The Construction And Fermentation Of Recombinant Escherichia Coli For Succinate Production

Succinic acid is an important chemical product, widely used in food, agriculture, medicine and fine chemical industry, and has a huge market prospect. Compared with the traditional production method in chemical way, microbial production of succinic acid by fermentation has a lot of advantages and huge potential. Many research institutes all over the world are paying great concern on its progress. Construction of recombinant Escherichia coli with higher yield succinate production by genetic engineering method is one of hotest topics in recent years research. There are a large number of by-products besides succinic acid in the component of wide type Escherichia coli anaerobic fermentation production. In order to achieve a higher succinate production, metabolic regulation must be carryed out on a strain of E. coli, blocking the metabolic bypass and leading carbon flow to the target product as much as possible.In this paper, metabolic pathway regulation was performed on a wild type strain of Escherichia coli CICIM B0013 by using Xer/dif recombinase system. In order to Eliminate the by-products of the fermentation process, we constructed a recombinant E. coli strain B0013-1040, of which the gene ackA-pta ,ldhA, pflB, adhE were deleted from chromosome. This strain can produce succinate as the major product in anaerobic fermentaion.Secondly, we carryed out a research about the regulation of glucose uptaking pathway in E. coli. To shut down the glucose phosphotransferase system (PTS), ptsG which encoding protein EIICBglc of the phosphotransferase system was deleted and made the galactose collaborative transportation systerm to be the main pathway for glucose uptaking in a recombinant strain of E. coli B0013-1050. This change has greatly increased the succinic acid biosynthesis precursor PEP. The fermentation experiments showed the succinate production of E. coli B0013-1050 has been improved significantly. A higher production of 30.5 g/L succinate was achieved in 36 h anaerobic fermentation, the productivity and yield was enhanced to 0.85 g/L·h and 65.2%, respectively. Additionally there was no lactate, acetate, formate and ethanol detected by high performance liquid chromatography (HPLC) in the fermentation production. The ptsG mutant also showed a disappearance of glucose metabolites inhibition to cells, and it can use a variety of carbon sources as substrate, this can help us to reduce the production costs and make contribution to industrial application.In order to strengthen the succinate synthesis pathway and guide more carbon flow to oxaloacetate which is very important to succinate productin. We enhanced the expression of PEP carboxylase by clone the ppc gene to plasmids with different copy numbers and then transform them into the recombinant strain E. coli B0013-1050. The result showed that the expression level of PEP carboxylase in succinate production process is not the higher the better. It requires a reasonable level for effectively promoting the accumulation of succinate. The recombinant E. coli B0013-1050 (pTH-ppc) has a highest production in all strains constructed. It can produce as much as 36.2 g/L succinate in 36 hours anaerobic fermentation, and the productivity and yield was enhanced to 1.01 g/L·h and 64.3%, respectively.Finally, the construction of glyoxylate cycle in E. coli anaerobic fermentation for producing succinic acid was discussed in this paper.