安普霉素生物合成相关基因的研究
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摘要
为了研究黑暗链霉菌安普霉素生物合成基因，首先通过鸟枪克隆的方法克隆其安普霉素抗性基因。提取黑暗链霉菌H6总DNA，经Sau3AI不完全酶切、凝胶电泳，收集6～15kb片段连接到载体pIJ702。连接液转化标准宿主菌S.lividans TK24原生质体，最终得到6个可以在安普霉素平板上生长的阳性克隆。阳性克隆中重组质粒pSYX1含有12kb黑暗链霉菌的DNA，对12kb片段进一步分析将安普霉素抗性基因定位在1.5kb SphI-KpnI片段上。
抗性基因上游2.0kb BamHI-SphI片段测序表明在该片段中有一个完整的ORF，位于877～1995bp之间，编码373个氨基酸。在这个ORF中，碱基组成为109A+388C+489G+136T，G+C含量为78.2％，密码子第三位碱基是G+C的占96.3％，符合链霉菌基因的特点。同源比较分析没有发现同源序列，推测这一序列可能是一未知的新基因，命名为aprA。
将克隆到的链霉菌DNA2.0kb片段以及报告基因ermE、xylE插入到具有oriT的大肠杆菌—链霉菌穿梭质粒pHZ132中构建接合转移质粒pZXB014，并将其转入大肠杆菌ET12567(pUZ8002)中，获得供体菌ET12567(pUZ8002，pZXB014)。通过接合转移的方法将质粒pZXB014导入黑暗链霉菌H6中，筛选基因发生重组的菌株。PCR实验证明基因重组菌株中接合转移质粒同源整合到黑暗链霉菌H6的染色体上。基因重组菌株不能合成安普霉素，证明克隆到的aprA基因参与黑暗链霉菌安普霉素的合成，同时也证明在黑暗链霉菌中安普霉
沈阳药科人学颀上学位论文 摘要
素生物合成基因与安普霉素抗性基因是连锁存在的。
To study the biosynthetics genes of apramycin in Streptomyces tenebrarius, the apramycin resistant gene was isolated by gunshot cloning firstly. S.tenebrarius chromosomal DNA , partially digested with Sau3Al to yield fragments of 6 ~ 15kb, was ligated with vector pIJ702. The ligation mixture was used to transform S.lividans TK24 protoplasts. Six colonies capable of growing in the presence of apramycin were isolated. Plasmid pSYXl, containing a 12kb insert of S.tenebrarius DNA, was isolated from one of six colonies. Apramycin resistant gene was located in 1.5-kb Sphl-Kpnl fragment of plasmid pSYXl though further studies.
The 2.0-kb BamHl-Sphl fragment, upstream fragment of the apramycin resistant gene, was sequenced. Analysis of the nt sequence revealed that it contained an ORF from nt positions 877 to 1995. Composed of 109A, 388C, 489G and 136T, the ORF encoded a protein of 373 amino acids. The G+C percentage of this ORF was 78.2% and that of the third base of the codons was 96.3%. No homologous sequence was found by comparing it with the sequences obtained from the net, so that the cloned gene was supposed to be an unknown new gene, designated as oprA.
A fragment, containing 2.0kb cloned 5. tenebrarius DNA and reported genes of ermE and xylE , was inserted in plasmid pHZ132 ( an
E.coli-Streptomyces shuttle plasmid incorporating oriT from RK2) to construct disruption plasmid pZXB0l4. The plasmid was transformed into E.coli ET12567(pUZ8002) to construct recombinant E.coli ET12867(pUZ8002, pZXB014 ). Recombinant S.tenebrarius was obtained by conjugation E.coli ET12867(pUZ8002, pZXB014) with S.tenebrarius H6 to scan strains of which plasmids integrated into S.tenebrarius H6 chromosomal DNA. The gene recombinant strain NO.42 can't generate ampramycin, which indicated that the cloned gene is involved in apramycin biosynthesis in S.tenebrarius. It was also proved that the biosynthestic genes of apramycin was linked to the apramycin resistant gene in S.tenebrarius.
