金霉素生物合成基因ctcK的研究
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摘要
为检测ctcK基因的功能,利用基因工程技术在金色链霉菌J13(Streptomyces aureofaciens J13)上构建ctcK基因缺失工程菌SK12(ΔctcK),并分析了其次级代谢产物的变化.经质谱分析显示,工程菌SK12代谢物中检测到去甲基金霉素m/z=465.10[M+H]~+的分子离子峰,但未检测到金霉素m/z=479.12[M+H]~+的分子离子峰,这与出发菌J13代谢物的检测结果相反.结果表明,ctcK基因的失活阻断了金霉素的生物合成代谢流,使工程菌SK12主要积累去甲基金霉素,显示ctcK基因参与金霉素C-6位甲基化.本研究初步阐明了ctcK基因的功能,同时获得了一株主产去甲基金霉素的工程菌.
In order to detect the function of ctcK,the ctcK deletion mutant S. aureofaciens SK12( ΔctcK) was constructed in S. aureofaciens J13 by genetic engineering. Changes of its secondary metabolites were analyzed. The molecular ion peak of demethylchlortetracycline( m / z = 465. 10 [M + H]~+) instead of chlortetracycline( m/z =479. 12 [M + H]~+) was detected in the metabolites of mutant strain SK12 by MS analyses. This is in contrast to the detection result of metabolites of parent strain J13. These results indicate that the inactivation of ctcK interdicts chlortetracycline biosynthesis flow and lead to the main accumulation of demethylchlortetracycline in mutant strain SK12,revealing the successful methylation of C-6 of chlortetracycline with ctcK involved. In this study,the function of the ctcK was preliminarily elucidated. An engineered bacteria that mainly produces monocomponent demethylchlortetracycline has been simultaneously obtained.
In order to detect the function of ctcK,the ctcK deletion mutant S. aureofaciens SK12( ΔctcK) was constructed in S. aureofaciens J13 by genetic engineering. Changes of its secondary metabolites were analyzed. The molecular ion peak of demethylchlortetracycline( m / z = 465. 10 [M + H]~+) instead of chlortetracycline( m/z =479. 12 [M + H]~+) was detected in the metabolites of mutant strain SK12 by MS analyses. This is in contrast to the detection result of metabolites of parent strain J13. These results indicate that the inactivation of ctcK interdicts chlortetracycline biosynthesis flow and lead to the main accumulation of demethylchlortetracycline in mutant strain SK12,revealing the successful methylation of C-6 of chlortetracycline with ctcK involved. In this study,the function of the ctcK was preliminarily elucidated. An engineered bacteria that mainly produces monocomponent demethylchlortetracycline has been simultaneously obtained.
引文
[1]NGUYEN F,STAROST A L,ARENZ S,et al.Tetracycline antibiotics and resistance mechanisms[J].Biol Chem,2014,395(5):559-575.
[2]NELSON K L,BROZEL V S,GIBSON S A,et al.Influence of manure from pigs fed chlortetracycline as growth promotant on soil microbial community structure[J].World J Microb Biot,2011,27(3):659-668.
[3]WEN B,LIU Y,WANG P,et al.Toxic effects of chlortetracycline on maize growth,reactive oxygen species generation and the antioxidant response[J].J Environ Sci China,2012,24(6):1099-1105.
[4]CHOPRA I,ROBERTS M.Tetracycline antibiotics:mode of action,applications,molecular biology,and epidemiology of bacterial resistance[J].Microbiol Mol Biol R,2001,65(2):232-260.
[5]PAUTKE C,VOGT S,KREUTZER K,et al.Characterization of eight different tetracyclines:advances in fluorescence bone labeling[J].J Anat,2010,217(1):76-82.
[6]余彦国,张瑾.四环素类药物的非抗菌作用机制与非抗感染临床应用[J].中国兽药杂志,2012,46(1):54-56.
[7]裴立忠,王勇平,张鹏,等.四环素类抗生素生产状况与市场发展前景[J].中国兽药杂志,2015,49(1):64-68.
[8]MCCORMICK J R,JENSEN E R.Biosynthesis of the Tetracyclines.VIII.1 Characterization of 4-hydroxy-6-methylpretetramid[J].J Am Chem Soc,1965,87(8):1794-1795.
[9]RYAN M J.Strain for the production of 6-dimethyltetracycline,method for producing the strain and vector for use in the method:US,5989903[P],1999-11-23[2015-12-26].http://www.dwz.cz/32xi.
[10]NAKANO T,MIYAKE K,ENDO H,et al.Identification and cloning of the gene involved in the final step of chlortetracycline biosynthesis in Streptomyces aureofaciens[J].Biosci Biotech Bioch,2004,68(6):1345-1352.
[11]ZHU T,DENG Z X,YOU D L,et al.Deciphering and engineering of the final step halogenase for improved chlortetracycline biosynthesis in industrial Streptomyces aureofaciens[J].Metab Eng,2013,19:69-78.
