Breeding Of A High-yield Strain From Bacillus Licheniformis For Bacitracin A

Antimicrobial peptides are small peptides which are widely distributed in nature.They play important role in innate immune system.Antimicrobial peptides have a unique mechanism with broad-spectrum and rare drug resistance.Some antimicrobial peptides have killing effect on tumor cells.Thus,antimicrobial peptides have high values in medicine,cultivation,food,and other industries.This study aims to obtain a strain with strong antibacterial activity of Bacillus licheniformis in nature,then isolate and purify its antibacterial peptides.Finally,using the genome shuffling technique,we hope to breed a new strain with enhanced production.The main results are as follows:1.Screening and identification of Bacillus with enhanced productivity:The strain Bacillus TJ12 was isolated from raw peach gum,which can produce antibacterial substances.Fermentation products have antibacterial activity on StapHylococcus aureus,Micrococcus luteus,Bacillus cereus and Fusarium moniliforme.Combined with colony morpHology,pHysiological and biochemical characterization,16S rRNA and gyrB sequence analysis,the strain was identified as Bacillus licheniformis.2.Isolation,purification and identification of the main antibacterial substance:The main active substance was separated and purified by ammonium sulfate precipitation,organic solvent extraction,SepHadex LH-20 and High pressure liquid chromatograpHy(HPLC).The main active substance has strong resistance to pH,temperature,and three kinds of protease(trypsin,proteinase K,pepsin).By electrospray ionization-mass spectrometry(ESI-MS)analysis,we preliminarily identified the main antibacterial component as bacitracin A.3.Breeding of high-yielded strains by genome shuffling:We used the genome shuffling technique to breed Bacillus licheniformis TJ12 to enhance bacitracin productivity.First,we used UV light,nitroso-guanidin(NTG),AtmospHeric,and Room Temperature Plasma(ARTP)to obtain a candidate mutant library.Based on the optimum conditions for protoplast preparation and regeneration,multi-parental whole genome shuffling was carried out with 4 mutant strains(U11?U23?H1?L71)by PEG mediated protoplasts fusion.The result shows that the shuffled strain F3 was identified by biparental inactivation method,and showed significantly improved antibacterial activity and good genetic stability.The maximum yield was as high as 760 mg/L,which was increased to 1.7 fold compared with the initial strain TJ12.Shuffled strain F3 got into the stable growth period beforehand,but the final cell growth was almost identical to the wild strain.There was little difference in pH variation during the fermentation process.Sugar and bacitracin production were higher than the wild strain.The synthesis and regulation of gene expression were higher than wild strain,wherein the synthetic gene comparatively increased more.With a higher productivity of bacitracin in the shuffled strain F3,we speculated that the shuffled strain had a more powerful self-tolerance mechanism for bacitracin and its relevant synthetic gene may have some modifications.