Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/91539
標題: 使用染色體工程技術構築低碳排放之重組大腸桿菌
Constructing Low-carbon Evolution by Chromosomal Engineering of Rubisco-based Engineered Escherichia coli
作者: Fang-Yu Ou-Yang
歐陽芳鈺
關鍵字: chromosomal engineering
pentose phosphate pathway
carbon recycling
Rubisco
engineered Escherichia coli
next generation sequencing
transcriptome sequencing
染色體工程
五碳糖磷酸途徑
碳回收
重組大腸桿菌
次世代定序
轉錄組定序
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摘要: 利用微生物製成合成特用化學品是替代能源發展方向之一,若能回收菌體代謝過程所產生的二氧化碳,發酵產物的產率能夠從回收的二氧化碳得到提升。先前研究將外源蛋白PrkA及Rubisco大量表現於大腸桿菌中,消耗1莫耳五碳糖僅排放0.621莫耳的CO2,能達到理論二氧化碳下降量的38%。相關研究也指出此重組菌株代謝六碳糖的同時,增強非氧化可逆的五碳糖磷酸途徑能夠回收二氧化碳。為了能在回收碳的過程中進一步控制碳流,本研究將zwf基因去除藉以破壞五碳糖磷酸途徑的氧化不可逆路徑,增大非氧化路徑的碳流,進而回收更多二氧化碳。進一步探討Rubisco轉型至大腸桿菌中對菌體的代謝所造成之影響。在厭氧環境的批次培養中,使用20 g/L葡萄糖為碳源於M9培養基之發酵數據得知,去除zwf基因,氧化的五碳糖磷酸路徑被破壞後,Rubisco的表現將碳通量導向非氧化的五碳糖代謝,加上PrkA的共表現後,成功構築碳回收路徑,使得基因剔除之重組大腸桿菌突變株的選擇率,即Total CO2/ PEtOH+PAcetate (mol/mol) 分率低於原生型E. coli BL21 (DE3) 共表現PrkA及Rubisco 有54%,顯示此基因改造後的重組大腸桿菌確實能夠有低二氧化碳排放的效能。將Rubisco轉型至E. coli中,於好氧環境培養會加速糖的消耗速率,於厭氧環境培養能增進菌體的生長,而Rubisco影響CsrA活性間接誘導CsrA調控相關基因,使gluconeogenic基因表現量增強進而伴隨著大量二氧化碳的排放,進而使合成PEP的速率提高。而五碳糖與PEP代謝物均可用於胺基酸與苯環化合物合成所需。因此Rubisco的表現使細胞的糖消耗速率增快,以作為肝糖儲存或供給於胺基酸等化合物合成。
The microbial conversion for bio-based chemical productions is one of the alternative sources of energy. If the development of the system which could recycle carbon dioxide before it is emitted during the fermentation process. While the CO2 emission is lowered, the benefit is that the yield of fermentation products can be increased by using the carbon in the carbon dioxide. In previous study, Rubisco-based engineered E. coli, containing heterologous Rubisco and PrkA, produced only 0.621 mol of CO2 per consumption with pentoses, achieves 38% of theoretical CO2 reduction. Besides, our parallel study demonstrates that Rubisco-based engineered E. coli can recycle evolved CO2 with hexoses by enhancing the function of the non-oxidative pentose phosphate pathway (NOPP pathway). To further control the carbon flow during the recycling of CO2, glycolysis and pentose phosphate pathway (PP pathway) have been destroyed by disrupting zwf gene. Therefore, the carbon flux through the NOPP pathway would be increased and then that is converted into the Rubisco-based engineered pathway to recycling more CO2. Beside, focus on the effect between Rubisco and host cell, E. coli BL21 (DE3), is another point of this study. The results shows that the M9 medium contained 20 g/L glucose as a sole carbon which in anaerobic batch culture, while E. coli BL21 (DE3) containing mutation of zwf genes (designated as MZ) and harboring PrkA and Rubisco grow normally similar to wild-type E. coli BL21 (DE3). It exhibited 54% decreased in Total CO2/PEtOH+PAcetate (mol/mol) fraction compared to the control E. coli strain containing only PrkA and Rubisco. However, Rubisco in E. coli would make response to some exclusively gluconeogenic genes due to the CsrA regulation. CsrA regulates gluconeogenic genes showed up-regulation responses, and improve PEP synthesis rate was associated with the increase of carbon dioxide evolution, which PEP is the intermediate of glycolysis. Pentose and PEP could be impact the biosynthesis of amino acids and aromatic compounds. Therefore, sugar consumption rate increase for energy storage and aromatic compounds during strain harboring Rubisco. In a few words, the performance of the destroyed the OPP pathway coupled with recombinant E. coli harboring PrkA and Rubisco, which is efficient and essential. The interaction of heterologous Rubisco with the intact metabolism of E. coli is profound and interesting. More discussion will be addressed later.
URI: http://hdl.handle.net/11455/91539
其他識別: U0005-2811201416190085
文章公開時間: 2016-08-31
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