透明颤菌血红蛋白基因在龟裂链霉菌中的表达及其对土霉素合成的影响
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摘要
目的考察透明颤菌血红蛋白基因(vgb)在大肠埃希菌(E.coli)和龟裂链霉菌(S.rimosus)中的表达,研究在不同溶氧条件下表达的透明颤菌血红蛋白(VHb)对龟裂链霉菌生长和土霉素合成的影响。方法将vgb插入表达型质粒pET-28a中,导入E.coli BL21(DE3)用以表达VHb并测定其活性。以整合型质粒pSET152为载体,利用erm E*启动子组成型表达vgb基因,将构建的质粒通过接合转移整合到S.rimosus M4018基因组上,利用CO差示光谱法和SDS-PAGE鉴定两个重组菌中的血红蛋白。在5L罐上考察不同溶氧条件下VHb的表达对重组龟裂链霉菌生长和土霉素合成的影响。结果重组大肠埃希菌和重组龟裂链霉菌都能够表达具有生物活性的VHb,发酵结果表明,在117h时与对照菌相比,重组龟裂链霉菌在高氧和低氧条件下的干重分别提高了6%和32%,同时单位干重土霉素的产量分别提高了41%和93%。结论 VHb在重组龟裂链霉菌中成功表达,改善了菌体的生长和土霉素的合成,为解决工业发酵过程中氧限制问题提供了策略。
Objective To investigate the expression of Vitreoscilla hemoglobin gene(vgb) in E. coli and Streptomyces and study the effects of expressed Vitreoscilla hemoglobin(VHb) on the growth and oxytetracycline(OTC) production in S. rimosus grown under different dissolved oxygen concentrations. Methods The vgb gene was inserted into the expression plasmid pET-28 a, and the generated plasmid was introduced into E. coli BL21(DE3) to express VHb. In addition, a pSET152-derived integrative plasmid was constructed with vgb gene expression under the control of a constitutive promoter Perm E*, and it was introduced into S. rimosus M4018 chromosome through conjugation. The VHb was identified with CO difference spectrum analysis and SDS-PAGE. The effects of VHb expression on the growth and OTC biosynthesis of recombinant S. rimosus grown under conditions with different dissolved oxygen were studied in a 5 L fermentor. Results Both recombinant E. coli and S. rimosus were able to express biologically active VHb. Compared with the control strain M4018, the dry weights of recombinant S. rimosus cultivated with high or low dissolved oxygen concentrations were increased by 6% and 32% at 117 h, respectively. Meanwhile, the OTC productions per dry cell weight were increased by 41% and 93% at 117 h, respectively. Conclusion The successful expression of VHb in the recombinant S. rimosus could improve its growth and OTC yield, providing an effective strategy to solve the oxygen limitation problem during the fermentation process.
Objective To investigate the expression of Vitreoscilla hemoglobin gene(vgb) in E. coli and Streptomyces and study the effects of expressed Vitreoscilla hemoglobin(VHb) on the growth and oxytetracycline(OTC) production in S. rimosus grown under different dissolved oxygen concentrations. Methods The vgb gene was inserted into the expression plasmid pET-28 a, and the generated plasmid was introduced into E. coli BL21(DE3) to express VHb. In addition, a pSET152-derived integrative plasmid was constructed with vgb gene expression under the control of a constitutive promoter Perm E*, and it was introduced into S. rimosus M4018 chromosome through conjugation. The VHb was identified with CO difference spectrum analysis and SDS-PAGE. The effects of VHb expression on the growth and OTC biosynthesis of recombinant S. rimosus grown under conditions with different dissolved oxygen were studied in a 5 L fermentor. Results Both recombinant E. coli and S. rimosus were able to express biologically active VHb. Compared with the control strain M4018, the dry weights of recombinant S. rimosus cultivated with high or low dissolved oxygen concentrations were increased by 6% and 32% at 117 h, respectively. Meanwhile, the OTC productions per dry cell weight were increased by 41% and 93% at 117 h, respectively. Conclusion The successful expression of VHb in the recombinant S. rimosus could improve its growth and OTC yield, providing an effective strategy to solve the oxygen limitation problem during the fermentation process.
引文
[1]Petkovic H,Cullum J,Hranueli D,et al.Genetics of Streptomyces rimosus,the oxytetracycline producer[J].Microbiol Mol Biol Rev,2006,70(3):704-728.
