Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/97737
標題: 具有還原性三羧酸循環酵素之化學自營性大腸桿菌代謝路徑分析
Metabolic Pathway Analysis of Chemoautotrophic Escherichia coli with Reductive Tricarboxylic Acid Enzymes
作者: 陳宥婕
You-Jie Chen
關鍵字: 還原性三羧酸循環
固碳作用
大腸桿菌
化學自營
rTCA cycle
carbon fixation
Escherichia coli
chemoautotrophic
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摘要: 全球暖化所造成之氣候異常現象日益嚴重,其中二氧化碳所造成之影響不容小覷,因此減碳相關之議題備受全世界關注。還原性三羧酸循環 (rTCA cycle)為自然界中固碳作用循環之一,前人探討將大腸桿菌中所缺乏之還原性三羧酸循環酵素補足,使大腸桿菌能夠利用rTCA cycle行固碳作用,結果發現大腸桿菌額外表現ATP-citrate lyase (ACL)以及α-ketoglutarate ferredoxin oxidoreductase (KOR)酵素後,菌株在厭氧無糖之培養下亦能夠生長,因此本研究之目的為探討大腸桿菌表現外源性ACL以及KOR酵素後,菌株進行化學自營之可行性,以及其基因轉錄體分析代謝路徑。本研究在先前培養條件是以M9作為厭氧基礎培養基,並額外添加電子接受者 (NaNO3)、提供者 (氫氣)以及casein,利用剔除與rTCA cycle相關之基因以推測其碳流方向,結果發現分別剃除glyoxylate shunt與GABA shunt之相關基因,皆對於菌株生長有負面影響。而在進一步探討單一氨基酸對於菌株生長之影響結果中,意外發現實驗菌株 (有外源酵素KOR/ACL)在完全不添加casein的條件下其生菌數有所增長,此結果更加確立菌株有額外添加外源酵素之條件下,亦能夠行化學自營模式並使生菌數增長,因此接下來之研究皆專注於觀察菌株在化學自營條件下生菌數之生長情況。首先在剃除rTCA cycle相關之基因後對於菌株生長影響之結果發現,剔除aceA基因後對於菌株生長有正面影響,這與先前有添加casein之培養結果相反。另一方面,在菌株分析之轉錄體結果中發現,實驗菌株 (分別為有額外添加KOR以及KOR/ACL菌株)在醣解作用以及rTCA cycle相關路徑之基因表現量與控制組比對後有所差異,KOR菌株在pyruvate與glutamate代謝相關路徑之基因表現量皆有提升,而KOR/ACL菌株在醣解作用、pyruvate以及glutamate相關路徑之基因表現量皆有提升。由結果推論,額外添加KOR以及KOR/ACL酵素後對於菌株之碳流走向略微不同,但pyruvate代謝與GABA shunt皆為兩者生長之關鍵。
The global warming are increasingly severe, carbon dioxide, one of greenhouse gases is the most influential gas. Nowadays, the issue of carbon reduction has received worldwide attention. Reductive tricarboxylic acid (rTCA cycle) is one of the carbon fixation cycle in nature. In previously studies, we complement the rTCA cycle enzymese that are missing in E.coli. It can make E.coli can use rTCA cycle to work carbon fixations. Than we found that when added ATP-citrate lyase (ACL) and α-ketoglutarate ferredoxin oxidoreductase (KOR) genes in E.coli, it could growth under anaerobic and sugar-free culture conditions. In this study, we focused on the chemoautotrophic feasibility of E.coli which expressed KOR and ACL enzymes under anaerobic and sugar-free culture conditions. And comparing the growth of the experiment strains with the gene transcriptome data to analyze its carbon metabolism pathway. We used M9 as an anaerobic medium, and added an electronic acceptor (NaNO3), a donor (hydrogen) and a nitrogen source (casein) in the medium. In the study of knockout single gene that related to rTCA cycl, we found knockout genes in glyoxylate shunt and GABA shunt had a negative impact on strains growth. In the experiment of changing nitrogen sourse to single amino acids, surprisingly, the CFU of the experiment strains (expressed KOR and ACL) would increase under anaerobic and amino acid-free culture. In further research, we will focus on the effect of the strains growth under anaerobic and amino acid-free culture. First, in the test of knockout aceA gene that is in the glyoxylate shunt show it had a positive impact on experiment strains growth. On the other hands, we found that the genes expression of the experiment strains (with additional expressed KOR and KOR/ACL enzymes, respectively) had up-regulation in glycolysis and rTCA cycle-related pathway after comparison with the control strain in the transcriptome analysis. Finally, we found that pyruvate metabolic and GABA shunt are both important to the KOR strain and the KOR/ACL strain to survive in anaerobic and amino acid-free culture.
URI: http://hdl.handle.net/11455/97737
文章公開時間: 10000-01-01
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