利用代謝工程在大腸桿菌中優化正丁醇與正丙醇的比例

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研究生：	陳世濟 Chen, Shih Chi
論文名稱：	利用代謝工程在大腸桿菌中優化正丁醇與正丙醇的比例 Engineering Higher 1-Butanol to 1-Propanol Ratio from Keto-Acid Pathway in Escherichia coli
指導教授：	沈若樸 Shen, Roa Pu
口試委員:	蘭宜錚彭惠玲
學位類別：	碩士 Master
系所名稱：	工學院 - 化學工程學系 Department of Chemical Engineering
論文出版年：	2016
畢業學年度：	104
語文別：	中文
論文頁數：	48
中文關鍵詞：	代謝工程、正丁醇、大腸桿菌
外文關鍵詞：	metabolic engineering, butanol, e.coli
相關次數：	點閱：4 下載：0
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正丁醇為現今最受矚目的一項生質能源，由於其具有和石油相當的能量密度，與生質酒精相比具有較低的親水性與蒸氣壓，提升儲存和運輸的便利性，因此我們認為正丁醇極具有成為石油替代品之潛能。
先前已有研究成功發展出改造生物本身的胺基酸合成代謝途徑製造keto-acids，再將keto-acids經酵素轉換後形成正丁醇。本研究即是以葡萄糖作為碳源並利用胺基酸合成代謝途徑中的citramalate pathway做為正丁醇的生產途徑，此代謝途徑除了正丁醇外，還會產出競爭副產物正丙醇，兩者主要在競爭中間產物2-ketobutyrate。先前研究顯示，正丁醇在產物中所佔的比例比正丙醇低(正丁醇：正丙醇= 1:6)，因此本研究希望藉由調控分叉點基因的表現強弱程度 (更換複製起始點、替換操縱子結構與核醣體結合位點)、降低正丁醇前驅物進入有毒副產品norvaline的可能性、提升反應酵素(Kivd)對於正丁醇前驅物之專一性，以及提高反應中的重要基質濃度等方式來將碳通量導入正丁醇的比例增高。
目前實驗結果顯示藉由調控分叉點基因的表現程度，順利提升了正丁醇對於正丙醇的比例，與先前文獻上研究結果相比約提升15倍，之後利用突變反應酵素提升對於正丁醇前驅物的專一性，得到突變酵素Kivd(V461A)可將正丁醇與正丙醇比例提升至30倍，卻造成正丁醇產量降低(約0.49 g/L)，同時也觀察到正戊醇的產量與未突變之Kivd相比提升了11%，代表此突變對於正戊醇的前驅物具有更高的專一性，在提高反應物中重要基質濃度的實驗中，於細胞誘導時添加5 g/L sodium acetate至培養液，發現不論Kivd是否突變正丁醇產量均有明顯提升，證實了添加sodium acetate確實對於正丁醇的產量有正向的幫助。在降低前驅物之副產物norvaline的毒性方面實驗結果則不如預期，正丁醇與正丙醇的產量皆大幅的下降(約0.08 g/L以及0.02 g/L)且對於兩者之間比例的提升也無正向效益，目前仍無明確的分析結果可以解釋此狀況。從目前結果顯示，如何在不降低正丁醇產量的情況下同時減少競爭副產物之生成，將變成未來值得探討的方向。

