常规诱变结合高通量筛选选育可利霉素高产菌株

Rational screen of high Kelimycin-producing strain by combined conventional mutagenesis and high-throughput screen method

摘要:

摘要:可利霉素是通过基因工程定向育种技术获得的新型大环内酯类抗生素，是国家一类新药。【目的】为满足工业化生产需要，其工程菌株的发酵水平有待提高。【方法】多种常规诱变技术交替处理和高通量筛选方法选育可利霉素高产菌株，处理方法包括原生质体紫外诱变、DES(硫酸二乙酯)诱变、紫外光复活诱变、缬氨酸抗性筛选和正突变菌株的富集。【结果】高产菌株WSJ-1-7-49-133-82-43的摇瓶生物效价比出发菌株WSJ-1-7-49提高56%，500L中试发酵罐突变菌株效价较出发株高61%。【结论】说明多轮常规诱变育种结合高通量的筛选方法可以用于工业生产菌株的高效筛选。

Abstract:

Abstract:Kelimycin，is a new macrolide antibiotic drug obtained through genetic engineering approaches．With 4 "-Oisovalerylspiramycins as the major components，was produced by genetically engineered Streptomyces spiramyceticus transformed with 4 " -O-acyltransferase gene from S． mycarofaciens．［Objective］ Improve the efficiency of strain fermentation，to meet the needs of industrial production．［Methods］ The enhanced kelimycin-producing strain was obtained by applying various conventional mutagenesis approaches，and high-throughput screen methods，including protoplast mutagenesis by ultraviolet，mutagenesis by diethyl sulfate and UV-reactivation，valine content resistance screen and enrichment of improved strains．A strategy for positive mutant enrichment was developed after mutagenesis and before high-throughput screen．［Results］ Finally，the high-producing strain WSJ-1-7-49-133-82-18-43 was obtained and its potency in shake flask increased by 56% compared to the original strain． The potency in 500 L pilot fermenter increased by 61%．［Conclusion］This study shows that the screening industrial production strains can be enhanced effectively by combining multiple conventional mutagenesis and high-throughput screen methods.