若干抗生素的生物合成基因研究与化学合成研究
|
摘要
氨基糖苷类抗生素(Aminoglycoside antibiotics)是一类结构中既含有氨基糖苷又含有氨基环醇的抗生素，主要包括链霉素、卡那霉素、阿泊拉霉素等，绝大多数是在链霉菌属中产生的。
黑暗链霉菌S.tenebrarius H6产生多种氨基糖苷类抗生素，主要有阿泊拉霉素、妥布霉素及卡那霉素B。为研究其生物合成基因，本实验室李天伯等构建了S.tenebrarius H6的基因文库，并设计探针，通过文库杂交从中得到完整基因orfE，推测为dTDP-葡萄糖-4，6-脱水酶基因。
通过进一步的亚克隆和测序，我们在orfE的下游又得到一个不完整的基因orfL，推测为dTDP-葡萄糖-4-脱水鼠李糖还原酶基因，和三个完整的基因orfG1，orfG2及orfM。orfG1，orfG2和orfM分别推测为葡萄糖转移酶基因和甘露糖转移酶基因(Bankit number：476862；收录号：AY131228)。
为了对orfE进行功能研究，我们进行了基因阻断实验。由于常规原生质体方法不能实现对S.tenebrarius H6的基因转移，我们在S.tenebrarius H6中建立了接合转移的基因转移系统。最终以温敏型穿梭质粒pHZ132为载体构建重组质粒，红霉素抗性基因ermE为选择标记，大肠杆菌ET12567(pUZ8002)为供体菌，S.tenebrarius H6为受体菌，在SM培养基上成功实现了接合转移，通过导入重组质粒，并在高温(42℃)、加入红霉素的选择性培养条件下与受体菌染色体发生单交换同源重组，实现orfE的基因阻断。阻断株经传代稳定性实验、游离质粒排除、PCR及PCR产物测序在基因水平确证后，发酵培养，结果发现阻断株不再产生妥布霉素。表明orfE基因是妥布霉素生物合成必须基因，与orfE连锁的基因簇可能与妥布霉素的生物合成有关。
Aminoglycoside antibiotics are mostly regarded as compounds mainly based on amino-N-containing-sugars and/or cyclitol derivatives, including streptomycins, kanamycins, apramycins, and so on. Most of them are produced by Streptomyces.
Streptomyces tenebrarius H_6 produces a complex of aminoglycoside antibiotics, such as apramycin, tobramycin and kanamycin B etc. To study the possible genes involved in the biosynthesis of aminoglycosides in Streptomyces tenebrarius H6 ,primers were designed according to the highly conserved sequence of the dTDP-glucose-4,6-dehydratse gene , and 0.6kb PCR product was obtained from S. tenebrarius H_6 genome. Using this fragment as probe, an ORF of putative dTDP-glucose-4,6-dehydratase gene consisted of 1,132bp, designated as orfE was obtained.
Based on the result, three other ORFs orfG1, orfG2 and orfM encoding the glycosyltransferase and mannosyltransferase linked to orfE were obtained by subcloning and sequencing (Bankit number:476862; accession number: AY131228) .
The function of the orfE gene was studied by gene disruption. The orfE gene was disrupted by inserting the erythromycin resistance gene (ermE) into the unique SalI site of orfE. An E.coli/Streptomyces shuttle vector pHZ132 was used in conjugation, which contains a temperature sensitive replicon that functions only at temperatures below 34℃. Gene disruption was done by conjugation between E.coli ET12567 (pUZ8002/pHZ132E0.6) and S.tenebrarius H_6. The conjugation was first carried out on SM medium at 37℃. Potential exconjugants then were picked and inoculated in selected medium containing nalidixic acid and Em and incubated at 42℃.
