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研究生:張樺雯
研究生(外文):Hua -Wen Chang
論文名稱:利用基因重組技術建構生產生質酒精之Escherichiacoli菌株
論文名稱(外文):Construction of recombinant Escherichia coli strain by genetic technique for bioethanol production
指導教授:魏毓宏
學位類別:碩士
校院名稱:元智大學
系所名稱:生物科技與工程研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:中文
論文頁數:69
中文關鍵詞:生質酒精Zymomonas mobilis重組大腸桿菌
外文關鍵詞:Bioethanolrecombinant Escherichia coligenetic techniques
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生質酒精在經濟與環保方面是極具潛力之再生能源,目前普遍使用的酒精發酵菌株為Zymomonas mobilis,只能發酵六碳糖(glucose)來生產酒精,卻不能利用五碳糖(xylose)進行發酵,因此使發酵原料種類受到限制。Escherichia coli本身同時具有能代謝六碳糖與五碳糖能力,卻無法合成酒精。究其原因,主要是大腸桿菌缺乏直接合成酒精之關鍵基因-丙酮酸脫羧&;#37238;(PDC)以及醇脫氫&;#37238;(alcohol dehydrogenase; ADH),因此,無法直接代謝碳水化合物,以合成酒精。基於上述原因,本研究擬利用選殖自Zymomonas mobilis之合成酒精之關鍵基因PDC及ADH,並將其轉殖於大腸桿菌E. coli DH5α,藉以建構可直接代謝五碳糖及六碳糖,以合成酒精之基因重組E. coli菌株。首先利用PCR的方法,自Zymomonas mobilis中選殖出合成酒精之關鍵基因PDC及ADH,接著設計專一性引子,將PDC及ADH進行放大,之後將其轉型至E. coli DH5α進行表現。研究證實:經SDS-PAGE分析實驗,確認PDC及ADH已成功被表現,且得一基因重組菌株E. coli ETOH1。研究進一步顯示,E. coli ETOH1的確已具有代謝碳水化合物,以合成酒精之能力。且E. coli ETOH1在以LB及5%Xylose為基質及溫度37℃與轉速100 rpm之培養下,可得最佳之酒精合成效率,其酒精濃度達3951 ppm。研究亦證實,以E. coli ETOH1代謝發酵酒精製程中,其產物酒精合成濃度,將隨LB之添加比例增加而增加。即E. coli ETOH1在上述培養條件下(37℃、100 rpm),將LB基質提高至2倍之情形下,其產物酒精產量可達7337 ppm。其次,研究亦證實:高厭氧條件,有助於E. coli ETOH1發酵合成酒精,即E. coli ETOH1在以LB及5%Xylose為基質,當溫度控制於37℃,且完全靜置(0 rpm)之培養條件下,其酒精合成濃度可高達9583 ppm。由於LB內含關鍵成分:Yeast extract可促進E. coli ETOH1代謝合成酒精,並提升其酒精產量。




Bioethanol is a potential and renewable energy. Although Zymomonas mobilis is suitable for ethanol fermentation, it is unable to metabolize pentose; therefore, the resources used for ethanol fermentation are limited. Escherichia coli are able to metabolize both hexose and pentose to growth under aerobic conditions. But, Escherichia coli could not be able to produce ethanol due to it is mainly lack of direct synthesis genes (e.g., pyruvate decarboxylase; PDC and lcohol dehydrogenase; ADH). For these reasons, this study aims to contract a recombinant Escherichia coli for the production of bioethanol by genetic techniques. We hound that the recombinant E. coli would be able to utilize pentoes and hexoses and produce ethanol. First, this study have used PCR to clone PDC and ADH genes from Z. mobilis, and then design specific primers to amplify the PDC and ADH, followed by its transformation into E. coli DH5α for expression. The analysis by SDS-PAGE experiments confirmed that PDC and ADH have been successfully performance. Hence, we got a recombinant strain named E. coli ETOH1. Further studies emphasized that E. coli ETOH1 do have a metabolism of carbohydrates and the ability of ethanol synthesis. When LB and 5% xylose were used as substrate; temperature was 37 ℃ and the speed of movement was 100 rpm, E. coli ETOH1 performed the best alcohol synthesis efficiency with the alcohol concentration of 3951 ppm. In addition, we demonstrated an increment of the initial LB concentration led to the enhancement in yield of alcohol. Moreover, optimal operations conditions (temperature and agitation speed) for the production of bioethanol were also identified. It was found that recombinant E. coli ETOH1 was able to produce 7337 ppm of bioethanol when it was grown under the optimal operation conditions of 37℃ and 100 rpm. Moreover, the study revealed the yield of product up to 9583 ppm was gained by recombinant E. coli ETOH1 in the anaerobic conditions (without shaking), at temperature of 37 ℃, in medium with LB and 5% of xylose. Furthermore, we confirm that the yeast is the key component for the production of bioethanol.

