Construction Of Genetically Engineered Bacteria Producing Fumaric Acid And Fumaric Acid Derivatives

Citric acid synthase and cis aconitase have a greater influence on the strength of the citric acid cycle.Therefore,the genes of the two enzymes were cloned and introduced into the fumarate-producing bacteria StrainABCDIA to further increase the yield of fumaric acid on the basis of the predecessors.To explore the influence of the ratio of intracellular NAD+/NADH,the heterologous NADH oxidase gene and the self-sourced transhydrogenase gene were introduced into the fumarate-producing bacteria StrainABCDIA respectively.The yield per unit mass of fumaric acid was 0.54 g/g and 0.67 g/g.Though the biomass was not as good as StrainABCDIA,the bacteria's acid production capacity was enhanced,indicating that the adjustment of cofactors is beneficial to the accumulation of fumaric acid.This study integrated and optimized the previous research and obtained EcS?EcN?CgS?CgN four Strains.Using modified M9 medium for shake flask fermentation of recombinant bacteria,in terms of biomass and fumaric acid production,the four strains were slightly higher than the control group StrainABCDIA,but the overall difference was not much.Among them,the yield of CgS fumaric acid was higher,reaching 0.511 g/L.However,due to poor growth,this yield did not meet expectations.Compared with the yield per cell mass,the yield of CgS fumaric acid can reach 0.937 g/g,which was 20%higher than that of the control StrainABCDIA,which was an advantage over other strains.From the perspective of intracellular cofactors NAD+/NADH,CgS were increased by 40.24%compared with StrainABCDIA,indicating that by overexpressing the transhydrogenase increased the ratio of intracellular NAD+/NADH,thereby increasing the TCA cycle rate.This study explored the production of fumaric acid esters by biological methods,heterologous expression of acyl ligase and acyl transferase.Using the ELLMAN method to measure protease activity,calculated according to the reduction of coenzyme A in the system,the enzyme activity was low,but the overall showed a trend of activity,and then the same method was used to explore the optimal reaction conditions of BafX.As a result,when the reaction temperature was 25?,the pH was 8.0,and the ATP concentration was 0.2 mmol/L,the enzyme reaction rate was maximum.