阴沟肠杆菌乳酸脱氢酶基因缺失突变株的构建及其生物学特性  

Lactic Dehydrogenase Gene Deletion Mutant of Enterobacter cloacae:Construction and Biological Characteristics

作　　者：石会玲 周宇航 何平 黄蒙蒙 邵帅 葛菁萍[1,2] 凌宏志 Shi Huiling;Zhou Yuhang;He Ping;Huang Mengmeng;Shao Shuai;Ge Jingping;Ling Hongzhi(Key Laboratory of Microbiology of Heilongjiang Province,Life Science College,Heilongjiang University,Harbin 150080;Agricultural Microorganisms Technology Education Engineering Research Center,Vlarhin 150500)

机构地区：[1]黑龙江大学生命科学学院微生物重点实验室,哈尔滨150080 [2]黑龙江大学农业微生物技术教育部工程研究中心,哈尔滨150500

出　　处：《中国农学通报》2021年第23期29-37,共9页Chinese Agricultural Science Bulletin

摘　　要：旨在应用自杀质粒重组技术,构建阴沟肠杆菌乳酸脱氢酶突变株,为进一步提高乙偶姻的产量和扩大菌株选择范围奠定基础。利用双酶切的方法将同源片段插入到自杀质粒p KR6K中,构建出ldh基因敲除质粒,然后利用细菌接合的方法敲除E. cloacae的ldh基因。成功克隆出两段E. cloacae乳酸脱氢酶基因的同源序列,长度分别为526 bp,通过序列比对分析,E. cloacae乳酸脱氢酶基因序列相似性为100%。通过对E. cloacae进行乳酸脱氢酶基因的敲除,成功构建一株ldh缺失重组菌株E. cloacae△ldh,同时2,3-丁二醇提高6.8%,乙酸提高了24.3%。E. cloacae乳酸脱氢酶缺失工程菌株构建成功,对利用微生物法工业化生产乙偶姻奠定基础。The aims are to construct a lactic dehydrogenase mutant of Enterobacter cloacae by suicide plasmid recombination technique, and lay a foundation for further increasing acetoin yield and expanding the range of strain selection. The homologous fragment was inserted into the suicide plasmid pKR6 K by double restriction endonuclease digestion, and the ldh gene knockout plasmid was constructed. Then the ldh gene of E.cloacae was knocked out by bacterial conjugation. Two homologous sequences of E.cloacae lactate dehydrogenase gene were successfully cloned with a length of 526 bp. Sequence alignment analysis showed that the sequence similarity of E.cloacae lactate dehydrogenase gene was 100%. By knocking out the lactate dehydrogenase gene of E.cloacae, a ldh deletion recombinant strain E.cloacae △ ldh, was successfully constructed, while 2,3-butanediol and acetic acid were increased by 6.8% and 24.3%, respectively. The E.cloacae lactic dehydrogenase deletion engineering strain was successfully created, which lays a foundation for the industrial production of acetoin by the microbial method.

关 键 词：阴沟肠杆菌 乳酸脱氢酶 自杀质粒 同源重组 基因敲除 

分 类 号：S182[农业科学—农业基础科学]