Design And Optimization Of Fumaric Acidresponsive Two-component Biosensor

As a natural organic acid,fumaric acid is not only an important organic chemical raw material,but also an intermediate of fine chemical products.At the same time,it is also widely used in food,coatings,resins,plasticizers and other fields.At present,biological fermentation is an important method for the production of fumaric acid,especially using Escherichia coli as chassis microorganism.Rapid and accurate detection of the production and accumulation of fumaric acid in fermentation broth is an urgent need in the fermentation process.However,due to the selective permeability of cell membrane,there is a great difference in the concentration of fumaric acid-producing strains inside and outside the cell.Therefore,the intracellular biosensor can not detect the real concentration of extracellular fumaric acid,but the detection efficiency of traditional high performance liquid chromatography is low.In order to overcome this limitation,based on the dcu S-dcu R two-component system in E.coli,a two-component biosensor which can respond to extracellular fumaric acid was designed,and our research contents are as follows:（1）Design,construction and performance characterization of fumaric acid-responsive biosensor.Using the two-component system dcu S-dcu R which can respond to fumaric acid in Escherichia coli and the downstream promoter regulated by this two-component system,a biosensor with basic response to fumaric acid was constructed.The performance of the biosensor was tested,and it was determined that the best detection time of fumaric acid sensor was 8 hours after induction,and the highest detection concentration was 30 m M.At the same time,the response specificity of the fumaric acid-responsive biosensor was determined.Under the induction of different structural analogues,the response of the biosensor to fumaric acid was only 1.7 times.（2）Detection conditions and structure optimization of fumaric acid-responsive biosensor.The biosensor elements were analyzed from the point of view of molecular biology.According to the transcription factor binding sites on different promoters used to construct the biosensor plasmid,the effects of corresponding factors glucose,nitrate and oxygen on the detection of the biosensor were determined.After the expression of dcu S-dcu R operon was regulated by medium intensity PUTR_crp,the effects of glucose and oxygen were eliminated.According to the metabolic pathway,the host strain was adjusted to E.coli BL21（DE3）Δsuc D,and the position of operon and downstream sfgfp gene on the plasmid was adjusted.The dynamic range（the ratio of maximum to minimum fluorescence signal）of fumaric acid-responsive biosensor was successfully increased to 4 times.（3）Optimization of expression proportion and strength of Dcu S and Dcu R.The structural peculiarity of the two-component system for constructing fumaric acid-responsive biosensor is that there is a 4-base overlap between the two genes of Dcu S and Dcu R.In order to determine the effect of expression level of Dcu S and Dcu R on the performance of the biosensor,five gradient intensities of PUTRs were used to express dcu S and dcu R genes in monocistron structure,respectively.The results showed that fumaric acid-responsive biosensor with the highest dynamic range of 4.9 times was obtained by the highest intensity of PUTR_{glp D} regulating dcu S and the moderate intensity of PUTR_{glt A} regulating dcu R.In order to solve the problem that there is no linear relationship between the dynamic range of the biosensor and the intensity of PUTR,the transcriptional expression levels of dcu S and dcu R were detected by real-time fluorescence quantitative PCR,and it was found that the best expression ratio of dcu S:dcu R was46:54.And on the premise of maintaining the best relative expression level,the higher absolute expression level is more beneficial to improve the dynamic range of fumaric acid-responsive sensor.（4）Modification of ATP binding site of the histidine kinase Dcu S.The process that Dcu S binds ATP and then phosphorylates with Dcu R is an important part of the work of fumaric acid-responsive biosensor.Therefore,modifying the ATP binding site of Dcu S is helpful to improve the enzyme activity to optimize the performance of fumaric acid-responsive biosensor.By mutating the known ATP binding site sequence,it is proved that the specific GXGXG amino acid motif can improve the dynamic range of the biosensor.After the saturation mutation of the first G site and the second and fourth X sites,the dynamic range of the best mutant of the biosensor reached 6.6 times,which was 34.7%higher than that of the previous round of optimization and 388%higher than that before optimization.