抗性基因上游2.0kb BamHI-SphI片段测序表明在该片段中有一个完整的ORF，位于877～1995bp之间，编码373个氨基酸。在这个ORF中，碱基组成为109A+388C+489G+136T，G+C含量为78.2％，密码子第三位碱基是G+C的占96.3％，符合链霉菌基因的特点。同源比较分析没有发现同源序列，推测这一序列可能是一未知的新基因，命名为aprA。
将克隆到的链霉菌DNA2.0kb片段以及报告基因ermE、xylE插入到具有oriT的大肠杆菌—链霉菌穿梭质粒pHZ132中构建接合转移质粒pZXB014，并将其转入大肠杆菌ET12567(pUZ8002)中，获得供体菌ET12567(pUZ8002，pZXB014)。通过接合转移的方法将质粒pZXB014导入黑暗链霉菌H6中，筛选基因发生重组的菌株。PCR实验证明基因重组菌株中接合转移质粒同源整合到黑暗链霉菌H6的染色体上。基因重组菌株不能合成安普霉素，证明克隆到的aprA基因参与黑暗链霉菌安普霉素的合成，同时也证明在黑暗链霉菌中安普霉
沈阳药科人学颀上学位论文 摘要
素生物合成基因与安普霉素抗性基因是连锁存在的。
To study the biosynthetics genes of apramycin in Streptomyces tenebrarius, the apramycin resistant gene was isolated by gunshot cloning firstly. S.tenebrarius chromosomal DNA , partially digested with Sau3Al to yield fragments of 6 ~ 15kb, was ligated with vector pIJ702. The ligation mixture was used to transform S.lividans TK24 protoplasts. Six colonies capable of growing in the presence of apramycin were isolated. Plasmid pSYXl, containing a 12kb insert of S.tenebrarius DNA, was isolated from one of six colonies. Apramycin resistant gene was located in 1.5-kb Sphl-Kpnl fragment of plasmid pSYXl though further studies.
The 2.0-kb BamHl-Sphl fragment, upstream fragment of the apramycin resistant gene, was sequenced. Analysis of the nt sequence revealed that it contained an ORF from nt positions 877 to 1995. Composed of 109A, 388C, 489G and 136T, the ORF encoded a protein of 373 amino acids. The G+C percentage of this ORF was 78.2% and that of the third base of the codons was 96.3%. No homologous sequence was found by comparing it with the sequences obtained from the net, so that the cloned gene was supposed to be an unknown new gene, designated as oprA.
A fragment, containing 2.0kb cloned 5. tenebrarius DNA and reported genes of ermE and xylE , was inserted in plasmid pHZ132 ( an
E.coli-Streptomyces shuttle plasmid incorporating oriT from RK2) to construct disruption plasmid pZXB0l4. The plasmid was transformed into E.coli ET12567(pUZ8002) to construct recombinant E.coli ET12867(pUZ8002, pZXB014 ). Recombinant S.tenebrarius was obtained by conjugation E.coli ET12867(pUZ8002, pZXB014) with S.tenebrarius H6 to scan strains of which plasmids integrated into S.tenebrarius H6 chromosomal DNA. The gene recombinant strain NO.42 can't generate ampramycin, which indicated that the cloned gene is involved in apramycin biosynthesis in S.tenebrarius. It was also proved that the biosynthestic genes of apramycin was linked to the apramycin resistant gene in S.tenebrarius.
