利用大肠埃希氏菌光控基因表达系统降解多菌灵农残
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摘要
利用一种新型的重组大肠埃希氏菌光控基因诱导表达系统来生产多菌灵水解酶,用于环境中多菌灵农药残留的降解。该光控系统基于光敏融合蛋白LEV1在光照条件下发生同源二聚化并抑制下游基因转录的原理。SDS-PAGE和酶联免疫实验结果表明,异源多菌灵水解酶基因可通过光控机制被超表达,生产的多菌灵水解酶在溶液和土壤样品中具有降解多菌灵农药残留的活性。本技术利用光作为诱导剂,具备廉价易得、无毒害、可随时添加或去除等优势,是一种有前景并可大规模生产生物酶产品的工具。
A newly developed Escherichia coli light-regulated gene expression system was used to produce methyl-1 h-benzimidazol-2-yl carbamate(MBC)hydrolyzing esterase for degrading residual pesticide MBC in environment. This light-regulated system is based on dimerization of photosensitive protein LEV1 upon light exposure to repress the expression of downstream gene. SDS-PAGE and ELISA results showed that the gene of MBC hydrolyzing esterase could be overexpressed in our light-regulated system,by which the MBC hydrolyzing esterase was produced and it had enzymatic activity of degrading pesticide MBC in both liquid solution and soil samples. Light as an inducer,given its many advantages such as cheap,easy to be obtained,non-toxic,and added/removed at any time,is a promising tool for the industrial production of enzymes.
A newly developed Escherichia coli light-regulated gene expression system was used to produce methyl-1 h-benzimidazol-2-yl carbamate(MBC)hydrolyzing esterase for degrading residual pesticide MBC in environment. This light-regulated system is based on dimerization of photosensitive protein LEV1 upon light exposure to repress the expression of downstream gene. SDS-PAGE and ELISA results showed that the gene of MBC hydrolyzing esterase could be overexpressed in our light-regulated system,by which the MBC hydrolyzing esterase was produced and it had enzymatic activity of degrading pesticide MBC in both liquid solution and soil samples. Light as an inducer,given its many advantages such as cheap,easy to be obtained,non-toxic,and added/removed at any time,is a promising tool for the industrial production of enzymes.
引文
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[11]Wang YS, Huang YJ, Chen WC, et al. Effect of carbendazim and pencycuron on soil bacterial community[J]. Journal of Hazard Materials, 2009, 172:84-91.
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[13]Lee JM, Lee J, Kim T, et al. Switchable gene expression in Escherichia coli using a miniaturized photobioreactor[J]. PLoS One,2013, 8(1):e52382.
[14]Cho HS, Seo SW, Kim YM, et al. Engineering glyceraldehyde-3-phosphate dehydrogenase for switching control of glycolysis in Escherichia coli[J]. Biotechnology Bioengineering, 2012, 109:2612-2619.
[15]Schein CH. Production of soluble recombinant proteins in bacteria[J]. Nat Biotech, 1989, 7:1141-1149.
[16]Menart V, Jevsevar S, Vilar M, et al. Constitutive versus thermoinducible expression of heterologous proteins in Escherichia coli based on strong PR, PL promoters from phage lambda[J].Biotechnology Bioengineering, 2003, 83:181-190.
[17]Rinas U. Synthesis rates of cellular proteins involved in translation and protein folding are strongly altered in response to overproduction of basic fibroblast growth factor by recombinant Escherichia coli[J]. Biotechnology Progress, 1996, 12:196-200.
[18]Lee SK, Keasling JD. Propionate-regulated high-yield protein production in Escherichia coli[J]. Biotechnology Bioengineering,2006, 93:912-918.
[19]Jana S, Deb JK. Strategies for efficient production of heterologous proteins in Escherichia coli[J]. Applied Microbiological Biotechnology, 2005, 67:289-298.
[20]Young DD, Deiters A. Photochemical activation of protein expression in bacterial cells[J]. Angewandte Chemistru International Edition, 2007, 46:4290-4292.
[21]Drepper T, Krauss U, Berstenhorst SMZ, et al. Lights on and action!Controlling microbial gene expression by light[J]. Applied Microbiology Biotechnology, 2001, 90(1):23-40.
[22]Kang DG, Lim GB, Cha, HJ. Functional periplasmic secretion of organophosphorous hydrolase using the twin-arginine translocation pathway in Escherichia coli[J]. Journal of Biotechnology, 2005,118:379-385.
[23]Yang C, Fredual F, Qiao C. Export of methyl parathion hydrolase to the periplasm by the Twin-arginine translocation pathway in Escherichia coli[J]. Journal of Agricultural Food Chemistry,2009, 57:8901-8905.
