The recycling of CO2 by Rubisco-based engineered Escherichia coli to make bio-based chemicals

• Main Authors: Cheng-Han Yang, 楊承翰
• Other Authors: Si-Yu Li
• Format: Others
• Language: zh-TW
• Published: 2015
• Online Access: http://ndltd.ncl.edu.tw/handle/57959394669365325152

碩士 === 國立中興大學 === 化學工程學系所 === 103 === In previous study, the zwf gene has been knocked out in Rubisco-based E. coli (designated as MZB) enhance the flux of non-oxidative pentose phosphate pathway. Compare with JB, the total CO2 / sugar consumption (mol/mol) decreased 58% in MZB but the carbon recovery was low (~70-75%) when PrkA and Rubisco was expressed in E. coli. In this study, since we knockout the ldh gene and frd gene, we still fail to get a good carbon recovery in the mutant strain with the expression of PrkA and Rubisco. Especially for the IPTG induction strain MZLFB+IP, the carbon recovery of MZLFB+IP is about 73%. However, we do see several important phenotypes of MZLFB+IP, including the increase in Biomass and the decrease in acetate. This is the result of anaplerotic reaction, glyoxylate shunt. At the same time we find an unknown peak in the HPLC analysis. Finally, we find that malate has the same retention time with the unknown peak. Malate was quantified and added into the calculation of carbon recovery. The result shows that the carbon recovery of MZLFB+IP is 93%. To make sure the glyoxylate shunt was induced by the presence of Rubisco, additional 2 g/L acetate and express the PrkA and Rubisco separately has been tested. The results show that the additional acetate will enhance the production of malate and overexpression of Rubisco alone can observe same phenotypes. The RNA-sequencing based on NGS (Next generation sequencing) of J3 and JB strain also shows the consistent results. The results show that the glyoxylate shunt relative genes including acs, aceA, aceB and glcB are increasing in both J3 and JB. Using equations based on mass balance and stoichiometry to simulate MZLFB+IP, the results indicate that the Rubisco pathway fraction is 10.6%. To enhance the carbon recycling effectiveness by Rubisco, introduce the NAD+-dependent formate dehydrogenase. The enzyme reaction is convert formate and NAD+ to CO2 and NADH. In the mutant strain MZLF the main product is ethanol, so the FDH is not only increasing the available CO2 for Rubisco but also regenerating NADH for redox balance. For the co-expression of PrkA, Rubisco and FDH, in MZLFB+FDH+IP strain the Total CO2 / EtOH (mol/mol) can be decreased substantially compared to MZLF+FDH+IP. Here also using equations to simulate MZLFB+FDH+IP, the results indicate that the Rubisco pathway fraction is 29.2%.