Construction Of High Tolerance Ethanologenic Escherichia Coli And Evaluation Of Its Fermentation Performance

With the gradual exhaustion of fossil resources and increasing attention to environmental problems,bioethanol has become the most promising alternative to fossil fuels.E.coli has become one of the new strains for ethanol production due to its clear research background,broad substrate range and mature fermentation technology.High concentration of monosaccharide in biomass hydrolysate and ethanol accumulated in fermentation process are the main factors restricting the production of bioethanol.Trehalose is one of the most important compatibility solutes synthesized and accumulated by E.coli under environmental stress.In this study,the trehalose metabolic pathway in E.coli was modified to enhance intracellular trehalose accumulation,the tolerance of the strains to substrates and product was improved,as well as the productivity increased.The main results are as follows:Mutants with genetically modified trehalose metabolic pathway were constructed and screened.A series of mutants were successfully constructed using Red homologous recombination:JC11(?treA).JC12(?treF)?JC13((?treC)?JC21((?treA,(?treF).JC22((?treF,(?treC)?JC23((?treC,(?treA)?JC31((?treA,(?treF,(?treC)?JC41((?treA,(?treF,(?treC,PostBA::PldhA)?The ability of trehalose synthesis and accumulation of all the mutants was higher than that of the original strain.JC31 intracellularly accumulated the trehalose up to 3 times,and JC41 11 times that of the original strain.The tolerance of the strains and its relationship with intracellular trehalose accumulation were investigated.The original strain B0013-1031H and the trehalose accumulation strains JC31 and JC41 showed same growth status under ordinary culture conditions.The results showed that intracellular trehalose accumulation significantly improved the tolerance of the strain to glucose,xylose and arabinose,and to lactic acid and ethanol,but not to butanol.The tolerance of the strain to glucose and ethanol was consistent with the level of the intracellular trehalose accumulation.The higher the intracellular trehalose content,the stronger the tolerance of the strain to glucose and ethanol.The fermentation performance of the ethanologenic recombinant E.coli was examined.The ethanologenic recombinant E.coli B0013-1031H-PA,JC31-PA and JC41.PA were obtained by engineering ethanol synthesis pathway.In shaking flask fermentation with 120 g/L glucose,JC31-PA showed the optimal fermentation performance.JC31-PA yielded 50.6 g/L of ethanol,which was 5.42%higher than that of the control.The conversion rate of ethanol from glucose was 48.72 g/100 g glucose,12.67%higher than that of the control,and 95%of the theoretical conversion rate.JC31-PA fermented in a 5-L bioreactor and yielded 48 g/L of ethanol.The average ethanol synthesis rate was 0.75 g/L·h,and the conversion rate of ethanol from glucose was 37.07 g/100 g glucose.