合成代謝途徑，藉由代謝物的生成以消耗NADH，變成氧化態輔因子NAD+，可以使NAD+能循環於糖解作用並再利用，菌株就能恢復無氧生長，達成生產目標物之代謝途徑的演化，提高目標物產量之目的。若因目標代謝途徑所消耗之NADH，與菌株體內代謝生成NADH之化學計量數目上，產生不平衡的情形，則可能使這演化平台受到限制而無法使用，因此本論文目的是將以氧化還原平衡為基礎之無氧篩選平台進行改善，建立新篩選平台，並用來探討大腸桿菌內源性生成2,3-丁二醇之可能性，並衍生作為蛋白質工程所需之篩選平台的應用。
本論文利用突變菌株JCL166於無氧環境，會累積NADH而無法生長的特性，在其菌體內表現任一可以消耗NADH以進行催化反應的基因，並在培養過程提供代謝中間物，作為第二碳源，使消耗NADH的代謝途徑，不再受限於與原本內源代謝途徑(糖解作用)所產生NADH化學計量數之比例限制，讓菌株在無氧環境恢復生長能力。因此，只要突變菌株JCL166額外所表現的基因(要篩選的目標基因)，能夠對於添加的代謝中間物，進行還原反應，消耗累積NADH，讓菌株原本不能在無氧環境生長，卻因正確基因表現氧化還原酵素活性，還原所添加的代謝中間物，去除NADH累積而修復無氧生長能力。因此，建立本論文之非單一碳源氧化還原篩選平台後，以此篩選平台成功確立大腸桿菌甘油去氫酶(glycerol dehydrogenase)GldA為主要具有還原acetoin活性的酵素，並且能讓大腸桿菌不表現任何異源基因下，以自身內源基因成功生產3 g/L meso-2,3-丁二醇。另外，為了提高2,3-丁二醇產量，大腸桿菌共同表現NADH專一性Kp-BudC (Klebsiella pneumoniae)與NADPH專一性Cb-Adh (Clostridium beijerinckii)之2,3-丁二醇去氫酶，在這2種酵素協同作用下，不但能讓菌株本身可以因應環境的變化，自行調整使用NADH及NADPH，維持高度酵素催化活性的表現，並且能幾乎不累積代謝中間產物acetoin情況下，有2,3-丁二醇之高產量生成。經過批次饋料發酵槽條件最適化探討，於發酵56小時後，菌株生產可達90 %皆為(R,R)形式之92 g/L 2,3-丁二醇。此外，本論文的篩選平台，亦能應用於改變氧化還源酵素輔因子專一性為NADH之蛋白質工程的篩選。將NADPH專一性2,3-丁二醇去氫酶，Cb-Adh，進行突變後，再以非單一碳源氧化還原篩選平台進行選別，成功篩選出NADH專一性之突變酵素Cb-Adh(M23-6)，kcat為約1 s-1。因此，本論文建立非單一碳源氧化還原篩選平台，將來能在學術或產業界，對於未知基因探勘或是蛋白質工程改質，具有實質應用價值，以利代謝工程或合成生物方法構築代謝途徑，讓菌株生產特用化學品。

Fermentative redox balance has long been utilized as a metabolic evolution platform to improve the efficiency of NADH-dependent pathways and bioprospect enzyme activity. However, such a system relies on the complete recycling of NADH and may become limited when the target pathway results in excess NADH stoichiometrically. The purpose of this study is to improve anaerobic selection platform by fermentative redox balance and increase the extensive application of this platform.
In this research, an endogenous capability of Escherichia coli for 2,3-butanediol (2,3-BD) synthesis was explored using the anaerobic selection platform based on redox balance. To address the issue of NADH excess associated with the 2,3-BD pathway, we devised a substrate-decoupled system where a pathway intermediate is externally supplied in addition to the carbon source to decouple NADH recycling ratio from the intrinsic pathway stoichiometry. Based on this substrate-decoupled selection scheme, we successfully identified the glycerol dehydrogenase (Ec-GldA) as the major enzyme responsible for the acetoin reducing activity observed in E. coli. The final strain demonstrated a meso-2,3-BD production titer of 3 g/L without the introduction of foreign genes.
Furthermore, This work also describes the engineering of cofactor versatility for 2,3-BD production by simultaneous overexpression of a NADH-dependent 2,3-BD dehydrogenase from Klebsiella pneumoniae (Kp-BudC) and a NADPH-specific 2,3-BD dehydrogenase from Clostridium beijerinckii (Cb-Adh). Co-expression of Kp-BudC and Cb-Adh improved the condition versatility for 2,3-BD synthesis via flexible utilization of NADH and NADPH. With optimization of medium and fermentation condition, the co-expression strain produced 92 g/L of 2,3-BD in 56 h with 90 % stereopurity for (R,R) isoform and 85 % of maximum theoretical yield.
Changing cofactor preference of oxidoreductases by protein engineering is oftentimes essential to make the pathway more suitable for industrial application. However, screening of the potential positives in a huge mutant library from rational design or random mutagenesis can be labor-intensive. In this work, we used the NADPH-dependent alcohol dehydrogenase from Clostridium beijerinckii (Cb-Adh) as an example and created a mutant library based on random mutagenesis using error prone PCR. Upon selection and enrichment of the mutant library using the redox balance platform, we successfully isolated Cb-Adh variants with cofactor preference changed from NADPH to NADH. After two rounds of mutagenesis and selection, the final Cb-Adh mutant displayed significant increase in its catalytic activity toward NADH and non-detectable activity toward NADPH. The substrate-decoupled selection system allows redox balance regardless of the pathway stoichiometry thus enables segmented optimization of different reductive pathways through enzyme bioprospecting and metabolic evolution.

