高产雷帕霉素吸水链霉菌的遗传改造和发酵过程优化
|
摘要
雷帕霉素(Rapamycin)又名西罗莫司(Sirolimus),是由吸水链霉菌(S. hygroscopicus)产生的新型三烯含氮大环内酯抗生素。雷帕霉素除作为免疫抑制剂外,目前还广泛应用于器官移植、皮肤病、心脏病等领域。本文以实验室保存的低产雷帕霉素产生菌为出发菌株,通过传统诱变育种技术,结合高通量筛选手段,辅以培养基优化,大幅度提高了雷帕霉素产生菌的生产能力,并在发酵罐进行了中试及放大。同时,采用原生质体育种及基因组重排等新技术对雷帕霉素产生菌进行选育,并初步探索了雷帕霉素的异源合成。
试验首先初步优化了雷帕霉素产生菌的培养基和培养条件,确定了最优的发酵培养基为：4%葡萄糖,1%甘油,1%黄豆饼粉,0.15%L-Lys,0.5%K2HPO4, 0.5%KH2PO4,0.5%氯化钠,0.2%硫酸镁,0.15%硫酸亚铁。较优的摇瓶发酵条件是：摇瓶装液量为20ml/250ml,培养温度为280C,接种后培养5天收获菌体。在以上最优条件下,原始菌株发酵液中雷帕霉素产量最高可达120mg/L。
为提高雷帕霉素产量,开展了雷帕霉素产生菌的诱变育种研究。首先考察了紫外线、DES、NTG等不同诱变剂的剂量效应。然后通过单一诱变剂连续诱变、不同诱变剂交替诱变、不同诱变剂连续诱变等多种诱变方式,筛选到产量为279mg/L的诱变菌株D7-804,比出发菌株提高了156%。进一步发展了基于96孔板培养的雷帕霉素突变株的高通量筛选新技术,通过对约100,000个单菌落的快速高效筛选,筛选到高产突变菌株N5632,其最高产量达到445mg/L
在以上高产菌选育和摇瓶发酵优化的基础上,开展了在120L发酵罐上的雷帕霉素发酵的放大研究。结果表明,初始搅拌转速设定为200rpm,在进入产物合成期后转速升至400rpm,控制整个发酵过程中溶氧不低于10%,雷帕霉素产量最高达到412mg／L,基本达到了摇瓶发酵的平均水平。在此基础上,开展了采用多种补料方式提高发酵水平的研究。结果表明,40h为最佳补料时间,采用多组分组合补料方式能够最大幅度提高发酵水平,其中组合补料甘油与磷酸盐的方式,不仅能够保持发酵液的pH值的相对稳定,而且能有效提高雷帕霉素产量,发酵水平最高可达800mg／L以上。最后,将这一新型补料发酵工艺在20吨发酵罐上进行工业放大,雷帕霉素发酵水平也达到了700mg／L,产量较本课题开始时的最初发酵水平提高了4倍以上。
为了最大程度地提高雷帕霉素高产菌选育的效率,开展了基因组重排技术改造吸水链霉菌的遗传性能,提高雷帕菌素发酵水平的研究。首先确定了雷帕霉素产生菌原生质体制备、再生和融合的较优条件；然后通过吸水链霉菌原生质体诱变、融合以及与红霉素生产菌原生质体的种间融合等技术,得到具有不同遗传特性的产雷帕菌素产量较高的突变株。最后,对7株雷帕霉素高产突变株进行一轮基因组重排育种,结果筛选到一株产量较高菌株(GS-1437),雷帕霉素产量达到445mg/L,比最初出发菌株产量提高了59.5%,充分显示了基因组重排技术在改造链霉菌遗传特性方面的巨大潜力,与传统诱变育种方法比较,具有快速高效等显著优点。。
开展了雷帕霉素生物合成基因镞在异源宿主菌中表达及生物合成雷帕霉素的研究。在已构建的三种雷帕霉素异源合成系统中,白色链霉菌S. albus 28-1-1发酵产物样品表现出与雷帕霉素标准品类似的抗菌生物活性。通过初步的结构鉴定和分析,该发酵产物样品中的未知生物活性物质是一种生物活性类似雷帕霉素但结构上有较大区别的新化合物。进一步从转录水平上对异源宿主菌中雷帕霉素生物合成基因镞中的各个基因进行了转录分析。通过提取发酵72h的菌体RNA,采用RT-PCR技术分析各基因在转录水平上的差异,S. albus 28-1-1中绝大部分雷帕霉素生物合成基因未能被正常转录,仅有下游的rapG,rapF和rapD显示与参照系统相同的转录信号。这一研究结果对在异源宿主菌中生物合成雷帕霉素及其他新颖抗生素具有重要的参考意义。
Rapamycin (Sirolimus) is a triene macrolide antibiotic produced by Streptomyces hygroscopicus. Besides its wide application as an effective immunosuppressive agent, other important bioactivities have made rapamycin a potential drug lead for novel pharmaceutical development. However, the low titer of rapamycin in the original producer strain limits further industrialization efforts and restricts its use for other applications.
