利用合成系統生物學發展耐受正丁醇之大腸桿菌

Abstract

丁醇所能提供的能量和汽油相當，為一重要的工業用溶劑，由於它的物理特性、安全性高，而且不需要改裝汽車引擎就可直接使用，所以它比乙醇更適合當燃料。然而，當細胞在生產丁醇的同時，其生長會受到丁醇嚴重的抑制。本論文的主要目的在於利用合成系統生物學法選殖抗丁醇大腸桿菌新菌株，以用於生質能開發之中。本論文首先利用系統性的單一基因剔除菌株集(the Keio collection)來篩選耐受丁醇之菌株。在3,985個非必需基因剃除株中，我們發現有85株具有耐受丁醇的表型(phenotype)，當中有兩株具有可性的丁醇耐受度。我們將四株變異株以Biolog分析，Biolog是一個高通量的技術，可以一次自動化偵測高達兩千種細胞的表型。再來，我們利用蛋白質學的技術找出可能參與耐受丁醇的蛋白質，並了解在丁醇刺激下細胞所產生的生理反應。以2 %丁醇處理大腸桿菌後，我們以二維電泳膠分離蛋白質並以質譜儀鑑定出三十三個具有表現差異的蛋白質。其中高度量增加者包括抗氧化酵素、伴護蛋白、膜運輸蛋白，而表現量下降者則為參與糖解作用、精胺酸降解、色胺酸降解、ATP合成的酵素、ATP 運輸蛋白以及訊息傳遞蛋白。合成生物學提供了很好且方便的研究方法來改良微生物，使之產生丁醇。我們將高度表現的十三個蛋白質轉殖到IPTG可誘導的載體中，並測定轉殖株之丁醇耐受性。在這些過度表現的菌株當中，有六株相較於對照組具有較高的丁醇耐受度。最後，我們將建構有PhoH、MdoG、YdfG、Hmp、YqhD和TolB的質體分別送入ydhF- 和yheT -變異株中，得到了十二株新的大腸桿菌菌株。在這些菌株當中，以有過度表現PhoH蛋白質的ydhF缺乏的變異株具最佳丁醇抗性，其對2 %丁醇的抗性高過對照組五倍，可耐受至5 %丁醇。本篇研究使用系統生物學的角度及方式來改良生物的功能，建構出耐丁醇之大腸桿菌新菌株，以利於將來應用在能源開發中，並且對於未來發展替代能源具有一定程度的影響力。之後對於本研究中所找出的基因和蛋白質做特性分析，能夠幫助我們更了解複雜的溶液抗性本質為何。

1-Butanol with energy content similar to gasoline is an important industrial solvent and potentially a better fuel substitution than ethanol. However, 1-butanol causes serious product inhibition on the cells themselves while producing the solvents. The aim of this study was to develop 1-butanol tolerant Escherichia coli for applying it in biofuel production. A systematic and comprehensive collection of gene-disrupted E. coli K-12 mutants (the Keio collection) was used to screen the butanol-tolerant bacteria. Of the 3,985 nonessential gene mutants, eighty-five were found to exhibit 1-butanol tolerant phenotype, and two of them were displayed reliable 1-butanol tolerance. Four strains were characterized by a novel high-throughput technique, Phenotype MicroArrays (PMs)-Biolog, which can test up to 2000 cellular phenotypes simultaneously. Furthermore, we used proteomics approach to reveal potential proteins involved in 1-butanol tolerance and to understand the physiological response induced by 1-butanol stimulus. After E. coli cells were treated with 0 or 2 % (v/v) 1-butanol, thirty-three differential expressed proteins were separated and identified by 2DE and Q-TOF MS/MS. Our results showed that the anti-oxidative enzymes, chaperones, membrane transporters were the major group increased and proteins participated glycolysis, arginine degradation, tryptophan degradation, ATP synthesis, ATP transport and membrane signaling transduction were down-regulated. Thirteen of them were independently cloned into an IPTG-inducible vector; furthermore, their 1-butanol tolerance was evaluated. Among these overexpressed strains, six had higher 1-butanol tolerance than non-induced control. By introducing the protective proteins, PhoH, MdoG, YdfG, Hmp, YqhD, and TolB, into the ydhF- and yheT- knockout mutants, twelve novel E coli mutants were created. Ultimately, ydhF- mutant overexpressed PhoH demonstrated high 1-butanol tolerance up to 5.5 folds of the control and can tolerate up to 5 % 1-butanol. From this study, we not only obtain the 1-butanol tolerant E. coli mutants, but also have some impact on the development of alternative energy by synthetic systems biology knowledge. Our serial enhancement approach provided a more integrated observation of the process under the 1-butanol treatment. Further characterization of the genes and proteins identified in this study will likely improve our understanding of the complicated nature of solvent tolerance.