Study On The Development Of Host-vector System In Bacillus Megaterium

There is a Gram-positive bacterium that is endotoxin-free and can grow from a variety of carbon sources and secrete proteins to the extracellular in Bacillus spp.It is a production strain of many important industrial enzymes.In recent years,it has become an excellent expression host for the expression and secretion of heterologous genes in prokaryotic expression systems in Bacillus spp.With the rapid development of molecular biology and genetic engineering,Bacillus spp,has shown good application prospects.At present,Bacillus subtilis is the main prokaryotic expression system,but it is limited by factors such as self-secreting protease and unstable construction of recombinant plasmid.Bacillus megaterium can overcome this problem,then it doesn't have extracellular alkaline protease,which is conducive to the stable accumulation of the expressed protein;the recombinant plasmid is stable.Therefore,this study is to establish and optimize B.megaterium expression system,the ability to select one individual plant protein secretion from habitat and low extracellular protease activity of B.megaterium,transformation,and the wild type strains further genetic operators to improve the expression of heterologous proteins,and to establish a highly efficient expression system of bacillus spp.In this study,a strain of wild-type B.megaterium strains with weak extracellular neutral protease was screened from the natural environment to establish a heterologous protein expression system.The specific research work is mainly divided into the following points:1.Screening a strain of excellent wild-type neutral protease with weak extracellular neutral protease.In this study,more than 60 samples were sampled from different environments such as Jiangsu and Shandong,and then firstly screened to the B.megaterium by PCR using the primers specific to the B.megaterium Spo0A gene;then the extracellular medium was obtained by rescreening the skim milk plate.The strain with weak proteases;finally,the material of the study was determined according to the growth of the strain and the antibiotic sensitivity test:B.megaterium H2.2.Determination and knockout of extracellular neutral protease gene.The extracellular neutral protease gene nprM(GO:0005576,ORF:02376₁)was predicted by sequencing the whole genome of strain H2,similar to the reported neutral protease nprM gene associated with B.megaterium QM B1551.The extracellular protease gene was knocked out by a no-knockout technique.It was further confirmed by the skimmed milk plate that the nprM gene was the extracellular protease gene of the strain H2,and the growth of the knockout strain H2N was not affected.3.Establishment and optimization of electroporation method for B.megaterium H2.Protoplast transformation is often used in the reported B.megaterium transformation method,but the method is cumbersome and difficult to transform.In this study,sorbitol and mannitol were added to the electroporation medium as a high-permeability stabilizer,and glycine was added to weaken the cell wall and improve the electrotransformation efficiency The results showed that the electroporation efficiency of B.megaterium was increased to 1.2×103 CFU/?g DNA,and the recombinant plasmid was successftully constructed into B.megaterium.4.Expression of heterologous protein MPH in B.megaterium H2.Based on the previous research in this laboratory,the mpd gene was used as the reporter gene,and the expression vector p19XM was constructed using the xylose promoter PxyA and the repressor gene xylR in B.subtilis,and transformed into B.megaterium H2 and H2N.The results showed that both strains successfully expressed the heterologous gene mpd.Moreover,the strain that knocked out the extracellular neutral protease finally achieved an extracellular enzyme activity of MPH of 25.8 U/mL,which was 60%higher than that of the wild type strain.In summary,the excellent host B.megaterium H2 screened in this study can express the heterologous gene mpd.Moreover,by sequencing the whole genome of the strain H2,the extracellular neutral protease gene nprM was predicted,and the knockout strain was obtained by the no-knockout technique.In genetic manipulation,the medium and electroporation conditions prepared by electroporation of competent cells were optimized,and the recombinant plasmid was successfully transferred to B.megaterium.The final knockout strain H2N can also express the heterologous gene mpd,and its extracellular protein yield is higher than that of the wild type strain.This study preliminarily established the expression system of B.megaterium,which provided a new method and approach for the establishment of the transformation system of wild type B.megaterium and the established new expression system in Bacillus spp.