尿黑酸双加氧酶基因工程菌的构建
|
摘要
为了构建不同表达类型的尿黑酸双加氧酶(Dio6)基因工程菌,本研究利用Over-lap PCR技术使双加氧酶基因在组成型启动子(P2和P_(spoⅠ-Ⅱ))的控制下表达;利用高效液相色谱(HPLC)技术测定XJ-6、XJ-6+P_(T7)、XJ-6+P2、XJ-6+P_(spoⅠ-Ⅱ)、P_(spoⅠ-Ⅱ)、P2、P_(T7)在7 d对芘的降解。研究结果表明:构建得到3种启动子控制表达的基因工程菌,协同效应研究表明工程菌在XJ-6的协助下可以在含芘的无机盐培养基中生长,并且组成型启动子工程菌的菌数远高于诱导型启动子工程菌的菌数。组成型启动子相较于诱导型启动子工程菌能更稳定表达外源双加氧酶基因,由于诱导型启动子工程菌需要诱导剂IPTG,并且表达双加氧酶基因会受到诱导剂浓度、诱导温度等条件的影响,所以在实际应用中组成型启动子工程菌优于诱导型启动子工程菌。
In order to construct different expression types of engineering bacteria that express homogentisate dioxygenase(Dio6 gene), Overlap PCR technology was used to make the expressions of dioxygenase under the control of the constitutive promoter(P2 and P_(spoⅠ-Ⅱ)), and then the degradation rates of pyrene by the strains of XJ-6, XJ-6+P_(T7), XJ-6+P2, XJ-6+P_(spo Ⅰ-Ⅱ), P_(spo Ⅰ-Ⅱ), P2 and P_(T7) were determined by HPLC. The research results showed that genetically engineered bacteria controlled by three promoters were successfully constructed in this research. Synergistic effects showed that genetically engineered bacteria with the help of XJ-6 strain could grow in inorganic salt medium containing pyrene, and the microbial content of the constitutive promoter genetically engineered bacteria are more than that of the inducible promoter's. Compared to the inducible promoter, the constitutive promoter genetically engineered bacteria could stably express exogenous dioxygenase more.Because inducible promoter genetically engineered bacteria needs IPTG and the expression of dioxygenase was affected by the concentration of IPTG, induction temperature and so on, therefore, the constitutive promoter genetically engineered bacteria should be superior to the inducible promoter genetically engineered bacteria in a practical application.
In order to construct different expression types of engineering bacteria that express homogentisate dioxygenase(Dio6 gene), Overlap PCR technology was used to make the expressions of dioxygenase under the control of the constitutive promoter(P2 and P_(spoⅠ-Ⅱ)), and then the degradation rates of pyrene by the strains of XJ-6, XJ-6+P_(T7), XJ-6+P2, XJ-6+P_(spo Ⅰ-Ⅱ), P_(spo Ⅰ-Ⅱ), P2 and P_(T7) were determined by HPLC. The research results showed that genetically engineered bacteria controlled by three promoters were successfully constructed in this research. Synergistic effects showed that genetically engineered bacteria with the help of XJ-6 strain could grow in inorganic salt medium containing pyrene, and the microbial content of the constitutive promoter genetically engineered bacteria are more than that of the inducible promoter's. Compared to the inducible promoter, the constitutive promoter genetically engineered bacteria could stably express exogenous dioxygenase more.Because inducible promoter genetically engineered bacteria needs IPTG and the expression of dioxygenase was affected by the concentration of IPTG, induction temperature and so on, therefore, the constitutive promoter genetically engineered bacteria should be superior to the inducible promoter genetically engineered bacteria in a practical application.
