Breeding Of A New Strain Of High-yield Antibacterial Lipopeptide By Bacillus Interspecies Protoplast Fusion

Lipopeptides are secondary metabolites synthesized by non-ribosomal pathways in Bacillus,mainly including Surfactin,Itutin and Fengycin.Lipopeptides have excellent physical and chemical activities such as high-efficiency bacteriostasis,anti-tumor and anti-virus,good stability,good biocompatibility,and easy to be naturally degraded.Therefore,they are widely used in agric?lture,medicine,food,animal husbandry,environmental protection and many other fields,Both have broad application prospects.However,the ability of wild-type strains to produce lipopeptides is very low.In order to solve the problem of low yield of lipopeptides in wild-type strains,this study performed protoplast fusion and bred high-producing lipopeptide strains on two lipopeptide-producing Bacillus strains preserved in the laboratory.The optimization of the fermentation system to improve the production of lipopeptides,Main results were as follows:(1)The 16S rDNA assay was performed on the two parental strains,and the two parental strains were identified as Bacillus natto and Bacillus Siamese.The growth curves of the two parental strains were drawn,and the c?lture time of the two parental strains was determined when the protoplasts were prepared.Bacillus natto was c?ltured for 6 hours,and Bacillus Siamese cultured for 5 hours.(2)The preparation conditions of protoplasts were studied,and SMM was used as a hypertonic solution to facilitate the formation of protoplasts.The optimal conditions for the formation of protoplasts of Bacillus natto were as follows:2%glycine was added during the c?ltivation of Bacillus natto,the enzymatic hydrolysis concentration was 0.25 mg/m L,the enzymatic hydrolysis time was 25 min,and the protoplast formation was carried out at38?.The formation rate was 85.3%and the regeneration rate was 34.8%.The optimal conditions for the formation of protoplasts of Bacillus Siamese were:1%glycine was added during the c?ltivation of Bacillus Siamese,the enzymatic hydrolysis concentration was 0.2mg/m L,the enzymatic hydrolysis time was 2 min,and the protoplast formation was carried out at 38?.The formation rate was 86.2%and the regeneration rate was 35.1%.(3)The regeneration of protoplasts was studied.The medium was determined to be:peptone 1%,yeast powder 0.5%,beef extract 0.5%,Na Cl 0.5%,0.3 mol/L mannitol,0.02mol/L Mg Cl₂·6H₂O,0.02 mol/L maleic acid,p H 7.0-7.2,and 1%bovine serum albumin was added after sterilization.The fusion conditions of protoplasts were:50% PEG6000,pH 9.0,38? water bath for 15 min,the fusion rate reached 1.73×10^-5,and the fusion of the potential fusions was identified by PCR using the Sp and It differential fragments of the two parental strains.(4)The fermentation medium of the two parental strains was screened,and the Landy medium was finally determined as the fermentation c?lture.The antibacterial ability of the fermentation broth of the two parent strains to 6 pathogenic bacteria was investigated.It was found that the bacteriostatic ability of Bacillus natto fermentation broth was stronger than that of Bacillus Siamese,and P.aeruginosa was finally identified as indicator bacteria.(5)A total of 215 fusion strains were screened.Finally,the preliminary strain and HPLC rescreening by CPC-BTB method confirmed that the ability of the fusion strain F8 to produce Surfactin was 33.3%higher than that of Bacillus natto,and 60%higher than that of Bacillus Siamese.and scanning by electron microscopy revealed that the morphology of the fusion strain F8 was lengthened.The fermentation system was optimized for the fusion strain F8.The optimal fermentation carbon source was fructose,the nitrogen source was ammonium nitrate,the Mn²⁺concentration was 0.01 mmol/L,the Fe²⁺concentration was1.0 mmol/L,and the fermentation time was 36 h.The initial p H 7.0,the inoc?lum amount was 3%,and the fermentation temperature was 30?,and Surfactin production increased by47%compared to pre-optimization.