Study On Bioactive Metabolites Of Brevibacillus Brevis B011

Brevibacillus brevis B011 is an endophyte isolated from the stem of healthy tobacco in the bacterial wilt area.Previous studies found that it has an inhibitory effect on a variety of plant pathogenic fungi and bacteria,and has a good biological control effect.However,its antibacterial bioactive metabolites and their biosynthesis gene clusters(BGCs)have not been identified.In this paper,we use the bioinformatics analysis of secondary metabolites BGCs of B011 strain,Red-ET homologous recombination technology to knockout B011 secondary metabolites BGCs,and the antimicrobial spectrum of the mutant strains respectively.The main acting antibacterial bioactive metabolites of B011 synthesis and their BGCs were identified.The main study results are as follows:1.The BGCs of 12 secondary metabolites were mined from the B011 genome based on anti SMASH analysis.Among them,there are secondary metabolites such as edeine,tyrocidine,gramicidin,etc.which have been shown to have good antibacterial activity and application prospects;2.The B011 gene knockout system was constructed and multiple mutants of the secondary metabolites gene clusters of knockout metabolites were obtained.A reasonable and feasible gene knockout scheme was designed: recombinant temperature-sensitive plasmid p BR322 was constructed by using Red/ET homologous recombination technology,and B011 electric shock conversion system was established by optimizing the shock conversion conditions.Single and double knockout of BGCs of the secondary metabolites edeine,tyrocidine and gramicidin.Three single knockout mutant strains of B011Δgra,B011Δede and B011Δtyr,and two double knockout deletion mutant strains of B011Δede::gra and B011Δede::tyr were obtained,successful knockout was verified by PCR and sequencing;3.The five mutant strains were tested with the pathogen,and we found that edeine and tyrocidine were the main antibacterial bioactive metabolites synthesized by biocontrol strain B011.B011 wild-type strain,B011Δgra,B011Δede,B011Δtyr and B011Δede::gra mutant strain had certain antibacterial activity on a variety of pathogenic fungi,and B011Δede::tyr mutant strain lost its inhibitory effect on all tested fungi;Through pepper potted test,found that B011 wild-type strain had 50%efficacy against pepper blight,and B011Δede mutant strain showed 9.38%efficacy,The biological efficacy of the B011Δede mutant strain was decreased by 40.62%compared to that of the B011 wild-type strain,indicating that edeine plays the main role in the control of pepper blight,and tyrocidine also has the effect of inhibiting Phytophthora capsici;4.The cultures of B011Δede,B011Δede::gra and B011Δede::tyr mutant strains were collected by centrifugation,After methanol extraction and HPLC analysis,B011Δede::tyr mutant had four retention peaks of 16.640 min,18.520 min,20.362 min and 22.604 min respectively;The HPLC-MS/MS analysis found that,The secondary MS ion peak sizes of the three compounds were 1275.6919,1259.6992,1236.6770,respectively,their molecular weight is not completely consistent with the reported molecular weight and ion fragmentation characteristics of tyrocidine family compounds,indicating that B011 may have synthesized new derivatives of tyrocidine;The molecular structure of the tyrocidine A derivative of size 1235.6 was predicted by bioinformatics prediction and ion fragment information analysis.