引文
Andrea S.D.S.,Flavio A.D.,Robson A.,Rodrigo J.S.,Daiana B.B.,and Fatima M.B.,2012,Enzymatic activity of catechol-1,2-dioxygenase and catechol-2,3-dioxygenase produced by Gordonia polyisoprenivorans,Quim.Nova.,35(8):1587-1592
Bai P.,Zhong M.,Zhao Y.Y.,Yang F.,Lin J.W.,Li H.G.,and Ma H.,2012,The culture conditions of pyrene degrading bacteria ZQ5 and the effect of inorganic elements on its degradation efficiency,Huanjinggongcheng Xuebao(China Journal Of Environmental Engineering),6(7):2411-2416(白鹏,钟鸣,赵媛媛,杨峰,林景卫,李浩戈,马慧,2012,芘高效降解菌ZQ5的培养条件及无机元素对其降解效率的影响,环境工程学报,6(7):2411-2416)
Chen K.,Zhu Q.,Qian Y.G.,Song Y.,Yao J.,and Martin M.F.C.,2013,Microcalorimetric investigation of the effect of non-ionic surfactant on biodegradation of pyrene by PAH-degrading bacteria Burkholderia cepacia,Ecotox.Environ.Safe.,98(12):361-367
Cheng Z.D.,Chao Q.F.,Yang B.Y.,Yao X.Y.,and Yin D.D.,2012,Degradation conditions optimization and degradation genes of a pyrene degrading bacterium B2,Huanjinggongcheng Xuebao(China Journal Of Environmental Engineering),6(10):3795-3800(陈志丹,晁群芳,杨滨银,姚小云,尹丹丹,2012,一株芘降解菌B2的降解条件优化及降解基因,环境工程学报,6(10):3795-3800)
Debajyoti G.,Shreya G.,Tapan K.D.,and Youngho A.,2016,Current state of knowledge in microbial degradation of polycyclic aromatic hydrocarbons(PAHs):A review,Front Microbiol.,7(386):1369
Diao S.,Wang H.Q.,Xue J.,and Zhao Y.C.,2017,Screening,identification and degradation characteristic of low temperature resistant pyrene degrading bacteria,Zhongguo Huanjingkexue(China Environmental Science),37(2):677-685(刁硕,王红旗,许洁,赵一村,2017,低温耐盐芘降解菌的筛选鉴定及降解特性研究,中国环境科学,37(2):677-685)
Huang L.,2016,Expression,purification and functional study of moderately gentisicacid1,2-dioxygenase of halophilic Martelella sp.AD-3,Thesis for M.S.,East China University of Science,Supervisor:Liu Y.D.,pp.12-20(黄玲,2016,中度嗜盐菌Martelella sp.AD-3龙胆酸1,2-双加氧酶的表达、纯化及功能研究,硕士学位论文,华东理工大学,导师:刘勇弟,pp.12-20)
Huang X.R.,Zhang C.W.,Zhang R.J.,Zhang J.,and Zhang X.X.,2016,Screening,identification and degradation characteristics of polycyclic aromatic hydrocarbons degrading bacteria,Weishengwuxue Tongbao(Microbiology China),43(5):965-973(黄兴如,张彩文,张瑞杰,章俊,张晓霞,2016,多环芳烃降解菌的筛选、鉴定及降解特性,微生物学通报,43(5):965-973)
Janine T.L.,and Ridwaan M.,2015,Purification and characterization of catechol 1,2-dioxygenase from Acinetobacter sp.Y64 Strain and Escherichia coli transformants,Protein J.,34(6):421-433
Katarzyna H.,Agnieszka S.,Danuta W.,and Urszula G.,2013,Cloning and mutagenesis of catechol 2,3-dioxygenase gene from the gram-positive Planococcus sp.strain S5,J.Mol.Microb.Biotech.,23(6):381-390
Li Q.X.,Fan B.Q.,Gong M.B.,and Yang H.,2008,Isolation,characterization and degradation characteristics of pyrene degrading Mycobacterium M11,Huanjingkexue(Environmental Science),29(3):763-768(李全霞,范丙全,龚明波,杨慧,2008,降解芘的分枝杆菌M11的分离鉴定和降解特性,环境科学,29(3):763-768)
