Breeding Of Strain Highly Producing Propionicin By Genome Shuffling

Propionicin are kind of polypeptides or proteins produced by dairy propionibacteria. They can restrain G-bacteria, some G+, fungi and yeast. In this study, genome shuffling was used to breed the producing strain to improve the propionicin production rate of fermentation to promote the development of the industry, increase the social and economic benefits. The traditional mutation breeding techniques and microbial protoplast fusion were successfully reorganized by Gene technology for combination to the reorganization of objects from a single gene to the entire genome, while many parent strains were excellent phenotype with a reorganization of the strains, which could be efficiently and quickly filter out the fermentation of mutant strains for positive good character, instead of the mutants that is difficult to improve the performance of the defects after a long tradition of mutagen glazing.This paper complex mutagenesis by physical and chemical Propionibacteriuin.feudenreichii CS01to UV mutagenesis and NTG mutagenesis get the starting mutant strains selected for genome restructuring in order to obtain high-yield strains of propionicin for propionicin industrial production has a value of strain.The main contents and results are as follows:(1)Propionibacterium.feudenreichii CS01as the research object, through the optimization of UV mutagenesis of single factor experiment time and the concentration of nitrosoguanidine mutagenesis by UV and nitrosoguanidine mutagenesis methods of two strains of the original mutation, the three UV and three NTG mutagenic compound, obtained two mutant strains N11and N42. The antimicrobial activity of N11and N42amount strains were28.63AU/mL and28.83AU/mL respectively,35.4%and36.4%times more than the original strain(21.14AU/mL). The strain N11and N42as breeding genome shuffling the starting propionicin yield strain.(2)Optimized by single factor experiments Propionibacterium.feudenreichii protoplasts conditions, test results showed that:the optimum age of bacteria2d; the best work of Lysozyme concentration20mg/mL, working concentration of Mutanolysin15μg/mL; best hydrolysis time2h; the best reaction temperature37℃.(3)Established the inactivated protoplasts of strains suitable conditions, test results showed that:the optimum heat-inactivated protoplasts time50min; UV inactivation of the optimum time5min; at37℃for PEG fusion.(4)UV and NTG mutagenesis from two mutant strains as parents for protoplast fusion, integration of children received regeneration coated plate screening, isolates were picked from the larger colonies on plates after subculture, the The screening of fermentation, to obtain a stable strain of6characters, the first round of restructuring of the strain F11, F12, F13, F14, F15, F16. F1generation of the6strains from the same strategy by culture, protoplast fusion, regeneration after regeneration screening, rescreening fermentation test, a second round of restructuring of the strain F21, F22, F23, F24; F2generation of the four fusion strains were screened to obtain strains F31, F32, F33, F34, F3again on behalf of four fusion strains were screened to obtain strains F41, F42. Including F41, F42propionicin higher yield of propionicin production were36.83AU/mL,36.77AU/mL, respectively compared with the original strain74.2%,73.9%; strain was passaged6times, pullulan yield stability, F41, F42is more stable traits good strains.(5)By determinated the G+Cmol%content and esterase isoenzyme electrophoresis for Propionibacterium.feudenreichii, F41, F42, identificated they are the different strains within the same kinds. Fusion strains produced enzyme higher than the Propionibacterium.feudenreichii at the19kD.