Metabolic Engineering Research For Succinic Acid Production In Escherichia Coli

A series of genetic engineering Escherichia coli was obtained by ptsG gene knockout and transforming plasmids. The effect of ptsG, which encodes the glucose-specific permease of the phosphotransferase transport system (PTS) protein EIICBGlc, expression of galactose permease and glucose dehydrogenase on succinate production under anaerobic conditions were researched in this study. A biochemical reaction network was assembled with metabolites, and then the analysis and comparison of metabolic fluxes under anaerobic conditions of three different engineering strains was obtained. The main results presented in this work are as follows:The ptsG knockout mutant (TUQ2) was obtained according to the phageλRed recombinase methods with pTP801 as the template from the wild type E. coli W1485. The ptsG mutant is different in physiological and fermentation products yields. The mutation of ptsG can produce about 5 fold succinate yields, however, under anaerobic conditions, can slow the glucose consumption and cell growth rates.The galp and glk genes were amplified from W1485 by PCR and ligated into the expression plasmid pET28a (+) with the suitable enzyme excision to give plasmid pQZ3 and pQZ4. In order to co-express the glk and galp, the ori of plasmid pQZ3 was replaced with the ori of plasmid pACYC184 to give plasmid pQZ5.Expression of glk has no effect on the mutant strain in glucose consumption and growth rates. When the galp and glk are expressed together, the result is almost the same as the single expression of galp. Based on the above, we conclude that galp expression can accelerate the glucose consumption in the ptsG mutant.The gdh gene from Bacillus subtilis, which encode was cloned and express in E.coli, provided more NADH after induced, but the production of succinic acid was a little lower.A biochemical reaction network of engineering E.coli was assembled with metabolites, and a stoichiometric model was used to analyze metabolic fluxes of different engineering strains. The result of metabolic fluxes analysis indicated that flux of PEP is easy to control, this is very important to the fermentation.