Study On Genome And Transcriptome Expression Of Bacillus Licheniformis DW2

With the rapid development of sequencing technology,the cost of sequencing has been declining.Through high-throughput sequencing technologies,it is possible to better study the mechanisms of synthesis regulation within microorganisms and to help find the causes of differences in phenotypic characteristics among microorganisms.It can also provide theoretical and data support for the transformation of some industrial strains.Bacillus licheniformis is a common industrial strain that can produce a variety of amino acid and peptide antibiotics,so it is widely used in agricultural production,biopharmaceutical and industrial manufacturing.The B.licheniformis DW2 strain in this study can synthesize and secrete a secondary metabolite bacitracin under normal conditions.Bacitracin is a polypeptide antibiotic synthetized by a non-ribosome synthetase and has activity of anti-Gram-positive and partially anti-Gram-negative bacteria by preventing cell wall synthesis.In addition,our previous studies have shown that knockout transcription factor AbrB can increase the production of bacitracin in B.licheniformis.Therefore,in this study,it is meaningful to understand its genome features,expression differences between wild and AbrB-knockout(?AbrB)strains,and the regulation of bacitracin biosynthesis,through the combination of genome and transcriptome.The main contents and results of this study are as follows: 1.Genome assembly and annotation of B.licheniformis DW2In this study,whole genome sequencing of Bacillus licheniformis DW2 was performed using PacBio SMRT sequencing technology.The original sequencing data was approximately 674 Mb,the sequencing data after filtration was approximately 500 Mb,and the genome sequencing depth was approximately 100×.The assembled genome was a circular chromosome of 4,468,952 bp containing 4,717 protein-coding genes,24 rRNA genes and 81 tRNA genes and the GC content was approximately 45.93%.In addition,according to PacBio sequencing data,the methylation modification of B.licheniformis DW2 was also analyzed and there were two 6 mA methylation motifs in the genome.The comparative genomic analysis of B.licheniformis DW2 of this study was performed with B.licheniformis DSM13,B.licheniformis WX-02 and B.licheniformis 9945 A.Genomewide linear analysis showed that the genomic alignment of B.licheniformis DW2 and B.licheniformis 9945 A was the best.At the same time,we found that there is a specific fragment of about 80 Kb in the 3.3Mb-3.4Mb genome of B.licheniformis DW2.According to the functional annotated results,this specific fragment may be related to the physiological mechanisms such as the high production peptide antibiotics of B.licheniformis DW2.2.Transcriptome analysis of B.licheniformis DW2In this study,straid-specific RNA-Seq technology was used to detect the three time points in the logarithmic phase(14 h),the transition phase(22 h),and the stationary phase(25 h)of the wild strain and the knockout strain of B.licheniformis DW2.A total of 42.94 Gb of raw sequencing data was obtained by ssRNA-Seq sequencing.Based on the transcriptome data,the differentially expressed genes of wild strains and knockout strains were identified by DEseq software.It was found that the total number of differentially expressed genes increased with the increase in culture time,whether it was a wild strain or a knockout strain.These founding indicated that more genes are activated or participate in more biological regulation during stationary phase.Through COG function classification and KEGG enrichment,the majority of differentially expressed genes were involved mainly in the metabolism and transport of amino acids,oxidative phosphorylation,carbon metabolism,and cell movement and other metabolic pathways.Based on antiSMASH,a complete bacitracin synthesis gene cluster was obtained in B.licheniformis DW2.By analyzing gene expression of clusters,it was found that the transporter gene bcrABC was significantly up-regulated in knockout strain.This not only supports the previous study that B.licheniformis transported bacitracin out of cells to mediate bacitracin resistance through transporter genes.And it was also consistent with the previous experimental result that ?AbrB could yield more bacitracin.