摘要 iii
Abstract v
致謝 vii
圖目錄 x
表目錄 xii
第一章、緒論 13
1-1 前言 13
1-2 研究動機與目的 13
1-3 Zymomonas mobilis 的發現 16
1-4 Zymomonas mobilis 的特性與特徵 16
1-5 Zymomonas mobilis 的代謝途徑 19
1-6 Zymomonas mobilis 與Yeast 的比較 20
第二章 文獻回顧 25
2-1 生質酒精 25
2-2纖維素的構造 26
2-2-1纖維素分解酵素種類之介紹 27
2-3 Saccharomyces cerevisiae 28
2-4 Escherichia coli 29
第三章 材料方法 30
3-1 實驗材料 30
3-1-1 菌株及表現質體 30
3-1-2 引子組 32
3-1-3 藥品及酵素 32
3-2 染色體DNA的萃取 32
3-3 限制酶作用 33
3-4聚合酶鏈鎖反應 (Polymerase chain reaction, PCR ) 33
3-5 TA cloning 33
3-5-1 TA ligation 33
3-5-2 轉型作用(transformation) 34
3-5-3 質體DNA的萃取 34
3-6 重組質體之篩選 34
3-7 SDS聚丙烯醯胺膠體電泳法(SDS-PAGE) 35
3-7-1 樣本處理 35
3-7-2 鑄膠 35
3-7-3 電泳 35
3-8 重組菌株的培養 36
3-9 微生物生長之分析 36
3-10 酒精濃度分析方法 36
3-11 實驗儀器 37
3-12 實驗藥品 38
第四章 結果與討論 39
4-1 論文研究架構 39
4-2 菌種改殖 41
4-2-1選殖PDC、ADH 41
4-2-2 構築表現質體pPDC、pADH、pETOH1 42
4-3表現質體的建構 45
4-4 確認pdc、adh否被表現 45
4-5 前培養液組成份對E. coli ETOH1生長及代謝合成酒精之影響 50
4-6 主培養基加入誘導劑對E. coli ETOH1生長及代謝合成酒精之影響 51
4-7 E. coli ETOH1生長主培養液之探討 53
4-8 不同溫度對重組菌株生長及酒精之探討 55
4-9 不同轉速對E. coli ETOH1生長及酒精代謝之影響 57
4-10 不同濃度LB對E. coli ETOH1酒精代謝之影響 59
4-11 LB內含成分對E. coli ETOH1酒精代謝之影響 61
第五章 結論 65
參考文獻 66
附錄 68


1Chen, J., Zhang, W., Tan, L., Wang, Y. & He, G. Optimization of metabolic pathways for bioconversion of lignocellulose to ethanol through genetic engineering. Biotechnol Adv 27, 593-598,
2Swings, J. & De Ley, J. The biology of Zymomonas. Bacteriol Rev 41, 1-46 (1977).
3Doelle, M. B., Millichip, R. J. & Doelle, H. W. Production of Ethanol from Corn Using Inoculum Cascading of Zymomonas-Mobilis. Process Biochem 24, 137-140 (1989).
4Demain, A. L. & Solomon, N. A. Biology of industrial microorganisms. (Benjamin/Cummings Pub. Co., Advanced Book Program, 1985).
