生質化學品的可持續性被視為解決當前環境問題的其中一個方案，化學產業正在嘗試使用生物精煉方法來生產替代石油基化學品的化學品。在眾多化學品中，衣康酸被美國能源部於2004年評選為十二種最具價值的生質平台化學品之一。衣康酸具有一個甲基和兩個羧基，使其可以透過化學方法生成多種衍生物，並應用於聚酯、塑膠等材料中，因此每年的衣康酸產量超過八萬噸。然而，使用原生菌種(Aspergillus terreus)生產衣康酸的製程成本相對於石化系化學品並不具有競爭力。
因此，本論文透過代謝工程建立了一株能夠生產衣康酸的大腸桿菌菌株。目前，該菌株是通過剔除icd基因並大量表達cad、acnB、gltA和pyc基因來達到最佳產量。同時，透過代謝演化提高生產效率，篩選出高效率的衣康酸生產菌株，並能夠利用多種碳源（如葡萄糖、木糖、甘油、粗甘油等）作為原料。
在以葡萄糖、木糖和甘油作為碳源的培養基中，衣康酸的平均產量在72小時內接近20-22 g/L，最終轉化率約為0.30-0.43 g/g(相對於最大理論轉化率72%的42-60%)。特別值得注意的是，在TB培養基中，相比於葡萄糖，甘油和木糖的生產速率、產量和轉化率更高，這表明它們在我們的生產系統中能夠有效代謝為所需的前驅物。衣康酸的最高生產速率和轉化率均出現在生產的最初24小時內，分別接近0.43-0.60 g/L/h和0.35-0.55 g/g(相對於最大理論轉化率的47-76%)。
在6000-L醱酵槽中，大腸桿菌生產衣康酸的累積產量在42小時內達到87 g/L，轉化率(甘油轉化為衣康酸)超過60%，生產速率為2.07 g/L/h。
本論文內容涵蓋了衣康酸生產菌株的構建，輔以定向進化和醱酵製程的開發，成功建立了高效率生產衣康酸的菌株和6000-L醱酵槽製程。

The sustainability of bio-based chemicals is considered one of the solutions to current environmental issues, and the chemical industry is exploring the use of biorefinery methods to produce chemicals as alternatives to petroleum-based ones. Among many chemicals, itaconic acid was selected as one of the twelve most valuable bio-based platform chemicals by the U.S. Department of Energy in 2004. Itaconic acid has one methyl and two carboxyl groups, allowing it to be chemically transformed into various derivatives and applied in materials such as polyesters and plastics. Therefore, the annual production of itaconic acid exceeds 80,000 tons. However, the production cost of itaconic acid using the native strain Aspergillus terreus is not competitive compared to petrochemical-based chemicals.
Therefore, in this study, an Escherichia coli strain capable of producing itaconic acid was developed through metabolic engineering. Currently, the strain achieves optimal production by deleting the icd gene and overexpressing the cad, acnB, gltA, and pyc genes. Additionally, directed evolution was employed to improve production efficiency and select highly efficient itaconic acid-producing strains capable of utilizing various carbon sources such as glucose, xylose, glycerol, and crude glycerol as substrates.
In culture media with glucose, xylose, and glycerol as carbon sources, the average itaconic acid titer reached approximately 20-22 g/L within 72 h, with a final conversion yield of about 0.30-0.43 g/g (42-60% of the maximum theoretical yield). Notably, in TB medium, glycerol and xylose showed higher production rates, yields, and conversion efficiencies compared to glucose, indicating their efficient metabolism into the required precursors in our production system. The highest production rates and conversion yields of itaconic acid were observed within the first 24 h, reaching approximately 0.43-0.60 g/L/h and 0.35-0.55 g/g, respectively (corresponding to 47-76% of the maximum theoretical conversion yield).
In a 6,000-L fermentor, the cumulative itaconic acid production by E. coli reached 87 g/L within 42 h, with a yield exceeding 60% and a productivity of 2.07 g/L/h.
This study covered the construction of itaconic acid-producing strains, accompanied by directed evolution and the development of fermentation processes. It successfully established strains and a 6,000-L fermentation process for efficient itaconic acid production.

中文摘要 i
Abstract ii
誌謝辭 iv
目錄 v
圖目錄 vii
表目錄 ix
第 1 章 緒論 10
前言 10
研究動機與策略 12
衣康酸 12
衣康酸的生產 13
策略 15
第 2 章 文獻回顧 17
衣康酸醱酵生產 17
衣康酸代謝路徑 19
在 A. terreus 的代謝路徑 19
原生菌U. maydis與A. terreus之差異 21
衣康酸在哺乳類細胞的作用 24
A. terreus生產衣康酸的困難與挑戰 25
代謝工程改質菌株 26
利用大腸桿菌系統生產衣康酸 27
利用酵母菌系統生產衣康酸 30
利用麩胺酸棒狀桿菌系統生產衣康酸 31
利用藍綠菌系統生產衣康酸 31
第 3 章 材料與方法 34
菌株 34
基因剔除方法 34
DNA 操作方法 35
PCR-based E. coli codon-optimized A. terreus cad 基因合成 35
質體建構 36
培養液、菌株培養方式與醱酵生產條件 45
以試管或搖瓶中生產衣康酸 46
以醱酵槽生產衣康酸 47
代謝產物和麩胺酸的定量分析 48
粗萃蛋白中順烏頭酸脫羧酶活性分析 48
大腸桿菌萃取物製備 48
第 4 章 結果與討論 50
建立帶有 A. terreus 順烏頭酸脫羧酶的大腸桿菌 50
饋養路徑中的中間物（檸檬酸）發現前驅物的供給受到限制 52
剔除icd 和大量表現acnB, gltA, ppc 顯著提升衣康酸產量 53
大量表現pyc 增加衣康酸生產速率 54
氧氣在衣康酸生產扮演重要角色 57
氮源組成與濃度對衣康酸的影響 58
剔除 glyoxylate shunt 對衣康酸生產沒有助益 59
調整培養液 pH 測試不同碳源長時間衣康酸產量的變化 60
以甘油為碳源於醱酵槽中利用批次饋料策略放大測試衣康酸的生產 62
持續性表達啟動子與誘導行啟動子比較 63
於搖瓶中測試不同啟動子對衣康酸的影響 63
於0.5-L醱酵槽中測試不同啟動子對衣康酸生產的影響 64
代謝演化 66
醱酵槽生產製程開發 69
醱酵槽生產條件探討 69
5-L醱酵槽生產 72
6000-L 醱酵槽放大測試 74
有機會取代酵母萃取物的氮源 76
胺基酸對菌株PCC553生產衣康酸的影響 76
代謝演化高麩胺酸利用率的衣康酸生產菌株 78
不同濃度的麩胺酸對代謝演化後的菌株PCC553-m-G4 衣康酸產量的影響 79
使用味精工廠副產物(CMS)補充麩胺酸生產衣康酸 80
使用廢棄大腸桿菌作為氮源 84
其他碳源可行性評估 87
粗甘油 87
第 5 章 結論與未來展望 90
結論 90
菌株建構 91
以醱酵槽進行衣康酸生產測試 92
替代氮源 93
未來展望 93
菌株改良 93
醱酵製程 96
第 6 章參考文獻 99

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