Study On The Expression Feasibility Of Self-inducible Promoter P_srfa In Escherichia Coli And Lactic Acid Bacteria

Lactic acid bacteria expression system,prominent for the food-grade host bacteria,has a long history of being used in food industry Escherichia coli is widely used as genetically engineered host bacteria owing to its clear genetic background and simple genetic operation.Using promoter PsrfA,a self-inducible Bacillus subtilis expression system was constructed which was efficient in expressing a variety of heterologous proteins.Therefore,in order to broad the host range of promoter PsrfA,lactic acid bacteria and E.coli expression systems were developed with promoter PsrfA.The feasibility of target proteins and host strains and the factors affecting the promoter expression activity were studied.The main results were as follows(1)Construction of E.coli expression system.Using green fluorescent protein(GFP)and ?-galactosidase as the reporter proteins,expression vectors were constructed by inserting the genes of GFP and ?-galactosidase downstream of the promoter PsrfA,respectively.And then the recombinant vectors were separately transferred into E.coli JM109 and E.coli BL21 to detect the expression of the reporter proteins.By the analysis of laser scanning confocal microscopy,fluorescence intensity detection,and SDS-PAGE,protein GFP could be expressed under the controlled of the promoter PsrfA in E.coli JM109 and E.coli BL21.Moreover,after mutating the core region-10(TAAACT)of the promoter PsrfA to a conserved sequence(TATAAT),the expression of GFP in recombinant E.coli JM109 and E.coli BL21 increased by 5.6 times and 2.8 times,respectively.The fluorescence intensity and corresponding protein bands could be detected in the fermentation supernatants of the four recombinant bacteria.The highest expression level of fluorescent protein GFP detected in the fermentation supernatant of E.coli BL21-02 was 24449.91.For recombinant bacteria containing ?-galactosidase as the reporter protein,the promoter PsrfA can express ?-galactosidase in E.coli JM109 and E.coli BL21 by detecting ?-galactosidase activity and SDS-PAGE analysis;Also,after mutating the core-10 region of the promoter PsrfA to a conserved sequence,?-galactosidase activity was increased by 1.2 times and 1.3 times in E.coli JM109 and E.coli BL21,respectively(2)Study on the expression characteristics of promoter PsrfA in lactic acid bacteria.The expression vector containing PsrfA-gfp was transferred into Lactobacillus casei 5257,L plantarum 97,L.fermentum 087,and Weissella confuse 10,respectively,and the factors effecting the promoter activity were studied including the carbon and nitrogen sources of the medium,the initial pH of the medium and the pH of the fermentation broth during fermentation.The results showed that the promoter PsrfA can express the fluorescent protein GFP in L.casei 5257,L.plantarum 97,L.fermentum 087 and W.confusa 10.Carbon and nitrogen sources not only affected the growth of the bacterial cells but also the expression activity of the promoter PsrfA.The promoter PsrfA performed the highest expression of the fluorescent protein GFP in the medium containing maltose as the solo carbon source using L plantarum 97 as the host strain.The initial pH of the medium showed slight effect on the activity of promoter PsrfA.Moreover,the expression activity of promoter PsrfA in L.plantarum 97 and L.casei 5257 was enhanced by maintaining the pH of the medium above 4.5 during fermentation.(3)Application of lactic acid bacteria expression system.(a)Using promoter PsrfA to express NADH oxidase in L.plantarum 97 and L.casei 5257 to regulate the fermentation pH.The NADH oxidase activity detected in the recombinant bacteria and the pH of the fermentation broth were higher than those of the wild bacteria.In addition,the core conserved regions-35,-10,the ribosome binding site,and the binding sites of inhibitor Cod Y of the promoter PsrfA were mutated.The results showed that the expression of NADH oxidase could be increased with the corresponding mutants of the promoter PsrfA,thereby increasing the pH of the fermentation broth.By simultaneously changing the core regions-35 and-10 of the promoter PsrfA the activity of NADH oxidase expressed by L.plantarum 97-09 was 42.65 U/mg and the pH of the fermentation broth was increased by 0.32 compared with that of the control L Plantarum 97.the activity of NADH oxidase detected in L.casei 5257-09 was 18.73 U/mg,and the pH of the fermentation broth was increased by 0.20 compared with that of the control L.casei 5257.(b)Using promoter PsrfA to express laccase CotA in L.plantarum 97 and L casei 5257 which was derived from B.subtilis.The results showed that the recombinant strains L.plantarum 97-06 and L.casei 5257-06 containing PsrfA-cotA were able to express laccase CotA and the enzyme activity detected in L.plantarum 97-06 was higher than that of L.casei 5257-06.Adjusting the pH of the fermentation broth during the cultivation,the laccase activity expressed by L.plantarum 97-06 and L.casei 5257-06 could be increased by 2.8 times and 1.6 times,respectively.