一种A40926高产菌株的高通量筛选策略  

High-throughput Screening for A40926 High Producing Strain

作　　者：刘志钰 李娟 朱兵峰 刘忠 张贵民 

机构地区：[1]鲁南制药集团股份有限公司,山东临沂273400 [2]哺乳动物高效表达国家工程实验室,山东临沂273400

出　　处：《中国医药工业杂志》2018年第3期316-321,共6页Chinese Journal of Pharmaceuticals

摘　　要：为提高糖肽类抗生素A40926的发酵效价,本研究以野野村放线菌(Nonomuraea sp.)HIPI-H01为出发菌株,采用常压室温等离子体诱变系统进行诱变,并选择链霉素及A40926脂肪酸侧链生物合成前体结构类似物乙酸钠为筛选压力,建立抗性筛选模型,以淘汰大部分不符合要求的突变菌株,使有效性状的突变株得以快速筛选出来,提高正突变率。在建立筛选抗性菌株方法的基础上,以抑菌圈筛选法进行初筛,初步筛得抑菌圈较大的168株突变株。结合出发菌株特性,选取24孔板进行复筛,并比较了微孔培养与摇瓶培养的相关性。结果表明,采用微孔板培养与摇瓶培养具有较好的一致性。与摇瓶培养相比,微孔板筛选体系具有效率高、可有效减少操作步骤及物料的使用量等优点。初筛菌株经微孔板复筛、抑菌圈法检测,筛选出24株发酵液抑菌圈较大的菌株,并以HPLC法检测发酵液中A40926效价,获得1株遗传性能稳定的突变株134#,其合成A40926的能力与原始菌株相比提高了17.6%,A40926效价达到809 mg/L。该菌株命名为野野村放线菌HIPI-M134。The screening of production strains with good performances is the foundation and key parts in fermentation industry, which could significantly enhance the yield and the quality of products. In order to improve the yield of glycopeptides antibiotic A40926, the wild Nonomuraea sp. HIPI-H01 was mutagenized by atmospheric and room temperature plasma （ARTP） system. Streptomycin and sodium acetate, which has the similar structural with the initiator of branched fat-acid chain of A40926, were used as screening pressure to exclude most of the negative mutants and increase positive mutation rate. For the prescreening procedure, the inhibition zone method was established and 168 mutants with large inhibition zone were obtained. According to the characteristics ofNonomuraea sp., 24-well microplates were employed in the secondary screening procedure, which exhibited excellent uniformity with shaking flasks. Compared with shaking flasks, the microplate screening system had many advantages, such as higher efficiency, less procedure and reduced material consumption. Based on the microplate screening system and the inhibition zone assay, there were 24 mutants whose fermentation broth gave larger inhibition zones. A40926 were detected by HPLC and a genetically stable mutant 134# was obtained. It could produce 809 mg/L A40926, which was 17.6 % higher than that of the wild. The mutant was named Nonomuraea sp. HIPI-M134.

关 键 词：达巴万星 A40926 推理选育 高通量 

分 类 号：TQ920.6[轻工技术与工程—发酵工程] TQ469