乙醯乙醇和2,3-丁二醇為工業上重要的化學品，乙醯乙醇和2,3-丁二醇和其衍生物已經被廣泛應用於化工、食品、醫藥、溶劑、燃料及航太領域上。許多微生物可以產生乙醯乙醇與2,3-丁二醇，其中克雷白氏肺炎桿菌可代謝的碳源較多樣、效率較高且易於培養，因此很適合用於工業生產。過去已有許多研究利用基因工程修飾克雷白氏肺炎桿菌的乙醯乙醇/2,3-丁二醇代謝途徑，以減少碳源的消耗，並提高乙醯乙醇與2,3-丁二醇生產基因的表現量。本研究首先選擇幾個文獻報導較佳的做法，重複類似的基因修改，同時也剔除數個基因，以提高克雷白氏肺炎桿菌在老鼠的LD50的量，最後嘗試建立自細菌培養液中取得2,3-丁二醇的技術。本研究最終得到六株克雷白氏菌的突變株，也測定這些菌株的生長速率、酸鹼值變化、和乙醯乙醇等相關代謝產物的產量等，所得到結果與過去文獻報導大致雷同。另外，本研究也測試了其他文獻萃取與純化2,3-丁二醇的技術，也得到與文獻相符的結果。綜合以上結果，本研究證明現有文獻上報導之技術，如利用基因工程技術修改克雷白氏肺炎桿菌乙醯乙醇/2,3-丁二醇合成途徑，以及以加入鹽類分層等技術，適合用於工業上大量生產乙醯乙醇與2,3-丁二醇。

Acetoin and butane 2,3-diol is an industrially important chemical. The compound and its derivatives are widely used in chemical engineering, food, medicine, solvent, fuel, aeronautical and several other industrial fields. A number of microorganisms are able to produce acetoin and butane 2,3-diol. Among them, Klebsiella pneumoniae is a potentially useful producer because of its wide substrate spectrum, high production efficiency, and cultural adaptability. Several previous studies have modified the K. pneumoniae butane 2,3-diol biosynthesis pathway by genetic engineering to reduce by-product generation and to enhance acetoin and butane 2,3-diol synthesis reactions. This study also conducted a similar approach to engineer the butane 2,3-diol biosynthesis pathways. This was followed by deleting several virulence genes to increase LD50 of the K. pneumoniae. The last goal of this study is to establish a method to extract butane 2,3-diol from culture medium. A total of 6 K. pneumoniae mutants were constructed and their growth rate, medium pH, and production rates of butane 2,3-diol and acetoin were determined. The results are very similar with those reported in literatures. Furthermore, this study evaluated the efficiency of several previously known extraction techniques to purify butane 2,3-diol from culture medium. Our result is comparable with those reported before. Overall, this study confirms the findings reported by other literatures that genetic engineering of the butane 2,3-diol synthetic pathway and salt-based extraction techniques are useful in industrial production of butane 2,3-diol from K. pneumoniae.

摘要 I
Abstract II
致謝 IV
縮寫字對照表 V
目錄 VII
表目錄 IX
圖目錄 X
附錄目錄 XI
壹、 前言 1
1.1. 2,3-丁二醇 1
1.2. 以微生物生產2,3-丁二醇 2
1.3. 利用基因工程提高微生物的2,3-丁二醇產量 3
1.4. 克雷白氏肺炎桿菌的致病因子 4
1.5. 萃取2,3-丁二醇 6
1.6. 研究目的 8
貳、 材料與實驗方法 9
2.1. 菌株與生長環境 9
2.2. 染色體DNA萃取 9
2.3. 聚合酶連鎖反應 10
2.4. 勝任細胞製備 10
2.5. 熱休克轉型法 11
2.6. 質體構築 11
2.7. 建構各突變株 12
2.8. 生長曲線量測 13
2.9. 酸鹼值分析 13
2.10. 2,3-丁二醇脫氫酶 (butanediol dehyrdrogenase, BDH) 定量2,3-丁二醇 14
2.11. VP測試 (Voges–Proskauer test) 定量乙醯乙醇 14
2.12. 薄膜層析法 (thin layer chromatography, TLC) 定量2,3-丁二醇 15
2.13. 以3M™吸油棉分離萃取2,3-丁二醇 15
2.14. 鹽析法萃取2,3-丁二醇 15
參、 結果 17
3.1. 建構不同的突變株 17
3.2. ΔcpxAR在低營養培養基中生長速率下降 20
3.3. 不同的突變株呈現不同的酸鹼值 20
3.4. 定量各突變株的2,3-丁二醇產量 21
3.5. 定量各突變株的乙醯乙醇產量 22
3.6. 萃取2,3-丁二醇 22
肆、 討論 25
伍、 參考文獻 30

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