Nadaf N.H.,and Ghosh J.S.,2011,Purification and characterization of catechol-1,2-dioxygenase from Rhodococcus sp.NCIM 2891,Res.J.Environ.Earth Sci.,3(5):608-613
Sen G.,Parth S.,Banerjee S.,and Bandyopadhyay A.K.,2013,Sequence,Structural and functional characterization of homogentisate-1,2-dioxygenase of homo sapiens:an in silico analysis,Am.J.Bioinfor.Res.,3(3):42-61
Si X.P.,2011,Growth characteristics of polycyclic aromatic hydrocarbons degrading bacteria and their application in soil remediation,Thesis for M.S.,Chongqing Technology and Business University,Supervisor:Shao C.B.,pp.48-51(思显佩,2011,多环芳烃降解菌的生长特性及其在土壤修复中的应用,硕士学位论文,重庆工商大学,导师:邵承斌,pp.48-51)
Suenaga H.,Mizuta S.,Miyazaki K.,and Yaoi K.,2014,Diversity of extradiol dioxygenases in aromatic-degrading microbial community explored using both culture-dependent and culture-independent approaches,FEMS Microbiol.Ecol.,90(2):367-379
Sun K.,Liu J.,Li X.,and Lin W.T.,2014,Isolation,screening of two endophytic Bacteria with pyrene degrading function,Shengtaixuebao(Acta Ecologica Sinica),34(4):853-861(孙凯,刘娟,李欣,凌婉婷,2014,两株具有芘降解功能的植物内生细菌的分离筛选及其特性,生态学报,34(4):853-861)
Wang H.,2011,Bioremediation of soil contaminated by polycyclic aromatic hydrocarbons(PAHs)in situ,Dissertation for Ph.D.,Northeastern University,Supervisor:Sun T.H.,pp.36-38(王洪,2011,多环芳烃污染农田土壤原位生物修复技术研究,博士学位论文,东北大学,导师:孙铁珩,pp.36-38)
Wei K.,Chen S.N.,Yi H.,Ye J.S.,Peng H.,and Liu Z.H.,2016,Study on degradation characteristics and degrading enzymes of Bacillus cereus to pyrene,Huanjingkexue Xuebao(Acta Science Circumstantiae),36(2):506-512(卫昆,陈烁娜,尹华,叶锦韶,彭辉,刘则华,2016,蜡状芽胞杆菌对芘的降解特性及降解酶研究,环境科学学报,36(2):506-512)
Wu Y.C.,Teng Y.,Li Z.G.,Liao X.W.,and Luo Y.M.,2008,Potential role of polycyclic aromatic hydrocarbons(PAHs)oxidation by fungal laccase in the remediation of an aged contaminated soil,Soil Biol.Biochem.,40(3):789-796
Xu L.,Xiong W.,and Yang J.K.,2016,Effect of four types of promoters on the growth rate of Escherichia coli under Campylobacter jejuni hemoglobin,Zhongguo Shengwu Gongcheng Zazhi(China Biotechnology),36(2):43-50(许力,熊炜,杨江科,2016,四种类型启动子控制下空肠弯曲杆菌血红蛋白对大肠杆菌生长速度的影响,中国生物工程杂志,36(2):43-50)
Yang Y.,Yang J.K.,Xiong W.,Liang J.F.,Duan W.W.,and Chao Q.F.,2016,Aeromonas sp.XJ-6 oxygenase gene cloning,expression and degradation of tyrosine,Zhongguo Shengwu Gongcheng Zazhi(China Biotechnology),36(5):59-67(杨杨,杨江科,熊炜,梁建芳,段魏魏,晁群芳,2016,Aeromonas sp.XJ-6双加氧酶基因的克隆、表达及对酪氨酸的降解,中国生物工程杂志,36(5):59-67)
Youmee H.,Younghee J.,Daeyoung K.,Youngsoo K.,Chykyung K.,and Kyunghee M.,2000,Reaction characteristics of4-methylcatechool 2,3-dioxygenase from Pseudomonas putida SU10,J.Microbiol.Biotechn.,10(1):35-42
Zhang N.,Yi H.,Pen H.,He B.Y.,Ye J.S.,Qin H.M.,and Long Y.,2010,Degradation characteristics of pyrene by Stenotrophomonas maltophilia J12,Huanjing Kexue Yu Jishu(Environmental Scinence&Technology),33(4):6-9(张娜,尹华,彭辉,何宝燕,叶锦韶,秦华明,龙焰,2010,嗜麦芽窄食单胞菌J12对芘的降解特性,环境科学与技术,33(4):6-9)
