Study on the Efficient Constitutive Expression of Phytase YiAPPA in Entercoccus faecalis

The aim was to obtain a strong constitutive promoter to realize the efficient constitutive expression of phytase YiAPPA in Enterococcus faecalis EXW27. In this study,the promoters of 16S rRNA in Enterococcus faecalis were compared and analyzed,and the non-consensus region of the promoters were replaced by the randomized sequences,so as to obtain the random synthetic promoter sequences. Then the synthetic promoter library was constructed by using the enzyme cleavage site of Escherichia coli/Lactobacillus shuttle vector pSIP409,and the high-throughput screening technique of phytase activity was used to screen the strong constitutive promoter that drived YiAPPA expression in Enterococcus faecalis EXW27. And the enzymatic properties of recombinant YiAPPA were studied. The results showed that under the control of the constituent promoter p10,recombinant YiAPPA was successfully expressed in Enterococcus faecalis EXW27,and the production of recombinant YiAPPA corresponded to approximately 15% of the total cellular protein. The optimal reaction pH of YiAPPA was 4.5,and it exhibited excellent pH stability in the range of pH1.0~10.0,its specific activity at 55 ℃ was up to 3900 U/mg. In this study,the recombinant Enterococcus faecalis with both probiotic characteristics and phytase activity was constructed,which laid a foundation for the development of new transgenic microecologics.