摘要
构建一种新型的含有大片段同源臂的重组质粒用来对节杆菌进行代谢工程的改造。使用PCR扩增线性化载体片段,并通过酶切获取同源重组大片段,利用一步克隆技术将上述两片段连接起来;通过PCR-targeting将目标基因替换,并利用酶切自连将插入的片段删除,最后成功得到目标质粒p UK-d IMP。构建的质粒通过2次同源重组将目的基因肌酐酸脱氢酶(IMPDH)成功敲除,且最后菌株不含有任何抗性,开辟了一种针对节杆菌而言新型的基因改造方法。其中IMPDH基因缺失的菌株积累目标产物环磷酸腺苷的能力比对照菌株高49.7%,达到了(9.01±0.20)g/L。
A new recombinant plasmid containing a large fragment homologous arm was constructed for genetic modification in Arthrobacter sp.. A linear vector was amplified by PCR and a large homologous recombination fragment was obtained by restriction enzyme digestion. The above two fragments were ligated together by one step cloning technology. Then the target gene was replaced by PCR-targeting and the inserted fragment was deleted by restriction enzyme digestion and self-ligation. Finally,the target plasmid p UK-d IMP was obtained. The target gene inosine 5＇-monophosphate dehydrogenase（ IMPDH） was successfully knockouted using the constructed plasmid by two-step homologous recombination,and the recombinant strains had no antibiotic resistance. We created a new type of genetically modified method for Arthrobacter sp.. For the fermentaion of IMPDH gene deletion strain,the yield of the taget product c AMP reached（ 9. 01±0. 20） g / L,which was 49. 7% higher than that of the control.
出处
《生物加工过程》
CAS
2016年第1期14-18,35,共6页
Chinese Journal of Bioprocess Engineering
基金
国家杰出青年科学基金(21025625)
国家重点基础研究发展计划(973计划)(2013CB733602)
国家高技术研究发展计划(863计划)(2012AA021203)
国家自然科学基金(21106070
21376118)
关键词
节杆菌
环磷酸腺苷
同源重组
基因敲除
Arthrobacter sp.
cyclic adenosine monophosphate（c AMP）
homologous recombination
gene knock-out
