| Summary: | 碩士 === 國立中興大學 === 化學工程學系所 === 102 === Since the 17th century, industrial revolution led to the excessive emission of carbon dioxide. It impacts on the environment seriously, such as greenhouse effect. Overdeveloped industrial activities that results in the shortage of oil have been attracted much attention in recent years. Therefore, it began to be focused on the development of new energy along with reducing carbon dioxide evolution. Transketolase A (TktA) plays an important role in non-oxidation pentose phosphate pathway (NOPPP). It rearranges carbons by catalyzing two chemical reactions in NOPPP. It converts fructose-6-phosphate together with glyceraldehyde-3-phosphate into xylose-5-phosphate and erythrose-4-phosphate. TktA also converts glyceraldehyde-3-phosphate and sedoheptulose-7-phosphate into ribose-5-phosphate and xylose-5-phosphate. Transaldolase B (TalB) in NOPPP converts erythrose-4-phosphate and fructose-6-phosphate onto glyceraldehyde-3-phosphate and sedoheptulose-7-phosphate. In the engineering perspective, one of the advantage of the NOPPP is that has no CO2 emission.
In this study, we used two key enzymes, phosphoribulokinase (PrkA) and ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), in the Calvin–Benson–Bassham cycle as the exogenous genes to create a new pathway in which it can recycling CO2 in E .coli. We called it is the carbon recycling system. Moreover, Overexpression of TktA and TalB in carbon rearrangement system could convert hexoses to pentoses, which can serve as the precursor for the carbon recycling system. Thus, combining two systems could use cheaper carbon source like glucose to ferment ethanol and acetate. In the same times, it could recycle inorganic carbon for reducing CO2 emission. The results indicated that the carbon rearrangement system could reduce CO2 emission when engineered E. coli cultured in minimal medium. Furthermore, it was found that two enzymes TktA and TalB that was overexpressed simultaneously had the best performance in terms of the CO2 reduction. Compare with wild-type E. coli BL21 (DE3), Total CO2/PEtOH+Pacetate (mole/mole) could decrease 28.4%. On the other hand, overexpression of PrkA and Rubisco had the best effect to reduce CO2 emission in carbon recycling system. Compare with wild-type E. coli BL21 (DE3), Total CO2/PEtOH+Pacetate (mole/mole) could decrease 8.6%. However, overexpression of Rubisco led to more CO2 evolution. It may Rubisco changed the distribution of carbon flow in E. coli. The data shows that Rubisco caused the mRNA of pckA and maeB to increase and these related to CO2 evolution. As we combined two systems, overexpression of PrkA, Rubisco, and TktA could reduce CO2 emission more. Compare with wild-type E. coli BL21 (DE3), Total CO2/PEtOH+Pacetate (mole/mole) could decrease 27.2%. Furthermore, compare with wild-type E. coli BL21 (DE3), overexpression of PrkA, Rubisco, TktA, and TalB could decrease 28.4% in Total CO2/PEtOH+Pacetate (mole/mole). The data show when it has strong gene overexpression downstream, TktA plays a key role in carbon rearrangement system. In order to improve the carbon recycling system, this study used MgSO4, NaHCO3 and K2HPO4 as the factor to investigate what is the rate-limiting step in this system. When Pi concentration decreased, ATP:AMP ratio will declined and resulting in the decreased the activity of Rubisco activase. In this study, it was found that when K2HPO4 concentration added, Total CO2/PEtOH+Pacetate (mole/mole) was reduced in JB system. Compare with wild-type E. coli BL21 (DE3), JB and JB/pTA could reduce about 50.7~53.5% in Total CO2/PEtOH+Pacetate (mole/mole). Therefore, the ATP is the rate-limiting step in carbon recycling system.
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