引文
[1] Chater K F. Microbial development. Cold Spring Harbor Laboratory, USA New York. Cold Spring Harbor,1984
[2] 张蔚文．放线菌次生代谢的分子遗传学研究进展(上)．国外医药抗生素分册，1997，18(1)：1-4
[3] Haruyasu K., Miyuki S. M., Physical characterization of SCP1, a linear plasmid from Streptomyces coelicolor. J. Bacteriology, 1991,173(4):1523-1529
[4] Sergey B. Z., Hildgund S., The linear Streptomyces plasmid pLBl: analyses of transfer functions. MGG, 1994, 242:274-382
[5] 肖湘，周秀芬，邓子新．线性质粒——链霉菌基础生物学研究的一个新热点．国外医药抗生素分册，1999，20(2)：49-53
[6] Hopwood D．A．．链霉菌抗生素生物合成基因的克隆和分析．国外医药抗生素分册，1987,8(2)：112-126
[7] 马云鹏．链霉菌抗生素生物合成基因的克隆策略．国外医药抗生素分册，1990，11(4)：246-248
[8] 肖勇立，袁丽蓉．链霉菌抗生素生物合成基因簇的克隆分析．国外医药抗生素分册，1991，12(4)：241-247
[9] George, H. J., Hopwood D. A.. Molecular cloning and Expression of the phenoxazinone synthase gene from streptomyces antibioticus. The Journal of biological chemistry, 1984(22):14151-14157
[10] Hoopwood D．A．，Chater K．F．．链霉菌分子遗传学研究新进展．国外药学抗生素杂志，1986，7(2)：195-201
[11] Malpartida F., Hopwood D. A.. Molecular cloning of the whole biosynthetic pathyway of a streptomyces antibiotic and it's expression in a heterologous host. Nature 1985, 309:462-464
[12] Ohnuki T., Katoh T., Lmanaka T. et., Molecular cloning of tetracycline resistance genes from Streptomyces rimosus in Streptomyces griseus and characterization of the cloned genes. J. Bacteriol, 1985(161), 3:1010-1016
[13] 王以光．抗生素产生菌链霉菌基因表达与调节．中国抗生素杂志，1988，13(5)：378-382
[14] Rhodes P. M., Hunter I. S., Friend E. J. et.. Recombinant DNA methods for the oxytetracycline producer streptomyces rimosus. Biochem Soc Trans, 1984(12), 4:586-587
[15] Fisherman S. E., Coxk, Larson J. L,et. Cloning genes from the biosynthesis of a warcrolide antibiotic. Pro Natl Acad Sci USA. 1987(84), 23:8248-8252
[16] Binnie C., Warren M., Butler M. J.. Cloning and heterologous expression in streptomyces lividans of streptomyces rimosus genes involved in oxytetracycline biosynthesis. Journal of Bacteriology. 1989,171(2):887-895
[17] 王以光．麦迪霉素生物合成基因克隆研究．生物工程学报，1989，5(4)：261-269
[18] 王冶．反义核酸——研究基因功能的有效工具．国外医学遗传学分册，2002，25(1)：1-3
[19] 纪宗玲，刘继中，陈苏明．基因功能的研究方法．生物工程学报，2002，18(1)：117-120
[20] Yasumasa, Ota, Hideyuki, Tamegai, Fumitaka, Kudo, et al. Butirosin-biosynthetic gene cluser from Bactillus circulans. J. Antibiotics 2000, 53:1158～1167
[21] Rinehart K.L., Jr. & R. M.. Stroshane Biosynthesis of aminocyclitol antibiotics. J. Antibiotics 1976, 29:319～353
[22] Rinehart K. L., Jr. & T Suami(Ed) aminocyclitol antibiotics. ACS Symposium
Series No. 125. American Chemical Society, Washington D. C., 1980
[23] 王启振，郑新立．生物药品化学，沈阳：沈阳药科大学出版社
[24] Juan F, Martin, Paloma Liras Organization and expression of genes involved in the biosynthesis of antibiotics and other secondary metabolites. Annu. Rev. Microbiol., 1989, 43:173～206
[24] Kenneth L. Rinehart, Jr. and Ronald M. Stroshane. Biosynthesis of aminocylitol antibiotics. J. Antibiotics APR. 1976, 319～353
[25] Klaus Pissowotzki, Kambiz Mansouri, Wolfgang Piepersberg Genetics of streptomycin production in Streptomyces griseus:molecular structure and putative function of genes strELMB2N. Mol Gen Genet, 1991, 231:113～123
