Breeding High-Yied Strains Of Avilamycin By Ribosomal Engineering Techniques

Avilamycin is an oligosaccharide antibiotic produced by aerobic fermentation of Streptomyces viridoehrongenes,it’s widely used as a veterinary feed additives because of its growth-promoting and high safety.At present,the products of avilamycin was only produced by Lilly,which leads to a monopoly of technology.The domestic production ability of avilamycin was low,and there was no manufacturer to produced avilamycin-related products and the research on the breeding of high-yield strains was still in primary stage.Therefore,there was needed a simple and effective method for breeding avilamycin-producing strains.In this study,the ribosomal engineering technique was used to transform the Streptomyces viridoehrongenes preserved in the laboratory by screening streptomycin resistant mutants.Under the same conditions,studied the growth morphology of the starting strain and the mutation strains,the rpsL gene involved in streptomycin resistance,whole protein and the intracellular metabolites,not only can provided theoretical support for the high yield of antibiotics of mutant strains,but also provided a new idea for the breeding of Streptomyces viridoehrongenes.Used the laboratory-preserved Streptomyces viridoehrongenes gs77 as the starting strain,the minimum inhibitory concentration(MIC)of streptomycin was determined to be 700 μg/mL.6 batches of coated plates were separated and 223 strains were obtained.The average lethality rate of resistant mutant strains was over 80%and 53 positive mutant strains were obtained by susceptibility test,and the positive variability was as high as 23.7%.The yield of 5 mutant strains of avilamycin was 30%higher than that of the original strain.The mutant strain S.viridoehrongenes gs77-54 avilamycin was 1359.79 mg/mL,which was 1.75 fold higher than that of the original strain,and was genetically stable.Analyzed the morphological characteristics of the strains before and after the mutation and the rpsL gene(encoding ribosomal protein S12),the result showed that the aerial hyphae of the mutant strain was long and straight,and the spores were elliptical with a diameter of about 1.1 μm × 0.6 μm,which was significantly different from the starting strain.The sequence analyzed of the rpsL gene revealed that a point mutation occurred in the gene fragment,the G mutation at position 356 was A,the arginine was mutated to glutamine,and the amino acid change caused by the rpsL gene mutation was whether the specific increase in avilamycin production or the acquisition of its high-yield performance was caused by mutations in the rsmG gene remains to be studied and SDS-PAGE results indicated that multiple mutant sites were up-regulated in the mutant strain.GC-MS studied the differences in intracellular metabolite levels before and after mutation.The results showed that the pentose phosphate pathway,fatty acid metabolism and partial amino acid metabolism of high-yield strains were strong.It is speculated that the enhancement of these pathways may provideed synthesis of avilamycin substances such as polyketones,acetyl CoA,and propionyl CoA.