Bai P.,Zhong M.,Zhao Y.Y.,Yang F.,Lin J.W.,Li H.G.,and Ma H.,2012,The culture conditions of pyrene degrading bacteria ZQ5 and the effect of inorganic elements on its degradation efficiency,Huanjinggongcheng Xuebao(China Journal Of Environmental Engineering),6(7):2411-2416(白鹏,钟鸣,赵媛媛,杨峰,林景卫,李浩戈,马慧,2012,芘高效降解菌ZQ5的培养条件及无机元素对其降解效率的影响,环境工程学报,6(7):2411-2416)
Chen K.,Zhu Q.,Qian Y.G.,Song Y.,Yao J.,and Martin M.F.C.,2013,Microcalorimetric investigation of the effect of non-ionic surfactant on biodegradation of pyrene by PAH-degrading bacteria Burkholderia cepacia,Ecotox.Environ.Safe.,98(12):361-367
Cheng Z.D.,Chao Q.F.,Yang B.Y.,Yao X.Y.,and Yin D.D.,2012,Degradation conditions optimization and degradation genes of a pyrene degrading bacterium B2,Huanjinggongcheng Xuebao(China Journal Of Environmental Engineering),6(10):3795-3800(陈志丹,晁群芳,杨滨银,姚小云,尹丹丹,2012,一株芘降解菌B2的降解条件优化及降解基因,环境工程学报,6(10):3795-3800)
Debajyoti G.,Shreya G.,Tapan K.D.,and Youngho A.,2016,Current state of knowledge in microbial degradation of polycyclic aromatic hydrocarbons(PAHs):A review,Front Microbiol.,7(386):1369
Diao S.,Wang H.Q.,Xue J.,and Zhao Y.C.,2017,Screening,identification and degradation characteristic of low temperature resistant pyrene degrading bacteria,Zhongguo Huanjingkexue(China Environmental Science),37(2):677-685(刁硕,王红旗,许洁,赵一村,2017,低温耐盐芘降解菌的筛选鉴定及降解特性研究,中国环境科学,37(2):677-685)
Huang L.,2016,Expression,purification and functional study of moderately gentisicacid1,2-dioxygenase of halophilic Martelella sp.AD-3,Thesis for M.S.,East China University of Science,Supervisor:Liu Y.D.,pp.12-20(黄玲,2016,中度嗜盐菌Martelella sp.AD-3龙胆酸1,2-双加氧酶的表达、纯化及功能研究,硕士学位论文,华东理工大学,导师:刘勇弟,pp.12-20)
Huang X.R.,Zhang C.W.,Zhang R.J.,Zhang J.,and Zhang X.X.,2016,Screening,identification and degradation characteristics of polycyclic aromatic hydrocarbons degrading bacteria,Weishengwuxue Tongbao(Microbiology China),43(5):965-973(黄兴如,张彩文,张瑞杰,章俊,张晓霞,2016,多环芳烃降解菌的筛选、鉴定及降解特性,微生物学通报,43(5):965-973)
Janine T.L.,and Ridwaan M.,2015,Purification and characterization of catechol 1,2-dioxygenase from Acinetobacter sp.Y64 Strain and Escherichia coli transformants,Protein J.,34(6):421-433
Katarzyna H.,Agnieszka S.,Danuta W.,and Urszula G.,2013,Cloning and mutagenesis of catechol 2,3-dioxygenase gene from the gram-positive Planococcus sp.strain S5,J.Mol.Microb.Biotech.,23(6):381-390
Li Q.X.,Fan B.Q.,Gong M.B.,and Yang H.,2008,Isolation,characterization and degradation characteristics of pyrene degrading Mycobacterium M11,Huanjingkexue(Environmental Science),29(3):763-768(李全霞,范丙全,龚明波,杨慧,2008,降解芘的分枝杆菌M11的分离鉴定和降解特性,环境科学,29(3):763-768)
Nadaf N.H.,and Ghosh J.S.,2011,Purification and characterization of catechol-1,2-dioxygenase from Rhodococcus sp.NCIM 2891,Res.J.Environ.Earth Sci.,3(5):608-613