[26] Mamoru Hasegawa, A novel highly efficient gene-cloning system in Micromonospora applied to the genetic analysis of fortimicin biosynthesis. Gene, 1992, 115:85～91
[27] Tohru Dairi, Toshio Ohta, Eri Hashimoto, et al. Self cloning in Micromonospora olivasterospora of fms genes for fortimicin A (astromicin) biosynthesis. Mol Gen Genet 1992, 232:262～270
[28] Tohru Dairi, Kazuo Yamaguchi, Mamoru Hasegawa. N-Formimidoyl fortimicin A synthsae, a unique oxidase involved in fortimicin A biosynthesis:purification, characterization and geng cloning. Mol Gen Genet 1992, 236:49～59
[29] ohru Dairi, Toshio Ohta, Eri Hashimoto, et al. Organization and nature of fortimicin A (astromicin) biosynehetic genes studies using a cosmid library of Micromonospora olivasterospora DNA. Mol Gen Genet (1992) 236:39～48
[30] Souichi Ikeno, Tomohiro Tsuji, Kenji Higashide, et al. A 7.6kb DNA Region from Streptomyces kasugaensis M338-MI Includes some gengs responsible for Kasugamycin biosynthesis. J. Antibiotics 1998, 51:341～352
[31] Suzukake, K.; K.Tokunaga, H. Hayashi, M. Hori, Y. Uehara, D. Ikeda & H. Umezawa Biosynthesis of 2-de oxystreptamine. J. Antibiotics 1985, 38: 1211～1218
[32] Lucher, L. A. ; Y-M. Chen & J. B. Walker Reactions catalyzed by purified L-glutamine: keto-scyllol-inisitol aminotransferase, an enzyme required for biosynthesis of aminocyclitol antibiotics. Antimicrob. Agents chemother. 1989, 33:452～459
[33] Elisabeth Aubert-Pivert, Julian Davies Biosynthesis of butirosin in Bacillus circulans NRRL B3312: identification by sequence analysis and insertional mutagenesis of the butB gene involved in antibiotic production. Gene 1994, 147:1～11
[34] Fumitaka Kudo, Hideyuki Tamegai, Taketomo Fujiwara, et al. Molecular cloning of the gene for the key carbocycli-forming enzyme in the biosynthesis of 2-Deoxystrptamine-containing Aminocyclitol antibiotics and its comparison with dehydroquinate synthase. J. Antibiotics 1999, 52:559～571
[35] 李天伯，尚广东，夏焕章，等．链霉菌S．tenebrarius H6中与抗生素有关糖生物合成基因的克隆．生物工程学报,2001,17(3)：329～331
[36] Stark, W.M., Hoehe, M.M., Knox, N.G. Nebramycin, a new broad spectrum antibotic complex. Antimicrob Agents Chemother, 1967, 7:314-323
[37] Stark,W.M., Knox,N.G., Wilgus,R.M. Strains of Streptomyces tenebrarius and biosynthesis of nebramycin. Folia Microbiol, 1971, 16(3):205-217
[38] 熊宗贵，周朝辉，刘晓辉，等．尼拉霉素单组分——安普霉素高产菌株的研究．中国抗生素杂志，1997，22(5)：334-339
[39] 李相丰，何建勇，田威，等．安普霉素生物合成途径的研究．中国抗生素杂志，2002，27(2)，97-100
[40] 金冬雁，黎孟枫，等译．分子克隆实验指南．第2版．北京：科学出版社，1993．801
[41] 鲍楷．博士学位论文．华中农业大学．1999
[42] Hopwood,D.A., Bibb,M.J., Chater,K.F., et al. Genetic Manipulation of Streptomyces. A Laboratory Manual. England: John Innes Foundation Press, 1985
[43] Vara, J., Lewandowsk-Skarbek, M., Yiguang, W. Cloning of genes governing the deoxysugar portion of the erythromycin biosynthesis pathway in Saccharopolyspora erythreus. J Bacteriol, 1989, 171:5872-5881
[44] 吴胜．硕士学位论文．沈阳药科大学．2001
[45] David J. H., Daniel D., Gerard T. et al. Cloning of an aminoglycoside-resistance-encoding gene, kamC, from Saccharopolyspora hirsute: comparison with kamB from Streptomyces tenebrarius. Gene,1991,102:19-26