We optimized the fermentation medium of the original high-yield mutant S. hygroscopicus with regard to the formation of rapamycin, and the optimal fermentation medium was formulated. The optimal medium:4% glucose,1% glycerol, 1% soybean meal,0.15% L-Lys,0.5% K2HPO4,0.5% KH2PO4,0.5% NaCl,0.2% MgSO4,0.15% FeSO4. Under these optimal fermentation conditions, shake bottle loading of 20ml/250ml, culture temperature of 28℃, and havest cell mass after cultivated for 5 days, the rapamycin production was improved to 120mg/L.
Predicated on knowledge of the metabolic pathways related to rapamycin biosynthesis in S. hygroscopicus, we have designed a rational screening approach to generate a rapamycin high producer strain. The breeding of the rapamycin producer was carried out by isolating high-producing strains from colonies after exposure to the mutagens of UV, NTG or/and DES. The resultant strain produced rapamycin at 279mg/L in the shake flask scale. Then a novel high-throughput cultivation method was developed to rapidly screen large numbers of rapamycin-producing mutants of Streptomyces hygroscopicus by duplicate culturing of isolates on the surfaces of agar-solidified 96 wells in microtiter plates. By integrating 96-well solid cultivation and the bioassay, we screened more than 100,000 isolates and one mutant produced 445mg rapamycin/L was screened out, which was double the yield of parent strain used in the submerged fermentation process.
The fermentation processes were further scaled up in 120 L and 20,000 L fermentors, respectively at the pilot plant. Selected fermentation factors including agitation speed, pH, and on-line supplementation were systematically evaluated. A fed-batch strategy was established to maximize rapamycin production. With these efforts, an optimized fermentation process in the larger scale fermentor was developed. In the final fermentation titer, the rapamycin productivity was 812 mg/L in the 120 L fermentor and 783 mg/L in the 20,000 L fermentor, respectively.
Several protoplasts-related techniques including protoplasts mutation, intraspecies, and interspecies protoplasts fusion were carried out to improve the rapamycin productivity in S. hygroscopicus. The interspecies fusion of protoplasts of rapamycin producer S. hygroscopicus D7-804 and erythreumycin producer S. erythreus ZJU325 could have brought about one high-yield (345 mg/L) rapamycin producer with 23.6% higher than that of the parental strain. After genome shuffling work, two high-yielding strains were isolated and the highest productivity of rapamycin attained 445mg/L. The systematic research of protoplast-related techniques has established an applicable way to generate high-yield strains from original microorganisms which can only produce low amount of expected natural products, without information of target gene clusters and gene sequences. Comparing with traditional screening method, genome shuffling is much rapid and higher efficiency to obtain higher productivity strain.
