近年來，許多研究開始著重於尋找石化原料的替代品，而其中又以FDCA最具有未來性，FDCA可替代塑膠的原料－聚對苯二甲酸乙二酯 (PET) 製程中所需的對苯二甲酸，對環境較PET更為友善。目前FDCA可由HMF氧化成HMFCA或DFF，再氧化成FFCA，最後氧化成FDCA，本研究以基因轉殖技術將文獻提及可生產FDCA的酵素HmfH、HMFO、AAO、UPO、CPO、PAMO及GOase基因分別接入載體，將其轉型入對有機溶劑具有高耐受性的Pseudomonas. putida S12表現不同的酵素，以全細胞生物轉換法將HMF轉化成FDCA。我們首先將含有酵素基因的質體分別轉型入P. putida中，並以強啟動子PHCE表現酵素與50 mM HMF反應，發現HmfH及HMFO可將HMF轉化成FDCA，其轉化率分別約為78.8 %及77.6 %，其餘酵素僅反應至HMFCA即終止。接著我們測試以ATc作為誘導劑的誘導型啟動子PTc表現前次結果無法生產FDCA的酵素，並以20及100 ng/ml的ATc誘導，但所有酵素反應依然止於HMFCA。由於目前的文獻僅GOase及HRP被提及可用E. coli生產，而P. putida與E. coli同為原核生物，因此接下來我們將GOase與HRP一同使用搭配不同的啟動子與信號肽，期望能解決HMFCA累積的問題，但仍然無法生產FDCA，因此我們推測這些酵素可能不適合於P. putida中表現。而為了將酵素基因嵌入P. putida染色體以降低篩選菌株所使用的抗生素成本，我們將三種CRISPR系統SpCas9、SaCas9及FnCas12a應用於P. putida中，發現其搭配λ-Red系統可成功嵌入外源基因，因此我們分別將可生產FDCA的HmfH及HMFO兩個酵素基因分別嵌入P. putida染色體中並與50 mM HMF反應，結果顯示HMF可成功被轉化成FDCA，其轉化率分別約為86.1 %及84.2 %。未來我們會繼續嘗試其他培養條件或酵素，期望能解決HMFCA累積的問題並提高FDCA產量，並設計去除CRISPR質體的系統，以增加菌株的穩定性。

Recently, FDCA becomes one of the most promising bio-based compound. It can be an alternative to the ingredient of PET and is more eco-friendly. FDCA can be produced by oxidizing HMF to HMFCA or DFF, then to FFCA, finally to FDCA. In this study, we expressed several enzymes such as HmfH, HMFO, AAO, UPO, CPO, PAMO and GOase for FDCA production in bacteria Pseudomonas putida S12 with high tolerance to organic solvent by bio-transformation. We first expressed enzymes by strong promoter P¬HCE and reacted with 50 mM HMF. HmfH and HMFO could successfully convert HMF to FDCA at the yield of ~78.8 % and 77.6 %, but others could only produce HMFCA. Next, we expressed enzymes that couldn’t work in previous result by inducible promoter PTc which could be induced by ATc and induced the expression by 20 and 100 ng/ml ATc, but the reaction still stopped at HMFCA. Because GOase and HRP were the only enzymes that could be expressed in E. coli in previous literatures, we then expressed GOase and HRP together combining different promoter and signal peptide. Unfortunately, HMFCA was still the main product. To integrate enzyme gene into the genome of P. putida, we applied 3 CRISPR system SpCas9, SaCas9 and FnCas12a in P. putida, and found that all of them could integrate foreign genes into the genome with the help of λ-Red system. Thus, we integrated HmfH and HMFO genes into its genome, and 50 mM HMF could be successfully transformed to FDCA at the yield of ~86.1 % and 84.2 %. For future work, we will try to optimize our culture condition or express other enzymes, hoping to solve the problem of HMFCA accumulation. Besides, we will design the plasmid curing system for elimination fo CRISPR plasmid to enhance the stability of strain.

誌謝 I
摘要 II
Abstract III
目錄 IV
表目錄 VII
圖目錄 IX
第一章 文獻回顧 1
1-1 前言 1
1-2 FDCA (2,5-furandicarboxylic acid) 1
1-3 HMF (5-hydroxymethyl-2-furfural) 3
1-4 HMF氧化還原酶 3
1-5 Pseudomonas putida S12 5
1-6 生物固定化技術與全細胞生物轉化法 (whole-cell biotransformation) 6
1-7 CRISPR/Cas系統 7
1-8 λ-Red系統 8
1-9 研究動機與目標 9
第二章 實驗材料與方法 19
2-1 建構質體方法 19
2-1-1 聚合酶連鎖反應 (polymerase chain reaction，PCR) 19
2-1-2 限制酶 (DNA restriction enzyme) 反應 20
2-1-3 凝膠電泳法 (gel electrophoresis) 20
2-1-4 瓊脂凝膠萃取DNA片段 21
2-1-5 接合酶 (DNA ligase) 反應 21
2-1-6 DNA引子黏合 (primer annealing) 與磷酸化反應 22
2-1-7 Gibson Assembly 23
2-1-8 以熱休克法 (heat-shock) 將質體轉型至E. coli 23
2-1-9 自E. coli萃取質體 24
2-2 質體建構 25
2-2-1 模板質體pBBR 27
2-2-2 含酵素基因之pBBR質體 28
2-2-3 含酵素基因及誘導型啟動子之pBBRTc質體 28
2-2-4 含GOase、HRP及強啟動子PHCE之pBBR-GH質體 28
2-2-5 含酵素基因及信號肽之質體 29
2-3 勝任細胞 (competent cell) 製備 30
2-4 以電穿孔法 (electroporation) 將質體轉型入P. putida 31
2-5 CRISPR及λ-Red系統嵌入模板DNA測試 32
2-6 轉殖菌株之HMF轉化反應 33
2-7 十二烷基硫酸鈉聚丙烯醯胺凝膠電泳 (SDS-PAGE) 35
2-8 西方墨點法 (Western blot) 36
2-9 LC-PDA分析 37
第三章 實驗結果與討論 51
3-1 實驗流程 51
3-2 更換質體之抗生素抵抗基因 51
3-3 以強啟動子PHCE表現各種酵素與HMF反應 52
3-4 以誘導型啟動子PTc表現各種酵素與HMF反應 53
3-5 同時表現GOase及HRP並加入信號肽與HMF反應 54
3-6 測試不同CRISPR系統是否可對P. putida造成雙股斷裂 55
3-7 以CRISPR系統將HmfH基因嵌入P. putida之染色體 57
3-8 以CRISPR搭配λ-Red系統將HmfH基因嵌入P. putida之染色體 58
3-9 將染色體嵌有HmfH或HMFO基因之P. putida與HMF反應 60
第四章 結論 71
第五章 未來工作 74
參考文獻 76

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