Screening High-yield Strains Producing Neomycin And Optimizing Its Fermentation Process

Neomycin is a widely used broad spectrum,water soluble aminoglycoside antibiotic,produced during the fermentation of Streptomyces fradiae.It inhibits the growth of some Gram-negative and Gram-positive bacteria.It has a narrow therapeutic range,is potentially toxic,like other aminoglycosides and may cause ototoxicity and nephrotoxicity.But as an ideal bactericide that interferes with protein synthesis,neomycin sulfate(the sulfate salts of neomycin)is widely used in clinic and veterinary medicine.In this study,in order to improve the screening efficiency of mutant strains of neomycin sulfate after mutagenesis,a high-throughput screening method based on well plates and microplate reader for the production of neomycin sulfate by Streptomyces fradiae was developed first.Then,the high-yield mutant Sf6-2 of Streptomyces fradiae was obtained by six rounds of ARTP mutagenesis and streptomycin resistance screening.Finally,the fermentation medium was optimized by response surface method to increase the fermentation production of the high-yield strain Sf6-2.The main research contents and results are as follows:(1)Development of a high-throughput screening method for the production of neomycin sulfate by Streptomyces fradiae.The addition(μL)of 1.0×10-4mol/L trypan blue solution which the main influencing factor of the method was optimized and the corresponding spectrophotometric method was established.Different well plate for high-throughput screening was studied and the fermentation conditions were optimized by orthogonal experiment.The results showed that the addition of 1.0×10-4 mol/L trypan blue was 500 μL and peak area of neomycin sulfate showed good linearity with concentrations in the range of 1.284～10.272 U/mL.The linear equation was Y=0.0320X-0.0262(R2=0.9988),the average recovery rate was 98.4684%,and the RSD of repeatability was 3.2700%.The 24-well plates suitable for high-throughput screening and a further optimization increased the potency of the mutant by 27.40%,i.e.6825±77 U/mL which occurred in the mutant cultivated in the optimal fermentation conditions composed of:220 r/min,2 mL and 8%for speed,medium volume and inoculum size.(2)Improved production of neomycin sulfate of Streptomyces fradiae by ARTP mutagenesis and screening.The distribution coefficient and inhibitory effect of different potency of mutant strains in different concentration of streptomycin plates were compared to determine the optimum resistance concentration of streptomycin.And the production of neomycin sulfate of Streptomyces fradiae was improved by ARTP mutagenesis and streptomycin resistance screening.The results showed that it appeared that the distribution fraction of neomyc:in sulfate of mutants having the higher potency of neomycin sulfate increased with increases of the concentration of streptomycin in the selection plate.Nevertheless,the quantity of colonies decrease dramatically and even becomes practically 0 when the concentration of streptomycin is above 10μg/mL.The research selects 8 μg/mL of streptomycin as the optimum concentration to acquire higher pre-screening efficiency.After six rounds of repeated ARTP mutagenesis and streptomycin resistance screening,the potency of neomycin sulfate was 7780 ± 110 U/mL of a mutant was obtained,which was 1.45 times higher than the original strain and named Sf6-2.(3)Optimization of fermentation medium for strains producing neomycin sulfate.Plackett-Burman test design was used to screen significant influencing factors,and the optimal fermentation medium composition was determined through Box-Behnken design results and data analysis.The results showed that among the components tested,soluble starch,peptone concentration and(NH4)2SO4 concentration were considered to be significant since their P-value<0.05 and the coefficient of these variables showed their positive effects on the response.The optimum values of three components were 73.98,9.23 and 5.99 g/L,respectively,calculated by response surface methodology,and the potency of neomycin sulfate could thereby reach 10849 ± 141 U/mL,about 99%of the predicted value.There was ideal agreement between the observed accuracy of the model.The potency of neomycin sulfate is almost 2 times of the original strain and 1.4 times of the mutant strain Sf6-2 after was optimized.