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Title: | 大腸桿菌的外源性正丁醇逆境之全基因組分析 Genome-wide analysis of exogenous 1-butanol stress in Escherichia coli |
Authors: | Hsuan-Kai Wang 王宣凱 |
Advisor: | 阮雪芬(Hsueh-Fen Juan) |
Keyword: | 丁醇,全基因組篩選,耐受,敏感,大腸桿菌, Butanol,Genome-wide screen,Tolerant,Sensitive,E. coli, |
Publication Year : | 2011 |
Degree: | 碩士 |
Abstract: | 丁醇相較於乙醇具有眾多的優點,例如:具有高能量、低含水比、低蒸氣壓、可完全取代汽油作為引擎燃料等,已被認定為具有潛力的生質能源。在操作容易的大腸桿菌中生產丁醇的代謝基因工程已經被完成;然而,丁醇進入工業化量產的主要問題在丁醇對於宿主具有毒性,並因此造成低的丁醇產量。為了瞭解丁醇毒性複雜的原理並且獲得一個關於丁醇逆境反應的完整的概念,我們運用陣列式定量篩選的程序,監控全基因組的細菌在不同丁醇濃度下的生長情況。經過篩選與分析的結果,初步鑑定出一群丁醇耐受性菌株以及一群丁醇敏感性菌株。在1%丁醇環境下,有兩株丁醇耐受性菌株acrA和acrB突變株的生長速率、存活率、細胞數目與細胞膜完整性皆比野生株顯著差異地好。直系同源基因的群集(COG)可以提供功能基因組分析的組織架構;因此,我們利用COG將敏感性菌株做分類,且發現最大的類別是COG C (能量轉換和生產)。並整合丁醇敏感性與丁醇耐受性菌株以鑑定丁醇逆境反應網路和其富含的生物功能。此丁醇逆境反應網路中顯示氧化磷酸化作用可能在丁醇逆境反應下扮演一個重要的角色。並且,缺乏會活化電子傳遞鏈中的複合體的轉錄因子的菌株對丁醇較為敏感,其轉錄因子包含Crp和fruR。在特定的突變株中大量表現Crp和FruR確實可增加丁醇耐受性。從以上分析結果顯示電子傳遞鏈與丁醇逆境反應有密切的關係。這些線索在未來的基因工程上將有助於發展丁醇耐受性菌株甚至是高丁醇產量的宿主。 1-butanol has been considered as a promising biofuel because of many advantages over ethanol, such as high energy content, low hygroscopicity, low vapor pressure, and allowing for a complete replacement of gasoline without modifications to the existing vehicle engines. Metabolic engineering of user-friendly host, Escherichia coli, for 1-butanol production has been accomplished. However, the pivotal problem associate with the industrial production of 1-butanol is that this product is toxic towards the host, resulting in a low 1-butanol yield. To comprehend the complex basis for its toxicity and obtain a global conception of 1-butanol stress response, we applied array-based, quantitative screening procedures for monitoring growth of bacterial in different 1-butanol concentration at genome-wide scale. After screening and analysis, we identified a group of 1-butanol sensitive strains and a group of tolerant strains. In 1% 1-butanol condition, growth rate, cell viability, cell number and integrity of cell membrane of these 1-butanol tolerant strains, acrA and acrB mutants, were significantly higher than wild type. The cluster of orthologous genes (COG) can provide the framework for functional genome analysis. Therefore, we used COG to categorize these 1-butanol sensitive strains, and found that COG C (energy conversion and production) was the major group. The sensitive and tolerant candidates and network analysis were integrated to identify the 1-butanol stress response networks and their enriched biological functions. The 1-butanol stress response network uncovered that oxidative phosphorylation might play important roles in 1-butanol stress response. Furthermore, mutants had increased sensitivity to 1-butanol because they had no these transcription factors, including Crp and FruR which activated complexes in oxidative phosphorylation. Overexpression of Crp and FruR in certain mutants could increase their 1-butanol tolerance. Taken together, these results reveal a key relationship between oxidative phosphorylation and 1-butanol stress response. Our studies also provide a valuable in-depth insight into development of a 1-butanol tolerant strain, even high-titer, 1-butanol-producing host. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/47075 |
Fulltext Rights: | 有償授權 |
Appears in Collections: | 分子與細胞生物學研究所 |
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