Study On The High Throughput Strategy To Screen Strains With High Polymyxin E-productivity

Polymyxin E is a kind of polymyxins, a generic name for a group of chemically related fatty acyl decapeptide cationic antibiotics, produced by strains of Paenibacillus Polymyxa. Polymyxin E displays excellent potency against Gram-negative pathogens. High throughput screening in the application of strain breeding of polymyxin E producer and fermentation of polymyxin E have been systematically studied in this paper. And finally several high-yielding mutants which showed superior ability of producing polymyxin E in shake flask scale were identified.The determination method of polymyxin E by HPLC was established. After the optimization of acetonitrile content and pH value in the mobile phase and detection wavelength, the major compoents of polymyxin E in the broth, named polymyxin El and polymyxin E2, could be successfully separated.The optimum medium compositions and culture conditions were determined both for seed culture and fermentation process. Under the optimum conditions, polymyxin E productivity increased by about 17% to 500 mg/L.A high-throughput cultivation method for P. polymyxa isolates in 96-well microtiter plates was established. The culture conditions including culture volume, running rate, were optimized for the production of polymyxin E. A piece of gauze with eight layers and a polythene lid perforated with 96 holes were used as a microtiter plate lid. Under above conditions, evaporation rate was reduced to 2% and no cross-contamination was observed. Moreover, a highly similar pattern of polymyxin E biosynthesis in microtiter plates and in shake flask was achieved.A microtiter plate-based bioassay method was establied for high throughput determination of polymyxin E. The inhibition effect of each sample against Escherichia coli JM109 was used for the evaluation of polymyxin concentration. In order to get comparable results, some culture conditions were optimized to develop a standard bioassay.According to the biosynthesis path of polymyxin E, a mutated library with about 13,613 isolates, obtained from rational breeding of the original wild type strain, were subjected to high throughput screening. Among these mutants, the best producer (N6-557) was identified with a high productivity of polymyxin E (1,200 mg/L) in shake flasks, about 140% improvement compared with that of the wild type strain.