由於化合物之碳鏈長度不同時，其性質也會跟著改變，而合成C-C鍵是構建碳框架和擴大生化多樣性之關鍵步驟。本論文主要針對α-酮酸(2-keto acids)與Acetyl-CoA之碳鏈延伸進行研究。一般而言，α-酮酸結合Acetyl-CoA碳鏈延伸途徑有二，其一為結合後形成α-羥基羧酸(α-hydroxy carboxylic acids)，屬於「重複碳鏈延伸系統」，本論文結合α-轉胺酶篩選平台，開發胺基酸碳鏈延伸途徑；另一為透過CoA參與反應途徑，本論文結合熱力學驅動力，開發新穎衣康酸合成途徑。
非天然胺基酸具有抗癌、抗菌之功效以及於藥物開發端亦扮演著要角，然而許多非天然胺基酸只能透過人工合成方式而得，因此，利用代謝工程創建合成途徑顯得相當重要。由於α-酮酸不僅作為胺基酸合成重要中間體，其結構上具有ketone group，使得其可透過結合不同的反應，如: 脫羧、氧化還原、胺化等，可獲得多樣的產物，應用潛力相當廣泛。因此，本論文欲利用大腸桿菌開發及優化α-酮酸之多樣性，以達到多元之胺基酸以及下游產物合成，透過開發不同生長篩選平台進行關鍵酵素之定向演化實現其目標。大腸桿菌中許多胺基酸是利用α-轉胺酶將α-酮酸轉化而得，因此，針對此特點對α-轉胺酶設計篩選平台，可望藉由各式天然與非天然胺基酸作為α-酮酸來源，以達到擴大、優化α-酮酸其來源多樣性。本論文成功利用此建立之篩選平台演化出α-轉胺酶突變體提升對目標胺基酸之活性，並進一步生產α-酮酸之下游產物，在產量方面也有所提升，其中亦結合碳鏈延伸系統，創建新的非天然胺基酸合成途徑，如Neopentylglycine、Homoserine以及Homoglutamate。另外，由於重複碳鏈延伸系統對帶有非烷烴基之α-酮酸其活性效率較差，因此本論文嘗試設計不同的篩選平台，以α-轉胺酶與碳鏈延伸系統之組合對營養缺陷株進行生長修復，進一步達到拓展α-酮酸合成酶對非天然受質之相容性，但目前尚未成功篩選到對目標α-酮酸活性提高之合成酶。
在合成途徑中結合不可逆的關鍵步驟通常被認為可有效地將碳通量導引到目標產物中，因此，本論文透過Thioesterase作為熱力學驅動力，實現逆轉衣康酸降解。以Pyurvate以及Acetyl-CoA作為主要前驅物，創建獨立於TCA cycle的新穎衣康酸合成途徑。透過生物探勘以及體外生產測試，證明該途徑之可行性，但由於體內生產衣康酸產量不理想，後續透過篩選適合的Thioesterase替換原途徑中CoA轉移酶、剔除競爭途徑基因加強前驅物之累積、降低生產環境溶氧效率以及強化醋酸同化，最終以高菌量生產下衣康酸產量於生產120小時可達1.15 g/L，相較於原途徑只有11 mg/L，有著相當大的差異。

As the length of the carbon chain of the compound is different, its properties will also change accordingly. Synthesis of C-C bonds is a key step in building carbon frameworks and expanding biochemical diversity. This study mainly focuses on the carbon chain extension between α-keto acids (2KA) and acetyl-CoA. Generally speaking, there are two ways for α-keto acids to combine with acetyl-CoA to extend the carbon chain. One is to form α-hydroxycarboxylic acids after combination, which belongs to " iterative carbon chain elongation ". In this study, we developed the synthetic amino acid elongation pathway based on α-transaminase selection platform; the other is through CoA participating in the reaction pathway, we constructed a novel itaconic acid synthesis pathway via thermodynamic driving force.
Unnatural amino acids (UAAs) have anti-cancer and antibacterial effects and the potential for drug development as building blocks. However, many unnatural amino acids can only be obtained through artificial synthesis. Therefore, it is quite important to create synthetic pathways by metabolic engineering. Since α-keto acids (2KA) not only serve as important intermediate in the amino acids synthesis, but have a ketone group in its structure, it can obtain various products by combining different reactions, such as decarboxylation, redox, amination, etc., resulting in its application potential is quite wide. This study aims to use Escherichia coli to diversify 2KAs, enabling the synthesis of a wide range of amino acids and related products. This goal will be achieved through the creation of growth-related selection platforms for the directed evolution of essential enzymes. Many amino acids in E. coli are obtained by converting 2KA through α-transaminase. Therefore, designing a selection platform for α-transaminase based on this characteristic is expected to use various natural and non-natural amino acids as sources of 2KA to expand and optimize the source diversity of 2KA. This study successfully used the established selection platform to evolve α-transaminase mutants to improve the activity toward target amino acids and further produce downstream products of 2KAs, the yield has also been improved. Furthermore, the evolved α-transaminases also combine chain elongation to create new non-natural amino acid synthesis pathways, such as Neopentylglycine, Homoserine and Homoglutamate. Synthetic unnatural amino acids are particularly important, especially for anticancer drugs and other high-value added chemicals. In addition, since the iterative carbon chain elongation system has poor activity efficiency for 2KAs with non-alkane groups, this study attempts to use a combination of α-transaminase and carbon chain elongation system to design selection platform to further expand the promiscuity of α-keto acid synthase with non-natural substrates. However, no synthase with improved activity toward the target 2KA has yet been successfully selected.
Development of an irreversible and committed step within the pathway is often seen as effective to direct higher carbon flux into the target product. Here, we demonstrate that enhancing of thermodynamic driving force by an irreversible reaction catalyzed by thioesterase may be beneficial to reverse itaconic acid degradation. Using pyruvate and acetyl-CoA as the main precursors, a novel itaconate synthesis pathway which is orthogonal to the TCA cycle was created. First, we bioprospected various heterologous enzymes and conducted the in vitro assay to demonstrate the pathway feasibility. However, due to suboptimal production of itaconate in vivo, we screened the suitable thioesterase to replace the CoA transferase to improve the thermodynamic feasibility. Subsequently, we deleted competing pathways to enhance the precursors pools, improved acetate assimilation and process optimization (pH level, dissolved oxygen amount), finally, with high cell density production, the IA titer can reach 1.15 g/L. Compared with the original route, which can only produce 11 mg/L IA, there is a considerable improvement.