The biosynthetic gene cluster of rapamycin(RAP) has been respectively integrated into three Streptomyces mode hosts S. albus, S. ceolicolor M512, and S. lividans K4-114 by BAC-based technology to generate the RAP heterologous production systems. The bioassay results suggested that olny S. albus 28-1-1 from the constructed three heterologous systems has clearly showed the antibiotic activity similar to RAP. But the parallel HPLC and LC-MS analyses did not detect any trace of the production. Subsequent RT-PCR analyses has indicated that the majority of RAP biosynthetic genes were not transcripted, only three downstream genes rapG, rapF and rapD showed transcription signal in S. albus. Thus, insufficient or improper regulation of RAP genes could be a critical reason that limited the normal gene transcription. This exploring study will be a base for efficient biosynthesis of rapamycin and its similarities in the future.
试验首先初步优化了雷帕霉素产生菌的培养基和培养条件,确定了最优的发酵培养基为：4%葡萄糖,1%甘油,1%黄豆饼粉,0.15%L-Lys,0.5%K2HPO4, 0.5%KH2PO4,0.5%氯化钠,0.2%硫酸镁,0.15%硫酸亚铁。较优的摇瓶发酵条件是：摇瓶装液量为20ml/250ml,培养温度为280C,接种后培养5天收获菌体。在以上最优条件下,原始菌株发酵液中雷帕霉素产量最高可达120mg/L。
为提高雷帕霉素产量,开展了雷帕霉素产生菌的诱变育种研究。首先考察了紫外线、DES、NTG等不同诱变剂的剂量效应。然后通过单一诱变剂连续诱变、不同诱变剂交替诱变、不同诱变剂连续诱变等多种诱变方式,筛选到产量为279mg/L的诱变菌株D7-804,比出发菌株提高了156%。进一步发展了基于96孔板培养的雷帕霉素突变株的高通量筛选新技术,通过对约100,000个单菌落的快速高效筛选,筛选到高产突变菌株N5632,其最高产量达到445mg/L
在以上高产菌选育和摇瓶发酵优化的基础上,开展了在120L发酵罐上的雷帕霉素发酵的放大研究。结果表明,初始搅拌转速设定为200rpm,在进入产物合成期后转速升至400rpm,控制整个发酵过程中溶氧不低于10%,雷帕霉素产量最高达到412mg／L,基本达到了摇瓶发酵的平均水平。在此基础上,开展了采用多种补料方式提高发酵水平的研究。结果表明,40h为最佳补料时间,采用多组分组合补料方式能够最大幅度提高发酵水平,其中组合补料甘油与磷酸盐的方式,不仅能够保持发酵液的pH值的相对稳定,而且能有效提高雷帕霉素产量,发酵水平最高可达800mg／L以上。最后,将这一新型补料发酵工艺在20吨发酵罐上进行工业放大,雷帕霉素发酵水平也达到了700mg／L,产量较本课题开始时的最初发酵水平提高了4倍以上。
为了最大程度地提高雷帕霉素高产菌选育的效率,开展了基因组重排技术改造吸水链霉菌的遗传性能,提高雷帕菌素发酵水平的研究。首先确定了雷帕霉素产生菌原生质体制备、再生和融合的较优条件；然后通过吸水链霉菌原生质体诱变、融合以及与红霉素生产菌原生质体的种间融合等技术,得到具有不同遗传特性的产雷帕菌素产量较高的突变株。最后,对7株雷帕霉素高产突变株进行一轮基因组重排育种,结果筛选到一株产量较高菌株(GS-1437),雷帕霉素产量达到445mg/L,比最初出发菌株产量提高了59.5%,充分显示了基因组重排技术在改造链霉菌遗传特性方面的巨大潜力,与传统诱变育种方法比较,具有快速高效等显著优点。。
开展了雷帕霉素生物合成基因镞在异源宿主菌中表达及生物合成雷帕霉素的研究。在已构建的三种雷帕霉素异源合成系统中,白色链霉菌S. albus 28-1-1发酵产物样品表现出与雷帕霉素标准品类似的抗菌生物活性。通过初步的结构鉴定和分析,该发酵产物样品中的未知生物活性物质是一种生物活性类似雷帕霉素但结构上有较大区别的新化合物。进一步从转录水平上对异源宿主菌中雷帕霉素生物合成基因镞中的各个基因进行了转录分析。通过提取发酵72h的菌体RNA,采用RT-PCR技术分析各基因在转录水平上的差异,S. albus 28-1-1中绝大部分雷帕霉素生物合成基因未能被正常转录,仅有下游的rapG,rapF和rapD显示与参照系统相同的转录信号。这一研究结果对在异源宿主菌中生物合成雷帕霉素及其他新颖抗生素具有重要的参考意义。
Rapamycin (Sirolimus) is a triene macrolide antibiotic produced by Streptomyces hygroscopicus. Besides its wide application as an effective immunosuppressive agent, other important bioactivities have made rapamycin a potential drug lead for novel pharmaceutical development. However, the low titer of rapamycin in the original producer strain limits further industrialization efforts and restricts its use for other applications.
