Regulation Mechanism Of Global Transcription Factor Cra In Escherichia Coli For Succinic Acid Biosynthesis

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 12chemical 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.A large number of genes and enzymes involved in the succinic acid metabolic process,among them the CO₂ fixation reaction and glyoxylate bypass are especially important,moderate expression of these genes is conducive to the production of succinic acid.In this paper,the global transcription factor Cra was first directed evolution to regulate the gene expression and enzyme metabolismprocess,the high yield strains were chosed.Then the relative expression of key genes and the activity of enzymes were determined and found mutant strain through enhancing the CO₂ fixation and activated the glyoxylate pathway to improve succinic acid production.Finally the structure and function of Cra was analysis,found that the combination between mutant Cra and activator is more easily,which can enhance the CO₂ fixation capacity and activate the glyoxylate pathway.First of all,through the expression of directed evolution global transcription factor Cra to regulate the carbon metabolism related genes and enzymes to improve the yield of succinic acid.The global transcription factor Cra was mutate by error prone PCR,connected to pTrc99A vector and transformed into Escherichia coli AFP111 to construct the mutant library.Through the rapid screen based on 96 well plate and HPLC,6 high yield strains were clected.Then the mutant sites were integrated to form4 strains,among which Tang1541 was the highest production strain,the production was78.6 g/L,it was 27%higher than the control strain Tang1533,the production of another intergrated strain Tang1544 was 74.4 g/L,it was 20%higher compared with the control strain Tang1533.Secondly,through the determination of key enzymes activity and key genes expression level,the relative expression of CO2 fixation genes ppc and pck did not changed significantly while the PPC activity of Tang1541 and Tang1544 both reached about 0.515 U/mg,which was 2 times higher than the control group Tang1533?0.175 U/mg?.At the same time,the PCK enzyme activity of the control group Tang1533 was 0.876 U/mg,the PCK enzyme activity of Tang1541 and Tang1544 were 1.214 U/mg and 1.143 U/mg,which compared with the control group were increased more than 30%,the mutant strain significantly enhance the ability of CO2 fixation.Meanwhile the glyoxylate pathway genes aceA,aceB expression level of Tang1541 increased 2.81 and 2.07 times respectively,the aceA,aceB expression level of Tang1544 upregulation 3.16 and 4.35 times,the increase rate is significant.The activity of isocitrate lyase and malate synthase of Tang1541 and Tang1544were increased more than 20%,the metabolic flux of the glyoxylate bypass upregulate significantly to enhance the yield of succinic acid.Finally,through the experiment of ITC mutation,the free binding energy of Tang1541 and Tang1544 were-60.263+0.5 KJ/mol and-69.722+0.8 KJ/mol is lower than that of Tang1534?-41.997 kJ/mol?,indicated that the mutation of transcription factor are more likely to combine with FBP to activate the Cra;The EMSA experiments also showed that the mutation of Cra factor enhanced the binding ability of Cra with aceABK promoter,which activated the glyoxylate pathway to produce more succinate.It is the first time through the expression of evoluted transcription factor Cra in the E.coli to regulate the global metabolic pathway of succinic acid.The ITC and EMSA experiments confirmed that mutation transcription factor was more likely to be activated and then enhance the CO₂ fixation capacity,improve the glyoxylate bypass fluxes and improve the succinic acid yield.The research results provide a new idea for the construction of genetic engineering bacteria of succinic acid,it also has a certain reference to the research of metabolism regulation.