At this temperature, only the strains in which the recombinated plasmid integrated into the chromosome of the wild-type strain by means of homologous recombination could grow. Five potential orfE gene disruptant stains B7-1~5, which did not show tobramycin and kanamycin B production were obtained. To confirm the result, B7-1~5 were incubated as liquid culture, the fermentation broth was analyzed by TLC
黑暗链霉菌S.tenebrarius H6产生多种氨基糖苷类抗生素，主要有阿泊拉霉素、妥布霉素及卡那霉素B。为研究其生物合成基因，本实验室李天伯等构建了S.tenebrarius H6的基因文库，并设计探针，通过文库杂交从中得到完整基因orfE，推测为dTDP-葡萄糖-4，6-脱水酶基因。
通过进一步的亚克隆和测序，我们在orfE的下游又得到一个不完整的基因orfL，推测为dTDP-葡萄糖-4-脱水鼠李糖还原酶基因，和三个完整的基因orfG1，orfG2及orfM。orfG1，orfG2和orfM分别推测为葡萄糖转移酶基因和甘露糖转移酶基因(Bankit number：476862；收录号：AY131228)。
为了对orfE进行功能研究，我们进行了基因阻断实验。由于常规原生质体方法不能实现对S.tenebrarius H6的基因转移，我们在S.tenebrarius H6中建立了接合转移的基因转移系统。最终以温敏型穿梭质粒pHZ132为载体构建重组质粒，红霉素抗性基因ermE为选择标记，大肠杆菌ET12567(pUZ8002)为供体菌，S.tenebrarius H6为受体菌，在SM培养基上成功实现了接合转移，通过导入重组质粒，并在高温(42℃)、加入红霉素的选择性培养条件下与受体菌染色体发生单交换同源重组，实现orfE的基因阻断。阻断株经传代稳定性实验、游离质粒排除、PCR及PCR产物测序在基因水平确证后，发酵培养，结果发现阻断株不再产生妥布霉素。表明orfE基因是妥布霉素生物合成必须基因，与orfE连锁的基因簇可能与妥布霉素的生物合成有关。
Aminoglycoside antibiotics are mostly regarded as compounds mainly based on amino-N-containing-sugars and/or cyclitol derivatives, including streptomycins, kanamycins, apramycins, and so on. Most of them are produced by Streptomyces.
Streptomyces tenebrarius H_6 produces a complex of aminoglycoside antibiotics, such as apramycin, tobramycin and kanamycin B etc. To study the possible genes involved in the biosynthesis of aminoglycosides in Streptomyces tenebrarius H6 ,primers were designed according to the highly conserved sequence of the dTDP-glucose-4,6-dehydratse gene , and 0.6kb PCR product was obtained from S. tenebrarius H_6 genome. Using this fragment as probe, an ORF of putative dTDP-glucose-4,6-dehydratase gene consisted of 1,132bp, designated as orfE was obtained.
Based on the result, three other ORFs orfG1, orfG2 and orfM encoding the glycosyltransferase and mannosyltransferase linked to orfE were obtained by subcloning and sequencing (Bankit number:476862; accession number: AY131228) .
The function of the orfE gene was studied by gene disruption. The orfE gene was disrupted by inserting the erythromycin resistance gene (ermE) into the unique SalI site of orfE. An E.coli/Streptomyces shuttle vector pHZ132 was used in conjugation, which contains a temperature sensitive replicon that functions only at temperatures below 34℃. Gene disruption was done by conjugation between E.coli ET12567 (pUZ8002/pHZ132E0.6) and S.tenebrarius H_6. The conjugation was first carried out on SM medium at 37℃. Potential exconjugants then were picked and inoculated in selected medium containing nalidixic acid and Em and incubated at 42℃.
At this temperature, only the strains in which the recombinated plasmid integrated into the chromosome of the wild-type strain by means of homologous recombination could grow. Five potential orfE gene disruptant stains B7-1~5, which did not show tobramycin and kanamycin B production were obtained. To confirm the result, B7-1~5 were incubated as liquid culture, the fermentation broth was analyzed by TLC
引文
【1】 顾觉奋 等，微生物药品化学与分析，军事医学科学出版社，1995年8月
【2】 方金瑞 等，抗生素，科学出版社，1988年
【3】 王启振 等．生物药品化学，沈阳药科大学，1996年
【4】 Stark. W. M, Hoehe. M. M, et al, Nebramycin, a new broad soectrum antibiotic complex, Antimicrob Agents Chemother, 1967, 7: 314-323
【5】 Stark. W. M, Knox. N. G, et al, Strains of Streptomyces tenebrarius and biosynthesis of nebramycin. Folia Microbiol, 1971, 16(3): 205-217
【6】 熊宗贵 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
【7】 刘诗通，妥布霉素，国外医药抗生素分册，1987，8(5)：346—354
【8】 李家斌等，硫酸妥布霉素治疗急性细菌性痢疾，中国抗生素杂志，1997．22(2)：159—160
【9】 Piepersberg W. (1997), Biotechnology of Antibiotics, Second Edition, Revised and Expanded, edited by William R. Strohl. The Ohio State University Columbus, Ohio, MARCEL DEKKER, INC, NEW YORK. BASEL. HONG KONG p. 81-163。
【10】 Mendez C. & Salas J. A. Altering the glycosylation pattern of bioactive compounds. TRENDS in Biotechnology, 2001, 19: 449-456.
