Application of metabolic and biochemical engineering techniques to enhance metabolic flux throughput and improve succinate production in Escherichia coli
Master of Science
Using biochemical engineering principles and metabolic engineering tools in tandem can considerably improve process productivity by manipulation of the throughput of certain pathways. Escherichia coli was chosen as the model system because of its vast available genetic information, rapid growth and the practice of standardized experimental protocols. The pathway leading to the production of succinic acid was examined for the effects of level of carbon substrate available to the system and overexpression of the phosphoenolpyruvate carboxylase (PEPC) enzyme system coupled with pathway manipulations and use of different carbon sources. Incorporation of the glcU gene from Staphylococcus xylosus encoding a novel glucose uptake protein resulted in enhancement of metabolic flux throughput in E. coli. This was further utilized to increase the productivity of a specific metabolite, acetoin, encouraging the use of this system to improve succinate levels. Further, overexpression of the feedback resistant Sorghum PEPC in the host was coupled to three pathway manipulations: ackA-pta mutant, ackA-pta-nuo mutant, and ackA-pta-nuo-ldh mutant, with a high yield value of 0.65 mole succinate per mole of glucose utilized obtained with the ackA-pta-nuo-ldh mutant. Using a reduced carbon source such as sorbitol increased the succinate production to approximately 16 g/l.
Microbiology; Biomedical engineering; Chemical engineering