Construction And Fermentation Of Genetically Engineered Escherichia Coli For The Production Of Succinic Acid

Succinic acid, a C4platform compound, which is widely used in food, medicineand chemicals, it has wide application. Succinic acid production by microbiologyfermentation has attracted much attention on its using of renewable resources, lowercost and greens environmental protection in recent years. But at present the mainlimiting factor is the lack of high yield succinic acid strain that can use cheap biomassfor fermentation. Therefore, we constructed a recombinant Escherichia coli strain forthe production of succinic acid, using a wild type Escherichia coli W as the startingstrain. By using λRed homologous recombination strategies, we deleted the ldhA gene,adhE gene, pflB gene, poxB gene and ackA gene respectively from E. coli W to obtaineda mutant E. coli WS100(△ldhA△adhE△pflB△poxB△ackA), then WS100wasdomesticated in anaerobic tube with mineral salt medium, and the resulted strain E. coliWS101mainly produced succinic acid.Further researchs were studied on WS101fermentation conditions such as initialglucose concentration, fermentation modes and feeding neutralizer. When WS101fermented with the initial concentration of6%glucose for30h, the consumption ofglucose and the production of succinic acid both are the highest, were59.81g/L and43.29g/L respectively. Succinic acid production in fed batch fermentation was exceeded11.62g/L than that in batch fermentation, and succinic acid productivity was also morethan0.16g/(L·h); Then, K+, NH4+and Ca2+as the neutralizer were investigatedrespectively in WS101fermentation. The yield of succinic acid was the highest usingK+as the neutralizer with the production of82.69g/L in72h, and succinic acidproduction was the least using NH4+as the neutralizer with the production of62.74g/L,while the production was74.26g/L using the Ca2+as the neutralizer.Finally, utilization of corn processing by-products by WS101were studied by fedbatch fermentation under anaerobic condition, using disused glucose solution as carbonsource, mineral salt, yeast and mineral salt, corn steep liquor as nitrogen source,respectively. The results demonstrated that succinic acid production were little differentbetween adding yeast or not in mineral salt medium of the fermentation, However, thefermentation time of the adding yeast mineral salt medium was shortened by18h,which illustrated that adding yeast in fermentation medium had significant effect on thefermentation time. Succinic acid production could reached83.13g/L in yeast mineralsalt medium, the productivity and yield reached1.54g/(L· h) and76%, respectivity. Inthe same conditions of fermentation. Succinic acid production in mineral salt mediumwas increased20.22g/L than that in corn steep liquor medium at72h, however,Succinic acid production in corn steep liquor medium also could reach60.86g/L, theproductivity and yield reached0.85g/(L· h) and66%, respectivity. Escherichia coliWS101showed greater potential on industrial application, owing to use cheap biomassraw material as substrate, such as corn steep liquor and disused glucose solution.