Study On The Omics Analysis And Application Of Bacillus Licheniformis BM 2709

B.licheniformis BM 2709 is an important industrial strain with high production of alkaline protease.The yield of B.licheniformis BM 2709 is up to 12000 U/mL±197 U/mL.However,the strain had the disadvantage of short production cycle of alkaline proteases,that is,the fermentation yield decreased greatly at the late stage of enzyme production.In order to modify the strain genetically,prolong the production cycle of protease and increase the yield of protease,B.licheniformis BM 2709 was studied by histology and application.First,the whole genome sequence of B.licheniformis BM 2709,an important industrial strain with high alkaline protease production,was sequenced,and the genomic sequence of BM 2709 with a content of 45.85%of 4,341,076 bp,GC was obtained.5166 coding genes,tRNA81 and rRNA80 were predicted.The genomic replication initiation site OriC of BM 2709 was predicted by Ori-Finder and GC-skew.It provides a clear genetic background for the transformation and optimization of the strain.Secondly,based on the measured BM 2709 genome,the whole genome transcription of BM 2709 in three stages(12 h,48 h,60 h)was analyzed by high throughput RNA-Seq sequencing technique.According to the analysis of FPKM value of bioinformatics data,267 and 173 genes were down-regulated in 48 h and 12 h respectively,and 182 and 16 genes were down-regulated in 60 h and 48 h,respectively.From these differentially expressed genes,24 candidate gene promoters were cloned and the gene expression regulatory elements(inducible promoters)were excavated.The shuttle vector pWH 1520,was used to construct the recombinant expression vector using alk as reporter gene and different promoter regions as expression regulatory elements.The recombinant expression vector was transformed into B.licheniformis ?2709-BM 2709 by electroporation.Empty vector alk-pWH1520 was used as control.Two new inducible promoters p707 and p1004 were obtained successfully.The results of fermentation test showed that the promoter p707 and p 1004 were 8.3 times stronger than the control.It can be seen that histology analysis is a powerful tool in biology such as genetic transformation and component mining.Finally,in order to study the effect of foreign gene location and integration direction on gene expression in BM 2709,we constructed a kan gene expression cassette(p43Kan),which is controlled by promoter p43,and inserted it into several sites at different distances from genomic OriC.Using shuttle plasmid pWH1520 as a vector,all regulatory elements are constructed on the vector.The regulatory elements include a kan gene controlled by a p43 promoter,a screening marker gene upp,controlled by a pS promoter,and an upstream and downstream homologue arm sequence.The electrotransformation was transformed into B.licheniformi ?upp-BM 2709 and integrated replacement by homologous recombination,which may be the result of breaking the structure of some operon and so on.Finally,only two recombinant genetically engineered strains in the same position and different directions were obtained.The results of antibiotic inhibition test show that the expression of this gene in different directions has no obvious effect.However,more experiments are needed to prove whether there is a general rule or not.