臺灣博碩士論文加值系統

從19世紀工業革命以來，全球暖化及氣候變遷的問題在地球各地蔓延開來，而這些問題都歸咎於溫室氣體。構築碳回收路徑可以減少二氧化碳在生產化學物品過程中或發酵工業中的生成，順道提升生物產業中的生產效率。而在先前的研究中，在大腸桿菌中表現Rubisco，可以提升菌株的生長狀況，但我們發現zwf、ldhA及frd的剔除使得糖耗越來越少，而Rubisco及PrkA的共表達也無法解決糖耗減少的問題。我們希望透過剔除pta使得菌體無法透過生產乙酸獲得ATP，必須藉由糖解作用來彌補這個缺口以達到提升糖耗的效果。在此研究中，雖然ppc的剔除沒有增加的葡萄糖消耗，但表達PrkA及Rubisco後可以提升耗糖量，這種效果在pta的剔除後依然存在著，CA1及CA3的糖耗為87 ± 2 及 88 ± 3 mM比CC的47 ± 2及CA的49 ± 1 mM還要多出許多，表示PrkA及Rubisco的表現是可以在ppc及pta的雙剔除中提升糖解作用。PrkA與Rubisco的共表現也可以提升耗糖量，CAB+IP的糖耗可以達到60 ± 6 mM。

From the industrial revolution in the 19th century, there had so many problems on earth like global warming and climate change because of greenhouse gases. Constructed a pathway to recycle the carbon dioxide is a way to reduce the carbon dioxide emission at chemical production or fermentation industries. Incidentally, it will also increases the efficiency of processes in related bio-industries. In previous study, when form I Rubisco is heterologously expressed in E. coli, we can see the enhanced biomass. But the glucose consumption of E. coli MZL and MZLF were less than E. coli MZ, and Rubisco-based engineered pathway was not rescuing this situation. In this study, the deletion of ppc was not increased the glucose consumption, but with induced by PrkA and Rubisco were extraordinarily increase of glucose consumption. This effect would further enhanced with deletion of pta. The glucose consumption of CA1 and CA3 were 87 ± 2 and 88 ± 3 mM much than CC and CA which glucose consumption were 47 ± 2 and 49 ± 1 mM. It proved that glycolysis was enhanced by double knockouts. The Rubisco-based engineered pathway can also rescue the low glucose uptake which glucose consumption of CAB+IP can reach 60 ± 6 mM.

Chapter 1. Introduction 1
1.1 Background 1
1.2 Motivation 3
1.3 Escherichia coli 4
1.4 Homologous recombination 6
1.5 Rubisco 6
1.6 PrkA 8
Chapter 2. Materials and Methods 9
2.1 Culture of strains 9
2.2 DNA purification 10
2.2.1 Purification of plasmid 10
2.2.2 DNA purification of gel extraction 11
2.2.3 Estimation of DNA concentration 12
2.3 Polymerase chain reaction 12
2.4 Agarose gel electrophoresis 12
2.5 Ethanol precipitation of DNA 13
2.6 Preparation of competent cells 14
2.6.1 Competent cells of chemical transformation 14
2.6.2 Competent cells of electroporation transformation 14
2.7 Transformation 15
2.7.1 Chemical transformation 15
2.7.2 Electroporation transformation 15
2.8 Recombination of E. coli 16
2.9 Analysis of metabolites 17
2.10 Analysis of CO2 concentration 18
2.11 Calculation of total CO2 concentration 18
Chapter 3. Results and discussion 21
3.1 Phenotype of E. coli strains CC and CA 21
3.2 Effects of PrkA on the phenotype of E. coli strains CC and CA 23
3.3 The glyoxylate shunt can be enhanced by the presence of Rubisco 25
3.4 Rubisco-based engineered pathway benefits the growth of CAB 27
Chapter 4. Conclusion 28
Chapter 5. Reference 29
Tables. 34
Figures. 52
Appendix I The list of chemicals 61
Appendix II The list of instruments 64
Appendix III Calibration curves 66

Alam, K. Y., & Clark, D. P. (1989). Anaerobic fermentation balance of Escherichia coli as observed by in vivo nuclear magnetic resonance spectroscopy. Journal of Bacteriology, 171(11), 6213-6217.
