摘要
本试验将橙色指包囊菌(Dactylosporangium aurantiacum)NRRL 18085中的非达霉素生物合成基因簇与本实验室保存的德干高原游动放线菌(Actinoplanes deccanensis) SIPI-XR01的测序结果进行对比,找到SIPI-XR01中含有与已报道的合成基因簇中编码LuxR家族调节因子的转录激活因子tiaR1基因类似的基因片段,并将其命名为xrr1,根据该基因设计引物,并通过PCR扩增和质粒构建的方法将其整合到菌株基因组上,成功构建了过量表达xrr1工程菌,命名为SIPIXR02。结果表明SIPI-XR02相比SIPI-XR01产量有明显提高,经过培养基碳氮源优化后摇瓶产量最高可达4592μg/ml,并随后在10 L发酵罐上进行验证,发酵后产量达到3 930μg/ml,具有工业应用价值。
Based on the biosynthetic pathway and mechanism of fidaxomicin reported in Dactylosporangium aurantiacum NRRL 18085 and sequencing results of Actinoplanes deccanensis SIPI-XR01 saved in our laboratory, we found a gene named xrr1 in SIPI-XR01, which was similar to tiaR1 encoding LuxR class regulators in NRRL 18085. According to the biosynthetic gene, xrr1 was obtained by PCR amplification using the primers designed. The engineering strain, named as SIPI-XR02, was constructed by integrating xrr1 into original strain genome. The results showed that the production of fidaxomicin by SIPI-XR02 was greatly improved compared with SIPI-XR01. Through optimizing the carbon sources and nitrogen sources of fermentation medium, the highest yield of fidaxomicin by SIPI-XR02 was 4 592 μg/ml in the shake flask, and 3 930 μg/ml in a 10 L fermentation tank, suggesting its prospect in industrial application.
作者
许睿
高萍
闵涛玲
陈昌发
胡海峰
XU Rui;GAO Ping;MIN Taoling;CHEN Changfa;HU Haifeng(School of Pharmacy,Fudan University,Shanghai 200120;Shanghai Institute of Pharmaceutical Industry,China State Institute of Pharmaceutical Industry,Shanghai 201203)
出处
《中国医药工业杂志》
CAS
CSCD
北大核心
2020年第2期204-210,共7页
Chinese Journal of Pharmaceuticals
关键词
非达霉素
正调控基因
工程菌
发酵优化
fidaxomicin
positive regulation gene
recombinant engineering strain
optimization of fermentation