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標題: 利用碳重組系統增強重組大腸桿菌回收二氧化碳之能力
The Enhancement of CO2 Recycling in Recombinant Escherichia coli by the Carbon Rearrangement System
作者: Ya-Han Li
關鍵字: Escherichia coli
carbon rearrangement system
CO2 recycling
Transketolase A
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摘要: 自17世紀工業革命之後,二氧化碳的過度排放已經影響到地球上的生態環境,如造成嚴重的溫室效應等,因此成為當務之急的問題,而近年來石油面臨短缺的問題也浮上檯面,因此開發以減碳為前期的新能源也開始備受矚目。在大腸桿菌當中tktA及talB扮演著重要的角色,此兩基因存在於non-oxidation pentose phosphate pathway (NOPPP)路徑當中,其功能是可以將碳數進行轉換,而轉換期間並不會造成碳的流失,即產生CO2。另外,從卡爾文循環中得知Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco)是回收CO2的重要酵素,但卡爾文循環中需要五碳糖作為基質反應,但五碳糖的價錢昂貴,因此希望利用便宜的六碳糖作為碳源,並藉由tktA及talB加強六碳糖轉化五碳糖之能力,以供給外源基因prkA及rbcLS使用。因此本研究於大腸桿菌中置入prkA、rbcLS此兩個外源基因,並同時大量表現大腸桿菌中的Transketolase A (TktA) 及 Transaldolase B (TalB),藉以利用tktA及talB所構築的碳轉化系統以及prkA及rbcLS構築的碳回收系統進行二氧化碳之回收,將此兩系統結合的優點在於不需要利用昂貴的五碳糖作為碳源,利用葡萄糖即可由碳轉化系統提供前驅物給碳回收系統利用,並構築新的代謝路徑,使大腸桿菌產生代謝物的同時,亦可減少二氧化碳之排放。結果顯示在minimal medium當中,碳轉化系統皆能產生減少CO2排放的效果,其中以TktA及TalB同時表現的效果最好,Total CO2/PEtOH+Pacetate (mole/mole)比wild-type E. coli BL21 (DE3)下降28.4%,而碳回收系統系統中,只有同時表現PrkA及Rubisco才會有減少CO2排放的效果,Total CO2/PEtOH+Pacetate (mole/mole)比wild-type E. coli BL21 (DE3)下降8.6%。然而,在單獨Rubisco表現之情形下,會有額外的CO2產生,推測是當rbcLS轉殖入大腸桿菌中,會影響到自身代謝路徑的分佈,而由NGS數據得知是影響pckA及maeB此兩個基因的路徑造成額外的CO2排放。當碳轉化及碳回收系統結合時,同時表現PrkA、Rubisco及TktA的Total CO2/PEtOH+Pacetate (mole/mole)比wild-type E. coli BL21 (DE3)下降27.2%,另外,PrkA、Rubisco、TktA及TalB同時表現時,Total CO2/PEtOH+Pacetate (mole/mole)比wild-type E. coli BL21 (DE3)下降28.4%,數據顯示當有強烈的下游基因表現時,tktA基因扮演著較為重要的角色。另外,利用MgSO4、NaHCO3及K2HPO4等鹽類探討碳回收系統之速率決定步驟發現,K2HPO4中的磷酸根與ATP:ADP ratio有關,當Pi濃度下降時,ATP:ADP ratio也隨之下降,進而造成Rubisco activase 的活性下降,結果顯示,隨著K2HPO4濃度的增加,碳回收系統的回收CO2的效果越好,當添加至6 g/L K2HPO4時,JB與JB/pTA的Total CO2 / PEtOH+Pacetate比起wild-type E. coli BL21 (DE3)約下降50.7~53.5%左右,因此ATP的供給為速率決定步驟。
Since the 17th century, industrial revolution led to the excessive emission of carbon dioxide. It impacts on the environment seriously, such as greenhouse effect. Overdeveloped industrial activities that results in the shortage of oil have been attracted much attention in recent years. Therefore, it began to be focused on the development of new energy along with reducing carbon dioxide evolution. Transketolase A (TktA) plays an important role in non-oxidation pentose phosphate pathway (NOPPP). It rearranges carbons by catalyzing two chemical reactions in NOPPP. It converts fructose-6-phosphate together with glyceraldehyde-3-phosphate into xylose-5-phosphate and erythrose-4-phosphate. TktA also converts glyceraldehyde-3-phosphate and sedoheptulose-7-phosphate into ribose-5-phosphate and xylose-5-phosphate. Transaldolase B (TalB) in NOPPP converts erythrose-4-phosphate and fructose-6-phosphate onto glyceraldehyde-3-phosphate and sedoheptulose-7-phosphate. In the engineering perspective, one of the advantage of the NOPPP is that has no CO2 emission. In this study, we used two key enzymes, phosphoribulokinase (PrkA) and ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), in the Calvin–Benson–Bassham cycle as the exogenous genes to create a new pathway in which it can recycling CO2 in E .coli. We called it is the carbon recycling system. Moreover, Overexpression of TktA and TalB in carbon rearrangement system could convert hexoses to pentoses, which can serve as the precursor for the carbon recycling system. Thus, combining two systems could use cheaper carbon source like glucose to ferment ethanol and acetate. In the same times, it could recycle inorganic carbon for reducing CO2 emission. The results indicated that the carbon rearrangement system could reduce CO2 emission when engineered E. coli cultured in minimal medium. Furthermore, it was found that two enzymes TktA and TalB that was overexpressed simultaneously had the best performance in terms of the CO2 reduction. Compare with wild-type E. coli BL21 (DE3), Total CO2/PEtOH+Pacetate (mole/mole) could decrease 28.4%. On the other hand, overexpression of PrkA and Rubisco had the best effect to reduce CO2 emission in carbon recycling system. Compare with wild-type E. coli BL21 (DE3), Total CO2/PEtOH+Pacetate (mole/mole) could decrease 8.6%. However, overexpression of Rubisco led to more CO2 evolution. It may Rubisco changed the distribution of carbon flow in E. coli. The data shows that Rubisco caused the mRNA of pckA and maeB to increase and these related to CO2 evolution. As we combined two systems, overexpression of PrkA, Rubisco, and TktA could reduce CO2 emission more. Compare with wild-type E. coli BL21 (DE3), Total CO2/PEtOH+Pacetate (mole/mole) could decrease 27.2%. Furthermore, compare with wild-type E. coli BL21 (DE3), overexpression of PrkA, Rubisco, TktA, and TalB could decrease 28.4% in Total CO2/PEtOH+Pacetate (mole/mole). The data show when it has strong gene overexpression downstream, TktA plays a key role in carbon rearrangement system. In order to improve the carbon recycling system, this study used MgSO4, NaHCO3 and K2HPO4 as the factor to investigate what is the rate-limiting step in this system. When Pi concentration decreased, ATP:AMP ratio will declined and resulting in the decreased the activity of Rubisco activase. In this study, it was found that when K2HPO4 concentration added, Total CO2/PEtOH+Pacetate (mole/mole) was reduced in JB system. Compare with wild-type E. coli BL21 (DE3), JB and JB/pTA could reduce about 50.7~53.5% in Total CO2/PEtOH+Pacetate (mole/mole). Therefore, the ATP is the rate-limiting step in carbon recycling system.
其他識別: U0005-2811201416180302
文章公開時間: 2017-08-31
Appears in Collections:化學工程學系所



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