[2]Pickens L B,Tang Y.Oxytetracycline biosynthesis[J].J Biol Chem,2010,285(36):27509-27515.
[3]唐振宇,郭美锦,张嗣良.土霉素生物合成研究进展[J].中国抗生素杂志,2009,34(10):581-587.
[4]Wakabayashi S,Matsubara H,Webster D A.Primary sequence of a dimeric bacterial haemoglobin from Vitreoscilla[J].Nature,1986,322(6078):481-483.
[5]Chi P,Webster D A,Stark B C.Vitreoscilla hemoglobin aids respiration under hypoxic conditions in its native host[J].Microbiol Res,2009,164(3):267-275.
[6]Park K,Kim K,Howard A J,et al.Vitreoscilla hemoglobin binds to subunit I of cytochrome bo ubiquinol oxidases[J].JBiol Chem,2002,277(36):33334-33337.
[7]Khosla C,Bailey J E.Heterologous expression of a bacterial haemoglobin improves the growth properties of recombinant Escherichia coli[J].Nature,1988,331(6157):633-635.
[8]Khosravi M,Webster D A,Stark B C.Presence of the bacterial hemoglobin gene improvesα-amylase production of a recombinant Escherichia coli strain[J].Plasmid,1990,24(3):190-194.
[9]Zhang L,Li Y,Wang Z,et al.Recent developments and future prospects of Vitreoscilla hemoglobin application in metabolic engineering[J].Biotechnol Adv,2007,25(2):123-136.
[10]Stark B C,Pagilla K R,Dikshit K L.Recent applications of Vitreoscilla hemoglobin technology in bioproduct synthesis and bioremediation[J].Appl Microbiol Biotechnol,2015,99(4):1627-1636.
[11]Stark B C,Dikshit K L,Pagilla K R.Recent advances in understanding the structure,function,and biotechnological usefulness of the hemoglobin from the bacterium Vitreoscilla[J].Biotechnol Lett,2011,33(9):1705-1714.
[12]文莹,宋渊,李季伦.透明颤菌血红蛋白在肉桂地链霉菌中的表达对其细胞生长及抗生素合成的影响[J].生物工程学报,2001,17(1):24-28.
[13]江曙,陈代杰,戈梅,等.透明颤菌血红蛋白基因在吸水链霉菌NND-52-C中的克隆与表达[J].南京农业大学学报,2004,27(4):60-63.
[14]马正,申屠旭萍,边亚琳,等.透明颤菌血红蛋白基因在淀粉酶产色链霉菌中的表达及对合成丰加霉素的影响[J].微生物学通报,2013,40(7):1175-1185.
[15]Wang S H,Liu F,Hou Z W,et al.Enhancement of natamycin production on Streptomyces gilvosporeus by chromosomal integration of the Vitreoscilla hemoglobin gene(vgb)[J].World J Microbiol Biotechnol,2014,30(4):1369-1376.
[16]余志拓,沈晓放,吴远杰,等.在吸水链霉菌中表达透明颤菌血红蛋白基因提高子囊霉素产量[J].中国医药工业杂志,2017,48(6):844-849.
[17]MacNeil D J,Gewain K M,Ruby C L,et al.Analysis of Streptomyces avermitilis genes required for avermectin biosynthesis utilizing a novel integration vector[J].Gene,1992,111(1):61-68.
[18]Rhodes P M,Hunter I S,Friend E J,et al.Recombinant DNA methods for the oxytetracycline producer Streptomyces rimosus[J].Biochem Soc Trans,1984,12(4):586-587.
[19]Sambrook J,Russell D W.Molecular cloning:a laboratory manual[M].3rd ed.New York:Cold Spring Harbor Laboratory press,2001.
[20]Hopwood D A,Bibb M J,Chater K F,et al.Genetic manipulation of Streptomyces:A laboratory manual[M].Norwich:John Innes Foundation,1985.
[21]朱怡非,朱春宝.透明颤菌血红蛋白基因在链霉菌中的克隆和表达[J].中国医药工业杂志,1998,29(6):253-258.
[22]李建武,肖能庚,余瑞元,等.生物化学实验原理和方法[M].北京:北京大学出版社,1994.