[2] 张蔚文．放线菌次生代谢的分子遗传学研究进展(上)．国外医药抗生素分册，1997，18(1)：1-4
[3] Haruyasu K., Miyuki S. M., Physical characterization of SCP1, a linear plasmid from Streptomyces coelicolor. J. Bacteriology, 1991,173(4):1523-1529
[4] Sergey B. Z., Hildgund S., The linear Streptomyces plasmid pLBl: analyses of transfer functions. MGG, 1994, 242:274-382
[5] 肖湘，周秀芬，邓子新．线性质粒——链霉菌基础生物学研究的一个新热点．国外医药抗生素分册，1999，20(2)：49-53
[6] Hopwood D．A．．链霉菌抗生素生物合成基因的克隆和分析．国外医药抗生素分册，1987,8(2)：112-126
[7] 马云鹏．链霉菌抗生素生物合成基因的克隆策略．国外医药抗生素分册，1990，11(4)：246-248
[8] 肖勇立，袁丽蓉．链霉菌抗生素生物合成基因簇的克隆分析．国外医药抗生素分册，1991，12(4)：241-247
[9] George, H. J., Hopwood D. A.. Molecular cloning and Expression of the phenoxazinone synthase gene from streptomyces antibioticus. The Journal of biological chemistry, 1984(22):14151-14157
[10] Hoopwood D．A．，Chater K．F．．链霉菌分子遗传学研究新进展．国外药学抗生素杂志，1986，7(2)：195-201
[11] Malpartida F., Hopwood D. A.. Molecular cloning of the whole biosynthetic pathyway of a streptomyces antibiotic and it's expression in a heterologous host. Nature 1985, 309:462-464
[12] Ohnuki T., Katoh T., Lmanaka T. et., Molecular cloning of tetracycline resistance genes from Streptomyces rimosus in Streptomyces griseus and characterization of the cloned genes. J. Bacteriol, 1985(161), 3:1010-1016
[13] 王以光．抗生素产生菌链霉菌基因表达与调节．中国抗生素杂志，1988，13(5)：378-382
[14] Rhodes P. M., Hunter I. S., Friend E. J. et.. Recombinant DNA methods for the oxytetracycline producer streptomyces rimosus. Biochem Soc Trans, 1984(12), 4:586-587
[15] Fisherman S. E., Coxk, Larson J. L,et. Cloning genes from the biosynthesis of a warcrolide antibiotic. Pro Natl Acad Sci USA. 1987(84), 23:8248-8252
[16] Binnie C., Warren M., Butler M. J.. Cloning and heterologous expression in streptomyces lividans of streptomyces rimosus genes involved in oxytetracycline biosynthesis. Journal of Bacteriology. 1989,171(2):887-895
[17] 王以光．麦迪霉素生物合成基因克隆研究．生物工程学报，1989，5(4)：261-269
[18] 王冶．反义核酸——研究基因功能的有效工具．国外医学遗传学分册，2002，25(1)：1-3
[19] 纪宗玲，刘继中，陈苏明．基因功能的研究方法．生物工程学报，2002，18(1)：117-120
[20] Yasumasa, Ota, Hideyuki, Tamegai, Fumitaka, Kudo, et al. Butirosin-biosynthetic gene cluser from Bactillus circulans. J. Antibiotics 2000, 53:1158～1167
[21] Rinehart K.L., Jr. & R. M.. Stroshane Biosynthesis of aminocyclitol antibiotics. J. Antibiotics 1976, 29:319～353
[22] Rinehart K. L., Jr. & T Suami(Ed) aminocyclitol antibiotics. ACS Symposium
Series No. 125. American Chemical Society, Washington D. C., 1980
[23] 王启振，郑新立．生物药品化学，沈阳：沈阳药科大学出版社
[24] Juan F, Martin, Paloma Liras Organization and expression of genes involved in the biosynthesis of antibiotics and other secondary metabolites. Annu. Rev. Microbiol., 1989, 43:173～206
[24] Kenneth L. Rinehart, Jr. and Ronald M. Stroshane. Biosynthesis of aminocylitol antibiotics. J. Antibiotics APR. 1976, 319～353
[25] Klaus Pissowotzki, Kambiz Mansouri, Wolfgang Piepersberg Genetics of streptomycin production in Streptomyces griseus:molecular structure and putative function of genes strELMB2N. Mol Gen Genet, 1991, 231:113～123
