摘要
【目的】脱氮副球菌(Paracoccus denitrificans)是一种环境友好的α-变形菌纲菌株,在有氧条件下也可进行反硝化过程,具有较好的脱氮能力。本研究以脱氮副球菌DYTN-1为底盘细胞,筛选氮素诱导型启动子用于强化硝化和反硝化途径,进而达到代谢工程强化脱氮副球菌DYTN-1去除氮素污染物的目的。【方法】通过接合转移的方法分别将过表达amoA、amoB、hao和nirS基因的重组质粒导入脱氮副球菌DYTN-1细胞中。经过荧光定量检测和氮素定量检测对脱氮副球菌DYTN-1的基因元件和氮去除能力进行表征。【结果】从基因组中挖掘了6个受NO_(2)^(‒)、NO_(3)^(‒)和NH_(4)^(+)诱导的启动子,诱导差异为2‒26倍;且过表达nirS的菌株用2 g/L KNO_(3)处理24 h后培养基中NO_(3)^(‒)的残余量为野生型菌株的67%。同时过表达hao和nirS基因的菌株在用1 g/L NH_(4)Cl和2 g/L KNO_(3)处理12 h后,其NO_(3)^(-)的剩余量仅为野生型菌株的50%,且最终总氮的降解效率达79.5%,剩余总氮仅为野生型菌株的一半。【结论】上述研究表明,利用筛选获得的启动子工具在P.denitrificans DYTN-1中进行代谢工程改造强化氮素污染物的去除具有可行性。
[Objective]Paracoccus denitrificans is an environment-friendly strain of α-Proteobacteria.It can also perform denitrification under aerobic conditions and has good denitrification ability.This study intended to use P.denitrificans DYTN-1 as the chassis cell to screen nitrogen inducible promoters for strengthening the nitrification and denitrification pathways,thus achieving the purpose of enhancing the removal of nitrogen pollutants by metabolic engineering.[Methods]Recombinant plasmids overexpressing amoA,amoB,hao and nirS genes were introduced into P.denitrificans DYTN-1 cells by conjugation.The gene elements and nitrogen removal ability of P.denitrificans DYTN-1 were characterized by fluorescence quantitative detection and nitrogen quantitative detection.[Results]Six promoters induced by NO_(2)^(-),NO_(3)^(-)and NH_(4)^(+) were obtained at 2 to 26-fold induction.The residual amount of NO in the medium of the nirS overexpressed strain was 67%of that of the wild-type strain after treatment with 2 g/L KNO_(3)for 24 h.The strains overexpressing both hao and nirs had only 50%of the residual NO;of the wild-type strain after treatment with 1 g/L NH.Cl and 2 g/L KNO;for 12 h.Furthermore,the final degradation efficiency of total nitrogen reached 79.5%and the residual total nitrogen was only half of that of the wild-type strain.[Conclusion]It is feasible to carry out metabolic engineering with the aboved screened promoters to enhance the removal of nitrogen pollutants in P.denitrificans DYTN-1.
作者
牛晓倩
陶楠
舒泉先
杨小雁
从瑞涛
周胜虎
邓禹
NIU Xiaoqian;TAO Nan;SHU Quanxian;YANG Xiaoyan;CONG Ruitao;ZHOU Shenghu;DENG Yu(National Engineering Research Center of Cereal Fermentation and Food Biomanufacturing,Jiangnan University,Wuxi 214122,Jiangsu,China;Jiangsu Provincial Research Center for Bioactive Product Processing Technology,Jiangnan University,Wuxi 214122,Jiangsu,China;Shandong Bohi Oils&Fats Industry Co.,Ltd.,Binzhou 256599,Shandong,China;DSM Jiangshan Pharmaceutical(Jiangsu)Company,Jingjiang 214500,Jiangsu,China)
出处
《微生物学报》
CAS
CSCD
北大核心
2022年第10期3932-3946,共15页
Acta Microbiologica Sinica
基金
国家重点研发计划(2019YFA0905502)
中央高校基本科研业务费专项资金(JUSRP12056,JUSRP51705A)
中国博士后科学基金(2021M690533)。
关键词
诱导型启动子
硝化途径
反硝化途径
脱氮副球菌DYTN-1
inducible promoter
nitrification pathway
denitrification pathway
Paracoccus denitrificans DYTN-1