We optimized the fermentation medium of the original high-yield mutant S. hygroscopicus with regard to the formation of rapamycin, and the optimal fermentation medium was formulated. The optimal medium:4% glucose,1% glycerol, 1% soybean meal,0.15% L-Lys,0.5% K2HPO4,0.5% KH2PO4,0.5% NaCl,0.2% MgSO4,0.15% FeSO4. Under these optimal fermentation conditions, shake bottle loading of 20ml/250ml, culture temperature of 28℃, and havest cell mass after cultivated for 5 days, the rapamycin production was improved to 120mg/L.
Predicated on knowledge of the metabolic pathways related to rapamycin biosynthesis in S. hygroscopicus, we have designed a rational screening approach to generate a rapamycin high producer strain. The breeding of the rapamycin producer was carried out by isolating high-producing strains from colonies after exposure to the mutagens of UV, NTG or/and DES. The resultant strain produced rapamycin at 279mg/L in the shake flask scale. Then a novel high-throughput cultivation method was developed to rapidly screen large numbers of rapamycin-producing mutants of Streptomyces hygroscopicus by duplicate culturing of isolates on the surfaces of agar-solidified 96 wells in microtiter plates. By integrating 96-well solid cultivation and the bioassay, we screened more than 100,000 isolates and one mutant produced 445mg rapamycin/L was screened out, which was double the yield of parent strain used in the submerged fermentation process.
The fermentation processes were further scaled up in 120 L and 20,000 L fermentors, respectively at the pilot plant. Selected fermentation factors including agitation speed, pH, and on-line supplementation were systematically evaluated. A fed-batch strategy was established to maximize rapamycin production. With these efforts, an optimized fermentation process in the larger scale fermentor was developed. In the final fermentation titer, the rapamycin productivity was 812 mg/L in the 120 L fermentor and 783 mg/L in the 20,000 L fermentor, respectively.
Several protoplasts-related techniques including protoplasts mutation, intraspecies, and interspecies protoplasts fusion were carried out to improve the rapamycin productivity in S. hygroscopicus. The interspecies fusion of protoplasts of rapamycin producer S. hygroscopicus D7-804 and erythreumycin producer S. erythreus ZJU325 could have brought about one high-yield (345 mg/L) rapamycin producer with 23.6% higher than that of the parental strain. After genome shuffling work, two high-yielding strains were isolated and the highest productivity of rapamycin attained 445mg/L. The systematic research of protoplast-related techniques has established an applicable way to generate high-yield strains from original microorganisms which can only produce low amount of expected natural products, without information of target gene clusters and gene sequences. Comparing with traditional screening method, genome shuffling is much rapid and higher efficiency to obtain higher productivity strain.
The biosynthetic gene cluster of rapamycin(RAP) has been respectively integrated into three Streptomyces mode hosts S. albus, S. ceolicolor M512, and S. lividans K4-114 by BAC-based technology to generate the RAP heterologous production systems. The bioassay results suggested that olny S. albus 28-1-1 from the constructed three heterologous systems has clearly showed the antibiotic activity similar to RAP. But the parallel HPLC and LC-MS analyses did not detect any trace of the production. Subsequent RT-PCR analyses has indicated that the majority of RAP biosynthetic genes were not transcripted, only three downstream genes rapG, rapF and rapD showed transcription signal in S. albus. Thus, insufficient or improper regulation of RAP genes could be a critical reason that limited the normal gene transcription. This exploring study will be a base for efficient biosynthesis of rapamycin and its similarities in the future.