5Buchholz, S. E., Dooley, M. M. & Eveleigh, D. E. Zymomonas - an Alcoholic Enigma. Trends in Biotechnology 5, 199-204 (1987).
6Swings, J., Kersters, K. & Deley, J. Numerical-Analysis of Electrophoretic Protein Patterns of Zymomonas Strains. Journal of General Microbiology 93, 266-271 (1976).
7Skotnicki, M. L., Lee, K. J., Tribe, D. E. & Rogers, P. L. Comparison of Ethanol-Production by Different Zymomonas Strains. Appl Environ Microb 41, 889-893 (1981).
8Altintas, M. M., Eddy, C. K., Zhang, M., McMillan, J. D. & Kompala, D. S. Kinetic modeling to optimize pentose fermentation in Zymomonas mobilis. Biotechnol Bioeng 94, 273-295, doi:10.1002/bit.20843 (2006).
9Dien, B. S., Cotta, M. A. & Jeffries, T. W. Bacteria engineered for fuel ethanol production: current status. Appl Microbiol Biotechnol 63, 258-266, doi:10.1007/s00253-003-1444-y (2003).
10Hamelinck, C. N., van Hooijdonk, G. & Faaij, A. P. C. Ethanol from lignocellulosic biomass: techno-economic performance in short-, middle- and long-term. Biomass Bioenerg 28, 384-410, doi:DOI 10.1016/j.biombioe.2004.09.002 (2005).
11Beguin, P. & Lemaire, M. The cellulosome: An exocellular, multiprotein complex specialized in cellulose degradation. Crit Rev Biochem Mol 31, 201-236 (1996).
12Bok, J. D., Yernool, D. A. & Eveleigh, D. E. Purification, characterization, and molecular analysis of thermostable cellulases CelA and CelB from Thermotoga neapolitana. Appl Environ Microbiol 64, 4774-4781 (1998).
13Lynd, L. R. & Zhang, Y. Quantitative determination of cellulase concentration as distinct from cell concentration in studies of microbial cellulose utilization: analytical framework and methodological approach. Biotechnol Bioeng 77, 467-475, doi:10.1002/bit.10142 [pii] (2002).
14Beguin, P., Millet, J. & Aubert, J. P. Cellulose degradation by Clostridium thermocellum: from manure to molecular biology. FEMS Microbiol Lett 79, 523-528 (1992).
15西澤俊ㄧ. 纖維素總論. 南山堂出版社, 24-25 (1981).
16Mandels, M., Andreotti, R. & Roche, C. Measurement of Saccharifying Cellulase. Biotechnology and Bioengineering, 21-33 (1976).
17Mandels, M., Andreotti, R. & Roche, C. Measurement of saccharifying cellulase. Biotechnol Bioeng Symp, 21-33 (1976).
18Bhat, M. K. & Bhat, S. Cellulose degrading enzymes and their potential industrial applications. Biotechnol Adv 15, 583-620, doi:S0734975097000062 [pii] (1997).
19Saloheimo, M. et al. Swollenin, a Trichoderma reesei protein with sequence similarity to the plant expansins, exhibits disruption activity on cellulosic materials. Eur J Biochem 269, 4202-4211, doi:3095 [pii] (2002).
20Goyal, A. K. & Eveleigh, D. E. Cloning, sequencing and analysis of the ggh-A gene encoding a 1,4-beta-D-glucan glucohydrolase from Microbispora bispora. Gene 172, 93-98, doi:0378-1119(96)00076-5 [pii] (1996).
21Bisaria, V. S. & Ghose, T. K. Biodegradation of Cellulosic Materials - Substrates, Microorganisms, Enzymes and Products. Enzyme Microb Tech 3, 90-104 (1981).
22Dien, B. S., Cotta, M. A. & Jeffries, T. W. Bacteria engineered for fuel ethanol production: current status. Appl Microbiol Biot 63, 258-266, doi:DOI 10.1007/s00253-003-1444-y (2003).


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