Objective To further increase the production of 166A and to obtain high-yield strains of 166A,Micromonospora sp.TMD166-MU1 was used as the starting strain to be investigated by low-energy ion implantation mutagenesis breeding technology.Through the analysis of the variable capacity parameters of the antibacterial activity of mutant strains,It was preliminarily explored that the mutagenic effects of different N+ion implantation parameters,including energy and doses,on the survival rate and positive and negative mutation rate of the strain TMD166-MU1,which combined with kanamycin resistance screening and Oxford Cup solid-state fermentation high-throughput screening,to obtain the high yield strains.Methods Strain TMD166-MU1 was implanted by Low-energy N+ion beam technology,the treated strain TMD166-MU1 was spread on agar medium plates containing kanamycin as selection pressure to obtain a single colony.The colonies were evaluated through the first high throughput screening with Oxford Cup method and the following second screening with flask culture,positive mutant strains were screened out by comparison of inhibitory zone against Bacillus subtilis with the starting strain.Finally,HPLC was used to determine the fermentation titer of positive mutant strains.Results By investigating the relationship between the implantation dose of 30 keV or 60 keV energy and the positive mutation rates of 166A producing strains,the parameters of N+ion implantation with the highest positive mutation rate(36.33％)were confirmed as 4×1013ions/cm2 of 30 keV nitrogen ions(N+)energy with 6 mg/L of kanamycin.Four high-yield mutant strains were obtained by the shake flask culture,and the 166A yields of these strains were at least 1.5-fold higher than that of the strain TMD166-MU1.A mutant strain coded as IK-3 was finally screened out and the 166A yield of the mutant strain IK-3 is 1.8-fold higher than that of TMD166-MU1 strain.Conclusion Low-energy N+ion implantation mutagenesis combined with antibiotic pressure and oxford cup high throughput screening model can effectively screen out 166A high-yield mutant strains,the integrated method was proved to be a simple and effective breeding approach to improve the yield of industrial microbes.
关键词
新硫肽类抗生素166A/低能离子注入/诱变/抗生素抗性筛选/高通量筛选/高产菌株
Key words
New thiopeptide antibiotic 166A/Low-energy ion implantation/Mutagenesis/Antibiotic resistance selection/High throughput screening/High-yield strains
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