Breeding Of Oxytetracycline High-producing Strains By ARTP Mutagenesis Techniques

Oxytetracycline,a kind of secondary metabolites from the fermentation process of Streptomyces rimosus,is widely used as broad-spectrum antibiotic.In recent years,the production capacity of traditional fermentation technology of oxytetracycline has been limited with the gradually matured of the production technology.Therefore,the whole production industry of oxytetracycline becomes focus on how to continue to improve the production capacity and reduce production cost.Excellent strain is the basic condition to guarantee the production of oxytetracycline while efficient mutation technology is significant to obtain the excellent strains.Atmospheric and Room Temperature Plasma(ARTP),a plasma source develops in the last few years,has had a good performance in the field of microbial breeding.In this study,separation and purification was given to the oxytetracycline-production strains A4-30-16 which was preserved in laboratory.The strain A4-30-16 was bred by naturally separation and classified into five groups according to the different colony morphology typical.The five groups were?type,?type,?type,?type and?type.Then this study measured the oxytetracycline yield of all the different groups.The results showed that?type strain was significantly better than other types on the number of colonies and the oxytetracycline yield.This strain was named F-8 which the oxytetracycline yield of strain increased about 7.7%compared with the original strain A4-30-16.This proved that it has some relationships between the colony morphology and oxytetracycline biosynthetic ability.Then started with the separated stable strain,draw the fatality rate curve by ARTP mutagenesis,ultimately determined that the best mutagenesis time was 60 s.Through the resistance tests of cerulenin and KH₂PO₄,the working concentration of screening reagents were confirmed.finally,the screening concentration of cerulenin was determined to be 35?g/m L and the screening concentration of KH₂PO₄was determined to be 36 mg/m L.The strain A4-30-16 was selected as an original strains for the mutation breeding,and three rounds of ARTP mutagenesis were used on the test strain.With the principle of metabolic-engineering directed screening,mutant strains were obtained by the directed breeding which used cerulenin and KH₂PO₄as the screening reagents.Through the three rounds of ARTP mutagenesis,the oxytetracycline high-producing strain P2-60-21 was obtained,and its oxytetracycline production of 250 m L fermentation flask achieved24685.5U/m L,which was increased by 26.8%to the original departure strain in the first round of ARTP mutagenesis,and was increased by 47.7%to the original departure strain from North China Pharmaceutical Factory.