Transcriptome Of Bacillus Amyloliquefaciens And Excavations And Application Of Novel Expression Elements

Bacillus amyloliquefaciens is an important industrial microbe for the production of many industrial enzymes such as?-amylase,levansucrase,and fibrinolytic enzymes and primary metabolites,like purine nucleosides and riboflavin.Although the complete genome sequence of B.amyloliquefaciens has been now published,the transcriptome of B.amyloliquefaciens has not been deeply studied.In this study,high-throughput RNA sequencing?RNA-seq?technology was applied to dissect the transcriptome of B.amyloliquefaciens strain XH7.The B.amyloliquefaciens genome-wide transcriptome map was depicted.The strain was grown in LB rich media at the late exponential growth phase was isolated for RNA sample preparation.The results showed that 3936 of the total of 4204 B.amyloliquefaciens genes?93.6%?were transcribed.Transcriptional start sites?TSS?of 1064 annotated genes and 749 operons were identified.Base on transcriptome and genomic information,the presented results indicated that intermediate IMP is converted to guanosine synthesis in the de novo purine nucleotides synthesis.This feature could make B.amyloliquefaciens XH7 accumulate guanosine.The results indicated that overexpression of purine operon elevate further the production of guanosine in B.amyloliquefaciens XH7.Based on the RN A-Seq data,we excavate the expression elements of B.amyloliquefaciens with high-throughput.Compared with the marker gene P43-bgaB which was inserted into B.amyloliquefaciens,288 genes demonstrated high expression based on the RPKM value.To screen for strong promoters,a beta-galactoside reporter was fused to eight candidate promoters from 288 genes with the highest expression levels?RPKM values?.The expression plasmids were constructed.The results illustrated that the candidate promoter P_r2?promoter for the sig W gene?displayed the strongest beta-galactosidase specific activity?5300 M u/mL?during the post-log phase.Using the promoter P_r2,we successfully expressed the xyn?encoding xylanase?gene.Using bga B gene?encoding heat-stable?-galatosidase?as reporter,a promoter probe plasmid p BE-bgaB was constructed.Using the vector,265 colonies containing likely promoters from B.amyloliquefaciens genomic DNA were obtained.Among them,the promoter P41 exhibited the strongest?-Gal activity?3017 Miller units?in B.amyloliquefaciens.The RBS?382#?of P41 was designed using the RBS Calculator on the web.The maximal?-Gal activity of plasmid pBEP41-bgaB?382?reached 6260 Miller units.Reconstructing the hybrid promoter improved the?-Gal production by 1.9 folds.It provides a new stronger promoter for the biotechnological application of Bacillus spp..A series of purine operon promoter mutants were constructed.The primary promoter of purine operon was truncated to relieve transcriptional attenuation.The primary promoter of purine operon was successfully substituted with the strong promoter P_r2 and P41 to remove the transcriptional repression.The results of shaking flask fermentation showed that the guanosine production yield of purE::P41 reached 16.25 g/L and enhanced 22.5%than WT.While pur E::P_r2 caused a significant decrease in guanosine production?65.78%than WT?and growth rate.The concentration of intracellular substances in fermentation broth was measured.The IMP concentration of purE::P41 was higher than WT.RN A samples of engineering strains on a time scale of the fermentation?12h,24h,46h,48h?were extracted.The transcriptional profile analysis showed that expression level of purine operon genes in purE::P41 were upregulated than WT.The overexpression of purine operon increased the guanosine biosynthesis.A series of?cyd engineering strains were constructed.The results of shaking flask fermentation showed that the guanosine production yield of?cyd engineering strains higher than WT.And the guanosine production yield of purE::P41?cyd reached 19 g/L and increased by 33.5%than WT.The concentration of organic acids in broth was measured.The results showed that the concentration of pyruvic acid and acetate in?cyd engineering strains were lower than WT.It seems that the fluxed in the EMP pathway are reduced and the fluxed in PP pathway are increased.And a low concentration of acetate was benefit for growth of?cyd engineering strains.The results showed that replacement of purine operon promoter and engineering of respiration improved the guanosine yild in B.amyloliquefaciens XH7.