【11】 李天伯，尚广东，夏焕章，王以光．黑暗链霉菌S．tenebrarius H6中与抗生素有关的糖生物合成基因的克隆．生物工程学报，2001，17：329-331．
【12】 Hopwood DA, BibbMJ, Cheer Kf, et al(2000), Practical Streptomyces Genetics. Norwich: Johnlnnes Foundation
【13】 37度医学网http://www.37c.com.cn/literature/library/theory/007/00705039.html
【14】 Trevor D. Lawley, et al, Bacterial conjugative transfer: visualization of successful mating pairs and plasmid establishment in live Escherichia coli, Moiecular Microbiology (2002) 44(4), 947-956
【15】 Trieu-cuot. P., Carlier. C., et al, Plasmid transfer by conjugation from Escherichia coli to gram-positive bacteria, FEMS Microbiol Lett, 1987, 48: 289-294
【16】 邓子新，周秀芬，质粒在大肠杆菌和链霉菌FR-008之间的属间接合转移，遗传??HEREDITAS (Beijing), 1994, 16(6): 7-10
【17】 覃重军，邓子新等，吸水链霉菌应城5变种的四个内源性质粒及其逐个消除的研究，微生物学报，1995，38(1)：14—20
【18】 Mazodier. P., Petter. R., et al, Intergeneria conjugation between Escherichia coli and Streptomyces species. J Bacteriol, 1989, 171: 3583-3585
【19】 Bierman. M., Logan. R., et al, Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp, Gene, 1992, 116: 43-49
【20】 Matsushima. P., et al, A gene cloning system for Streptomyces toyocaensis, Microbiology, 1996, 142: 261-267
【21】 Voeikova. T. A, conjugative transfer of a plasmid from E. coli to various strains of the order Actinomycetales, Genetika, 1999, 35(12): 1626-1633.
【22】 鲍锴，胡志浩等，一个可在大肠杆菌和链霉菌之间进行属间接合转移的柯斯质粒，科学通报，1995，40：1440
【23】 Bibb M. J., Janssen G. R. and Ward J. M. (1985)Cloning and analysis of the promoter region of the erythromycin resistance gene (ermE) of Streptomyces erythraeus Gene, 38: 215-226【1】 Wehrli W. Ansamycins: chemistry, biosynthesis and biological activity. Top Cuur. Chem. 1977, 72: 22-49.
【2】 Lancini G. Ansamycins. In: Vining LC (ed) Biochemistry and genetic regulation of commercially important antibiotics. Addision-Wesley, Reading, Mall., USA, 1983, pp231-254.
【3】 Rinehart K. L., Shield L. S. Chemistry of the ansamycinantibiotics. In: Herz W., Grisbach H., Kirby G. W. (eds) Progress in the chemistry of organic natural products, vol 33, Springer, Vienna New York, 1976, pp. 232-307
【4】 Sasaki K, Rinehart KL. Jr., Slomp G, et al. Geldanamycin. 1: Structure assignment. J. AM. Chem. 1970, Soc. 92: 7591-7593.
【5】 Redeuilh G. J Biol. Chem, 1997, 262: 6969-6975.
【6】 Veldscholte J. Biochemistry, 1992, 31: 2393-2399.
【7】 Sausville E. The National Cancer Institute Decision Network Meeting, Oct. 6, 1997, Bethesda, MD.
【8】 陶佩珍，娄志贤，姚天爵，等．广谱抗病毒抗生素17997体内外抗病毒活性研究．中国抗生素杂志，1997，22：368．
【9】 陶佩珍，杨茂，王淑琴，等．抗生素17997抗病毒作用机制的研究．中国抗生素杂志，1998，23：154—157．
【10】 陶佩珍，章天．抗病毒抗生素17997体外联合用药研究．中国抗生素杂志，2001，26：292．
【11】 August P. R., Tang L., Yoon Y. J., et al. Biosynthesis of the ansamycin antibiotic rifamycin: deductions from the molecular analysis of the rif biosynthetic gene cluster of Amycolatopsis mediterranei S699. Chemistry&Biology, 1998, 5: 69-79.
【12】 Chert S., Bamberg D. V., Hale V., et al. Biosynthesis of ansatrienin (mycotrienin) and naphthomycin Identification and analysis of two separate biosynthetic gene clusters in Streptomyces collinusTu 1892. Eur. J. Biochem., 1999, 261: 98-107.[13] Yu T. W., Bai L Q., Clade D., et al. The biosynthetic gene cluster of the maytansinoid antitumor agent ansamitocin from Actinosynnema pretiosum. PNAS. 2002, 99: 7968-7973.
[14] Chiao J. S., Xia T. H., Mei B. G., et al. Rifamycin SV and related ansamycins. In: Genetics and Biochemistry of Antibiotic Production, Vining L.C., Stuttard C. Eds. , Butterworth Heinemann, Boston, pp. 477-498.
[15] Lancini G. & Cavalleri B. Rifamycins. In Biotechnology of Antibiotics, 2~(nd) Ed., Strohl W. R. Ed. Dekker, New York, pp. 521-549.
[16] Katz L. & Donadio S. Polyketide synthesis: prospects for hybrid antibiotics. Annu. Rev. Microbiol. 47:875-912.
[17] (a) Karlsson A., Sartori G., White R., Eur J. Biochem. 1974 251. (b) White R. J., Martinelli E. FEBS Lett. , 1974, 49:233. (c) Haber A., Johnson R. D., Rinehart K. L., Jr. J. Am. Chem. 1997, 99:3541.
