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研究生:楊承翰
研究生(外文):Cheng-Han Yang
論文名稱:利用重組大腸桿菌回收二氧化碳生產生質化學品
論文名稱(外文):The recycling of CO2 by Rubisco-based engineered Escherichia coli to make bio-based chemicals
指導教授:李思禹
指導教授(外文):Si-Yu Li
口試委員:黃介辰陳炳宇趙雲鵬張嘉修
口試委員(外文):Chieh-Chen HuangPing-Yu ChenYun-Peng ChaoJo-Shu Chang
口試日期:2015-07-30
學位類別:碩士
校院名稱:國立中興大學
系所名稱:化學工程學系所
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:中文
論文頁數:74
中文關鍵詞:重組大腸桿菌二氧化碳回收生質化學品
外文關鍵詞:Engineered E. coliCarbon dioxide recyclingbiochemicalsRubisco
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  • 被引用被引用:2
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先前研究中將Rubisco-based E. coli剔除zwf基因,增進Non-oxidative Pentose phosphate pathway的碳流,在MZB菌株中其Total CO2 / sugar consumption (mol/mol)相較控制組JB下降58%,但是卻發現表現Rubisco的菌株具有較低碳回收率約70-75%。在本研究中為了進一步了解Rubisco固定二氧化碳後的碳的流向並增進二氧化碳回收效能,首先針對產乳酸路徑上之ldh基因及產琥珀酸路徑上的frd基因剔除,使系統簡化,經剔除後的菌株並表現PrkA及Rubisco的MZLFB+IP僅能得到73%的碳回收率,但是卻發現了乙酸減少以及biomass提升等現象並在HPLC分析中得到未知peak,經分析後得知此peak為蘋果酸,將蘋果酸納入碳回收率計算後可得93%的碳回收率,此現象可能為可能為乙醛酸途徑(Glyoxylate shunt)的互補反應(anaplerotic reaction),後續額外添加2 g/L乙酸的實驗證實了乙酸的添加會使蘋果酸產率提升,並且單獨大量表現Rubisco時也可觀察到此現象,再來以NGS為基礎的RNA-seq對帶有Rubisco的菌株分析得到的結果顯示,在乙醛酸途徑上的基因acs (acetyl-coenzyme A synthetase), aceA (isocitrate lyase), aceB (malate synthase)及glcB (malate synthase G)其基因表現量皆大於控制組E. coli BL21(DE3),接著利用基於質量守恆及化學計量之方程式以線性規劃模擬Rubisco路徑所佔比例,結果顯示MZLFB+IP菌株Rubisco路徑所佔比例為10.6%。再來為增加Rubisco固碳效能導入外源蛋白NAD+-dependent formate dehydrogenase濃縮CO2並再生NADH,結果顯示MZLFB+FDH+IP菌株能有效回收CO2其Total CO2/EtoH (mol/mol)數值相較於控制組MZLF+FDH+IP有大幅度的下降,再來同樣以線性規劃模擬Rubisco路徑所佔比例可得在MZLFB+FDH+IP菌株中Rubisco路徑所佔比例為29.2%。

