Genome Mining And Isolation Of Antibacterial Substance From Bacillus Subtilis BSD-2

Bacillus subtilis is an important microorganism of resisting to disease and promoting growth.And its resistance mechanism is mainly through the secretion of various antimicrobial substances to complete,lantibiotic is one of the important antipeptides.B.subtilis BSD-2,with plant pathogenic fungi of Botrytis cinerea and fusarium wilt antimicrobial activity,was isolated and screened in precedent experiments.To mine the potential biosynthesis gene clusters of novel antipeptides,B.subtilis BSD-2 was used.The structure and function of the potential synthetases were predicted and analyzed,the function of the synthetase gene was verified.Furthermore,the antipeptides of B.subtilis BSD-2 were isolated and purified.1.The complete biosynthesis gene clusters of subtilosin A,surfactin,bacilysin and tasA and theirs synthetic pathways were mined in B.subtilis BSD-2 via the software of antiSMASH,BAGEL 3 and local Blast.Furthermore,the partial biosynthesis gene clusters of fengycin,bacillaene and bacillibactin and theirs synthetic pathway were mined too.The six potential biosynthesis gene clusters of Class IV Lanthipeptide were mined in B.subtilis BSD-2 firstly.Theirs biosynthesis gene clusters and synthetic pathway was complete.2.The structures of potential Class IV Lanthipeptide synthetases from B.subtilis BSD-2 were predicted and analyzed using the software of ProtParam,KinasePhos,and VMD.The potential Class IV Lanthipeptide synthetases from B.subtilis BSD-2 and other seven studied proteins had the similar physical and chemical properties that they were hydrophilicity,and most proteins were acidic and stable.Alpha helix and coil were the basic secondary structure.The kinasephos of serine kinase and threonine kinase were contained.The evolutionary relationships of LANC-like domains were similar to total synthetases,which was different from STYKc/S_TKc domains.The Class IV Lanthipeptide synthetase was found in B.subtilis BSD-2 via tertiary structure analysis and genome mining.3.The two T4 LSL genes(Type IV Lanthipeptide synthetase like gene)named LanLA and LanLB were amplificated used PCR.The cloning vector and knock-out vector of LanLA/LanLB were constructed.Kana resistance gene was inserted to LanLA and LanLB respectively.And pB-LanLA-Kana and pB-LanLB-Kana recombinant plasmids were constructed.4.Antimicrobial substance which inhibited the growth of Botrytis cinerea was isolated via the methods of ammonium sulfate precipitation,weak anion chromatography and HPLC.A 3.0kDa protein band appeared after SDS-PAGE electrophoresis,and the band inhibited the growth of Botrytis cinerea which was inspected at original position.The results indicated that there were antimicrobial peptides with the molecular weight of about 3.0 kDa in BSD-2 metabolite.