Study On The High Throughput Strategy To Screen Strains With High Nosiheptide-Productivity

Nosiheptide is a kind of sulfur-containing peptide antibiotics, mainly produced by a microogranism named Streptomyces actuosus (S. actuosus). It has a strong inhibitory effect on Gram-positive cocci and bacilli, with the advantages of promoting animal growth, improving feed efficiency, and enhancing animal’s ability of resistance to disease. It is safe to human and animals, with little pollution to the environment. Hence, it is an ideal replacement of antibiotics. However, the overall level of industrial fermentation units is still low. It leaves plenty of scope for improvement of the performance of production strains.To obtain S. actuosu mutants with improved nosiheptide production, an atmospheric and room temperature plasma (ARTP) jet was used with 48-well microplates and inhibition zone method for high throughput screening, and the fermentation conditions of 48-well microplates were also optimized. The results showed that there were relatively good correlation among the diameter of inhibition zone of mutant colonies, the A408 of 48-well microplates and the A408 of shake flask. The correlation coefficient R between the size of inhibition zone diameter and the A408 of 48-well microplates is 0.534, while it could reached to 0.896 for the value between the A408 of 48-well microplates and the A408 of shake flask. The A408 of 48-well microplates can be improved by 96h fermentation with 0.5 mL mixed medium. Comparing with the traditional shake flask screening platform,48-well microplates and inhibition zone method could significantly improve the screening efficiency of high nosiheptide-producing strains.Although, A408 of 48-well microplates has been increased after optimized, but oxygen may still be a major limiting factor.Hence, we investigate different numbers of well microplates compared with flask titer.To optimize the fermentation condition of Streptomyces actuosu XW producing nosiheptide in 24-well microplates. The fermentation condition in the 24-well microplates was determined by using the single-factor experiments and the response surface method. The optimized fermentation condition of Streptomyces actuosu XW in 24-well microplates was obtained as follows:the liquid volume of seed medium 2.55 mL, the liquid volume of fermentation medium 0.93 mL and fermentation time 121 h respectively. The production of nosiheptide in 24-well microplates was improved significantly under the optimal condition, reaching 882.25 ug/mL, the experimental value was comparable to model prediction, having high inosculating degree, which was increased by 30.58% compared with the original condition. The result showed that there was a highly significant correlation relationship between the A408 of 24-well microplates and the A408 of shake flasks. The correlation coefficient R between the A408 of 24-well microplates and the A408 of shake flasks was 0.883.To obtain S. actuosu mutants with improved nosiheptide production, an atmospheric and room temperature plasma (ARTP) jet was used. After ARTP treatment 150s the mortality rate can reached 98.15%. It is found that the nosiheptide production of the 3 mutants can be increased to above 150% in the same fermented condition. This research indicated that ARTP mutagenesis techniques is an effective way to obtain strains with high yield of nosiheptide.The optimized the conditions of the formation and regeneration of the protoplasts, including concentration of glycine in seed medium, enzymolysis time, concentration of lysozymewere studied. The optimum conditions of preparing protoplast were as follows:the mycelia of Streptomyces actuosu were processed with 50mg/mL lysozyme for 70min and the seed medium containing 0.5% glycine. Through several rounds of protoplasts of ARTP mutation and high-throughput screening, the strain of Y2-47 with increased by 15.02% relative contents of nosiheptide were obtained than that of the control strain of 3-53 finally.The protoplasts were inactivated by heating at 65℃ for about 50min and UV treatment about 40min, respectively. The inactivated protoplasts were fused and regenerated with the strains of 3-53 and Y2-47 as a parent. Two high yielding strains RH-4 and RH-5 was isolated and the increased yield was about 5% higher than that of the parent strain Y2-47. The titer of mutant increased 70.4% campared with the original strain.