[18] Hornemann U., Kehrer J. P., Eggert J. H. J. Chem. Soc. Chem. Commun., 1974, 1045-1046.
[19] Gygax D., Ghisalba O., Treichler H., et al. J. Antibiot. 1990, 43:324.
[20] Anderson M. G., Kibby J. J., Rickards. R. W., et al. J. Chem. Soc. Chem. Commun. 1980, 1227-1278.
[21] Ghisalba O. & Nuesch J. J. Antibiot. (Tokyo)., 1981, 34:64-71.
[22] Becker a. M., Herlt a. J., Hilton G. L, et al. J. Antibiot. (Tokyo)., 1983,36:1323-1328.
[23] Kibby J. J., McDonald I. A., Rickards R. W. J. Chem. Spc. Chem. Commun. 1980, 768-769.
[24] Ghisalba O. & Nuesch J. J. Antibiot. (Tokyo) , 1978, 31:202-214.
[25] Ghisalba O. & Nuesch J. J. Antibiot. (Tokyo) , 1978, 31: 215-225.
[26] Ghisalba O., Fuhrer H., Richter W. J., et al. J. Antibiot. (Tokyo), 1981,34:58-63.
[27] Gygax D., Ghisalba O., Treichler H., et al. J. Antibiot. (Tokyo), 1990, 43:324-326.
[28] Harber A., Johnson R. D., Rinehart K. L. Jr. J. Am. Chem. Soc, 1977,??99: 3541-3544.
【29】 Meier R. M., Tamm C. J. Antibiot. (Tokyo), 1992, 45: 400-410.
【30】 Kim C. G., Kirschning A., Bergon P., et al. Biosynthesis of 3-amino-5-hydroxybenzoic Acid, the Precursor of mC7N Units in Ansamycin Antibiotics. J. Am. Chem. Soc., 1996, 118: 7486-7491.
【31】 Moore B. S.& Hertweck C. Biosynthesis and attachment of novel bacterial polyketide synthase starter units. The Royal Society of Chemistry, 2002, 19: 70-99.
【32】 Tang L., Fu H., McDaniel R. Formation of functional heterologous complexes using subunits from the picromycin, erythromycin and oleandomycin polyketide synthases. Chemistry & Biology, 2000, 7: 77-84.
【33】 Rodicio M. R., Bruton C. J. et al, New derivatives of the Streptomyces temperate phage C31 useful for the cloning and functional analysis of Streptomyces DNA. Gene, 1985, 34: 283-292.
【34】 Sladkova. I. Alomovaskaya, N. D et, al. The structure and size of the genome of actinophage C31 of Streptomyces coelicolor A3(2). Genetika (Moscow). 1977, 13: 342-344.
【35】 Hopwood DA, Bibb MJ, Chater Kf, et al (2000), Practical Streptomyces Genetics. Norwich: John Innes Foundation
【36】 Sambrook J, Fritscb EF, Maniatis T(1989), Molecular cloning: a laboratory manual, 2rid ed. Cold Spring Harbor Laboratory, NY: Cold Spring Harbor Laboratory Press.
【37】 Abbadi A, Brummel M, Spener F. Knockout of the regulatory site of 3-ketoacyl-ACP synthase Ⅲ enhances short- and medium-chain acyl-ACP synthesis Plant J 2000 24(1): 1-9.
【38】 Allen I. W., Ritchie D. A. Cloning and analysis of DNA sequences from Streptomyces hygroscopicus encoding geldanamycin biosynthesis. Mol. Gen. Genet., 1994, 243: 593-599.
【39】 Rascher A., Hu Zhihao, Viswanathan N., et al. Cloning and Characterization of a Gene Cluster forGeldanamycin Production in Streptomyces hyggroscopicus.??FEMS Microbiology Letters, 10788(2002), 1-8.
【1】 Chu D. T. W, Plattner J. J. Katz. L. New directions in antibacterial research. J Med Chem, 1996, 39: 3853-3874
【2】 Pharmacotherapy Perspectives。http://pswi.org/communications/PDFs/linezolid.pdf.
【3】 (a)Lin. A. H., Murray R. W, Vidmar. T. J, et al. The oxazolidinone eperezolid binds to the 50s ribosomal subunit and competes with binding of chloramphenicol and lincomycin. Antimicrob. Agent Chemother, 1997, 41: 2127-2131.
(b) Eustice D. C., Feldam P. A., Zajac. I, et al. Mechanism of action of DuP721: Inhibition of an early event during initiation of protein synthesis. Agents Chemother, 1988, 32: 1218-1222.
