Metabolic Engineering Of Escherichia Coli For Improved Succinic Acid Production

Succinate had drawn much interest because it's potential to be used as commodity chemicals. The fermentation strains are one of the keys to produce succinate from renewable feedstocks. A series of genetic modified Escherichia coli were constructed based on the principles of metabolic engineering. The succinic acid producing capabilities of these strains were studied systematically and the major findings are:Compared with the wild type, ptsG knock out Escherichia coli TUQ2 is different in physiological and fermentation products yields. TUQ2 produced about 4 fold succinate yields, however, it also showed very slow growth and low glucose consumption rates. TUQ2 increased the generation time to approximate 4 h and reached higher final cell density of 2.5 g/L by expression of galP. However, glk expression had no effect on the mutant. After expression of pyruvate carboxylase (PYC), TUQ2 can further improve the succinate yields to 1.2 mol/mol glucose. After the coexpressiong of PYC and galactose permease, TUQ2 showed higher succinate yield (1.15 mol/mol glucose) and the specific rate of glucose consumption from 0.33 g/L·h to 0.6 g/L·h.The formate dehydrogenase (FDH) of Candida boidini expression plasmid pQZ8 was constructed. After expression of FDH, the wild type E.coli consumed glucose quickly and the ethanol yield was increased sharply, however, the yield of succinate was almost not changed. The yields of succinate were decreased after the expression of FDH in TUQ2 and TUQ3.In this thesis, based on the genome-scale E. coli stoichiometric model iJR904, through the GAMS linear programming package, the in silico maximum succinic acid yield estimation is 1.71mol succinate/mol glucose. One optimal succinate producing pathway was also found. After comparisons with the metabolic flux analysis (MFA) results computed from the wet experiments data of the wild type, TUQ2, W1485/pQZ6 and TUQ2/pQZ6, two potential improvement targets, pyruvate carboxylase and glyoxylate shunt were identified and selected for the genetic modification.The strains TUQ8 and TUQ19 were constructed by disruption of iclR. The studies of them revealed that iclR knock out strains can reached higher cell density...