[24]Pandey G, Dorrian SJ, Russell RJ, et al. Cloning and biochemical characterization of a novel carbendazim(methyl-1H-benzimidazol-2-ylcarbamate)-hydrolyzing esterase from the newly isolated Nocardioides sp. strain SG-4G and its potential for use in enzymatic bioremediation[J]. Applied Environmental Microbiology, 2010,76(9):2940-2945.
[25]马正才,刘韧玫,杨弋.光激活基因表达系统在大肠杆菌中的应用[J/OL].中国科技论文在线, 2014.
[26]Chen X, Liu R, Ma Z, et al. An extraordinary stringent and sensitive light-switchable gene expression system for bacterial cells[J].Cell Research, 2016, 26(7):854-847.
[27]Shimizu-Sato S, Huq E, Tepperman JM, et al. A light-switchable gene promoter system[J]. Nat Biotech, 2002, 20:1041-1044.
[28]Levskaya A, Chevalier AA, Tabor JJ, et al. Synthetic biology:Engineering Escherichia coli to see light[J]. Nature, 2005, 438:441-442.
[29]Liang B, Lu P, Li HH, et al. Biodegradation of fomesafen by strain Lysinibacillus sp. ZB-1 isolated from soil[J]. Chemosphere,2009, 77:1614-1619.
[30]Mulbry WW, Karns JS. Purification and characterization of three parathion bacterial strains[J]. Applied Environmental Microbiology, 1989, 55(2):289-293.
[31]Munnecke DM. Enzymatic detoxification of waste organophosphate pesticides[J]. Agricultural Food Chemistry, 1980, 28:105-111.
[32]Munnecke DM, Hsieh PD. Microbial decontamination of parathion and p-nitrophenol in aqueous media[J]. Applied Microbiology,1974, 28(2):212-217.
[33]Fang H, Wang YQ, Gao CM, et al. Isolation and characterization of Pseudomonas sp. CBW capable of degrading carbendazim[J].Biodegradation, 2010, 21(6):939-946.
[34]Richins RD, Kaneva I, Mulchandani A, et al. Biodegradation of organophosphorus pesticides by surface-expressed organophosphorus hydrolase[J]. Nat Biotech, 1997, 15:984-987.
[35]王德正,吴辉,李志敏,叶勤.重组大肠杆菌发酵生产谷胱甘肽的氨基酸添加策略优化[J].生物技术通报, 2015, 31(9):197-203.
[36]Jongbloed JDH, Grieger U, Antelmann H, et al. Two minimal Tat translocasesin in Bacillus[J]. Molecular Microbiology. 2004,54:1319-1325.
[2]Sandahl M, Mathiasson L, Jonsson, JA. Determination of thiophanatemethyl and its metabolites at thrace level in spiked natural water using the supported liquid membrane extraction and the microporous membrane liquid-liquid extraction techniques combined on-line with high performance liquid chromatography[J]. Journal of Chromatography Application, 2000, 893:123-131.
[3]Boudina A, Emmelin C, Baaliouamer A. Photochemical behaviour of carbendazim in aqueous solution[J]. Chemosphere, 2003, 50:649-655.
[4]Kiigemagi U, Inman RD, Mellenthin WM, et al.. Residues of benomyl(determined as carbendazim)and captan in postharvesttreated pears in cold storage[J]. Journal of Agricultural Food Chemistry, 1991, 39:400-403.
[5]华小梅,单正军.我国农药的生产、使用状况及其污染环境因子分析[J].环境科学进展, 1996, 4(2):33-44.
[6]Lei J, Ren L, Hu, SB. The Characterization and bioremediation potential of degradation-enzyme from a newly isolated carbendazimdegrading Pseudomonas putida strain djl-1B[J]. International Journal of Simulation System, Science&Technology, 2015, 16(4B):178.
[7]张桂山,贾小明,马晓航,等.一株多菌灵降解细菌的分离、鉴定及系统发育分析[J].微生物学报, 2004, 44(4):417-421.
[8]田连生,陈菲. T2-2菌株对多菌灵的降解特性及生物修复试验[J].微生物学报, 2009, 49(7):925-930.
[9]Zhang XJ, Huang YJ, Harvey PR, et al. Isolation and characterization of carbendazim-degrading Rhodococcus erythropolis djl-11[J].PLoS One, 2013, 8(10):e74810.
[10]Wang Z, Wang Y, Gong F, et al. Biodegradation of carbendazim by a novel actino bacterium Rhodococcus jialingiae djl-6-2[J].Chemsphere, 2010, 81(5):639-644.
[11]Wang YS, Huang YJ, Chen WC, et al. Effect of carbendazim and pencycuron on soil bacterial community[J]. Journal of Hazard Materials, 2009, 172:84-91.
[12]MazellierP,LeroyE,LaatJT,LegubeB.Degradationof carbendazim by UV/H2O2investigatedbykineticmodelling[J].Environmental Chemistry Letter, 2003, 1:68-72.