【4】 Fuigitt R. B., Luckenbaugh R. W., 5-Halomethyl-3-phenyl-2-oxazolidinnones. US. Patent. 4, 128, 654, Dec. 5, 1978(CA. 1979, 90, 147009b)
【5】 Bricker S. J., Hutchinson D. K., Barbachyn. M. R, et al. Synthesis and antibacterial activity of U-100592 and U-100766, two oxazolidinone antibacterial agents for the potential treatment of multidyug-resistant Gram-positive bacterial infections. J. Med. Chem. 1996, 39: 673-679
【6】 Spangler S. K., Jacobs M. R., Appelbaum. P. C. Activities of RPR-106972(a new oral streptogramin), cefditoren(a new oral cephalosporin), two new oxazolidinones(U-100592 and U-100766), and other oral parenteral agents against 203 penicillin-susceptible and resistant pneumococci. Antimicrob. Agenta Chemother. 1996, 40: 481-484
【7】 Mulazimoglu L., Drenning S. D., Yu V. L. In vitro activities of two novel oxazolidinones(U-100592 and U-100766), a new fluoquinolone(trovafloxacin), and dalfopristin-quinupristin against Staphylococcus aureus and Staphylococcus epidermidis. Antimicrob. Agenta Chemother. 1996, 40: 2428-2430
【8】 London New Drugs Group, http://www.ukmi.nhs.uk/NewMaterial/html/docs/08050101.pdf.【9】 张致平，噁唑烷酮类抗菌药研究进展
【10】 Seneci P., Caspani M., Ripamonti F, et al. Synthesis and antimicrobial activity of oxazolidin-2-ones and related heterocycles. J Chem Soc, Perkin 1, 1994: 2345-2351
【11】 Gregory W. A, Brittelli D. R., Wang C. L. J., et al. Antibacterial. synthesis and structure-activity studies of 3-aryl-2-oxazolidinones, 1. the "B" group. J. Med Chem. 1989, 32: 1673-1681
【12】 Park C. H, Brittelli D. R., Wang C. L. J., et at. Antibacterial. synthesis and structure-activity studies of 3-aryl-2-oxazolidinones. 4. multiply-substituted-aryl derivatives.. J. Med. Chem. 1992, 35: 1156--1165
【13】 (a)(WO 1993 09103) CA 1993; 119: 160265u;
(b) (WO 1995 14684) CA 1995; 123: 314020g
【14】 Gregory W. A., Brottelli D. R., Wang C. L. J, et al. Antibacterial. synthesis and structure-activity studies of3-aryl-2-oxazolidinones, 2. the "A" group. J. Med. Chem. 1990, 33: 2569-2578.
【15】 (US 1987: 4705799) CA 1989: 110: 8198f【1】 朱玉洁，郭惠元．喹诺酮类抗菌药的历史与概况．国外医药抗生素分册，1999，20(3)：109—110．
【2】 刘明亮，郭惠元．喹诺酮类抗菌药的结构特征及构效关系．国外医药抗生素分册，1999，20(3)：116—120．
【3】 刘九雨．田治明，郭惠元．格雷沙星合成路线图解．中国医药工业杂志，待发表．
【4】 Miyamoto H, Yamashita H, Ueda H, et al. Synthesis and Biological Properties of Substituted 1,4-Dihydro-5-methyl-4-oxo-3-quinolinecarboxylic Acids. Bioorg Med Chem, 1995, 3: 1699-1706
【5】 郭惠元，田治明，苗华．2，4-二氯氟苯的合成[J]．中国医药工业杂志，1992，23：464-465．
【6】 (Vorozhtsov NN, Yakobson GG, Krizhechkovskaya NI. Nitration of fluorochlorobenzenes Zhur Obshchei Khim, 1961, 31: 1227-1229.) CA 1961, 55: 24606c～e
【7】 (Shi Yujun, Zhang huaixiang, Xu Yurong. Preparation of 2,4-dichloro-5-fluorobenzoic acid. CN: 1,031,074, 1989-2-15) CA 1990, 113: 77918q
【8】 郭惠元，顾慧儿．吡酮酸化学[J]．中国抗生素杂志，1995，20(2)：85-90
【2】 方金瑞 等，抗生素，科学出版社，1988年
【3】 王启振 等．生物药品化学，沈阳药科大学，1996年
【4】 Stark. W. M, Hoehe. M. M, et al, Nebramycin, a new broad soectrum antibiotic complex, Antimicrob Agents Chemother, 1967, 7: 314-323
【5】 Stark. W. M, Knox. N. G, et al, Strains of Streptomyces tenebrarius and biosynthesis of nebramycin. Folia Microbiol, 1971, 16(3): 205-217
【6】 熊宗贵 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
【7】 刘诗通，妥布霉素，国外医药抗生素分册，1987，8(5)：346—354
【8】 李家斌等，硫酸妥布霉素治疗急性细菌性痢疾，中国抗生素杂志，1997．22(2)：159—160
【9】 Piepersberg W. (1997), Biotechnology of Antibiotics, Second Edition, Revised and Expanded, edited by William R. Strohl. The Ohio State University Columbus, Ohio, MARCEL DEKKER, INC, NEW YORK. BASEL. HONG KONG p. 81-163。
【10】 Mendez C. & Salas J. A. Altering the glycosylation pattern of bioactive compounds. TRENDS in Biotechnology, 2001, 19: 449-456.
