Abstract:To improve the screening efficiency of the high - yielding mutant strains of natamycin，a high -throughput screening strategy was developed for the initial screening of 24-well deep-well plate/enzymatic assay（UV spectrophotometry）fermentation and the re-screening of shake flask（HPLC method）fermentation.The results obtained in the 24-well deep-well plate fermentation were in good agreement with that in the shake flask fermentation，confirming that the enzymatic assays can replace the HPLC assay for high - throughput screening of the mutant strains. With Streptomyces gilvosporeus TUST01 as the starting strain，high-yielding strain S. gilvosporeus Y-4-75 was selected from 887 mutant strains by multiple rounds of ultraviolet（UV），atmospheric and room temperature plasma（ARTP），diethyl sulfate（DES）mutagenesis，initial screening and rescreening of shake flask，and its natamycin yield in shake flasks（5.95 g/L）and biomass（29.19 g/L）were increased by 31.93% and 15.19%，respectively compared with those of the starting strain.