[13]Lee JM, Lee J, Kim T, et al. Switchable gene expression in Escherichia coli using a miniaturized photobioreactor[J]. PLoS One,2013, 8(1):e52382.
[14]Cho HS, Seo SW, Kim YM, et al. Engineering glyceraldehyde-3-phosphate dehydrogenase for switching control of glycolysis in Escherichia coli[J]. Biotechnology Bioengineering, 2012, 109:2612-2619.
[15]Schein CH. Production of soluble recombinant proteins in bacteria[J]. Nat Biotech, 1989, 7:1141-1149.
[16]Menart V, Jevsevar S, Vilar M, et al. Constitutive versus thermoinducible expression of heterologous proteins in Escherichia coli based on strong PR, PL promoters from phage lambda[J].Biotechnology Bioengineering, 2003, 83:181-190.
[17]Rinas U. Synthesis rates of cellular proteins involved in translation and protein folding are strongly altered in response to overproduction of basic fibroblast growth factor by recombinant Escherichia coli[J]. Biotechnology Progress, 1996, 12:196-200.
[18]Lee SK, Keasling JD. Propionate-regulated high-yield protein production in Escherichia coli[J]. Biotechnology Bioengineering,2006, 93:912-918.
[19]Jana S, Deb JK. Strategies for efficient production of heterologous proteins in Escherichia coli[J]. Applied Microbiological Biotechnology, 2005, 67:289-298.
[20]Young DD, Deiters A. Photochemical activation of protein expression in bacterial cells[J]. Angewandte Chemistru International Edition, 2007, 46:4290-4292.
[21]Drepper T, Krauss U, Berstenhorst SMZ, et al. Lights on and action!Controlling microbial gene expression by light[J]. Applied Microbiology Biotechnology, 2001, 90(1):23-40.
[22]Kang DG, Lim GB, Cha, HJ. Functional periplasmic secretion of organophosphorous hydrolase using the twin-arginine translocation pathway in Escherichia coli[J]. Journal of Biotechnology, 2005,118:379-385.
[23]Yang C, Fredual F, Qiao C. Export of methyl parathion hydrolase to the periplasm by the Twin-arginine translocation pathway in Escherichia coli[J]. Journal of Agricultural Food Chemistry,2009, 57:8901-8905.
[24]Pandey G, Dorrian SJ, Russell RJ, et al. Cloning and biochemical characterization of a novel carbendazim(methyl-1H-benzimidazol-2-ylcarbamate)-hydrolyzing esterase from the newly isolated Nocardioides sp. strain SG-4G and its potential for use in enzymatic bioremediation[J]. Applied Environmental Microbiology, 2010,76(9):2940-2945.
[25]马正才,刘韧玫,杨弋.光激活基因表达系统在大肠杆菌中的应用[J/OL].中国科技论文在线, 2014.
[26]Chen X, Liu R, Ma Z, et al. An extraordinary stringent and sensitive light-switchable gene expression system for bacterial cells[J].Cell Research, 2016, 26(7):854-847.
[27]Shimizu-Sato S, Huq E, Tepperman JM, et al. A light-switchable gene promoter system[J]. Nat Biotech, 2002, 20:1041-1044.
[28]Levskaya A, Chevalier AA, Tabor JJ, et al. Synthetic biology:Engineering Escherichia coli to see light[J]. Nature, 2005, 438:441-442.
[29]Liang B, Lu P, Li HH, et al. Biodegradation of fomesafen by strain Lysinibacillus sp. ZB-1 isolated from soil[J]. Chemosphere,2009, 77:1614-1619.
[30]Mulbry WW, Karns JS. Purification and characterization of three parathion bacterial strains[J]. Applied Environmental Microbiology, 1989, 55(2):289-293.
[31]Munnecke DM. Enzymatic detoxification of waste organophosphate pesticides[J]. Agricultural Food Chemistry, 1980, 28:105-111.
[32]Munnecke DM, Hsieh PD. Microbial decontamination of parathion and p-nitrophenol in aqueous media[J]. Applied Microbiology,1974, 28(2):212-217.
[33]Fang H, Wang YQ, Gao CM, et al. Isolation and characterization of Pseudomonas sp. CBW capable of degrading carbendazim[J].Biodegradation, 2010, 21(6):939-946.
[34]Richins RD, Kaneva I, Mulchandani A, et al. Biodegradation of organophosphorus pesticides by surface-expressed organophosphorus hydrolase[J]. Nat Biotech, 1997, 15:984-987.
[35]王德正,吴辉,李志敏,叶勤.重组大肠杆菌发酵生产谷胱甘肽的氨基酸添加策略优化[J].生物技术通报, 2015, 31(9):197-203.
[36]Jongbloed JDH, Grieger U, Antelmann H, et al. Two minimal Tat translocasesin in Bacillus[J]. Molecular Microbiology. 2004,54:1319-1325.