Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/91548
標題: 利用重組大腸桿菌回收二氧化碳生產生質化學品
The recycling of CO2 by Rubisco-based engineered Escherichia coli to make bio-based chemicals
作者: Cheng-Han Yang
楊承翰
關鍵字: Engineered E. coli
Carbon dioxide recycling
biochemicals
Rubisco
重組大腸桿菌
二氧化碳回收
生質化學品
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摘要: 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%.
URI: http://hdl.handle.net/11455/91548
其他識別: U0005-1708201510050400
文章公開時間: 10000-01-01
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