Screening Of High Yield Salinomycin Producing Strain And Optimization Of Its Fermentation Process With Novel Oil Feeding Strategy

Salinomycin is a kind of polyether antibiotics which is produced by Streptomyces albums.It is widely used as poultry anti-coccidia agent because of its high antimicrobial activity against Gram-positie organisms, antibacterial action against some fungi. It shows some advantages such as no drug-resistant, no cross drug resistence and no obvious accumulation phenomenon. Recent research discovered that cancer stem cell can be directly killed by salinomycin, which attracts widespread attention from various circles.In this thesis, the following contents were studied. Firstly, screening of high-salinomycin-yield mutants of Streptomyces albus was carried out. A mutant, designated S-E-6, was successfully selected by self-resistance methodology. The mean salinomycin titer of S-E-6 was 38% higher than that of the parent strain in flask culture. The optimum dose of mutagen is 0.12mL and 20g/L salinomycin on the alinomycin gradient plates. The production level of the mutant S-E-6 was stable after successive 3-generation subcultures, indicating that the selected mutant has a good genetic stability for salinomycin biosynthesis. Besides, the correlation between the radius of colonies grown on salinomycin gradient plates and their corresponding titers was analyzed, the results showed that there was a linear relationship with a correlation coefficient (R2) of 0.9546. So the conclusion could be drawn that the colony with large diameter and full in shape could bring high yield production.Then, S-13 was chosen as a start strain. A new recipe of culture medium was obtained through single factor experiment, Box-Behnken design. The titer of salinomycin was 24% higher using the new recipe than that of the old recipe and the amount of the oil used was 2.3 g/L less than the original medium. The new medium of and culture conditions are as follows:Seed medium:glucose 39g/L, soybean meal 24g/L, yeast powder9g/L, the shaking speed of 250 rpm,,temperature 32-34℃, pH 6.8-7.0 and fermentation duration of 2d were determined. Fermentation medium:germ flour 8 g/L, soybean meal 4.7 g/L, KCl2.2 g/L, ammonium tartrate 2 g/L, urea 1.6 g/L, NaCl 1g/L, MgSO40.1 g/L, K2HPO40.2 g/L, CaCO35 g/L, oil 118 g/L, inoculation 9%, the shaking speed of 250 rpm,,temperature 32-34℃, pH 6.8-7.0 and fermentation duration of 10d were determined.Finally, the feeding strategy was developed in 2 m3 fermenter in the process of enlargement according to the law of parameter correlation. The optimum rate of feeding oil was 1.3g/L/h and residual concentration of oil was 2.0-2.5g/L. The transition between sugar and oil utilization was analyzed in the term of RQ. It was found that the process of conversion between sugar and oil had similar tendency by the association analysis of online and offline parameters. Also the regulation of RQ and pH was studied. The key enzymes play a decisive role in the process of fermentation. The activity of these enzymes might be affected by pH. When the initial pH was reduced, the titer and the activity of the key enzymes were improved in some way. It still needs further study on the fed batch of oleic acid methylester.