| Summary: | 碩士 === 國立中興大學 === 化學工程學系所 === 106 === In this study, we used engineered Escherichia coli as our material and strengthen carbon utilization in E. coli metabolism. Within the original tricarboxylic acid cycle (TCA), we added the following genes: kor, acl, frd and sdh, thus modified the mentioned cycle into a plasmid using reductive tricarboxylic acid cycle (rTCA). The modified plasmid will absorb carbon dioxide during its metabolism, so we can achieve the effect of carbon recovery, as well as effective utilization of carbon sources. We transform the rTCA plasmid by electroporation method into Escherichia coli MZLF (BL21 (DE3) Δzwf, Δldh, Δfrd) with the co-expression of Rubisco. In this master''s thesis, we mainly to explore the supply of energy and carbon flow part. We can see from the rTCA net reaction that the supply of energy is indispensable while fixing carbon dioxide, so this study provides an energy supply system, the energy pump. By formate dehydrogenase to convert formate to produce carbon dioxide and NADH and then enhance the synthesis of ATP via the MNF system, we found that the C-2 / C-1 ratio can be increased from the original 1.03 ± 0.01 to 1.48 ± 0.12. In the regulation of carbon flux, we introduced a plasmid pLOI295, which enhances the conversion of C-2 carbon flux via pyruvate to ethanol by the pyruvate decarboxylase and alcohol dehydrogenase II. We named this process the Pdc-based carbon tap valve (CTV). After adding the Pdc-based carbon tap valve we can see the decline in pyruvate yield and the improvement of ethanol yield, the yield of ethanol can be raised from the original 0.92±0.02 to 1.65±0.10. So the final C-2/C-1 ratio can reach 1.90±0.19.
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