1-Butanol is the most popular one of biomass energy because its energy content is similar with petroleum. On the other hand, 1-butanol is hydrophobic and the vapor pressure is lower than ethanol, so 1-butanol has the higher convenience of transport and storage. We think 1-butanol has the potential to become a substitute for petroleum.
The previous study has successfully developed a synthetic amino acid metabolic pathways to transform itself to the production of 1-butanol from ketoacids. This study uses glucose as carbon source and citramalate pathway which is one of synthetic amino acid metabolic pathway as 1-butanol production pathway. In this pathway, 1-propanol is an inevitable byproduct competing for the precursor (2-ketobutyrate) needed for 1-butanol. In previous study, we found the ratio of 1-butanol and 1-propanol is low (Butanol：Propanol = 1:6). We hope to regulate the gene at pathway branch point expression (ex. change the replication origin, operon structure and ribosomal binding site), reduce the toxicity of norvaline which derived from precursor of 1-butanol , promote the specificity of reactive enzyme to the precursor of 1-butanol and add acetate to medium when cell induced to promote the ratio of 1-butanol to 1-propanol.
The experimental results show the extent of our regulation of gene at pathway branch-point expression can enhance the ratio of 1-butanol to 1-propanol about 15 times higher than the previous study. The ratio of 1-butanol to 1-propanol promoted from 15 times to 30 times by promoting the specificity of reactive enzyme to 1-butanol precursor and we found the titer of 1-butanol is low to 0.49 g/L. We observed when utilizing Kivd mutant (V461A) produce 1-pentanol compared to wild-type Kivd improved by 11%, so we think Kivd (V461A) has the higher specificity of the precursor of 1-pentanol than 1-butanol. The experiment result of adding acetate into medium show the titer of 1-butanol significantly enhance whatever wild-type Kivd or Kivd(V461A), so we can confirm adding sodium acetate has a positive effect to 1-butanol production. The experiment results of reducing the toxicity of norvaline are not good because the titers of 1-butanol and 1-propanol are low to 0.08 g/L and 0.02 g/L and the ratio is not promotion. We don’t have a good reason to explain these results. From the results, we think how can the same time enhance the titer of 1-butanol production and reduce the amounts of by-products become an important issue on the future.

摘要.........................................................    i
Abstract......................................................    iii
誌謝.........................................................    iv
目錄.........................................................    v
圖目錄.......................................................    vii
表目錄.......................................................    viii
第一章 文獻回顧..............................................    1
  1-1研究背景及目的.....................................................................................    1
1-2菌種及目標產物介紹.............................................................................    4
    1-2-1大腸桿菌簡介.................................................................................    4
    1-2-2正丁醇簡介......................................................................................    4
  1-3正丁醇生產途徑簡介.............................................................................    5
    1-3-1 CoA dependent pathway(ABE fermentation)..................................    5
    1-3-2 Keto-acid pathway...........................................................................    6
  1-4 Norvaline生成及對於大腸桿菌影響....................................................    7
  1-5調控kivd對於2-ketovalerate的專一性...............................................    8
  1-6額外添加Acetate對於正丁醇產量之影響..........................................    9
第二章 實驗材料及方法........................................    16
  2-1菌株介紹.................................................................................................    16
  2-2建立質體.................................................................................................    16
  2-3將建構之質體植入菌株.........................................................................    20
    2-3-1 Competent cell製作........................................................................    20
    2-3-2藉由電穿孔進行轉植.....................................................................    20
    2-3-3藉由Heat Shock進行轉植.............................................................    20
  2-4利用P1 Transduction進行knock out....................................................    21
  2-5正丁醇生產程序.....................................................................................    22
    2-5-1製備正丁醇使用的培養液.............................................................    22
    2-5-2正丁醇生產條件.............................................................................    22
    2-5-3樣品抽取.........................................................................................    22
2-6添加Acetate之劑量及方式..................................................................    22
2-7代謝物偵測............................................................................................    23
    2-7-1正丁醇產率偵測..............................................................................    23
     2-7-1-1儀器使用....................................................................................    23
     2-7-1-2標準品配置................................................................................    23
     2-7-1-3氣相層析儀樣品和標準品注射瓶配置....................................    23
    2-7-2 2-ketovalerate產率偵測..................................................................    23
     2-7-2-1儀器使用....................................................................................    23
     2-7-2-2標準品配置................................................................................    24
     2-7-2-3液相層析儀樣品和標準品注射瓶配置....................................    24
  2-8數據整理.................................................................................................    24
第三章 實驗結果與討論........................................    25
  3-1強化leuA相對於kivd的表現..............................................................    25
  3-2將質體轉殖進入菌株進行正丁醇生產測試.........................................    26
  3-3利用knock out genes克服norvaline毒性...........................................    28
3-4測試突變kivd(V461A)對於正丁醇與正丙醇比例的影響.................    30
3-5運用Saturated Mutagenesis進行kivd突變之質體建立與正丁醇生
產測試..........................................................................................................    
32
3-6誘導時添加Sodium Acetate進行正丁醇生產測試............................    34
3-7結論.......................................................................................................    35
參考文獻.....................................................    37
附錄.........................................................    40
A.    圖表附件................................................................................................    40
B.    圖形附件................................................................................................    47

                                

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