大量燃燒石化燃料促成大氣中二氧化碳濃度的遽增造就了溫室效應顯著的增幅為影響氣候變遷的主要因素，透過藍綠菌天然固二氧化碳能力生產石油衍生化學品為生質技術能源領域一重要研究項目。另一方面於回收廢棄物中具高含量木糖的木質纖維素作為碳源生產木糖衍生化學品矣為一重點發展項目，因此我們於藍綠菌中建構異源木糖代謝途徑生產具有高經濟價值的木糖醇及1,4-丁二醇。藍綠菌中可直接使用二氧化碳作為生長所需碳源及供給生產衍生物所需之酵素及cofactor之調控，希望藍綠菌藉由無天然代謝木糖機制將木糖直接轉換成木糖衍生化學品。
於藍綠菌中轉植來自E. coli中xylose transporter(xylE, xylFGH)將木糖有效運送至胞內後經由Candidia boidinii xylose reductase(CbXR)或是E. coli的xylose isomerase (xylA)及Pichia Stipitis的xylitol dehydrogenase(PsXDH)兩不同木糖醇代謝路徑生產木糖醇，經由調控Bicarbonate IPTG及木糖添加濃度等方式，藍綠菌表現xylE及CbXR於240小時可生產最高1.47g/L木糖醇。利用木糖生產1,4-丁二醇方面，木糖酸為其前驅物，先經由自Caulobacter crescentus的xylose dehydrogenase (xylBC)將木糖一步代謝成木糖酸測試異源基因的表現，目前木糖酸可於192小時生產最高至365mg/L。於藍綠菌中生產木糖衍生化學品過程，發現藍綠菌中表現xylE具有較好的木糖轉運效率，另外提高木糖濃度有利於提高木糖衍生化學品的產量但其yield(g/g xylose)降低，最後異源酵素cofactor類型的使用對於藍綠菌在生產及生長上具有一定的影響，NADPH-dependent酵素相較於NADH-dependent具有相對較好的產量及生長狀況。

The major factor that affecting the climate changed is the increasing green house effect which is attributed to the rising level of atmospheric carbon dioxide causing by burning fossil fuels. So nowadays producing petroleum-derived chemicals through the natural fixing carbon dioxide ability in cyanobacteria for reducing oil dependency is an important research project for bio-energy technologies. On the other hand , Recycling the waste of lignocellulose with high content of xylose for using as a carbon source to produce high economic value xylose-derived commodity products is the another key development project. Therefore we established the heterologous xylose metabolic pathways for producing xylitol and 1,4-Butandiol (1,4-BDO) in the Synechococcus elongatus PCC 7942.
In the engineered cyanobacteria fix CO2 as the carbon source for growth and protein synthesis, and provide the enzyme and cofactor for xylose-derived chemicals production. The carbon flux from heterologous xylose metabolic pathway in cyanobacteria could produce xylitol and 1,4-BDO directly and completely due to the characteristic of cyanobacteria haven’t the natural ability of xylose metabolism.
Integrated the heterologous xylose transporter (xylE, xylFGH) from E. coli to help xylose uptake then xylose metabolize to xylitol via xylose reductase from Candidia Boidinii (CbXR) or xylose isomerase(xylA) of E.coli and xylitol dehydrogenase (PsXDH) of Phichia Stipitis. The xylE assorted with CbXR can produce xylitol 1.47g/L in 240hr. In 1,4-BDO production, the first step is producing xylonic acid via xylose dehydrogenase (xylBC). We found xylE and xylBC arranged in groups can produce about 365mg/L in 192hr.
In the process of using cyanobacteria produce xylose-derived compounds we found xylE has the higher xylose uptake efficiency. Enhancing the xylose concentration can increase the titer of xylose-derived compounds (g/L) but decrease the yield(g/g xylose). Final, the cofactor type of heterologous enzymes used has the certain correlation with the production and the growth of the strains. The NADPH-dependent enzymes cause the strains get higher production yield and grow better than the strains with the NADH-dependent enzymes .

摘要 1
Abstract 3
誌謝 5
第一章 研究背景及目的 10
1-1用生物方式生產1.4丁二醇 11
1-2利用生物方式生產木糖醇 12
1-3利用藍綠菌生產化學品 14
1-4 菌種及目標產物介紹 16
1-4-1 藍綠菌簡介 16
1-4-2 1,4-丁二醇簡介 17
1-4-3 木糖醇簡介 18
1-5木糖衍生物生產途徑簡介 19
1-5-1 1,4-丁二醇生產途徑簡介 19
1-5-2 木糖醇生產代謝途徑簡介 22
1-6 研究動機 24
第二章 實驗材料及方法 26
2-1 菌株介紹 26
Synechococcus elongatus PCC 7942 26
Escherichia coli XL-1 Blue 26
2-2 建立質體 27
2-3 將建構之質體植入菌株 34
2-3-1 Competent cell製作 34
2-3-2電穿孔進行質體轉植 34
2-3-3 Heat Shock質體轉植 35
2-4藍綠菌質體轉殖 35
2-5利用P1 transduction進行基因剔除 35
2-6 木糖衍生化學品生產程序 37
2-6-1 製備木糖衍生物化學品使用的培養液 37
2-6-2木糖衍生化學品生產條件 37
2-6-3樣品抽取 38
2-7 代謝物偵測 39
2-7-1 木糖酸產率偵測 39
2-7-2木糖醇產率偵測 40
2-8 S. elongatus PCC7942 crude cell extract的製備 40
2-9酵素於體外活性測試 41
2-10 P. stipites (ATCC 58785) 及C. boidinii (ATCC 18810) Genomic DNA 製備 41
第三章 實驗結果與討論 42
3-1建構質體於藍綠菌中生產木糖衍生物 42
3-1-1建構質體於藍綠菌中生產木糖酸及1,4-丁二醇 42
3-1-2建構質體於藍綠菌中生產木糖醇 43
3-2藍綠菌進行木糖酸的產量測試 46
3-3測量xylBC於藍綠菌中的酵素活性 49
3-4藍綠菌進行木糖醇生產的測試 51
3-5 測量CbXR於藍綠菌中的酵素活性 57
3-6 CbXR codon optimization 58
3-7 木糖醇於生產木糖醇菌株的毒性測試 61
3-8 調控Bicarbonate 、IPTG及xylose於木糖衍生化學品的生產 62
3-8-1調控Bicarbonate於木糖酸的生產 64
3-8-2 調控IPTG於木糖酸的生產 65
3-8-3 調控木糖濃度於木糖酸的生產 67
3-8-4調控Bicarbonate於木糖醇的生產 68
3-8-5調控IPTG於木糖醇的生產 70
3-8-6調控木糖濃度於木糖醇的生產 71
3-9 尋找最高參與生產1,4-丁二醇代謝途徑的adh 73
結論 75
參考文獻 76

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