【11】 李天伯，尚广东，夏焕章，王以光．黑暗链霉菌S．tenebrarius H6中与抗生素有关的糖生物合成基因的克隆．生物工程学报，2001，17：329-331．
【12】 Hopwood DA, BibbMJ, Cheer Kf, et al(2000), Practical Streptomyces Genetics. Norwich: Johnlnnes Foundation
【13】 37度医学网http://www.37c.com.cn/literature/library/theory/007/00705039.html
【14】 Trevor D. Lawley, et al, Bacterial conjugative transfer: visualization of successful mating pairs and plasmid establishment in live Escherichia coli, Moiecular Microbiology (2002) 44(4), 947-956
【15】 Trieu-cuot. P., Carlier. C., et al, Plasmid transfer by conjugation from Escherichia coli to gram-positive bacteria, FEMS Microbiol Lett, 1987, 48: 289-294
【16】 邓子新，周秀芬，质粒在大肠杆菌和链霉菌FR-008之间的属间接合转移，遗传??HEREDITAS (Beijing), 1994, 16(6): 7-10
【17】 覃重军，邓子新等，吸水链霉菌应城5变种的四个内源性质粒及其逐个消除的研究，微生物学报，1995，38(1)：14—20
【18】 Mazodier. P., Petter. R., et al, Intergeneria conjugation between Escherichia coli and Streptomyces species. J Bacteriol, 1989, 171: 3583-3585
【19】 Bierman. M., Logan. R., et al, Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp, Gene, 1992, 116: 43-49
【20】 Matsushima. P., et al, A gene cloning system for Streptomyces toyocaensis, Microbiology, 1996, 142: 261-267
【21】 Voeikova. T. A, conjugative transfer of a plasmid from E. coli to various strains of the order Actinomycetales, Genetika, 1999, 35(12): 1626-1633.
【22】 鲍锴，胡志浩等，一个可在大肠杆菌和链霉菌之间进行属间接合转移的柯斯质粒，科学通报，1995，40：1440
【23】 Bibb M. J., Janssen G. R. and Ward J. M. (1985)Cloning and analysis of the promoter region of the erythromycin resistance gene (ermE) of Streptomyces erythraeus Gene, 38: 215-226【1】 Wehrli W. Ansamycins: chemistry, biosynthesis and biological activity. Top Cuur. Chem. 1977, 72: 22-49.
【2】 Lancini G. Ansamycins. In: Vining LC (ed) Biochemistry and genetic regulation of commercially important antibiotics. Addision-Wesley, Reading, Mall., USA, 1983, pp231-254.
【3】 Rinehart K. L., Shield L. S. Chemistry of the ansamycinantibiotics. In: Herz W., Grisbach H., Kirby G. W. (eds) Progress in the chemistry of organic natural products, vol 33, Springer, Vienna New York, 1976, pp. 232-307
【4】 Sasaki K, Rinehart KL. Jr., Slomp G, et al. Geldanamycin. 1: Structure assignment. J. AM. Chem. 1970, Soc. 92: 7591-7593.
【5】 Redeuilh G. J Biol. Chem, 1997, 262: 6969-6975.
【6】 Veldscholte J. Biochemistry, 1992, 31: 2393-2399.
【7】 Sausville E. The National Cancer Institute Decision Network Meeting, Oct. 6, 1997, Bethesda, MD.
【8】 陶佩珍，娄志贤，姚天爵，等．广谱抗病毒抗生素17997体内外抗病毒活性研究．中国抗生素杂志，1997，22：368．
【9】 陶佩珍，杨茂，王淑琴，等．抗生素17997抗病毒作用机制的研究．中国抗生素杂志，1998，23：154—157．
【10】 陶佩珍，章天．抗病毒抗生素17997体外联合用药研究．中国抗生素杂志，2001，26：292．
【11】 August P. R., Tang L., Yoon Y. J., et al. Biosynthesis of the ansamycin antibiotic rifamycin: deductions from the molecular analysis of the rif biosynthetic gene cluster of Amycolatopsis mediterranei S699. Chemistry&Biology, 1998, 5: 69-79.
【12】 Chert S., Bamberg D. V., Hale V., et al. Biosynthesis of ansatrienin (mycotrienin) and naphthomycin Identification and analysis of two separate biosynthetic gene clusters in Streptomyces collinusTu 1892. Eur. J. Biochem., 1999, 261: 98-107.[13] Yu T. W., Bai L Q., Clade D., et al. The biosynthetic gene cluster of the maytansinoid antitumor agent ansamitocin from Actinosynnema pretiosum. PNAS. 2002, 99: 7968-7973.
