自杀质粒同源重组介导阴沟肠杆菌adh基因缺失突变体构建及其生物学特性  

Alcohol Dehydrogenase Gene Deletion Mutant of Enterobacter cloacae by Suicide Plasmids Homologous Recombination:Construction and Biological Characteristics

作　　者：谢乐乐 何平 唐小越 葛菁萍[1] 凌宏志[1] XIE Lele;HE Ping;TANG Xiaoyue;GE Jingping;LING Hongzhi(Engineering Research Center of Agricultural Microbiology Technology,Ministry of Education/Heilongjiang Provincial Key Laboratory of Plant Genetic Engineering and Biological Fermentation Engineering for Cold Region/Key Laboratory of Microbiology,College of Heilongjiang Province/School of Life Sciences,Heilongjiang University,Harbin 150080)

机构地区：[1]黑龙江大学,生命科学学院,农业微生物技术教育部工程研究中心,黑龙江省寒区植物基因与生物发酵重点实验室,黑龙江省普通高校微生物重点实验室,哈尔滨150080

出　　处：《中国农学通报》2024年第18期96-104,共9页Chinese Agricultural Science Bulletin

基　　金：国家自然科学基金(31570492)。

摘　　要：本研究旨在探究敲除阴沟肠杆菌(Enterobacter cloacae)代谢通路上乙醇脱氢酶基因对乙偶姻代谢合成的影响,进一步提高乙偶姻产量。实验基于E.cloacae SDM中的乙醇脱氢酶adh基因序列设计特异性引物,并构建adh基因自杀质粒pKR6K-∆adh。利用热激法将自杀质粒转入E.cloacae∆budCldh,成功得到多基因缺失菌株E.cloaca∆budC-ldh-adh,并进行发酵性能测试。研究结果显示,与出发菌株相比,新构建的重组菌株在敲除了负责乙醇合成的关键酶乙醇脱氢酶adh基因后,其乙偶姻的生产强度提高了20.6%,产量提高了22.6%,同时乙醇产量提高了92.8%。此外,由于通过基因改造阻断了多条分支代谢路径,流向丁二酸代谢路径的碳通量明显变多,丁二酸产量提高101.3%。本研究成果不仅为构建高效乙偶姻生产菌株提供了有价值的参考,也为乙偶姻的大规模工业生产奠定了基础。The aim of this study was to investigate the effects of knockout of ethanol dehydrogenase gene in Enterobacter cloacae metabolic pathway on the metabolism and synthesis of acetoin,and further improve the production of acetoin.Based on the ethanol dehydrogenase(adh)gene sequence from E.cloacae SDM,a primer was designed to construct the adh gene suicide plasmid pKR6K-∆adh.Subsequently,this suicide plasmid was introduced into E.cloacae∆budC-ldh through heat shock method.The resulting polygene deletion strain E.cloaca∆budC-ldh-adh was successfully constructed and subjected to fermentation analysis.The findings revealed that the recombinant strain exhibited a 20.6%increase in acetoin production intensity,a 22.6%increase in yield,and a remarkable 92.8%increase in ethanol yield compared to control strains due to successful knockout of ethanol dehydrogenase(adh)gene which in charge of microbial ethanol synthesis pathways.Furthermore,as a consequence of blocking multiple branch metabolic pathways through genetic modification,carbon flux towards succinic acid metabolic pathway increased significantly leading to an impressive 101.3%enhancement in succinic acid yield.This experiment has provided valuable insights for constructing high-yielding strains for acetoin production and establishing large-scale industrial manufacturing processes.

关 键 词：乙偶姻 乙醇脱氢酶 同源重组 阴沟肠杆菌 基因缺失 基因敲除 代谢工程 

分 类 号：S182[农业科学—农业基础科学]