Engineering Pseudomonas putida KT2440 for simultaneous degradation of organophosphates and pyrethroids and its application in bioremediation of soil
Engineering Pseudomonas putida KT2440 for simultaneous degradation of organophosphates and pyrethroids and its application in bioremediation of soil
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工程恶臭假单胞菌KT2440同时降解有机磷和拟除虫菊酯及其在土壤生物修复中的应用
DOI：
10.1007/s10532-015-9729-2
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发表时间：
2015-04
期刊：
影响因子：
3.6
通讯作者：
Yang Chao
中科院分区：
文献类型：
--
作者：
Zuo Zhenqiang;Gong Ting;Che You;Liu Ruihua;Xu Ping;Jiang Hong;Qiao Chuanling;Song Cunjiang;Yang Chao
农业土壤通常同时受到有机磷和拟除虫菊酯类农药的污染。为了开发一个稳定的和无标记的恶臭假单胞菌共表达两种农药降解酶，我们构建了一个自杀质粒与表达盒含有组成型启动子J23119，OP降解基因（MPD），拟除虫菊酯水解羧酸酯酶基因（pytH），利用upp基因作为upp缺陷恶臭假单胞菌的反向选择标记。通过引入自杀质粒和两步同源重组，将mpd和pytH基因整合到一株健壮的土壤细菌P. putida KT 2440的染色体上，染色体上没有选择标记。通过Western印迹分析和酶活性测定证明mpd和pytH在恶臭假单胞菌KT 2440中的功能性表达。液体培养降解实验表明，甲基对硫磷、杀螟松、毒死蜱、氯菊酯、甲氰菊酯和氯氰菊酯（各0.2mM）的混合农药在48 h内完全降解。将该工程菌接种到经上述混合农药处理的土壤中（106个/g），其降解率高于未接种的土壤。在熏蒸和非熏蒸土壤中接种15天内，6种农药均能完全降解。这些结果突出了工程菌株用于OP和拟除虫菊酯农药共污染土壤的原位生物修复的潜力。
Agricultural soils are usually co-contaminated with organophosphate (OP) and pyrethroid pesticides. To develop a stable and marker-free Pseudomonas putida for co-expression of two pesticide-degrading enzymes, we constructed a suicide plasmid with expression cassettes containing a constitutive promoter J23119, an OP-degrading gene (mpd), a pyrethroid-hydrolyzing carboxylesterase gene (pytH) that utilizes the upp gene as a counter-selectable marker for upp-deficient P.putida. By introduction of suicide plasmid and two-step homologous recombination, both mpd and pytH genes were integrated into the chromosome of a robust soil bacterium P. putida KT2440 and no selection marker was left on chromosome. Functional expression of mpd and pytH in P. putida KT2440 was demonstrated by Western blot analysis and enzyme activity assays. Degradation experiments with liquid cultures showed that the mixed pesticides including methyl parathion, fenitrothion, chlorpyrifos, permethrin, fenpropathrin, and cypermethrin (0.2 mM each) were degraded completely within 48 h. The inoculation of engineered strain (106 cells/g) to soils treated with the above mixed pesticides resulted in a higher degradation rate than in noninoculated soils. All six pesticides could be degraded completely within 15 days in fumigated and nonfumigated soils with inoculation. Theses results highlight the potential of the engineered strain to be used for in situ bioremediation of soils co-contaminated with OP and pyrethroid pesticides.
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使用侧接短同源区域的 PCR 产物对假单胞菌进行无痕连续基因修饰
影响因子：
4.2
作者：
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影响因子：
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作者：
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作者：
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通讯作者：
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DOI：
10.1016/j.ibiod.2006.07.008
发表时间：
2006-09-01
影响因子：
4.8
作者：
Lan, W. S.;Gu, J. D.;Qiao, C. L.
通讯作者：
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