[14] Chiao J. S., Xia T. H., Mei B. G., et al. Rifamycin SV and related ansamycins. In: Genetics and Biochemistry of Antibiotic Production, Vining L.C., Stuttard C. Eds. , Butterworth Heinemann, Boston, pp. 477-498.
[15] Lancini G. & Cavalleri B. Rifamycins. In Biotechnology of Antibiotics, 2~(nd) Ed., Strohl W. R. Ed. Dekker, New York, pp. 521-549.
[16] Katz L. & Donadio S. Polyketide synthesis: prospects for hybrid antibiotics. Annu. Rev. Microbiol. 47:875-912.
[17] (a) Karlsson A., Sartori G., White R., Eur J. Biochem. 1974 251. (b) White R. J., Martinelli E. FEBS Lett. , 1974, 49:233. (c) Haber A., Johnson R. D., Rinehart K. L., Jr. J. Am. Chem. 1997, 99:3541.
[18] Hornemann U., Kehrer J. P., Eggert J. H. J. Chem. Soc. Chem. Commun., 1974, 1045-1046.
[19] Gygax D., Ghisalba O., Treichler H., et al. J. Antibiot. 1990, 43:324.
[20] Anderson M. G., Kibby J. J., Rickards. R. W., et al. J. Chem. Soc. Chem. Commun. 1980, 1227-1278.
[21] Ghisalba O. & Nuesch J. J. Antibiot. (Tokyo)., 1981, 34:64-71.
[22] Becker a. M., Herlt a. J., Hilton G. L, et al. J. Antibiot. (Tokyo)., 1983,36:1323-1328.
[23] Kibby J. J., McDonald I. A., Rickards R. W. J. Chem. Spc. Chem. Commun. 1980, 768-769.
[24] Ghisalba O. & Nuesch J. J. Antibiot. (Tokyo) , 1978, 31:202-214.
[25] Ghisalba O. & Nuesch J. J. Antibiot. (Tokyo) , 1978, 31: 215-225.
[26] Ghisalba O., Fuhrer H., Richter W. J., et al. J. Antibiot. (Tokyo), 1981,34:58-63.
[27] Gygax D., Ghisalba O., Treichler H., et al. J. Antibiot. (Tokyo), 1990, 43:324-326.
[28] Harber A., Johnson R. D., Rinehart K. L. Jr. J. Am. Chem. Soc, 1977,??99: 3541-3544.
【29】 Meier R. M., Tamm C. J. Antibiot. (Tokyo), 1992, 45: 400-410.
【30】 Kim C. G., Kirschning A., Bergon P., et al. Biosynthesis of 3-amino-5-hydroxybenzoic Acid, the Precursor of mC7N Units in Ansamycin Antibiotics. J. Am. Chem. Soc., 1996, 118: 7486-7491.
【31】 Moore B. S.& Hertweck C. Biosynthesis and attachment of novel bacterial polyketide synthase starter units. The Royal Society of Chemistry, 2002, 19: 70-99.
【32】 Tang L., Fu H., McDaniel R. Formation of functional heterologous complexes using subunits from the picromycin, erythromycin and oleandomycin polyketide synthases. Chemistry & Biology, 2000, 7: 77-84.
【33】 Rodicio M. R., Bruton C. J. et al, New derivatives of the Streptomyces temperate phage C31 useful for the cloning and functional analysis of Streptomyces DNA. Gene, 1985, 34: 283-292.
【34】 Sladkova. I. Alomovaskaya, N. D et, al. The structure and size of the genome of actinophage C31 of Streptomyces coelicolor A3(2). Genetika (Moscow). 1977, 13: 342-344.
【35】 Hopwood DA, Bibb MJ, Chater Kf, et al (2000), Practical Streptomyces Genetics. Norwich: John Innes Foundation
【36】 Sambrook J, Fritscb EF, Maniatis T(1989), Molecular cloning: a laboratory manual, 2rid ed. Cold Spring Harbor Laboratory, NY: Cold Spring Harbor Laboratory Press.
【37】 Abbadi A, Brummel M, Spener F. Knockout of the regulatory site of 3-ketoacyl-ACP synthase Ⅲ enhances short- and medium-chain acyl-ACP synthesis Plant J 2000 24(1): 1-9.
【38】 Allen I. W., Ritchie D. A. Cloning and analysis of DNA sequences from Streptomyces hygroscopicus encoding geldanamycin biosynthesis. Mol. Gen. Genet., 1994, 243: 593-599.
【39】 Rascher A., Hu Zhihao, Viswanathan N., et al. Cloning and Characterization of a Gene Cluster forGeldanamycin Production in Streptomyces hyggroscopicus.??FEMS Microbiology Letters, 10788(2002), 1-8.
