Continuous Mutation Breeding Of High ε-poly-L-lysine-producing Streptomyces Albulus And Optimization Of Its Fermentation Conditions

ε-poly-L-lysine（ε-PL）is a natural polymer composed of 25～35 L-lysine monomers produced by Streptomyces sp.Because of its broad antimicrobial activities,ε-PL is widely used in food preservation industry.Japan is the first country to study and successfully achieve industrial production with an annual output of 1,000 tons.However,China starts its research late.It has not fully been the industrial production due to the low production and fermentation limitation the strain.Therefore,it is of great significance to studyε-PL-producing strains to increase itsε-PL production.In order to improve the fermentation level ofε-PL,the breeding of Streptomyces albulus producingε-PL and the fermentation process were explored in this paper.The main research contents of this paper are as follows:1.S.albulus was used as the original strain which was bred by atmospheric and room temperatureplasma（ARTP）mutagenesis,diethyl sulfate（DES）mutagenesis,nitroguanidine（NTG）mutagenesis and nitrite（HNO₂）mutagenesis.The effects of four kinds of mutagens were compared with the positive mutation rate and the highestε-PL yield.It was showed that DES mutagenesis and ARTP mutagenesis were better conditions.Theε-PL yield increased by 16.3%and 14.8%compared with the original strain,respectively.Six antibiotics five other substances were selected as"sieves"for mutant strains screening.S albulus was treated by ARTP mutagenesis combined with different"sieves".The positive mutation rate of streptomycin resistance（Str^r）mutants were the highest（25%）,and the maximumε-PL production（0.7 g/L）of the mutant strain was obtained,which was 17.5%higher than that of the original strain.2.ARTP-DES continuous mutagenesis was first employed on the basis of the best mutagenesis method with S.albulus as the starting strain.The best conditions of the ARTP mutagenesis system were as follow:110 W power,2 mm exposure distance and a mutagenesis time of 30 s,60 s,105 s;The concentration of DES was 0.6%and the reaction time was 20 min.And a rapid screening method was improved combined with streptomycin resistance,methylene blue transparent ring,48 microporous plate culture and rapidlyε-PL productivity by ELIASA.Three high yieldε-PL strains（S.albulus AD-1,S.albulus AD-7 and S.albulus AD-9）with excellent performance were finally obtained after several rounds of initial screening and rescreening.One strain designated S.albulus AD-9was selected with aε-PL yield of 1.25 g/L in shake-flask fermentation,which was 2.08times of the parent strain.3.The high-yielding mutant S.albulus AD-9 was studied by several fermentation media.Theε-PL yield of S.albulus AD-9 reached the maximum value of 2.1 g/L after 84 h of fermentation in the optimized culture medium,3.5 times that of the original strain.It was found that the nutritional requirements of S.albulus AD-9 and shape of mycelium pellets during fermentation all changed compared to the physiological characteristics（Shape of mycelium pellets during fermentation and parameters of natural fermentation）of S.albulus AD-9 with the original strain.The results indicated that the metabolism of S.albulus AD-9 and its ability to synthesizeε-PL were enhanced.4.The optimum pH of S.albulus AD-9 compositeε-PL was determined to be 4.0 in a1-L bioreactor.On this basis two-stage batch fermentation（pH3.5-4.0）was carried out in a5-L fermentation tank.After 54 h of fermentation,the biomass reached 18.56 g/L,and theε-PL production was 7.98 g/L,which was nearly 3 times higher than that of shaking flask fermentation.After fed-batch fermentation for 144 h,theε-PL production reached 26.56g/L and the biomass was 34.58 g/L,which increased by 232.8%and 86.3%,respectively,than those of batch fermentation.The pH two-stage fermentation process was adjusted to solve the problem of low synthesis rate ofε-PL at the later stage of fermentation.The three-stage（pH5.0-3.3-4.0）fed-batch fermentation technology was established.Theε-PL production reached 30.12 g/L after 168 h fed-batch fermentation,which was 13.4%higher than pH two-stage batch-feed fermentation.The unit synthesis amount ofε-PL reached0.963 g/g,25.4%higher than that of two-stage fermentation（0.768 g/g）.The morphology of mycelium pellets was observed during the fermentation,and it was found that the tighter the mycelium pellets wound,the higher the synthesis rate ofε-PL produced.The establishment of pH three-stage fermentation technology compensated for the deficiency of pH two-stage fermentation and laid a foundation for further optimization and expansion of culture in the later stage.