Construction Of Host Strains In Bacillus Subtilis ATCC6051 And Study On Expression Elements,Protein Fold-related Functional Genes

Bacillus subtilis expression system is widely used in the production and expression of various foreign proteins,as well as the study of protein expression and secretion mechanisms;because of the characteristics of biosafety,clear genetic background,strong protein secretion.The aim of this study is to construct an unmarked expression host by using traceless knockout techniques and extracellular protein profiling,use the technique of histology and signal peptide libraries to screen and mine the best expression originals,analyze the folding-related genes in Sec pathway;and to establish a B.subtilis expression system with application potentials.In this paper,we obtained the non-resistant marker,no sporulation,few by-products express hosts of B.subtilis by using the temperature-sensitive plasmid pKS2 to knocked out the system-related genes seamlessly for B.subtilis ATCC 6051?including the eight protease-coding genes:nprE?nprB?aprE?mpr?bpf?epr?vpr?wprA,the sporogenic factor F encoding gene spollAC,surfactin synthase encoding gene srfAC?,and the corresponding study on B.subtilis 168.Then,the constructed strains were applied to expression of S.mobaraensis transglutaminase zymogen?proMTG?and G.kaustophilus heat-stable galactosidase?BgaB?.The ten-gene-deletion engineering strain B.subtilis ATCC6051?10was the best host and the extracellular secretion protein profile of this host was established.With analysis of the RNA-Seq datas from three strains of Bacillus spp.?B.subtilis 168,B.licheniformis ATCC14580,B.megaterium DSM319?,the promoter fragments of the top ten genes highly expressed in three Bacillus strains were screened,and 31 promoter candidates were obtained.Using BgaB as the reporter protein,a promoter screening plasmid was constructed.The results showed that the BgaB activity of two promoters(P_glvA and P_hag)were higher than that of the traditional strong promoter P43 in the late logarithmic growth phase.Three Bacillus expression genes were ranked by homolog gene family analysis?Orthomcl?and the RPKM values,the promoter fragments from the top ten common high-expressing genes,30 promoter candidates were obtained.The results showed that the BgaB activity of five promoters was higher than that of P43.The promoter activity of P_ydzA?BS168 strain?was up to 44.3 U/mL,which was 3.41 folds that of P43.Then seven strong promoters were used to recombinantly express proMTG and pullulanase?PUL?from B.naganoensis.The results showed that P_sodA?DSM319?expressed proMTG enzyme activity up to 82.6 U/mL,1.49 folds that of P43;and The P_spoVG?BS168?expressed PUL enzyme activity up to 328.4 U/mL,1.47folds that of P43.A high-throughput screening plasmid for signal peptides was constructed from 173 Sec signal peptide libraries.A total of 48 efficiently secreted signal peptides were obtained by screening the target protein BgaB,in which the secretory effect of signal peptide LytF?cell wall hydrolases?was extremely significant.Furthermore,the signal peptide library was used for signal peptide screening of proMTG and B.stearothermophilus maltogenic amylase?AmyM?.ProMTG obtained 30 effective signal peptides,AmyM obtained 27 effective secreted signal peptides;both of these two extracellular enzymes have the best secretion-promoting effect in signal peptide AprE.The results confirmed that different signal peptides have significant differences in the secretion efficiency of different proteins,and specific screening of target proteins is required.Recombinant expression ProMTG was constructed with the best expression host6051?10,the strongest promoter P_sodA and the best signal peptide S_aprE.The recombinant strain M5-1 was obtained and the highest enzyme activity was 126.55 U/mL in shaking flask culture for 48 h,and the enzyme activity of the fermentation after purification by affinity chromatography was 53.06 U/mg,which was 1.74 folds higher than that reported in the same period.M5-1 was fermented in 5 L fermentor and the highest enzyme activity was 855.54U/mL in 54 h,which was 6.76 folds that of the shake flask.The enzyme activity after purification by affinity chromatography was 171.98 U/mg,which was 3.24 folds that of the shake flask.The results showed that the expression system successfully achieved highly efficient recombinant expression of proMTG and had good potential for application.Using 6051?10 as the starting strain,four strains of engineered strains lacking folding-related genes?prsA,htrA,and htrB?and three strains of genetically engineered strains that integrate different PrsA proteins of Bacillus were constructed.The engineered bacteria with inactivated PrsA had the lowest enzymatic activity,only 12.5%of M5-1.The recombinant strains?integration and co-expression of PrsA protein?improved the secretory expression of proMTG in B.subtilis,which inactivated its own PrsA protein.Using 6051?10as the host,three recombinant strains of co-expressed PrsA?from three different Bacillus spp.?and proMTG were constructed.The results showed that overexpression of PrsA promoted the secretion of proMTG.The recombinant strain co-expressed with PrsA protein from B.megaterium showed the best secretion of proMTG.