【1】 Chu D. T. W, Plattner J. J. Katz. L. New directions in antibacterial research. J Med Chem, 1996, 39: 3853-3874
【2】 Pharmacotherapy Perspectives。http://pswi.org/communications/PDFs/linezolid.pdf.
【3】 (a)Lin. A. H., Murray R. W, Vidmar. T. J, et al. The oxazolidinone eperezolid binds to the 50s ribosomal subunit and competes with binding of chloramphenicol and lincomycin. Antimicrob. Agent Chemother, 1997, 41: 2127-2131.
(b) Eustice D. C., Feldam P. A., Zajac. I, et al. Mechanism of action of DuP721: Inhibition of an early event during initiation of protein synthesis. Agents Chemother, 1988, 32: 1218-1222.
【4】 Fuigitt R. B., Luckenbaugh R. W., 5-Halomethyl-3-phenyl-2-oxazolidinnones. US. Patent. 4, 128, 654, Dec. 5, 1978(CA. 1979, 90, 147009b)
【5】 Bricker S. J., Hutchinson D. K., Barbachyn. M. R, et al. Synthesis and antibacterial activity of U-100592 and U-100766, two oxazolidinone antibacterial agents for the potential treatment of multidyug-resistant Gram-positive bacterial infections. J. Med. Chem. 1996, 39: 673-679
【6】 Spangler S. K., Jacobs M. R., Appelbaum. P. C. Activities of RPR-106972(a new oral streptogramin), cefditoren(a new oral cephalosporin), two new oxazolidinones(U-100592 and U-100766), and other oral parenteral agents against 203 penicillin-susceptible and resistant pneumococci. Antimicrob. Agenta Chemother. 1996, 40: 481-484
【7】 Mulazimoglu L., Drenning S. D., Yu V. L. In vitro activities of two novel oxazolidinones(U-100592 and U-100766), a new fluoquinolone(trovafloxacin), and dalfopristin-quinupristin against Staphylococcus aureus and Staphylococcus epidermidis. Antimicrob. Agenta Chemother. 1996, 40: 2428-2430
【8】 London New Drugs Group, http://www.ukmi.nhs.uk/NewMaterial/html/docs/08050101.pdf.【9】 张致平，噁唑烷酮类抗菌药研究进展
【10】 Seneci P., Caspani M., Ripamonti F, et al. Synthesis and antimicrobial activity of oxazolidin-2-ones and related heterocycles. J Chem Soc, Perkin 1, 1994: 2345-2351
【11】 Gregory W. A, Brittelli D. R., Wang C. L. J., et al. Antibacterial. synthesis and structure-activity studies of 3-aryl-2-oxazolidinones, 1. the "B" group. J. Med Chem. 1989, 32: 1673-1681
【12】 Park C. H, Brittelli D. R., Wang C. L. J., et at. Antibacterial. synthesis and structure-activity studies of 3-aryl-2-oxazolidinones. 4. multiply-substituted-aryl derivatives.. J. Med. Chem. 1992, 35: 1156--1165
【13】 (a)(WO 1993 09103) CA 1993; 119: 160265u;
(b) (WO 1995 14684) CA 1995; 123: 314020g
【14】 Gregory W. A., Brottelli D. R., Wang C. L. J, et al. Antibacterial. synthesis and structure-activity studies of3-aryl-2-oxazolidinones, 2. the "A" group. J. Med. Chem. 1990, 33: 2569-2578.
【15】 (US 1987: 4705799) CA 1989: 110: 8198f【1】 朱玉洁，郭惠元．喹诺酮类抗菌药的历史与概况．国外医药抗生素分册，1999，20(3)：109—110．
【2】 刘明亮，郭惠元．喹诺酮类抗菌药的结构特征及构效关系．国外医药抗生素分册，1999，20(3)：116—120．
【3】 刘九雨．田治明，郭惠元．格雷沙星合成路线图解．中国医药工业杂志，待发表．
【4】 Miyamoto H, Yamashita H, Ueda H, et al. Synthesis and Biological Properties of Substituted 1,4-Dihydro-5-methyl-4-oxo-3-quinolinecarboxylic Acids. Bioorg Med Chem, 1995, 3: 1699-1706
【5】 郭惠元，田治明，苗华．2，4-二氯氟苯的合成[J]．中国医药工业杂志，1992，23：464-465．
【6】 (Vorozhtsov NN, Yakobson GG, Krizhechkovskaya NI. Nitration of fluorochlorobenzenes Zhur Obshchei Khim, 1961, 31: 1227-1229.) CA 1961, 55: 24606c～e
【7】 (Shi Yujun, Zhang huaixiang, Xu Yurong. Preparation of 2,4-dichloro-5-fluorobenzoic acid. CN: 1,031,074, 1989-2-15) CA 1990, 113: 77918q
【8】 郭惠元，顾慧儿．吡酮酸化学[J]．中国抗生素杂志，1995，20(2)：85-90