Sen G.,Parth S.,Banerjee S.,and Bandyopadhyay A.K.,2013,Sequence,Structural and functional characterization of homogentisate-1,2-dioxygenase of homo sapiens:an in silico analysis,Am.J.Bioinfor.Res.,3(3):42-61
Si X.P.,2011,Growth characteristics of polycyclic aromatic hydrocarbons degrading bacteria and their application in soil remediation,Thesis for M.S.,Chongqing Technology and Business University,Supervisor:Shao C.B.,pp.48-51(思显佩,2011,多环芳烃降解菌的生长特性及其在土壤修复中的应用,硕士学位论文,重庆工商大学,导师:邵承斌,pp.48-51)
Suenaga H.,Mizuta S.,Miyazaki K.,and Yaoi K.,2014,Diversity of extradiol dioxygenases in aromatic-degrading microbial community explored using both culture-dependent and culture-independent approaches,FEMS Microbiol.Ecol.,90(2):367-379
Sun K.,Liu J.,Li X.,and Lin W.T.,2014,Isolation,screening of two endophytic Bacteria with pyrene degrading function,Shengtaixuebao(Acta Ecologica Sinica),34(4):853-861(孙凯,刘娟,李欣,凌婉婷,2014,两株具有芘降解功能的植物内生细菌的分离筛选及其特性,生态学报,34(4):853-861)
Wang H.,2011,Bioremediation of soil contaminated by polycyclic aromatic hydrocarbons(PAHs)in situ,Dissertation for Ph.D.,Northeastern University,Supervisor:Sun T.H.,pp.36-38(王洪,2011,多环芳烃污染农田土壤原位生物修复技术研究,博士学位论文,东北大学,导师:孙铁珩,pp.36-38)
Wei K.,Chen S.N.,Yi H.,Ye J.S.,Peng H.,and Liu Z.H.,2016,Study on degradation characteristics and degrading enzymes of Bacillus cereus to pyrene,Huanjingkexue Xuebao(Acta Science Circumstantiae),36(2):506-512(卫昆,陈烁娜,尹华,叶锦韶,彭辉,刘则华,2016,蜡状芽胞杆菌对芘的降解特性及降解酶研究,环境科学学报,36(2):506-512)
Wu Y.C.,Teng Y.,Li Z.G.,Liao X.W.,and Luo Y.M.,2008,Potential role of polycyclic aromatic hydrocarbons(PAHs)oxidation by fungal laccase in the remediation of an aged contaminated soil,Soil Biol.Biochem.,40(3):789-796
Xu L.,Xiong W.,and Yang J.K.,2016,Effect of four types of promoters on the growth rate of Escherichia coli under Campylobacter jejuni hemoglobin,Zhongguo Shengwu Gongcheng Zazhi(China Biotechnology),36(2):43-50(许力,熊炜,杨江科,2016,四种类型启动子控制下空肠弯曲杆菌血红蛋白对大肠杆菌生长速度的影响,中国生物工程杂志,36(2):43-50)
Yang Y.,Yang J.K.,Xiong W.,Liang J.F.,Duan W.W.,and Chao Q.F.,2016,Aeromonas sp.XJ-6 oxygenase gene cloning,expression and degradation of tyrosine,Zhongguo Shengwu Gongcheng Zazhi(China Biotechnology),36(5):59-67(杨杨,杨江科,熊炜,梁建芳,段魏魏,晁群芳,2016,Aeromonas sp.XJ-6双加氧酶基因的克隆、表达及对酪氨酸的降解,中国生物工程杂志,36(5):59-67)
Youmee H.,Younghee J.,Daeyoung K.,Youngsoo K.,Chykyung K.,and Kyunghee M.,2000,Reaction characteristics of4-methylcatechool 2,3-dioxygenase from Pseudomonas putida SU10,J.Microbiol.Biotechn.,10(1):35-42
Zhang N.,Yi H.,Pen H.,He B.Y.,Ye J.S.,Qin H.M.,and Long Y.,2010,Degradation characteristics of pyrene by Stenotrophomonas maltophilia J12,Huanjing Kexue Yu Jishu(Environmental Scinence&Technology),33(4):6-9(张娜,尹华,彭辉,何宝燕,叶锦韶,秦华明,龙焰,2010,嗜麦芽窄食单胞菌J12对芘的降解特性,环境科学与技术,33(4):6-9)