In previous study, the zwf gene has been knocked out in Rubisco-based E. coli (designated as MZB) enhance the flux of non-oxidative pentose phosphate pathway. Compare with JB, the total CO2 / sugar consumption (mol/mol) decreased 58% in MZB but the carbon recovery was low (~70-75%) when PrkA and Rubisco was expressed in E. coli. In this study, since we knockout the ldh gene and frd gene, we still fail to get a good carbon recovery in the mutant strain with the expression of PrkA and Rubisco. Especially for the IPTG induction strain MZLFB+IP, the carbon recovery of MZLFB+IP is about 73%. However, we do see several important phenotypes of MZLFB+IP, including the increase in Biomass and the decrease in acetate. This is the result of anaplerotic reaction, glyoxylate shunt. At the same time we find an unknown peak in the HPLC analysis. Finally, we find that malate has the same retention time with the unknown peak. Malate was quantified and added into the calculation of carbon recovery. The result shows that the carbon recovery of MZLFB+IP is 93%. To make sure the glyoxylate shunt was induced by the presence of Rubisco, additional 2 g/L acetate and express the PrkA and Rubisco separately has been tested. The results show that the additional acetate will enhance the production of malate and overexpression of Rubisco alone can observe same phenotypes. The RNA-sequencing based on NGS (Next generation sequencing) of J3 and JB strain also shows the consistent results. The results show that the glyoxylate shunt relative genes including acs, aceA, aceB and glcB are increasing in both J3 and JB. Using equations based on mass balance and stoichiometry to simulate MZLFB+IP, the results indicate that the Rubisco pathway fraction is 10.6%. To enhance the carbon recycling effectiveness by Rubisco, introduce the NAD+-dependent formate dehydrogenase. The enzyme reaction is convert formate and NAD+ to CO2 and NADH. In the mutant strain MZLF the main product is ethanol, so the FDH is not only increasing the available CO2 for Rubisco but also regenerating NADH for redox balance. For the co-expression of PrkA, Rubisco and FDH, in MZLFB+FDH+IP strain the Total CO2 / EtOH (mol/mol) can be decreased substantially compared to MZLF+FDH+IP. Here also using equations to simulate MZLFB+FDH+IP, the results indicate that the Rubisco pathway fraction is 29.2%.

誌謝 i
中文摘要 ii
Abstract iii
目錄 v
表目錄 viii
圖目錄 ix
第一章 緒論 1
1.1研究背景 1
1.2研究動機 2
第二章 文獻回顧 3
2.1二氧化碳 3
2.1.1 二氧化碳捕捉及封存技術 3
2.1.2 生物固碳 4
2.2 卡爾文循環 ( Calvin-Benson-Bassham cycle ) 4
2.3 Rubisco 5
2.4 大腸桿菌 6
2.4.1 大腸桿菌介紹 6
2.4.2大腸桿菌代謝產物 7
2.5 五碳糖磷酸路徑 8
2.5 染色體工程-基因剔除技術 8
第三章 實驗材料與方法 10
3.1 實驗菌株與質體 10
3.2 實驗藥品與實驗器材 10
3.2.1 實驗藥品 10
3.2.2引子序列 10
3.2.3 實驗器材 10
3.3 菌種培養 11
3.3.1 培養基配置 11
3.3.2活化 12
3.3.3 菌種保存 12
3.3.4厭氧批次培養 12
3.4 DNA純化方法 13
3.4.1質體DNA純化 13
3.4.2膠體純化DNA 15
3.4.3 DNA濃度分析 15
3.5聚合酵素連鎖反應 (Polymerase Chain Reaction) 16
3.6 膠體電泳法 17
3.7 酒精沉澱 18
3.8 勝任細胞製備 18
3.8.1 CaCl2化學法 18
3.8.2電擊穿透法勝任細胞製作 18
3.9 基因轉殖 19
3.9.1化學法製備勝任細胞之基因轉殖 19
3.9.2以電擊穿透法製備勝任細胞之基因轉殖 19
3.10 重組菌種建構 20
3.10.1 同源重組 20
3.10.2 ldh knock-out線性DNA製備 21
3.10.3 frd knock-out線性DNA製備 21
3.10.4置換染色體上基因 22
3.11 代謝物分析 23
3.12 二氧化碳濃度分析 23
3.13 批次培養實驗之二氧化碳濃度計算 23
3.14 質體構築 24
3.14.1 PCR 25
3.14.2 剪切反應 25
3.14.3 連接反應 26
3.14.4 轉型作用 26
3.15蛋白質定性分析 27
第四章 結果與討論 29
4.1 Rubisco誘導乙醛酸途徑 (Glyoxylate shunt) 29
4.2 添加乙酸之發酵實驗證實Rubisco誘導乙醛酸途徑 30
4.3 以NGS分析基因表現量佐證Rubisco誘導乙醛酸途徑 31
4.4 以線性規劃模擬基因剔除突變株中代謝產物碳的流向 32
4.5 表現外源蛋白Formate dehydrogenase濃縮CO2及再生NADH 34
第五章 結論 37
第六章 參考文獻 58
第七章 附錄 65


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