Halomonas sp.QHL1四氢嘧啶合成基因簇ectABC与上游调控序列的克隆及其功能分析
|
摘要
目的克隆中度嗜盐菌Halomonas sp.QHL1中的四氢嘧啶合成基因簇及上游调控序列并进行功能分析。方法通过PCR技术从Halomonas sp.QHL1中克隆获得ectABC基因簇及上游启动子序列(promoter+ectABC),将其与表达载体pColdⅠDNA连接,转化至E.coil BL21(DE3),用SDS-PAGE分析目的基因的异源表达、盐耐受实验检测重组菌株的耐盐能力。结果克隆得到大小为3335 bp的promoter+ectABC基因序列,并在E.coil BL21(DE3)中成功异源表达,重组菌株盐耐受能力明显提高。结论四氢嘧啶生物合成基因操纵子序列能够在E.coil BL21(DE3)中异源表达,且含有该序列的工程菌株耐盐能力显著提高。
Objective To clone and characterize the genes involved in the biosynthetic pathway of ectoine and its upstream regulation sequence of Halomonas sp.QHL1.Methods PCR was used to obtain the ectABC gene cluster and its upstream promoter(promoter+ectABC),and then connected with expression vector pColdⅠDNA and transformed into E.coil BL21(DE3).The heterologous expression of recombinant strain in E.coil BL21(DE3)was confirmed by SDS-PAGE.The ability of salt tolerance of recombinant strain was detected by salt tolerance experiments.Results The ectABC gene cluster and its upstream promoter 3 335 bp was cloned and heterologously expressed successfully in E.coil BL21(DE3).Compared with the native E.coil BL21,the ability of recombinant strains resistant to salt was significantly improved.Conclusion The operon sequences of ectoine biosynthesis gene could be heterologously expressed in E.coil BL21(DE3)and the salt tolerance of the engineered strain was significantly improved.
Objective To clone and characterize the genes involved in the biosynthetic pathway of ectoine and its upstream regulation sequence of Halomonas sp.QHL1.Methods PCR was used to obtain the ectABC gene cluster and its upstream promoter(promoter+ectABC),and then connected with expression vector pColdⅠDNA and transformed into E.coil BL21(DE3).The heterologous expression of recombinant strain in E.coil BL21(DE3)was confirmed by SDS-PAGE.The ability of salt tolerance of recombinant strain was detected by salt tolerance experiments.Results The ectABC gene cluster and its upstream promoter 3 335 bp was cloned and heterologously expressed successfully in E.coil BL21(DE3).Compared with the native E.coil BL21,the ability of recombinant strains resistant to salt was significantly improved.Conclusion The operon sequences of ectoine biosynthesis gene could be heterologously expressed in E.coil BL21(DE3)and the salt tolerance of the engineered strain was significantly improved.
引文
[1]Ning Y,Wu X,Zhang C,et al.Pathway construction and metabolic engineering for fermentative production of ectoine in Escherichia coli[J] .Metabolic Engineering,2016,36:10-18.
[2]朱德锐,韩睿,沈国平,等。青海湖盐单胞菌Ectoine合成基因簇ectABC的重组共表达[J]。水生生物学报,2015,39(2):358-367。
[3]He Y Z,Gong J,Yu H Y,et al.High production of ectoine from aspartate and glycerol by use of whole-cell biocatalysis in recombinant Escherichia coli[J].Microbial Cell Factories,2015,14(1):55.
[4]Dwivedi M,Backers H,Harishchandra R K,et al.Biophysical investigations of the structure and function of the tear fluid lipid layer and the effect of ectoine.Part A:Natural meibomian lipid films[J].Biochimica et Biophysica Acta(BBA)-Biomembranes,2014,1838(10):2708-2715.
[5]Sonnemann U,Scherner O,Nina Werkh?user.Treatment of Rhinitis Sicca Anterior with Ectoine Containing Nasal Spray[J].Journal of Allergy,2014,2014:273219.
[6]Eichel A.Meta-Analysis of the Efficacy of Ectoine Nasal Spray in Patients with Allergic Rhinoconjunctivitis[J].Journal of Allergy,2014,2014(6):292545.
