Study On The Breeding Of High-yield Strain For Single Component And Fermentation Scan-up Of Milbemycin

Milbemycin is a kind of polyketone compound which is a 16-membered ring macrolide.According to the compound has a hydrogenated benzofuran structure or not,it can be simply divided into α-form and β-form.Since the activity of a-type is much higher than that of β-type,the research of α-type all over the world is more than of β-type.At present,the mainstream product in the commercial market is c-5 oximedoxomycin,which is a mixture of A3 oxime(<20%)and A4 oxime(>80%).Besides,it has high activity,low toxicity,safety and reliability,easy degradation and excellent prevention and treatment effect on parasites,so it is widely used in anti-parasite drugs.The industrial production of the mixture of Milbemycin oxime was obtained by semi-fermentation and semi-synthesis.The proportion of Milbemycin oxime has strict requirements.Because the structure and physical and chemical properties of the two components are similar,and the extraction process is difficult to control,the breeding and fermentation culture optimization of single-component strains with high yield are of great significance for improving the production level of Milbemycin oxime.Streptomyces milbemycinicus CGMCC 7677 was the original strain in this study,the strain had the productivity of 1056 mg/L with A4 content of 74.7%which obtained by natural isolation.Then single or combination of physiochemical mutation of UV,atmospheric pressure room temperature plasma,nitrosoguanidine,ethyl methanesulfonate,and screened with resistant plates containing glycine,sodium acetate and rifamycin,the milbemycin fermentation potency increased by more than three times.Two colonies different from colonies of original strain were screened by a resistant plate containing 3-bromopyruvate,and then the milbemycin single-component high-yield strains 75-22(A4 high yield)and 95-23(A3 high yield)were obtianed by further mutagenized and resistance screening.The mutant strain of 75-22 has a flask titer(A3+A4)of more than 3500 mg/L,and the content of A4 is more than 80%.The mutant strain 95-23 flask titer(A3+A4)was 3000 mg/L,and the content of A3 was more than 70%.The genetic stability of the two mutant strains was investigated,and the genetic stability was good.The seeding conditions,culture temperature,and medium loading of the newly selected strains were investigated.The fermentation medium was optimized by single factor screening and orthogonal experiment of carbon source and nitrogen source,and fermentation medium suitable for different high-yield single component strain were obtained.The fermentation titer(A3+A4)of 75-22 could reach 3700 mg/L and the A4 content was more than 80%after the optimized medium and culture conditions were used for 12 days.After feeding 0.2%sodium acetate and 0.2%sodium propionate at 72 hours for 12 days,the 95-23 fermentation titer(A3+A4)reached 3100 mg/L,and the A3 content was more than 70%.The two strains were amplified in a 50L fermentation tank,and the feeding process was added by analyzing and simulating the metabolism curve of shaking flask test.75-22 was cultured in a 50L fermenter for 12 days.By adding fructose,the fermentation titer(A3+A4)reached 3600 mg/L,and the A4 content was over 80%.95-23 was cultured in a 50L fermenter for 12 days.By adding glucose and precursors,the fermentation titer(A3+A4)reached 3000 mg/L,and the A3 content was 70%.Breeding and fermentation amplification studies of high-yield single-component strains of milbemycin provides a good basis for the reduction of industrial production cost and quality of milbemycin.