[26] Mamoru Hasegawa, A novel highly efficient gene-cloning system in Micromonospora applied to the genetic analysis of fortimicin biosynthesis. Gene, 1992, 115:85～91
[27] Tohru Dairi, Toshio Ohta, Eri Hashimoto, et al. Self cloning in Micromonospora olivasterospora of fms genes for fortimicin A (astromicin) biosynthesis. Mol Gen Genet 1992, 232:262～270
[28] Tohru Dairi, Kazuo Yamaguchi, Mamoru Hasegawa. N-Formimidoyl fortimicin A synthsae, a unique oxidase involved in fortimicin A biosynthesis:purification, characterization and geng cloning. Mol Gen Genet 1992, 236:49～59
[29] ohru Dairi, Toshio Ohta, Eri Hashimoto, et al. Organization and nature of fortimicin A (astromicin) biosynehetic genes studies using a cosmid library of Micromonospora olivasterospora DNA. Mol Gen Genet (1992) 236:39～48
[30] Souichi Ikeno, Tomohiro Tsuji, Kenji Higashide, et al. A 7.6kb DNA Region from Streptomyces kasugaensis M338-MI Includes some gengs responsible for Kasugamycin biosynthesis. J. Antibiotics 1998, 51:341～352
[31] Suzukake, K.; K.Tokunaga, H. Hayashi, M. Hori, Y. Uehara, D. Ikeda & H. Umezawa Biosynthesis of 2-de oxystreptamine. J. Antibiotics 1985, 38: 1211～1218
[32] Lucher, L. A. ; Y-M. Chen & J. B. Walker Reactions catalyzed by purified L-glutamine: keto-scyllol-inisitol aminotransferase, an enzyme required for biosynthesis of aminocyclitol antibiotics. Antimicrob. Agents chemother. 1989, 33:452～459
[33] Elisabeth Aubert-Pivert, Julian Davies Biosynthesis of butirosin in Bacillus circulans NRRL B3312: identification by sequence analysis and insertional mutagenesis of the butB gene involved in antibiotic production. Gene 1994, 147:1～11
[34] Fumitaka Kudo, Hideyuki Tamegai, Taketomo Fujiwara, et al. Molecular cloning of the gene for the key carbocycli-forming enzyme in the biosynthesis of 2-Deoxystrptamine-containing Aminocyclitol antibiotics and its comparison with dehydroquinate synthase. J. Antibiotics 1999, 52:559～571
[35] 李天伯，尚广东，夏焕章，等．链霉菌S．tenebrarius H6中与抗生素有关糖生物合成基因的克隆．生物工程学报,2001,17(3)：329～331
[36] Stark, W.M., Hoehe, M.M., Knox, N.G. Nebramycin, a new broad spectrum antibotic complex. Antimicrob Agents Chemother, 1967, 7:314-323
[37] Stark,W.M., Knox,N.G., Wilgus,R.M. Strains of Streptomyces tenebrarius and biosynthesis of nebramycin. Folia Microbiol, 1971, 16(3):205-217
[38] 熊宗贵，周朝辉，刘晓辉，等．尼拉霉素单组分——安普霉素高产菌株的研究．中国抗生素杂志，1997，22(5)：334-339
[39] 李相丰，何建勇，田威，等．安普霉素生物合成途径的研究．中国抗生素杂志，2002，27(2)，97-100
[40] 金冬雁，黎孟枫，等译．分子克隆实验指南．第2版．北京：科学出版社，1993．801
[41] 鲍楷．博士学位论文．华中农业大学．1999
[42] Hopwood,D.A., Bibb,M.J., Chater,K.F., et al. Genetic Manipulation of Streptomyces. A Laboratory Manual. England: John Innes Foundation Press, 1985
[43] Vara, J., Lewandowsk-Skarbek, M., Yiguang, W. Cloning of genes governing the deoxysugar portion of the erythromycin biosynthesis pathway in Saccharopolyspora erythreus. J Bacteriol, 1989, 171:5872-5881
[44] 吴胜．硕士学位论文．沈阳药科大学．2001
[45] David J. H., Daniel D., Gerard T. et al. Cloning of an aminoglycoside-resistance-encoding gene, kamC, from Saccharopolyspora hirsute: comparison with kamB from Streptomyces tenebrarius. Gene,1991,102:19-26