Genetics Breeding Of High Yielding Strain For Antimicrobial Peptide In Paenibacillus Polymyxa JSA-9

Paenibacillus polymyxa, which can produce a variety of antimicrobial substances, including peptides, proteins, nucleosides, pyrazine and phenolic, etc., has an effective activity against bacteria, fungi and virus. It can prevent various kinds of plant diseases. Paenibacillus polymyxa JSa-9, an antimicrobial peptides producing strain preserved in our laboratory, can yield lipopeptide antibacterial substance which natural safe, stable, and broad-spectrum antimicrobial. This antimicrobial substance has a huge potential application in the field of biological medicine, food, cosmetics and agriculture. However, lower yield of wild strains makes production and recycling costs increase, which results in its industrial production limited. Therefore, looking for the method to increase production, reduce cost of production and recycle costs is imperative. The purpose of this study is enhancing the production of antibacterial lipopeptide by antibiotics mutagenesis and protoplast fusion which can breed high-yield strains of the antimicrobial lipopeptide. The main content of this study includes the following aspects:1. In the study, single resistance selection and combinatory resistance selection by using antibiotics have been proceed.88high-yield strains were screened by antibiotics mutagenesis. Among88strains, the best combined mutagenesis was obtained by gentamycin which peak area increased by40%compared with control group during retention time of25-30min. By stability experiments, eight stable high-yield strains were obtained for protoplast fusion experiments.2. Proper incubating conditions of parent strains were determined, Paenibacillus polymyxa C-2-1-102and C-2-1-113were taken as example.Growing curve was drawn to determine the best culture condition:First, the strains activated in culture slant for24h, then transferred into the liquid culture broth to shake cultivate under the condition of30℃,180rpm for22h. At last,2.5mL bacterial fluid transferred to50mL cell collective broth and incubated for14-15h. Bacteria was collected and treated by lysozyme. In this experiment, optimum conditions of lysozyme were considered. The rate of protoplast formation and regeneration showed that parent strains should be treated by lysozyme at concentration of0.1mg/mL for7min at37℃.3Through both inactivated protoplast fusion, the best fusion condition was determined through fusion rate. The best fusion condition was:parent strains were inactivated by boiling water for90min and parent strains were inactivated by ultraviolet treatment for250min(30w,20cm). Both inactivated protoplast were fused by PEG6000at concentration of40%, pH9.0, water bath in37℃for15min. The fusion rate was up to3.05×10-4.4. After fusion,360strains of fusants were tested by fermentation productivity. Through which, two fusants were obtained. The bacteriostasis test showed that the lipopeptide productivity increased. HPLC analysis showed that the absorption peak area of the best fusant2-16was41.01%higher than Paenibacillus polymyxa JSa-9. The better fusants screened were transferred for10generations, and the lipopeptide productivity was certified to be stable through bacteriostasis test.