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標題: 以代謝工程技術對大腸桿菌三羧酸循環代謝產物之影響
The Effect of Tricarboxylic Acid Cycle Metabolites in Metabolic Engineering Escherichia coli.
作者: 林姿余
Tzu-Yu Lin
關鍵字: 基因重組大腸桿菌;還原性三羧酸循環;琥珀酸;化學自營;二氧化碳固碳;E. coli recombination;Reductive tricarboxylic acid cycle;Succinate;Chemoautotroph;Carbon fixation
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全球暖化及氣候變遷是由於人們過度使用石化燃料,例如煤和石油,產生大量二氧化碳、甲烷、氧化亞氮等溫室氣體,因而造成過度的溫室效應所導致。二氧化碳減量議題不僅是地區甚至是全球議題,若是能透過微生物回收二氧化碳之時,產生替代石化工業化合物及具有經濟效益之產品,便可減少非再生能源之利用。在前人研究中,已透過轉殖綠硫菌之還原性三羧酸循環之關鍵酵素,包括α-ketoglutarate:ferredoxin oxidoreductase(KOR)、ATP citrate lyase(ACL)、fumarate reductase(FR)及succinate dehydrogenase(SDH),開發出混營性大腸桿菌,而前人進一步剔除生成其他副產物的基因藉此來增加琥珀酸,以單一剔除methylglyoxal synthase (mgsA)基因後之琥珀酸產量為最高。而本實驗利用此固碳優勢的能力,於Echerichia coli K-12 BW25113利用基因重組系統探討多重基因剔除E.coli中代謝支線之基因,有效提高特殊代謝產物之產率,並增加固碳作用所需的前驅物(琥珀酸)。當BW25113以葡萄糖作為碳源時,所生產代謝產物當中以乳酸含量佔大部分,而同時剔除mgsA及aldehyde-alcohol dehydrogenase(adhE),能有效促進琥珀酸以及酸類化合物含量,另外在後續實驗中於ΔmgsA ΔadhE菌株上剔除DNA-binding transcriptional repressor(iclR) gene,以達到活躍乙醛酸路徑並促進琥珀酸生產,為本實驗缺陷株當中最佳產酸菌株,此外,在本實驗菌株轉殖rTCA酵素後以葡萄糖作為碳源的情況下,會對琥珀酸進行消耗,有效提升甲酸含量,部分菌株甚至能生產較高的總酸類化合物。在無糖代謝結果中發現,B-K(BW25113轉殖pGETS-K)菌株將無機碳作為碳源的情況下能產生高達1.21 g/L之醋酸,推測 KOR轉植菌株能固定無機二氧化碳將碳回推至Pyruvate及Acetyl-CoA,而藉由轉化成醋酸過程中獲得能量,來提供細胞存活。

Global warming and climate change are caused by overuse of fossil fuels, such as coal and petroleum, producing such as carbon dioxide, methane, and nitrous oxide, resulting in greenhouse effect. The issue of carbon dioxide reduction has not only been a regional or even global issue. In recent years, the issue of carbon dioxide emission is not only a regional but even global issue. If carbon dioxide can be recycled through microorganisms and convered to petrochemicals alternatives , the use of non-renewable energy can be reduced. In previous studies, a set of reductive tricarboxylic acid (rTCA) cycle genes including α-ketoglutarate:ferredoxin oxidoreductase, ATP citrate lyase, fumarate reductase, and succinate dehydrogenase were introduced from Chlorobium tepidum TLS to Escherichia coli . Mixotroph E. coli was developed, in which the yield of succinic acid was the highest after knock-out of mgsA gene. Taking advantage of the CO2 fixation, this experiment explores the multi-metabolic gene knock-out of mixotroph E. coli, and effectively increases the yield of specific metabolites and precursor (succinate) for carbon fixation. When BW25113 uses glucose as a carbon source, most of the produced metabolites are lactate. At the same time knock-out mgsA and adhE gene, the amount of succinate and acid compounds are increased. In this experiment, iclR gene was knocked out on ΔmgsA ΔadhE strain, active glyoxylate pathway and promote succinic acid production, the strain is the best acid-producing strain among knock-out strains in the experiment. In addition, the rTCA transgenic strain consumes succinate to increase the formate content, and some strains can produce higher acid compounds. Finally, under the sugar-free anaerobic condition, the acetic acid level of the B-K strain can be as high as 1.21 g/L. Therefore, in the anaerobic and sugar-free state, E. coli prefer to consume and convert carbon dioxide to pyruvate and Acetyl-CoA, and cell survival is provided by the energy obtained during the conversion to acetic acid.
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