Study On Efficient Transformation Of Uridine By Escherichia Coli

Uracil is an important active substance involved in RNA synthesis in organisms,encoding protein sequence information required by biology,and is also an important intermediate of fine chemical pesticides and medicine.Uracil has a wide range of applications.At present,uracil is mainly synthesized by chemical methods.Strong alkali and organic solvent are used in the synthesis process,which produces wastewater difficult to treat and causes certain harm to the environment.Therefore,this paper studies the conversion of substrate uridine into uracil through biological methods to reduce the harm to the environment by chemical methods.In this study,Escherichia coli BL21(DE3)was used as the expression host of Pyrimidine-specific ribonucleoside hydrolase RihA(RihA),and the substrate uridine was converted to uracil by whole cell biocatalysis.It is expected that the soluble expression of RihA can be improved by optimizing the signal peptide and its co-expression with molecular chaperone and knocking out the carboxypeptidase gene related to cell wall synthesis,thus increasing the conversion rate of uridine.The main research contents and results are as follows:1.The plasmid p ET22b-RihA was constructed and recombinantly expressed in Escherichia coli BL21(DE3).The results showed that the extracellular enzyme activity was 0.77 U/m L and the intracellular enzyme activity was 1.48 U/m L,indicating that most soluble enzyme proteins existed in the intracellular.SDS-PAGE protein electrophoresis showed that a large number of inclusion bodies were produced.2.To optimize the signal peptide,Fhud,Omp A,Omp C,Omp F,Omp T,Pho A and Pho E signal peptides were used to replace Pel B signal peptides in the original p ET22b-RihA plasmid.The transformation of uridine was determined by substrate fermentation.When the initial uridine content added in the fermentation broth was about 55g/L,the original strain A with Pel B signal peptide and strain B with Pho A signal peptide were almost all transformed all uridine into uracil after 10 h of substrate application,while in the fermentation broth of the remaining strains contained there were residual uridines.Therefore,the strains containing these two signal peptides were selected for further research.3.The molecular chaperone plasmid was constructed using p CDM4 as the skeleton,and the optimized signal peptide plasmid was co-expressed with the molecular chaperone plasmid in E.coli BL21.The results of substrate fermentation showed that strain F had the best conversion effect on uridine,which was co-expressed by Pho A signal peptide derived from alkaline phosphatase and molecular chaperone Gro ES-Gro EL.When the initial uridine content in fermentation broth was about 65 g/L,the uridine conversion rate was98.9% after substrate inoculation for 15 h.The uridine conversion rate of the original strain A was 80.2%.The results of enzyme activity assay showed that the highest extracellular RihA activity was found in strain C co-expressed by Pel B signal peptide and molecular chaperone Gro ES-Gro EL,and its RihA activity was 7.70 U/m L,10.0 times that of original strain A.Strain G,which co-expressed Pho A signal peptide and molecular chaperone Dna K-Dna J-Grp E,had the highest total soluble RihA activity,and its RihA activity was9.99 U/m L,4.45 times that of original strain A.SDS-PAGE showed that the target bands of broken precipitates(inclusion bodies)in strain C,D,E,F and H were significantly smaller than those in strain A,while there were obvious target bands in the supernatants of fermentation.These results suggest that chaperone can increase the soluble expression of RihA enzyme and decrease the production of inactive inclusion bodies.4.Dac A,dac B and dac C in E.coli BL21(DE3)were respectively knocked out by Red recombinant technology to obtain three single knockout strains and one dac B/dac C double knockout strain.The plasmid combinations with the best uridine transformation in Step 3were transformed into these strains.Substrate fermentation results showed that these knockout strains had no significant change in the transformation rate of uridine compared with strain F,so strain F was selected for further research.5.To study the effects of different substrate concentrations on uridine transformation.From an economic perspective,the best feeding concentration was the substrate twice the volume of input and fermentation broth: substrate transformation time was about 53 h,the yield of uracil was 73.45 g/L,and the uracil productive rate was 98.16%.In this study,whole cell biocatalysis was used to convert uridine into uracil using Escherichia coli as host.The production process is relatively simple and environmentally friendly,which lays a foundation for the preparation of uracil by biological transformation.