摘要..............................................................I
Abstract.......................................................III
謝誌..............................................................V
目錄.............................................................VI
圖表...........................................................VIII
第一章 緒論......................................................1
1-1 前言.........................................................1
1-2 研究動機與策略................................................3
第二章 文獻回顧...................................................5
2-1 輔因子(cofactor)在代謝工程重要性與應用性.......................5
2-1-1 輔因子參與代謝反應的重要性...................................5
2-1-2 NAD(P)H專一性氧化還源酵素之簡介..............................6
2-1-3 蛋白質工程應用於氧化還源酵素對輔因子專一性的改變..............11
2-1-4 輔因子於代謝工程的應用性....................................13
2-2 2,3-丁二醇合成途徑之簡介與應用................................15
2-2-1 生產菌株介紹..............................................15
2-2-2 合成途徑簡介..............................................15
2-2-3 可能具有的生理功能.........................................17
2-2-4 2,3-丁二醇的應用..........................................17
2-3 微生物合成2,3-丁二醇的代謝工程策略............................18
2-3-1 原生菌株之代謝工程.........................................18
2-3-2 量產發酵策略..............................................21
第三章 材料與方法................................................25
3-1 試劑與化學品................................................25
3-2 代謝工程改質菌株生產2,3-丁二醇................................25
3-2-1 製備實驗菌株...............................................25
3-2-2 建構質體...................................................26
3-3 培養基與發酵生產條件.........................................32
3-4 無氧條件之生長復原測試Anaerobic growth rescue................33
3-5 利用ASKA質體基因庫篩選二級醇去氫酶............................33
3-6 代謝產物分析方法.............................................34
3-7 酵素純化....................................................35
3-8 酵素活性測試................................................35
3-9 發酵槽試驗..................................................36
3-10 Error prone PCR技術建立突變質體庫...........................37
第四章 結果與討論(1)-以發酵氧化還原平衡建立非單一碳源氧化還原篩選平台38
4-1 添加代謝中間物acetoin建立非單一碳源氧化還原篩選平台(substrate-decoupled redox selection platform).............................38
4-2 篩選平台對於不同輔因子(cofactor)之反應靈敏度探討...............42
第五章 結果與討論(2)-非單一碳源氧化還原篩選平台的應用(I)—探尋具氧化還原酵素活性之未知基因：大腸桿菌可能存在之內源性2,3 -丁二醇代謝途徑.......49
5-1 由ASKA質體基因庫篩選大腸桿菌具有2,3-丁二醇去氫酶活性之可能基因..49
5-2 具有2,3-丁二醇去氫酶活性之GldA酵素活性特徵探...................51
5-3 大腸桿菌是以經由diacetyl代謝途徑生成acetoin...................54
5-4 大腸桿菌表現特定內源基因之以生產2,3-丁二醇.....................57
第六章 結果與討論(3)-大腸桿菌自發性調節利用NADH與NADPH以增進2,3-丁二醇產量.............................................................60
6-1 共同表現NADH專一性與NADPH專一性丁二醇去氫酶之改質菌株以適應環境變化及提升2,3-丁二醇產量..............................................60
6-2 細胞粗酵素活性分析(Enzyme activity in crude extracts)........63
6-3 長時間發酵生產2,3-丁二醇.....................................64
6-4 發酵槽生產條件探討...........................................66
第七章 結果與討論(4)- 非單一碳源氧化還原篩選平台的應用(II)—蛋白質工程(protein engineering)：轉換氧化還原酵素輔因子專一性................70
7-1 建立突變基因質體庫以篩選輔因子專一性改變之突變酵素..............70
7-2 已改變輔因子專一性之酵素活性特徵探討...........................74
7-3 再次利用突變基因庫篩選以恢復原有酵素催化活性...................76
7-4 再次突變篩選之酵素活性特徵探討................................79
7-5 利用篩選平台進行蛋白質工程改質其他氧化還原酵素之應用............81
7-5-1 蛋白質工程改質木糖還原酶 (xylose reductase).................81
7-5-1-1 調整非單一碳源氧化平衡篩選平台之培養條件...................81
7-5-1-2 以木糖還原酶突變基因質體庫進行非單一碳源氧化還原平台篩選....85
7-5-2 羧酸還原酶 (carboxylate reductases)之蛋白質工程............86
第八章 結論與未來展望............................................90
8-1 結論........................................................90
8-2 未來展望.....................................................92
參考文獻.........................................................94

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