Metabolic Engineering Of Escherichia Coli For The Production Of Succinic Acid

Succinic acid is an important four-carbon platform chemical that is widely used in pharmaceutical,degradable plastics,dietary,fiber,metal processing,and pesticides.Because of the depletion of fossil fuels and the need for sustainable development,the biosynthesis of succinic acid from renewable raw materials has become a worldwide concern.The production of succinic acid by Escherichia coli is limited by the imbalance of intracellular cofactors,resulting in low yield,low titer,and many by-products.In this study,based on the yield of succinic acid under different fermentation conditions,the stoichiometric analysis showed that efficient production of succinic acid by E.coli FMME-N-26 under two-stage fermentation conditions required the oxidative tricarboxylic acid（TCA）cycle to provide sufficient ATP and NADH for the reducing tricarboxylic acid（r-TCA）cycle.The intracellular ATP and NADH contents were regulated by metabolic engineering strategy to build engineering strain E.coli FW-17.Finally,through the fermentation optimization strategy,the production of succinic acid by E.coli FW-17 was promoted.The main contents of this study are as follows:1.Chemometric analysis of succinic acid production:First of all,the production of succinic acid by E.coli FMME-N-26 was analyzed under aerobic,anaerobic and two-stage fermentation conditions.It was found that TCA cycle was required to provide a reasonable amount of NADH and ATP for the r-TCA pathway during the efficient production of succinic acid.Secondly,the effects of intracellular NADH and ATP content on the production of succinic acid by E.coli FMME-N-26 strain were studied.The process of succinic acid production by E.coli was analyzed by stoichiometry.Thus,the problem of reduced synthesis of succinic acid by r-TCA caused by insufficient energy and cofactor was relieved.2.Increase intracellular ATP and NADH levels to promote succinate production:First,to increase intracellular ATP levels,we heteronomically expressed Zmglf gene encoding the glucose-diffusing protein from Zymomonas mobilis,overexpressed pgm gene encoding phosphoglycerate mutase and Aspck gene encoding phosphoenolpyruvate kinase from Actinobacillus succinogenes.E.coli FW-1,E.coli FW-2,and E.coli FW-3 were constructed,respectively.The results of fermentation showed that the intracellular ATP content of E.coli FW-1,E.coli FW-2 and E.coli FW-3 were increased by 17.95%,100.00%and 47.44%,respectively.Succinic acid titer of E.coli FW-1 and E.coli FW-3 was 9.25%and 6.55%higher than that of the parent strain E.coli FMME-N-26,respectively.However,there was no significant change in the succinic acid titer of E.coli FW-2.Secondly,to increase the intracellular NADH level,the adh E gene encoding alcohol dehydrogenase was knocked out to construct E.coli FW-5,E.coli FW-6 and E.coli FW-7 strains were constructed by overexpressing the gap A gene encoding glyceraldehyde phosphate dehydrogenase and pnc B gene encoding nicotinic phosphoribosyltransferase,respectively.The results of fermentation showed that the intracellular NADH content of E.coli FW-5,E.coli FW-6,and E.coli FW-7were 28.13%,8.57%,and 17.20%higher than that of E.coli FMME-N-26,respectively.Succinic acid titer was increased by 6.26%,10.50%,and 13.09%,respectively.Finally,in order to coordinate the balance of ATP and NADH supply,the expression levels of pgm and Zmglf genes,gap A and pnc B genes were optimized by RBS combinations of different strength,and the optimal engineering strain E.coli FW-17 was obtained.The succinic acid titer reached 85.67g·L^-1.Compared with the original strain E.coli FMME-N-26,it was increased by 34.80%.3.Fermentation optimization to promote succinic acid production:First,the two-stage fermentation process of E.coli FW-17 was optimized in terms of the type and concentration of corn pulp,p H neutralizer in the anaerobic stage,and glucose concentration in the anaerobic stage.The results of 5 L fermenter showed that the titer and productivity of succinic acid increased to 144.36 g·L^-1 and 1.99 g·L^-1·h^-1,respectively,when 5 g·L^-1 Tongsheng corn pulp was used as nitrogen source,the p H was adjusted by magnesium carbonate and sodium carbonate,and the concentration of glucose was 1 g·L^-1.Secondly,when the optimal fermentation conditions were scaled up to 1000 L fermenter,the titer and productivity of succinic acid reached 143.58 g·L^-1·h^-1 and 1.99 g·L^-1·h^-1,respectively.