Berg, I. A., Kockelkorn, D., et al. (2010). Autotrophic carbon fixation in archaea. Nature Reviews Microbiology, 8(6), 447-460.
Butler, J. N. (1991). Carbon Dioxide Equilibria and Their Applications Michigan, USA: LEWIS PUBLISHERS, Inc.
Carter, D. M., & Radding, C. M. (1971). The role of exonuclease and beta protein of phage lambda in genetic recombination. II. Substrate specificity and the mode of action of lambda exonuclease. J Biol Chem, 246, 2502–2512.
Castaño-Cerezo, S., Pastor, J. M., et al. (2009). An insight into the role of phosphotransacetylase (pta) and the acetate/acetyl-CoA node in Escherichia coli. Microbial cell factories, 8(1), 1.
Chang, D.-E., Shin, S., et al. (1999). Acetate metabolism in a pta mutant ofescherichia coli w3110: Importance of maintaining acetyl coenzyme a flux for growth and survival. Journal of Bacteriology, 181(21), 6656-6663.
Chang, Y. Y., Wang, A. Y., et al. (1994). Expression of Escherichia coli pyruvate oxidase (PoxB) depends on the sigma factor encoded by the rpoS (katF) gene. Molecular microbiology, 11(6), 1019-1028.
Chen, S.-K., Chin, W.-C., et al. (2013). Fermentation approach for enhancing 1-butanol production using engineered butanologenic Escherichia coli. Bioresource technology, 145, 204-209. doi: http://dx.doi.org/10.1016/j.biortech.2013.01.115
Cherepanov, P. P., & Wackernagel, W. (1995). Gene disruption in Escherichia coli: Tc R and Km R cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant. Gene, 158(1), 9-14.
Cherrington, C. A., Hinton, M., et al. (1991). Short‐chain organic acids at pH 5.0 kill Escherichia coli and Salmonella spp. without causing membrane perturbation. Journal of Applied Bacteriology, 70(2), 161-165.
Cox, S. J., Levanon, S. S., et al. (2006). Development of a metabolic network design and optimization framework incorporating implementation constraints: a succinate production case study. Metabolic engineering, 8(1), 46-57.
Datsenko, K. A., & Wanner, B. L. (2000). One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proceedings of the National Academy of Sciences, 97(12), 6640-6645. doi: 10.1073/pnas.120163297
Dharmadi, Y., Murarka, A., et al. (2006). Anaerobic fermentation of glycerol by Escherichia coli: a new platform for metabolic engineering. Biotechnology and bioengineering, 94(5), 821-829.
Dittrich, C. R., Bennett, G. N., et al. (2005). Characterization of the Acetate‐Producing Pathways in Escherichia coli. Biotechnology progress, 21(4), 1062-1067.
Eiteman, M. A., & Altman, E. (2006). Overcoming acetate in Escherichia coli recombinant protein fermentations. Trends Biotechnol, 24(11), 530-536.
Gong, F., Liu, G., et al. (2015). Quantitative analysis of an engineered CO 2-fixing Escherichia coli reveals great potential of heterotrophic CO 2 fixation. Biotechnology for biofuels, 8(1), 1.
Gosset, G. (2005). Improvement of Escherichia coli production strains by modification of the phosphoenolpyruvate: sugar phosphotransferase system. Microbial cell factories, 4(1), 1.
Hallenbeck, P. L., & Kaplan, S. (1987). Cloning of the gene for phosphoribulokinase activity from Rhodobacter sphaeroides and its expression in Escherichia coli. Journal of Bacteriology, 169(8), 3669-3678.
Hallenbeck, P. L., Lerchen, R., et al. (1990). Phosphoribulokinase activity and regulation of CO2 fixation critical for photosynthetic growth of Rhodobacter sphaeroides. Journal of Bacteriology, 172(4), 1749-1761.
Kai, Y., Matsumura, H., et al. (2003). Phosphoenolpyruvate carboxylase: three-dimensional structure and molecular mechanisms. Archives of Biochemistry and Biophysics, 414(2), 170-179.
