Selective Breeding Of Salinomycin High-yielding Strain By High-throughput Screening

Salinomycin is a member of the spiroketal class of polyether antibiotics produced by submerged fermentation of Streptomyces albus. Due to its anticoccidial and antibacterial activity, less toxic for human, effective and broad-spectrum properties, salinomycin is widely used as an effective veterinary drug. To obtain high salinomycin-producing strains, the non-rational strain breeding strategy was employed. There are two main technologies used in strain breeding:one was to establish a mutation platform to obtain a large number of mutants; the other was to establish a screening platform to screen prospective mutants exactly and rapidly.Firstly, a high-throughput screening (HTS) method was investigated. The HTS platfrom inculded two technical systems:one was the micro-cultivation technique based on24-square deep-well microtiter plate (MTP), and the other is the high-throughput assay technique based on microplate reader. The two techniques supplemented each other to form a complete screening system to lay a foundation for large scale non-rational strain selection.Secondly, an atomspheric and room temperature plasma (ARTP) mutation method was implemented. The mutagenic mechanisms and effect of ARTP on Streptomyces albus were investigated. The plasma jet yielded high total (54.8%) and postive (26.0%) mutation rates after the spores of Streptomyces albus was treated. Combined with the UTS platform, three high yield strains, designated S-3, S-9and S-11, respectively, with stable inheritance were obtained, the salinomycin productivity of which increased by about45%,66.7%and70%, respectively, compared to the original strain under the same culture conditions.Thirdly, the mutation platform based on protoplast technology was conducted. Effects of medium, the cultivation time of mycelia, the concentration of sucrose and glycine, the concetration of lysozyme, lytic time and temperature on protoplast formation and regeneration were investigated. The protoplast formation rate and regeneration rate could respectively approach95%and17%under the optimal conditions. On the basis of protoplast preparement and regeneration, the protoplasts of Streptomyces albus was treated by UV, and as high as24.5%postive mutation rate was obtained. Combined with the HTS platform, three high yield strains, S-612, S-620and S-622with stable inheritance were selected out, the salinomycin productivity of which increased by57.6%,45.5%and69.7%, respectively, compared to the original strain on the same culture conditions.