引文
[1]杨灿,刘宝山.新型高效多活性低毒免疫抑制剂一西罗莫司研究新进展.海峡药学,2003,15(5)：1-4.
[2]季新燕,温耀明,陈振伟,郑卫.环孢素的生物学活性及其衍生物的研究进展.国外医药：抗生素分册,2006,27(3).
[3]陈忠华.第十七届国际器官移植会议纪要(一).中华器官移植杂志,1999,1：59-60.
[4]Zyriakides G, Miller J. Use of cyclosporine in renal transplantation. Transplantation Proceedings,2004,36(2):167-172.
[5]Kino T, Hatanaka H, Hashimoto H, et al. FK506, a novel immunosuppressant isolated from a Streptomyces. I. Fermentation, isolation, and physicochemical and biological characteristic. J Antibiot,1987,40(9):1249.
[6]力弘,贾永锋.雷公藤多甙的抗炎和免疫抑制作用.上海医科大学学报,2000,27(6)：502-505.
[7]Lazary S, Stahelin H. Immumosuppressive and specific antibiotic effects of ovalicin. Experientia,1968,24:1171.
[8]Vezina C, Kudelski, Sehgal SN. Rapamycin (AY-22,989), a new antibiotic:I. Taxonomy of the producing Streptomycete and isolation of the active principle. J Antibiot,1975,28:721.
[9]Martel RR, Klicius J, Galet S. Inhibition of the immune response by rapamycin, a new antifungal antibiotic.1977. Can J Physiol Pharmacol,55(1):48-51.
[10]Goggins WC, Fisher RA, Dattilo JB, et al. Analysis of functional renal allograft tolerance with single-dose rapamycin based induction immunosuppression. Transplantatin,1997,63:310-314.
[11]Balter M. Protein matchmaker may lead new gene therapy to the alter. Nature, 1996,273:183.
[12]Cheng, Y.R., Huang, J., Qiang, H., Lin, W.L., Demain, A.L. Mutagenesis of the rapamycin producer Streptomyces hygroscopicus FC904. J Antibiot,2001,54: 967-972.
[13]Chen YL, et al. New process control strategy used in a rapamycin fermentation. Process Biochemistry,1999,34:383-389.
[14]Sehgal SN. Rapamycin (Sirolimus):An overview and mechanism of action. Ther Drug Monil,1995,17:660.
[15]Betetta L, Gingras AC, Svitkin YV et al. Rapamycin blocks the phosphorylation of 4E-BP1 and inhibits cap-dependent iniation of translation. EMBO J.1995, 15:658.
[16]Sabatini DM, Pierchala BA, Barrow RK et al. The Rapamycin and FKBP12 traget (RAFT) displays phospatidylinositol 4-kinase activity. J Biol Chern, 1995,270:20875.
[17]夏国伟,夏穗生,张元芳.雷帕霉素研究进展.中华器官移植杂志,2002,23(1)：60-62.
[18]董济民,慕为民.西罗莫司抗肿瘤研究的进展.湖南中医药大学学报,2009,29(8)：65-67.
[19]Harrison DE, et al. Rapamycin fed late in life extends lifespan in genetically heterogeneous mice. NATURE,2009,460(16):392-396.
[20]涂响安.雷帕霉素及其在肾移植中的应用.国外医学泌尿系统分册,2003,23(1)：14-16.
[21]赵武述.免疫抑制剂的研究进展及在器官移植中的应用.中国处方药,2003.4：66-69.
[22]陈纪林,高润霖,杨跃进等.雷帕霉素药物洗脱支架在冠心病复杂病变中应用的近远期疗效.中华心血管病杂志,2004,32(4)：867-869.
[23]杨培增,张震.葡萄膜炎免疫治疗的进展与展望.中华眼科杂志,2002,38(9)：574-576.
[24]杨震坤,沈卫峰,张瑞岩等.雷帕霉素洗脱支架治疗冠状动脉慢性完全性闭塞病变.中国循环杂志,2006.
