Modification Of Escherichia Coli Fumaric Acidbiosynthesis System

Fumaric acid is an intermediate product of the TCA cycle,and has a wide range of applications in many fields.At present,the synthesis of fumaric acid is mainly based on the chemical synthesis of petroleum-based products.However,due to the non-renewability of petroleum resources and the environmental pollution of chemical synthesis,there is an urgent need to develop an alternative method for fumaric acid production.And the biological fermentation method has attracted more and more attention because of the characteristics of low cost,easy operation and environmental friendliness.In recent years,with the development of genetic engineering and metabolic engineering technology,researchers have begun to artificially design and transform the fermentation and metabolism processes of various natural products in order to further improve the fermentation yield.The natural strain producing fumaric acid is Rhizopus,because of its unclear genetic and metabolic background,most of the current work on fumaric acid metabolism is based on the model organism E.coli as a platform for the research of metabolism and transformation.In the pre-laboratory work,based on strain E coli JM109,Strain ABCDIA was constructed through knockout of fumarate reductase gene(frdABCD),fumarase gene(fumA,fumB and fumC),local transcription factor(iclR)and global transcription factor(arcA).The yield of fumaric acid by fermentation of this strain was obviously improved.However,due to excessive knockout of genes during the work,the biomass of bacteria decreased significantly and we found that the knockout of certain genes had no positive effect on the yield of fumaric acid.In summary,the work of this paper mainly about the following aspects:(1)Based on the Strain ABCDIA,the GalP transport system was constructed comparing with PTS glucose transport system,the fumaric acid production ofthe Strain ABCDIA was increased by 50%.On this basis,the malate dehydrogenase gene(mdh)-knockout strain was constructed,and the production of fumaric acid was increased by 16.85%.After further expression of the acetyl-CoA synthetase gene(acs),the accumulation of fumaric acid per unit cell was further improved,which was 115.49%higher than Strain ABCDIA.Finally,using acetic acid as carbon source to ferment the above recombinant bacteria,it provided a feasible basis for fermenting fumaric acid with crude acetic acid.(2)The problem of serious decline in biomass caused by excessive knockout of genes in the previous work was analyzed,and we found that only the knockout of the fumB and arcA genes has a positive effect on fumaric acid production.Therefore,the previous work was optimized and integrated,and the strain E.coli JM109 was used as the starting strain to knock out the fumB and arcA genes.In combination with the construction of Galp transport system,co-expressing malate dehydrogenase gene(mdh)and citrate synthase gene(gltA),and constructing an acetic acid replenishment pathway.the yield of fumaric acid was increased from 5 mg/L of the original strain E.coli JM109 to 77.9 mg/L.(3)Studying the effects of energy metabolism on the production of fumaric acid and regulating the level of intracellular cofactors.The ratio of NAD+/NADH was increased by overexpressing the transgenase gene(sthA)of E.coli and the NADH oxidase gene(noxE)of Streptococcus pneumoniae.After the verification of fermentation experiments,the production of fumaric acid was increased by 93.5%and 19.5%,respectively.