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The Construction and Fermentation of Recombinant Escherichia Coli for Succinate Production

Author: CaoJianLei
Tutor: ShiGuiYang
School: Jiangnan University
Course: Fermentation Engineering
Keywords: Succinate Recombinant E. coli Metabolic regulation Knockout Xer / dif recombinase system
CLC: TQ921.7
Type: Master's thesis
Year: 2010
Downloads: 150
Quote: 0
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Abstract


Succinic acid is an important chemical products, widely used in the field of food, agriculture, pharmaceutical and fine chemical, huge market prospects. Compared to traditional chemical production method, microbial fermentation production of succinic acid has unparalleled advantages and potential, many research institutions at home and abroad. Which means of the use of genetic engineering to build a yield of succinic acid recombinant E. coli is a research hotspot in recent years, due to the wild-product of anaerobic fermentation of E. coli succinic acid content is low, and there is a lot of byproducts. To achieve a high yield of succinic acid purpose, it is necessary to carry out the transformation of the metabolic pathways, blocking metabolic slip, guide carbon flow faster and more flow to the target product. This paper Xer / dif recombinase system, the regulation of the metabolic pathways in a wild Escherichia coli Escherichia coli CICIM B0013. First, in order to eliminate a heteroaryl acid byproducts produced during fermentation, the knock in addition to E. coli CICIM B0013 succinic metabolic competition pathway include acetate kinase and phosphate acetyltransferase gene (ackA-pta), lactate dehydrogenase gene (ldhA ), and pyruvate formate lyase gene (pflB) ethanol dehydrogenase gene (adhE), including a key enzyme gene, the recombinant strain E. coli B0013-1040 obtained with efficient conversion of glucose under anaerobic conditions generate succinic capacity, the fermentation broth, no acetic acid, formic acid and other byproducts. Secondly, this paper on the basis of the strain of E. coli has been blocked in succinic acid metabolic branch adjustment of glucose absorption. Close glucose phosphate transferase system (PTS), knocking addition to the system the key enzyme Ⅱ CBGlc encoding gene ptsG, prompting the cooperative transportation systems galactose become coli glucose transport pathway. This change has greatly increased the succinic acid precursor, the PEP fermentation experiment results show that the ability of producing succinic acid, the resulting recombinant strains of E. coli B0013-1050 has been improved significantly. 36 h anaerobic conversion of succinic acid production reached 30.5 g / L, and the intensity of production of 0.85 g / L · h, glucose - succinic sugar acid conversion was 65.2%, the fermentation broth was not detected by HPLC Titanium heteropoly acid. In addition, the ptsG gene deletion also lifted the inhibition of glucose metabolites on cell, E. coli can also use a variety of carbon sources other than glucose, largely broaden the substrate spectrum of a succinic acid fermentation to reduce production costs , laid the foundation for the large-scale industrial production. Strengthen succinic acid biosynthetic pathway, guided metabolic flow more flow to oxaloacetate this critical nodes, this paper strengthen the expression of PEP carboxylase. By E. coli endogenous ppc gene and the connection of different copy number plasmid, introducing a recombinant bacteria, thereby to achieve the control of the ppc gene expression in the host body dose. The study found that succinic acid production process in the expression of PEP carboxylase dose is not as high as possible, but need to be controlled at a reasonable level in order to effectively promote the accumulation of succinate. Experiment recombinant bacteria E. coli B0013-1050 (pTH-ppc) The production capacity of the strongest one in the recombinant bacteria, anaerobic conversion of glucose 36 h succinic acid production reached 36.2 g / L, the intensity of production was 1.01 g / (L · h), glucose - succinic acid conversion rate of 64.3% at the advanced level in the public similar literature. Finally, we also explore and study the building of E. coli glyoxylate cycle feasibility of anaerobic fermentation of succinic acid.

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