摘要 i
Abstract ii
目錄 iv
第一章 緒論 1
1-1 前言 1
1-2 研究動機與策略 2
第二章 文獻回顧 3
2-1 合成Unnatural amino acids (UAAs)之重要性與應用 3
2-2 α-酮酸 (2-keto acid)在代謝工程重要性與應用性 3
2-3 2-keto acid生產及轉胺酶介紹 4
2-4 蛋白質工程 (Protein engineering) 5
2-5 碳鏈延伸 (Carbon chain elongation) 9
2.5.1 碳鏈延伸介紹 9
2.5.2 α-酮酸合成酶之簡介 13
2-6 衣康酸合成途徑之簡介與應用 18
2.6.1 合成途徑簡介 18
2.6.2 降解途徑簡介 19
2-7 合成驅動力之簡介與重要性 20
第三章 實驗材料及方法 21
3-1 化學藥品及試劑 21
3-2 菌株介紹 21
3-3 質體建構 21
3-4 培養基與生產條件 21
3-4-1 α-轉胺酶篩選平台所使用之培養液 21
3-4-2 KS篩選平台所使用之培養液 21
3-4-3 利用經篩選後α-轉胺酶之生產條件 22
3-4-4 胺基酸碳鏈延伸之生產條件 22
3-4-5 逆衣康酸降解生產條件 22
3-6 代謝產物分析方法 23
3-7 酵素純化 24
3-8 酵素活性分析 24
3.8.1 KS酵素活性分析 24
3.8.2 α-轉胺酶酵素活性分析 25
3.8.3 逆衣康酸降解途徑酵素活性分析 25
3-9 體外生產衣康酸 26
第四章 結果與討論 (一) 27
4-1利用α-轉胺酶篩選平台拓展2-keto acids多元性 27
4-1-1 利用已建構完成的α-轉胺酶篩選平台找尋非內源之2-keto acids 27
4-1-2 進行α-轉胺酶酵素動力學及突變位點分析 30
4-1-3 利用經篩選後之α-轉胺酶突變體生產2-keto acids之下游產物 34
第五章 結果與討論 (二) 36
5-1 利用2-keto acid synthase (KS)篩選平台拓展α-酮酸合成酶對非天然受質之相容性 36
5-1-1 探勘2-keto acid synthase (KS)之特性 36
5-1-2 結合α-轉胺酶突變體進行胺基酸碳鏈延伸 39 40
5-2於大腸桿菌內建構2-keto acid synthase (KS)篩選平台 44
5-2-1以Threonine/Methionine營養缺陷株建立KS篩選平台 45
5-2-2 開發3-OH-pyr、4-OH-2KB下游產物 46
5-2-3 建立Threonine/Methionine營養缺陷株 47
5-2-4 測試KS對3-OH-pyr活性及對RJ01生長修復狀況 47
5-2-5 以Methionine/Threonine營養缺陷株對KS基因突變庫進行篩選 48
5-2-6 優化以Threonine/Methionine營養缺陷株建立之KS篩選平台 48
5-3 以Phenylalanine營養缺陷株做為KS篩選平台 50
5-4 以Tyrosine營養缺陷株做為KS篩選平台 53
第六章 結果與討論 (三) 58
6-1 構建逆衣康酸降解途徑 58
6-2 鑑定逆衣康酸途徑具高效率之酵素組合 59
6-3 逆衣康酸降解途徑之體內試驗 60
6-4 提高熱力學驅動力以提升衣康酸產量 61
6-5 氧氣限制對逆衣康酸降解途徑生產之影響探討 62
6-6 具有衣康酰輔酶A硫酯酶活性之硫酯酶特徵探討 64
6-7 透過增加前驅物及降低競爭以優化衣康酸生產 67
6-8 嘗試以發酵槽進行生產放大測試 69
6-9 以高菌量條件生產衣康酸 71
6-10 透過生產條件優化提升衣康酸產量 73
第七章 結論與未來工作 75
7-1 結論 75
7-2 未來工作 77
7-2-1 New route for itaconate biosynthesis through transcriptional factor based screening 77
參考文獻 79

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