Streptomyces Viridochromogene Mutagenesis And Screening Of High Yield Avilamycin

Avilamycin, known as Surmax, which was obtained from the culture of Streptomyces viridochromogene under aerobic fermentation condition. Avilamycin exhibited obvious cell growth-promoting and disease-preventing effect on animal. It has very higher safety and lower residue in practice and mainly used as feed additive for pigs and chickens. China approved its use as an imported veterinary drug in 2000. Currently, avilamycin production techniques and products have been monopolized by Eli Lilly. There also have some domestic research achievements in this field, but the strain produce low avilamycin concertration in fermentation.In this research, firstly, Streptomyces viridochromogene CGMCC 4.1119 was selected as the original strain. The spores suspension was mutated by UV-Li Cl, atmospheric temperature plasma(ARTP) and nitrosoguanidine(NTG) for breeding avilamycin high-producing strains. Statistical method was applied to analyze three kinds of screening factors including calcium chloride, streptomycin and aminobutyric acid: we count respectively numerical value of the mutant strains, then use the Gaussian distribution curve fitting method analysis. Using biostatistics method to analyze three parameters: positive mutant number(steep grade value on Y axis, A), the mean diameter squared value of inhibition zone(mathematical expectation, μ), the probability of obtain positive mutant(The variance, σ). α-amino butyric acid was selected as the best screening factor that mutagenizing S. viridochromogene to increase avilamycin yield.The mutation spores were smeared on the agar plate containing α-amino butyric acid, streptomycin or calcium chloride as the selective pressure. Use agar block screening method and cylinder plate rescreening. Subsequently, a genetically stable mutant strain SVN-2 was obtained and the avilamycin production reached 545 mg·L-1, which was 4.87 times compared with the parent strain(112 mg·L-1).Fermentation medium components and culture conditions of strain SVN-2 culture in flasks were optimized by single factor test and response surface optimization. The optimum fermentation medium was soluble starch 40.2 g·L-1, soybean meal 25.2 g·L-1, soy peptone 6.90 g·L-1, Mg SO4 2 g·L-1, Mn Cl2 0.5 g·L-1, Ca CO3 0.5 g·L-1; valine 2 g·L-1. p H 7.5, temperature 28℃, shaking speed 200 r·min-1, liquid volume 50 m L/250 m L, inoculation 5%, inoculum age 48 h. The production of avilamycin reached 628 mg·L-1Finally, Fed-batch fermentation and the fermentation process were optimized. In batch fermentation, wet cell weight was 101 g·L-1 and the avilamycin production was 628 mg·L-1. After 56 h supplemented with soluble starch to 20 g·L-1 and Cl-1 to 10 mmol·L-1 in the fermentation medium, wet cell weight was 128 g·L-1, the avilamycin concentration reached 691 mg·L-1. Avilamycin production and wet cell weight were further improved.