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Breeding ε-poly-L-lysine High-producing Strain Through Combined Mutation Strategy

Posted on:2019-10-09Degree:MasterType:Thesis
Country:ChinaCandidate:J J ZhaoFull Text:PDF
GTID:2371330548982882Subject:Fermentation engineering
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ε-poly-L-lysine is multiple lysine polymers,and mainly used as food preservative.The important characteristics are mainly reflected in three aspects:thermal stability、degradability and antimicrobial effect.Many countries have already recognized the use ofε-poly-L-lysine,such as Japan、America and China.The purpose of this study was to increase the yield ofε-PL producing strain named Streptomyces albulus M-Z18.Using the combined breeding strategy,aε-PL mutant S.albulus RA-G16 was obtained.The research contents mainly include three aspects:(1)Breedingε-PL hyper-yield strains by ribosome engineering technique.The commonly used antibiotics include streptomycin,geneticin,gentamycin,rifampin,paromomycin,lincomycin,thiostrepton and fusidic acid.Different kinds of microorganisms,the tolerance to antibiotics exist difference.First,the effective antibiotics were screened to find out the antibiotics with inhibitory effect on M-Z18.The results showed that the other six antibiotics except thiostrepton and fusidic acid could obviously inhibit the growth of M-Z18;then determined the minimum inhibitory concentration(MIC).The screening concentration was determined and the mutant library was constructed.Multiple hyper-yield strains were obtained.The genetic stability of the mutant was investigated through multiple subcultures.After five times subcultures,only strains resistant to streptomycin、geneticin and paromomycin were still maintained good sporulating capacity and fermentation performance.They were S.albulus R-S6,S.albulus R-G8 and S.albulus R-P9,respectively,as the starting strain for the next strain transformation.(2)Breedingε-PL hyper-yield strains by atmospheric pressure room temperature plasma mutagenesis technique.First,the lethal rate of ARTP was determined,and choosed the treatment time 6 min as the irradiation dose to start breeding experiment.However,the screening results were not satisfactory.Only the production of S.albulus R-P9 increased,but the increase was not very high.Two mutants obtained were S.albulus RA-1(2.23 g L-1)and S.albulus RA-14(2.28 g L-1).(3)Breedingε-PL high-producing strains by genome shuffling.The fermentation performance and unit cell synthesis ability of the mutant S.albulus RA-1 and S.albulus RA-14 were examined.The results showed that the traits were stable and superior to the original strains.The two strains were used as starting strain of the genome shuffling.This study optimized the conditions for protoplast preparation,then determined the inactivation time of protoplasts and the conditions for fusion regeneration.The protoplast fusion experiment was conducted under optimal conditions.Finally,a high-producing fusion S.albulus RA-G16(2.76 g L-1)was screened,and theε-PL yield was compared with that of the original strain M-Z18 increased 72.50%.
Keywords/Search Tags:ε-poly-L-lysine, ribosome engineering, atmospheric pressure room temperature plasma mutagenesis, genome shuffling
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