[12]张南燕,洪文荣,林玉双,等.打靶载体p IJ792的构建[J].海峡药学,2010,20(4):164-167.
[13]方志锴,洪文荣,严凌斌,等.金色链霉菌接合转移体系的构建[J].福建农林大学学报(自然科学版),2011,40(5):521-524.
[14]陈梁军.金霉素发酵工艺研究[J].海峡药学,2010,22(6):23-25.
[15]刘慰,秦浩,刘霜,等.一株枯草芽胞杆菌的分离鉴定及其杀虫活性的研究[J].湖南师范大学自然科学学报,2014,37(5):14-20.
[16]TOBIAS K,BIBB M,MARK J B,et al.Practical streptomyces genetics[M].Norwich:The John Innes Foundation,2000.
[17]SAMBROOK J F,RUSSELL D W.Molecular Cloning:A Laboratory Manual[M].New York:Cold Spring Harbor Laboratory Press,2001.
[18]程惠芳,刘金玲,啜淑英.产去甲基金霉素突变株的选育Ⅱ.金霉素链霉菌635突变株的选育[J].微生物学报,1974,14(1):74-76.
[19]ZHANG W J,WATANABE K,CLAY C,et al.Investigation of early tailoring reaction in the oxytetracycline biosynthetic pathway[J].J Biol Chem,2007,282(35):25717-25725.
[2]NELSON K L,BROZEL V S,GIBSON S A,et al.Influence of manure from pigs fed chlortetracycline as growth promotant on soil microbial community structure[J].World J Microb Biot,2011,27(3):659-668.
[3]WEN B,LIU Y,WANG P,et al.Toxic effects of chlortetracycline on maize growth,reactive oxygen species generation and the antioxidant response[J].J Environ Sci China,2012,24(6):1099-1105.
[4]CHOPRA I,ROBERTS M.Tetracycline antibiotics:mode of action,applications,molecular biology,and epidemiology of bacterial resistance[J].Microbiol Mol Biol R,2001,65(2):232-260.
[5]PAUTKE C,VOGT S,KREUTZER K,et al.Characterization of eight different tetracyclines:advances in fluorescence bone labeling[J].J Anat,2010,217(1):76-82.
[6]余彦国,张瑾.四环素类药物的非抗菌作用机制与非抗感染临床应用[J].中国兽药杂志,2012,46(1):54-56.
[7]裴立忠,王勇平,张鹏,等.四环素类抗生素生产状况与市场发展前景[J].中国兽药杂志,2015,49(1):64-68.
[8]MCCORMICK J R,JENSEN E R.Biosynthesis of the Tetracyclines.VIII.1 Characterization of 4-hydroxy-6-methylpretetramid[J].J Am Chem Soc,1965,87(8):1794-1795.
[9]RYAN M J.Strain for the production of 6-dimethyltetracycline,method for producing the strain and vector for use in the method:US,5989903[P],1999-11-23[2015-12-26].http://www.dwz.cz/32xi.
[10]NAKANO T,MIYAKE K,ENDO H,et al.Identification and cloning of the gene involved in the final step of chlortetracycline biosynthesis in Streptomyces aureofaciens[J].Biosci Biotech Bioch,2004,68(6):1345-1352.
[11]ZHU T,DENG Z X,YOU D L,et al.Deciphering and engineering of the final step halogenase for improved chlortetracycline biosynthesis in industrial Streptomyces aureofaciens[J].Metab Eng,2013,19:69-78.
[12]张南燕,洪文荣,林玉双,等.打靶载体p IJ792的构建[J].海峡药学,2010,20(4):164-167.
[13]方志锴,洪文荣,严凌斌,等.金色链霉菌接合转移体系的构建[J].福建农林大学学报(自然科学版),2011,40(5):521-524.
[14]陈梁军.金霉素发酵工艺研究[J].海峡药学,2010,22(6):23-25.
[15]刘慰,秦浩,刘霜,等.一株枯草芽胞杆菌的分离鉴定及其杀虫活性的研究[J].湖南师范大学自然科学学报,2014,37(5):14-20.
[16]TOBIAS K,BIBB M,MARK J B,et al.Practical streptomyces genetics[M].Norwich:The John Innes Foundation,2000.
[17]SAMBROOK J F,RUSSELL D W.Molecular Cloning:A Laboratory Manual[M].New York:Cold Spring Harbor Laboratory Press,2001.
[18]程惠芳,刘金玲,啜淑英.产去甲基金霉素突变株的选育Ⅱ.金霉素链霉菌635突变株的选育[J].微生物学报,1974,14(1):74-76.
[19]ZHANG W J,WATANABE K,CLAY C,et al.Investigation of early tailoring reaction in the oxytetracycline biosynthetic pathway[J].J Biol Chem,2007,282(35):25717-25725.