Collaborative Metabolic Regulation Of CO₂ Transportation And Fixation During The Succinate Production In E. Coli

As typical four-carbon platform compound, succinate used as precursor to various commodity chemicals in industries like pharmaceutical, biodegradable polymers and so on, US Department Energy classified it as most potential compound among the top 12 chemical buiding blocks. Traditionally succinate is produced by chemical synthesis from petroleum feedstocks, Biosynthesis of succinate using microorganisms is more competitive because its green, cost effective and energy-saving. During the process of biosynthesis of succinate, CO2 was entered into the cell by passive diffusion and then generate succinate by fixed reaction, so the transportation and fixation of CO2 are decisive roles for biosynthesis of succinate.In this paper, HCO3-transporters BicA and SbtA from cyanobacteria have been expressed in the membrane of AFP111, so the AFP111 can transport HCO3- form extracellular into intracellular initiatively.Then overexpressed the ppc from cyanobacteria to enhance of the ability of CO2 fixation in AFP111. At last, combine the transportation and fixtion to enhance the succinate production and analyze the collaborative metabolic regulation of CO2 transportation and fixation during the succinate production in E.coli AFP111.First, construct plasmids pTrc-sbtA pTrc-bicA, pTrc-bica-sbtA and transferred into E. coli AFP111, successfully achieved the HCO3- transporters SbtA and BicA from Cyanobacteria alone and in combination expression on the cell membrane of E. coli, so that AFP111 have the capacity.of transporting HCO3- from the extracellular into intracellular initiatively. To test the impact of HCO3- transportation on succinic acid production, the batch fermentation of strains were carried out in the bioreactor.The results show that when expression SbtA in E. coli, the succinic acid production is 21.24 g/L, 24% higher than the control AFP111/pTrc99A; when expressed BicA alonely the succinic acid production is 12% higher than the control. When express the transporters BicA and SbtA simultaneously, the succinic acid production is 28% higher than the control. So that enhanced the CO2 transport and improve the HCO3- supply in cells were favor to succinic acid production.Secondly, in order to fixed HCO3- in time, at the same time of enhancing CO2 transportation, enhance the fixation reaction by overexpression of phosphoenolpyruvatecarboxylase(PPC) in order to achieve collaborative metabolic regulation of CO2 transport and fixed. According to CO2 transport-function and fixed-function gene combinations, built the the four plasmid pTrc-ppc, pTrc-ppc-sbtA. pTrc-ppc-bicA and pTrc-ppc-bica-sbtA. Through the results of batch fermentation, when express the PPC,BicA and SbtA simultaneously,the succinic acid production is 25.25 g/L,49%higher than the control.For the first time, heterologous HCO3- transports have been expressed in the membrane of E. coli to realize the initial HCO3- transportation of in E. coli; and by the coordinately expression of the transit and fixed-function gene,HCO3- have been fixationed in time, and ultimately the production of succinic acid and yield been improved. The results provide a new idea on engineered bacteria of succinic acid, also has a certain significance on coordinately regulation of metabolic pathways.