Mutation Breeding And Fermentation Conditions Of L-methionine Producing Strain

In this study,AS1.299 was used as the starting strain,and the mutants with high L-methionine production were obtained by three rounds of alternate mutagenesis with ultraviolet light and nitrosoguanidine?NTG?;the nutritional conditions of the mutants were optimized by single factor and orthogonal design methods;and the single factor and response surface methodology were used to design the mutants.The fermentation conditions of mutant strains were optimized.First of all,ultraviolet and nitrosoguanidine?NTG?were alternatively mutated to treat AS1.299strain.L-methionine production was used as the index to study the optimal concentration of UV mutagenesis and UV irradiation time,NTG mutagenesis best concentration and mutagenesis time,and the yield of L-methionine was used as the index to test and verify the genetic stability of 5 passages.Secondly,single factor and orthogonal design were used to optimize the nutritional conditions of the mutant and determine the most suitable carbon and nitrogen sources.The optimum concentration of glucose,urea,MgSO₄.7H₂O and biotin was obtained by using L-methionine content as index.The optimum nutrient conditions for L-methionine fermentation were obtained by orthogonal design on the basis of single factor experiment.Finally,single factor and response surface design methods were used to optimize the fermentation conditions of mutant strains.On the basis of optimizing nutrient conditions and taking L-methionine content as an index,the optimum pH,12 h seed age,inoculation,liquid loading,fermentation temperature and fermentation time were obtained by single factor experiment.On the basis of single factor experiment,three significant factors were selected for response surface methodology design to determine the fermentation conditions for L-methionine production by Corynebacterium.The following conclusions are drawn:1.The L-methionine-producing strain,AS1.299,was treated by UV and NTG alternately.The highest L-methionine yield of the mutant N3-11 was 1.02±0.05 g/L,which was 0.59 g/L higher than that of the original strain.2.The optimum nutritional condition of mutant is glucose as the most suitable carbon source and urea as the most suitable nitrogen source.The optimum nutrient combination was 8%glucose,0.6%urea,0.07%MgSO₄.7H₂O,and 2.326 g/L L-methionine when biotin was 5 ug/100ml.3.Under the optimum nutrient conditions,the fermentation conditions of L-methionine production were as follows:pH value was 6.95,seed age was 12 hours,inoculation was 8%,liquid loading was 20%,fermentation temperature was 32.38 C and fermentation time was 74.41 H.