[23]Yu L,Cao N,Wang L,et al.Oxytetracycline biosynthesis improvement in Streptomyces rimosus following duplication of minimal PKS genes[J].Enzyme Microb Technol,2012,50(6-7):318-324.
[24]Khosla C,Bailey J E.The Vitreoscilla hemoglobin gene:Molecular cloning,nucleotide sequence and genetic expression in Escherichia coli[J].Mol Gen Genet,1988,214(1):158-161.
[25]Liu C Y,Webster D A.Spectral characteristics and interconversions of the reduced,oxidized,and oxygenated forms of purified cytochrome o[J].J Biol Chem,1974,249(13):4261-4266.
[26]Dubendorf J W,Studier F W.Controlling basal expression in an inducible T7 expression system by blocking the target T7 promoter with lac repressor[J].J Mol Biol,1991,219(1):45-59.
[27]张锋,赵晓瑜,周艳芬.低氧诱导透明颤菌血红蛋白基因在大肠埃希菌中的表达[J].华北农学报,2006,21(S1):20-22.
[28]祁艳霞,杨明明,高文,等.透明颤菌血红蛋白基因(vgb)在大肠埃希菌中的高效表达[J].黑龙江畜牧兽医,2005,(8):7-9.
[29]Geckil H,Stark B C,Webster D A.Cell growth and oxygen uptake of Escherichia coli and Pseudomonas aeruginosa are differently effected by the genetically engineered Vitreoscilla hemoglobin gene[J].J Biotechnol,2001,85(1):57-66.
[30]谢昌贤,刘运添,王鹏飞.高表达透明颤菌血红蛋白的金色链霉菌工程菌的生长特性研究[J].当代畜牧,2012,(1):44-46.
[31]谢帅,周海娇,田威,等.透明颤菌血红蛋白基因(vgb)在那西肽产生菌-活跃链霉菌中的表达[J].沈阳药科大学学报,2011,28(6):490-494.
[32]高峰,陈太春,苟丽霞,等.透明颤菌血红蛋白基因(vgb)表达对委内瑞拉链霉菌秦岭变种生长代谢及瑞拉菌素效价的影响[J].农业生物技术学报,2010,18(6):1182-1188.
[2]Pickens L B,Tang Y.Oxytetracycline biosynthesis[J].J Biol Chem,2010,285(36):27509-27515.
[3]唐振宇,郭美锦,张嗣良.土霉素生物合成研究进展[J].中国抗生素杂志,2009,34(10):581-587.
[4]Wakabayashi S,Matsubara H,Webster D A.Primary sequence of a dimeric bacterial haemoglobin from Vitreoscilla[J].Nature,1986,322(6078):481-483.
[5]Chi P,Webster D A,Stark B C.Vitreoscilla hemoglobin aids respiration under hypoxic conditions in its native host[J].Microbiol Res,2009,164(3):267-275.
[6]Park K,Kim K,Howard A J,et al.Vitreoscilla hemoglobin binds to subunit I of cytochrome bo ubiquinol oxidases[J].JBiol Chem,2002,277(36):33334-33337.
[7]Khosla C,Bailey J E.Heterologous expression of a bacterial haemoglobin improves the growth properties of recombinant Escherichia coli[J].Nature,1988,331(6157):633-635.
[8]Khosravi M,Webster D A,Stark B C.Presence of the bacterial hemoglobin gene improvesα-amylase production of a recombinant Escherichia coli strain[J].Plasmid,1990,24(3):190-194.
[9]Zhang L,Li Y,Wang Z,et al.Recent developments and future prospects of Vitreoscilla hemoglobin application in metabolic engineering[J].Biotechnol Adv,2007,25(2):123-136.
[10]Stark B C,Pagilla K R,Dikshit K L.Recent applications of Vitreoscilla hemoglobin technology in bioproduct synthesis and bioremediation[J].Appl Microbiol Biotechnol,2015,99(4):1627-1636.
[11]Stark B C,Dikshit K L,Pagilla K R.Recent advances in understanding the structure,function,and biotechnological usefulness of the hemoglobin from the bacterium Vitreoscilla[J].Biotechnol Lett,2011,33(9):1705-1714.
[12]文莹,宋渊,李季伦.透明颤菌血红蛋白在肉桂地链霉菌中的表达对其细胞生长及抗生素合成的影响[J].生物工程学报,2001,17(1):24-28.
