Effects Of Bacillus Amyloliquefaciens Type Ⅰ Signal Peptidase On The Secretion And Expression Of Signal Peptides And Proteins

Signal peptidase can cleave the signal peptide to promote the release of mature protein into the extracellular space.Type I signal peptidase mainly cleaves the signal peptides of the Sec pathway and Tat pathway,and the Sec and Tat secretion pathways are the main pathways of exogenous protein secretion in Bacillus.sp,so It is necessary to study the function of type I signal peptidase and its effect on the secretion of foreign proteins.In this project,the laboratory-preserved strain Bacillus amyloliquefaciens TCCC19030 was used as the starting strain,and the transcriptome data was compared with the NCBI database to determine the existence of four type I signal peptidases on the genome: Sip A,Sip S,Sip W and Sip V,and using molecular docking technology to conduct molecular simulation docking between four type I signal peptidases and 50 signal peptides on the genome,and analyze the interaction between signal peptidases and signal peptides.The average value of energies indicated that the order of binding ability was as follows: Sip A(-5.11)>Sip S(-3.12)>Sip W(-2.74)>Sip V(-0.43).In order to further evaluate the relationship between signal peptidase and signal peptide,three strains with signal peptidase deficiency were constructed in B.amyloliquefaciens:19030-ΔsipA,19030-Δsip S and 19030-Δsip W.Combined with the simulation docking results,8 different signal peptides were selected:Ydb K,Nuc B,Phr F,Yhd C,Yol C,Ywj E,Ypj P and Ykn X,24 recombinant strains were constructed,using alkaline protease as the reporter protein and the enzyme activity of extracellular enzymes as the evaluation index.Different signal peptidases and signal peptides work together in the secretion of foreign proteins.In order to explore the effect of type I signal peptidase on the secretion of different exogenous proteins,different recombinant strains were constructed using fusion protein,green fluorescent protein,amino peptidase and alkaline protease as reporter proteins respectively.The results showed that the deletion of signal peptidase genes sip S,sip W and sipA had no significant effect on the secretion of green fluorescent protein and fusion protein,but has a certain effect on the secretion of amino peptidase and alkaline protease.Among them,the lack of signal peptidase sip W gene reduces the enzyme vitality ratio of amino enzymes by 18.8%,the lack of signal peptidase sipA gene reduces the enzyme vitality ratio of amino enzymes by 21.2%,which indicating that signal peptidases have key effects on the secretion of different exogenous proteins are different.Since the deletion of the signal peptidase gene sipA will have a greater impact on the secretion of alkaline protease,this study integrated the gene into the position of the amy E gene in the genome of the starting strain TCCC 19030 in order to improve the enzymatic activity of alkaline protease.The results showed that the enzymatic activity of the integrated strain 19030-Δamy E::sipA was increased by about 19.9% compared with the original strain,which provided a reference for the construction of high-yielding foreign protein strains.