[7]周洋,张学俊,王红,等。四氢嘧啶对人冰冻红细胞质量影响的初步研究[J]。中国输血杂志,2017,30(3):271-275。
[8]徐蕊。四氢嘧啶合成工程菌株构建[D]。大连:大连海事大学,2012。
[9]朱德锐。青海湖嗜盐菌多样性与四氢嘧啶的生物合成机制研究[D]。武汉:华中师范大学,2014。
[10]Forster D,Behnke A,Stoeck T.Meta-analyses of environmental sequence data identify anoxia and salinity as parameters shaping ciliate communities[J].Systematics and Biodiversity,2012,10(3):277-288.
[11]PedrósAlió,Carlos.Trophic ecology of solar salterns[M].Halophilic Microorganisms.Springer Berlin Heidelberg,2004.
[12]Roesser M,V Müller.Osmoadaptation in bacteria and archaea:common principles and differences[J].Environmental Microbiology,2002,3(12):743-754.
[13]Bownik A,St?pniewska,Zofia.Ectoine as a promising protective agent in humans and animals[J].Arhiv Za Higijenu Rada I Toksikologiju,2016,67(4):260.
[14]Galinski E A,Pfeiffer H P,Trüper H G.1,4,5,6-Tetrahydro-2-methyl-4-pyrimidinecarboxylic acid.A novel cyclic amino acid from halophilic phototrophic bacteria of the genus Ectothiorhodospira[J].European Journal of Biochemistry,2010,149(1):135-139.
[15]Swan B K,Ehrhardt C J,Reifel K M,et al.Archaeal and bacterial communities respond differently to environmental gradients in anoxic sediments of a California hypersaline lake,the Salton Sea[J].Applied & Environmental Microbiology,2010,76(3):757-768.
[16]Grammann K,Volke A,Kunte H J.New type of osmoregulated solute transporter identified in halophilic members of the bacteria domain:TRAP transporter TeaABC mediates uptake of ectoine and hydroxyectoine in Halomonas elongata DSM 2581T[J].Journal of Bacteriology,2002,184:3078-3085.
[17]Street TO,Krukenberg KA,Rosgen J,t al.Osmolyte-induced conformational changes in the Hsp90 molecular chaperone[J].Protein Science,2010,19(11):57-65.
[18]Tanimura K,Nakayama H,Tanaka T,et al.Ectoine production from lignocellulosic biomass-derived sugars by engineered Halomonas elongata[J].Bioresource Technology,2013,142(8):523-529.
[19]Reshetnikov A S,Khmelenina V N,Mustakhimov I I,et al.Diversity and phylogeny of the ectoine biosynthesis genes in aerobic,moderately halophilic methylotrophic bacteria[J].Extremophiles Life Under Extreme Conditions,2011,15(6):653-656.
[20]Schwibbert K,Marinsanguino A,Bagyan I,et al.A blueprint of ectoine metabolism from the genome of the industrial producer Halomonas elongata DSM 2581T[J].Environmental Microbiology,2011,13(8):1973-1994.
[21]唐娜,曹姣,马怀远,等。Halomonas socia NY-011～T中四氢嘧啶合成基因簇ectABC和ectD的克隆及其功能鉴定[J]。应用与环境生物学报,2017,23(1):140-145。
[22]张慧艳,刘芝瑾,徐同,等。易变链霉菌TRM 45540四氢嘧啶合成相关基因的克隆[J]。西北农业学报,2017,26(3):471-476。
[23]高波,马怡茗,洪煜,等。密旋链霉菌Act12中四氢嘧啶合成基因簇的鉴定及其功能分析[J]。西北农林科技大学学报(自然科学版),2017,45(6):213-220。
[24]Louis P,Galinski E A.Characterization of genes for the biosynthesis of the compatible solute ectoine from Marinococcus halophilus and osmoregulated expression in Escherichia coli[J].Microbiology,1997,143(4):1141-1149.
[2]朱德锐,韩睿,沈国平,等。青海湖盐单胞菌Ectoine合成基因簇ectABC的重组共表达[J]。水生生物学报,2015,39(2):358-367。
[3]He Y Z,Gong J,Yu H Y,et al.High production of ectoine from aspartate and glycerol by use of whole-cell biocatalysis in recombinant Escherichia coli[J].Microbial Cell Factories,2015,14(1):55.
[4]Dwivedi M,Backers H,Harishchandra R K,et al.Biophysical investigations of the structure and function of the tear fluid lipid layer and the effect of ectoine.Part A:Natural meibomian lipid films[J].Biochimica et Biophysica Acta(BBA)-Biomembranes,2014,1838(10):2708-2715.
