低能离子注入诱变法选育新硫肽类抗生素166A高产菌株  

Breeding of new thiopeptide antibiotics 166A high-yield strains by low-energy ion implantation mutagenesis method

作　　者：戴剑漉[1,2] 李艳青 李欣 蒋忠科[1,3] 赫卫清[1,2] 孙承航[1,3] Dai Jianlu;Li Yanqing;Li Xin;Jiang Zhongke;He Weiqing;Sun Chenghang(State Key Laboratory of Bioactive Substances and Functions of Natural Medicines,Institute of Medicinal Biotechnology,Chinese Academy of Medical Sciences&Peking Union Medical College,Beijing 100050;Key Laboratory of Antibiotic Biotechnology,National Health Commission of the People's Republic of China,Institute of Medicinal Biotechnology,Chinese Academy of Medical Sciences&Peking Union Medical College,Beijing 100050;Department of Microbial Chemistry and Beijing Key Laboratory of Antimicrobial Agents,Institute of Medicinal Biotechnology,Chinese Academy of Medical Sciences&Peking Union Medical College,Beijing 100050;Southern Marine Science and Engineering Guangdong Laboratory(Zhanjiang),Guangdong Medical University,Zhanjiang 524023)

机构地区：[1]中国医学科学院&北京协和医学院医药生物技术研究所“天然药物活性物质与功能”国家重点实验室,北京100050 [2]中国医学科学院&北京协和医学院医药生物技术研究所国家卫生健康委员会抗生素生物工程重点实验室,北京100050 [3]中国医学科学院&北京协和医学院医药生物技术研究所微生物化学研究室和抗感染药物研究北京市重点实验室,北京100050 [4]广东医科大学南方海洋科学与工程广东省实验室,湛江524023

出　　处：《中国抗生素杂志》2024年第9期995-1003,共9页Chinese Journal of Antibiotics

基　　金：中国医学科学院医学与健康科技创新工程项目(No.2021-1-I2M-028)。

摘　　要：目的为进一步提高产量,以小单孢菌Micromonospora sp.TMD166-MU1为出发菌株,采用低能离子注入技术,研究不同离子注入参数对菌株存活率、正负突变率的影响,通过突变株抑菌活性变势参数分析,初步探讨N+离子注入对166A生产菌所产生的诱变效果,并结合卡那霉素抗性筛选及牛津杯固体发酵高通量筛选,获得高产菌株。方法利用低能N+离子注入技术对出发菌株TMD166-MU1的孢子进行辐照诱变,以卡那霉素耐受性为选择压力,不同诱变条件处理的菌悬液涂布在含有卡那霉素培养基平板上培养后获得单菌落,并以牛津杯固体发酵高通量筛选方法进行高产菌株初筛,之后对高产菌株进行摇瓶复筛,利用高效液相法检测突变株摇瓶发酵的化学效价。结果通过研究30和60 keV能量下不同注入剂量与小单孢菌正突变率的生物学效应关系,确定了在注入能量为30 keV、注入剂量4×10^(13)ions/cm^(2)、卡那霉素抗性筛选浓度为6 mg/L条件下,可获得最高达36.33%的正突变率。复筛效价是出发菌株1.5倍以上的有4株,其中突变株IK-3的166A产量是菌株TMD166-MU1的1.8倍。结论采用低能N+离子注入诱变方式,再结合抗生素抗性筛选及牛津杯固体发酵高通量筛选的集成方法能简单、高效地获得166A高产突变菌株。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 4×10^(13)ions/cm^(2) 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 thro

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 

分 类 号：R978[医药卫生—药品]