arcA基因提高大肠杆菌对有机溶剂的耐受性

Enhanced organic solvent tolerance of Escherichia coli by arc A

【目的】将来源于恶臭假单胞菌(Pseudomonas putida JUCT1)的基因arc A(编码精氨酸脱亚胺酶)整合到Escherichia coli JM109(DE3)基因组中,以提高该菌对有机溶剂的耐受性。【方法】以P.putida JUCT1的基因组为模板扩增基因arc A,并与p ET-20b(+)连接后导入E.coli JM109(DE3)中,验证该基因提高E.coli JM109(DE3)对有机溶剂的耐受性。利用Red同源重组的方法将arc A整合到E.coli JM109(DE3)基因组中。【结果】E.coli JM109(DE3)/p ET-20b(+)-arc A在添加了2.0%(体积比)环己烷、0.1%(体积比)甲苯、4.0%(体积比)萘烷和0.1%(体积比)丁醇的培养基中培养8 h后,其OD660由初始的0.2分别上升到0.8、0.9、1.8和1.3。将arc A成功整合到E.coli JM109(DE3)基因组中,获得了具有较好遗传稳定性的溶剂耐受E.coli JM109(DE3)宿主菌株。【结论】外源基因arc A能提高大肠杆菌菌株的有机溶剂耐受性,为工业化应用中耐溶剂微生物菌株的构建提供了实验依据和理论基础。

[Objective] Gene arc A encoding arginine deiminase from Pseudomonas putida JUCT1 was integrated into the genome of Escherichia coli JM109（DE3） to enhance its organic solvent tolerance（OST）. [Methods] Using genome of P. putida as template, we amplified arc A gene and expressed it in strain E. coli JM109（DE3）. Subsequently, arc A gene was integrated into the genome of E. coli JM109（DE3） by red-mediated recombination to obtain a strain with an inheritable OST phenotype. [Results] When growing in 2.0%（V/V） cyclohexane, 0.1%（V/V） toluene, 4.0%（V/V） decalin and 0.1%（V/V） butanol, the OD660 values of recombinant E. coli JM109（DE3）/p ET-20b（＋）-arc A could reach about 0.8, 0.9, 1.8 and 1.3, respectively. [Conclusion] Our results demonstrate that microbial OST could be enhanced by the expression of arc A gene. Importantly, this work provides experimental data and molecular basis for constructing OST bacteria for industrial applications.