Metabolic Engineering Of E.coli For The Fermentative Production Of Itaconate

Itaconic acid is an important chemical in a wide range of materials such as plastics,resins,and coatings.The current production is mainly produced by fermentation of Aspergillus terreus,but this method also has some shortcomings such as slow growth and complex nutrition requirements,and genetically engineered bacteria can solve these problems well.However,in the research of metabolic engineering reforming antibiotics to produce itaconic acid,there is still lack of effective means to increase the production of itaconate.In order to construct high-yield itaconic acid engineering bacteria with fast growth and easy cultivation,Escherichia coli BL21(DE3)which has a good match with the recombinant plasmid was used to over-express the recombinant strain of shake flask fermentation overexpressing the cad gene encoding cis aconitate decarboxylase Itaconic acid produced 36 mg/L at 24 h.Under the optimized optimal conditions,the content of itaconic acid in the 24 hour shake flask fermentation can reach 85 mg/L,an increase of 136%.By further expressing the glt gene encoding citrate synthase and using the ACS gene encoding acetyl-CoA synthetase to construct the acetate supplement pathway,the itaconic acid content was increased by 54%to 131 mg/L.It was also found that the expression of only the cad gene and the acn gene encoding aconitase would lead to a decrease in the production of itaconic acid and a significant increase in biomass.In addition,it was found that exogenous addition of sodium citrate and cis-aconitic acid significantly promoted the synthesis of itaconic acid.Engineering bacteria co-expressing cad,glt and ACS added 12 g/L sodium citrate at 0 h after induction,and cultured in a shake flask at 18℃ for 24 h.The yield of itaconic acid and the utilization rate of citrate reached 317 mg/L and 66%,only a small amount of acetic acid(0.579 g/L)accumulated in the fermentation broth.Finally,a certain amount of material transport in the fermentation strain was investigated,and it was found that the addition of surfactant had no obvious effect on the yield of itaconic acid,while the expression of the MFS gene had a certain effect on the fermentation process,but the effect was poor and required further exploration.In the end,the constructed genetically engineered bacteria could produce375.1 mg/L itaconic acid under optimal conditions for 24 h,which was a 941.9%increase compared to when only the cad gene was expressed.The above results indicate that the ability to produce itaconic acid in E.coli can be effectively improved by strengthening the itaconic acid anabolic pathway and strengthening the supply of itaconic acid precursors.