[13]江曙,陈代杰,戈梅,等.透明颤菌血红蛋白基因在吸水链霉菌NND-52-C中的克隆与表达[J].南京农业大学学报,2004,27(4):60-63.
[14]马正,申屠旭萍,边亚琳,等.透明颤菌血红蛋白基因在淀粉酶产色链霉菌中的表达及对合成丰加霉素的影响[J].微生物学通报,2013,40(7):1175-1185.
[15]Wang S H,Liu F,Hou Z W,et al.Enhancement of natamycin production on Streptomyces gilvosporeus by chromosomal integration of the Vitreoscilla hemoglobin gene(vgb)[J].World J Microbiol Biotechnol,2014,30(4):1369-1376.
[16]余志拓,沈晓放,吴远杰,等.在吸水链霉菌中表达透明颤菌血红蛋白基因提高子囊霉素产量[J].中国医药工业杂志,2017,48(6):844-849.
[17]MacNeil D J,Gewain K M,Ruby C L,et al.Analysis of Streptomyces avermitilis genes required for avermectin biosynthesis utilizing a novel integration vector[J].Gene,1992,111(1):61-68.
[18]Rhodes P M,Hunter I S,Friend E J,et al.Recombinant DNA methods for the oxytetracycline producer Streptomyces rimosus[J].Biochem Soc Trans,1984,12(4):586-587.
[19]Sambrook J,Russell D W.Molecular cloning:a laboratory manual[M].3rd ed.New York:Cold Spring Harbor Laboratory press,2001.
[20]Hopwood D A,Bibb M J,Chater K F,et al.Genetic manipulation of Streptomyces:A laboratory manual[M].Norwich:John Innes Foundation,1985.
[21]朱怡非,朱春宝.透明颤菌血红蛋白基因在链霉菌中的克隆和表达[J].中国医药工业杂志,1998,29(6):253-258.
[22]李建武,肖能庚,余瑞元,等.生物化学实验原理和方法[M].北京:北京大学出版社,1994.
[23]Yu L,Cao N,Wang L,et al.Oxytetracycline biosynthesis improvement in Streptomyces rimosus following duplication of minimal PKS genes[J].Enzyme Microb Technol,2012,50(6-7):318-324.
[24]Khosla C,Bailey J E.The Vitreoscilla hemoglobin gene:Molecular cloning,nucleotide sequence and genetic expression in Escherichia coli[J].Mol Gen Genet,1988,214(1):158-161.
[25]Liu C Y,Webster D A.Spectral characteristics and interconversions of the reduced,oxidized,and oxygenated forms of purified cytochrome o[J].J Biol Chem,1974,249(13):4261-4266.
[26]Dubendorf J W,Studier F W.Controlling basal expression in an inducible T7 expression system by blocking the target T7 promoter with lac repressor[J].J Mol Biol,1991,219(1):45-59.
[27]张锋,赵晓瑜,周艳芬.低氧诱导透明颤菌血红蛋白基因在大肠埃希菌中的表达[J].华北农学报,2006,21(S1):20-22.
[28]祁艳霞,杨明明,高文,等.透明颤菌血红蛋白基因(vgb)在大肠埃希菌中的高效表达[J].黑龙江畜牧兽医,2005,(8):7-9.
[29]Geckil H,Stark B C,Webster D A.Cell growth and oxygen uptake of Escherichia coli and Pseudomonas aeruginosa are differently effected by the genetically engineered Vitreoscilla hemoglobin gene[J].J Biotechnol,2001,85(1):57-66.
[30]谢昌贤,刘运添,王鹏飞.高表达透明颤菌血红蛋白的金色链霉菌工程菌的生长特性研究[J].当代畜牧,2012,(1):44-46.
[31]谢帅,周海娇,田威,等.透明颤菌血红蛋白基因(vgb)在那西肽产生菌-活跃链霉菌中的表达[J].沈阳药科大学学报,2011,28(6):490-494.
[32]高峰,陈太春,苟丽霞,等.透明颤菌血红蛋白基因(vgb)表达对委内瑞拉链霉菌秦岭变种生长代谢及瑞拉菌素效价的影响[J].农业生物技术学报,2010,18(6):1182-1188.