弗氏链霉菌双拷贝串联基因tylF、tylE和tylCV工程菌的构建

Construction of a Double-copy tylF、tylE and tylCV Streptomyces fradie strain

关键词:  泰乐菌素  弗氏链霉菌  tylE基因  tylF基因  tylCV基因

Key words:  tylosin  Streptomyces ffradie  tylE gene  tylF gene  tylCV gene

摘要:

为提高弗氏链霉菌对泰乐菌素的产素能力，构建了弗氏链霉菌双拷贝串联基因tylF、tylE和tylCV基因工程菌。本研究以弗氏链霉菌作为出发菌株，并以其全基因组DNA为模板分别扩增tylF和tylE以及tylCV基因、以质粒pSET152为模板扩增强启动子基因permE，采用重叠延伸PCR技术和EcoRV、XbaI双酶切结合的连接方法，构建弗氏链霉菌表达载体pSET152-permE-tylF-tylE-tylCV，并转化至大肠杆菌ET12567，利用大肠-链霉菌属间接合转移技术获得工程菌株。研究结果表明，工程菌株摇瓶效价(14580 u/mL)比出发菌株(11318 u/mL)提高了29%；液相色谱结果显示，工程菌株的泰乐菌素A和泰乐菌素C相对峰面积显著(P≤0.05)高于出发菌株。增加弗氏链霉菌tylF、tylE和tylCV的拷贝数可有效提高泰乐菌素的产量，以及优化组分，为泰乐菌素生产菌的遗传改造提供参考。

Abstract:

In order to improve the production capacity of Streptomyces fradiae to tylosin, the engineering strains with double copy tandem genes tylF, tylE and tylCV of Streptomyces fradiae were constructed. In this study, Streptomyces fradiae was used as the original strain andwhole genome DNA was used as template to amplify tylF, tylE, tylCV gene, and the plasmid pSET152 was used as template to amplify the promoter permE, respectively. The expression vector pSET152-permE-tylF-tylE-tylCV was constructed and transformed into E.coli ET12567. The engineered strain was obtained by indirect co-transfer of E.coli and Streptomyces. The results showed that the shaker titer of the engineering strain (14580 u/mL) was 29% higher than that of the original strain (11318 u/mL). The HPLC results showed that the relative peak area of the engineering strain was significantly higher than that of the original strain (P≤0.05). Increasing the copy number of tylF, tylE and tylCV of Streptomyces fradiae could effectively improve the yield of tylosin and optimize the composition, providing reference for the genetic modification of tylosin-producing bacteria.