Kellogg, E. A., & Juliano, N. D. (1997). The structure and function of RuBisCO and their implications for systematic studies. AMERICAN JOURNAL OF BOTANY, 84(3), 413-428. doi: 10.2307/2446015
Kmiec, E., & Holloman, W. (1981). Beta protein of bacteriophage lambda promotes renaturation of DNA. Journal of Biological Chemistry, 256(24), 12636-12639.
Kunze, M., Pracharoenwattana, I., et al. (2006). A central role for the peroxisomal membrane in glyoxylate cycle function. Biochimica Et Biophysica Acta (BBA)-Molecular Cell Research, 1763(12), 1441-1452.
Li, Y.-H., Ou-Yang, F.-Y., et al. (2015). The coupling of glycolysis and the Rubisco-based pathway through the non-oxidative pentose phosphate pathway to achieve low carbon dioxide emission fermentation. Bioresource Technology, 187, 189-197. doi: http://dx.doi.org/10.1016/j.biortech.2015.03.090
Little, J. W. (1967). An exonuclease induced by bacteriophage lambda. II. Nature of the enzymatic reaction. J. Biol. Chem., 242, 679-686.
Marsić, N., Roje, S., et al. (1993). In vivo studies on the interaction of RecBCD enzyme and lambda Gam protein. Journal of Bacteriology, 175(15), 4738-4743.
Murphy, K. C. (1991). Lambda Gam protein inhibits the helicase and chi-stimulated recombination activities of Escherichia coli RecBCD enzyme. Journal of Bacteriology, 173(18), 5808-5821.
Nishitani, Y., Yoshida, S., et al. (2010). Structure-based Catalytic Optimization of a Type III Rubisco from a Hyperthermophile. Journal of Biological Chemistry, 285(50), 39339-39347. doi: 10.1074/jbc.M110.147587
Parikh, M. R., Greene, D. N., et al. (2006). Directed evolution of RuBisCO hypermorphs through genetic selection in engineered E. coli. Protein Engineering Design and Selection, 19(3), 113-119.
Peterhansel, C., Krause, K., et al. (2013). Engineering photorespiration: current state and future possibilities. Plant Biol (Stuttg), 15(4), 754-758. doi: 10.1111/j.1438-8677.2012.00681.x
Rubin, E., & De Coninck, H. (2005). IPCC special report on carbon dioxide capture and storage. UK: Cambridge University Press. TNO (2004): Cost Curves for CO2 Storage, Part, 2.
Shinozaki, K., Yamada, C., et al. (1983). Molecular cloning and sequence analysis of the cyanobacterial gene for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase. Proceedings of the National Academy of Sciences of the United States of America, 80(13), 4050-4054.
Sydney, E. B., Sturm, W., et al. (2010). Potential carbon dioxide fixation by industrially important microalgae. Bioresource technology, 101(15), 5892-5896.
Tamoi, M., Murakami, A., et al. (1998). Lack of light/dark regulation of enzymes involved in the photosynthetic carbon reduction cycle in cyanobacteria, Synechococcus PCC 7942 and Synechocystis PCC 6803. Bioscience, biotechnology, and biochemistry, 62(2), 374-376.
Trueba, F., & Woldringh, C. (1980). Changes in cell diameter during the division cycle of Escherichia coli. Journal of Bacteriology, 142(3), 869-878.
Wedel, N., & Soll, J. (1998). Evolutionary conserved light regulation of Calvin cycle activity by NADPH-mediated reversible phosphoribulokinase/CP12/glyceraldehyde-3-phosphate dehydrogenase complex dissociation. Proceedings of the National Academy of Sciences, 95(16), 9699-9704.
Yang, C.-H., Liu, E.-J., et al. The comprehensive profile of fermentation products during in situ CO2 recycling by Rubisco-based engineered Escherichia coli. Microbial cell factories.
Zhu, Y., Eiteman, M., et al. (2007). Homolactate fermentation by metabolically engineered Escherichia coli strains. Appl Environ Microbiol, 73(2), 456-464.
Zhuang, Z.-Y., & Li, S.-Y. (2013). Rubisco-based engineered Escherichia coli for in situ carbon dioxide recycling. Bioresource technology, 150, 79-88.
楊承翰. (2015). 利用重組大腸桿菌回收二氧化碳生產生質化學品. 國立國立中興大學化學工程學系碩士學位論文, 1-74.