[5]Sonnemann U,Scherner O,Nina Werkh?user.Treatment of Rhinitis Sicca Anterior with Ectoine Containing Nasal Spray[J].Journal of Allergy,2014,2014:273219.
[6]Eichel A.Meta-Analysis of the Efficacy of Ectoine Nasal Spray in Patients with Allergic Rhinoconjunctivitis[J].Journal of Allergy,2014,2014(6):292545.
[7]周洋,张学俊,王红,等。四氢嘧啶对人冰冻红细胞质量影响的初步研究[J]。中国输血杂志,2017,30(3):271-275。
[8]徐蕊。四氢嘧啶合成工程菌株构建[D]。大连:大连海事大学,2012。
[9]朱德锐。青海湖嗜盐菌多样性与四氢嘧啶的生物合成机制研究[D]。武汉:华中师范大学,2014。
[10]Forster D,Behnke A,Stoeck T.Meta-analyses of environmental sequence data identify anoxia and salinity as parameters shaping ciliate communities[J].Systematics and Biodiversity,2012,10(3):277-288.
[11]PedrósAlió,Carlos.Trophic ecology of solar salterns[M].Halophilic Microorganisms.Springer Berlin Heidelberg,2004.
[12]Roesser M,V Müller.Osmoadaptation in bacteria and archaea:common principles and differences[J].Environmental Microbiology,2002,3(12):743-754.
[13]Bownik A,St?pniewska,Zofia.Ectoine as a promising protective agent in humans and animals[J].Arhiv Za Higijenu Rada I Toksikologiju,2016,67(4):260.
[14]Galinski E A,Pfeiffer H P,Trüper H G.1,4,5,6-Tetrahydro-2-methyl-4-pyrimidinecarboxylic acid.A novel cyclic amino acid from halophilic phototrophic bacteria of the genus Ectothiorhodospira[J].European Journal of Biochemistry,2010,149(1):135-139.
[15]Swan B K,Ehrhardt C J,Reifel K M,et al.Archaeal and bacterial communities respond differently to environmental gradients in anoxic sediments of a California hypersaline lake,the Salton Sea[J].Applied & Environmental Microbiology,2010,76(3):757-768.
[16]Grammann K,Volke A,Kunte H J.New type of osmoregulated solute transporter identified in halophilic members of the bacteria domain:TRAP transporter TeaABC mediates uptake of ectoine and hydroxyectoine in Halomonas elongata DSM 2581T[J].Journal of Bacteriology,2002,184:3078-3085.
[17]Street TO,Krukenberg KA,Rosgen J,t al.Osmolyte-induced conformational changes in the Hsp90 molecular chaperone[J].Protein Science,2010,19(11):57-65.
[18]Tanimura K,Nakayama H,Tanaka T,et al.Ectoine production from lignocellulosic biomass-derived sugars by engineered Halomonas elongata[J].Bioresource Technology,2013,142(8):523-529.
[19]Reshetnikov A S,Khmelenina V N,Mustakhimov I I,et al.Diversity and phylogeny of the ectoine biosynthesis genes in aerobic,moderately halophilic methylotrophic bacteria[J].Extremophiles Life Under Extreme Conditions,2011,15(6):653-656.
[20]Schwibbert K,Marinsanguino A,Bagyan I,et al.A blueprint of ectoine metabolism from the genome of the industrial producer Halomonas elongata DSM 2581T[J].Environmental Microbiology,2011,13(8):1973-1994.
[21]唐娜,曹姣,马怀远,等。Halomonas socia NY-011～T中四氢嘧啶合成基因簇ectABC和ectD的克隆及其功能鉴定[J]。应用与环境生物学报,2017,23(1):140-145。
[22]张慧艳,刘芝瑾,徐同,等。易变链霉菌TRM 45540四氢嘧啶合成相关基因的克隆[J]。西北农业学报,2017,26(3):471-476。
[23]高波,马怡茗,洪煜,等。密旋链霉菌Act12中四氢嘧啶合成基因簇的鉴定及其功能分析[J]。西北农林科技大学学报(自然科学版),2017,45(6):213-220。
[24]Louis P,Galinski E A.Characterization of genes for the biosynthesis of the compatible solute ectoine from Marinococcus halophilus and osmoregulated expression in Escherichia coli[J].Microbiology,1997,143(4):1141-1149.