[25]Yu K, Toral-Barza L, Discafani C, et al. mTOR, a novel target in breast cancer: the efect of CCI-779, an mTOR inhibitor, in preclinical models of breast cancer. Endocr Relat Cancer,2001,8(3):249-258.
[26]Hidalgo M, Buckner JC, Erlichman C, et al. A phase Ⅰ and pharmacokinetic study of temsirolimus(CCI-779) administered intravenously daily for 5 days every 2 weeks to patients with advanced cancer. Clin Cancer Res,2006,12(19): 5755-5763.
[27]Oza AM, Elit L, Provencher D, et al. A phase Ⅱ study of Temsirolimus(CCI-779) in patients with metastatic and/or recurrent endometrial cancer. J Clin Oncol,2008,26(15):5516.
[28]Fasolo A, Sessa C. mTOR inhibitors in the treatment of cancer. Expert Opin Investig Drugs,2008,17(11):1717-1734.
[29]Mabuchi S, Altomare DA, Cheung M, et al. RAD001 inhibits human ovarian cancer cell proliferation, enhances cisplatin-induced apoptosis and prolongs survival in an ovarian cancer model. Clin Cancer Res,2007,13(14): 4261-4270.
[30]Lane HA, Wood JM, McSheehy PM. mTOR inhibitor RAD00 1(Everolimus) has antiangiogenic/vascular properties distinct from a VEGFR tyrosine kinase jnhibitor. Clin Cancer Res,2009,15(5):1612-1622.
[31]李秀营,李东吉.免疫抑制剂-艾罗莫司.齐鲁药事,2007,26(12)：759-760.
[32]Hartfdrd CM, Desai AA, Janisch L. A phase I trial to determine the safety, tolerability, and maximum tolerated dose of deforolimus in patients with advanced malignan cies. Clin Cancer Res,2009,15(4):1428-1434.
[33]Meyer SD, Miwa T, Nakatsuka M, Schreiber SL. Synthetic investigations of rapamycin.1. Synthesis of a C10-C21 fragment. J Org Chem,1992,57: 5058-5060.
[34]Romo D, Johnson D, Plamondon L, Miwa T, Schreiber SL. Synthetic investigations of rapamycin.2. Synthesis of a C(22)-C(42) fragment. J Org Chem,1992,57:5060-5063.
[35]Meyer SD, Romo D, Johnson DD, Schreiber SL. Total synthesis of (-)-rapamycin using an Evans-Tishchenko fragment coupling. J Am Chem Soc, 1993,115:7906-7907.
[36]Kojima N, et al. NOVEL RAPAMYCIN PRODUCER. PA/US 1993/001534.
[37]林文良,吴晓华等.新型强效免疫抑制剂F904的结构测定.中国抗生素杂志2002,27(12)：713-716.
[38]Istvan Mlonar, Jesus F.Apraicio, Stephen. Organisation of the biosynthetic gene cluser for rapamycin in Streptomyces hgroscopicus:analysis of genes flanking the polyketide synthase. Gene,1996,169:1-7.
[39]Jesus F, et al. Organisation of the biosynthetic gene cluser for rapamycin in Streptomyces hgroscopicus:analysis of the enzymatic domains in the modular polyketide synthase.Gene,1996,169:9-16.
[40]Staunton J, Wilkinson B. Biosynthesis of Erythromycin and Rapamycin. Chem Rev,1997,97:2611-2629.
[41]Cheng YR, et al. Mutagenesis of the Rapamycin Producer Streptomyces hygroscopicus FC904. J Antibiotics,2001,54 (11):967-972.
[42]Tsuchida T, et al. Production of L-Leucine by a Mutant of Brevibacterium lactofermentum 2256. Agricultural and Biological Chemistry,1974,38(10): 1907-1911.
[43]李寅,陈坚,陈燕,黄敏,芮新生,潘春.丙酮酸高产芮株的选育及中试研究.工业微生物,2001,31(2).
[447 http://www.ncbi.nlm.nih.gov/genomes.
[45]Stephanopoulos G. Metabolic engineering:perspective of a chemical engineer. AIChE Journal.2002,48(5):920-926.
[46]Stemmer WPC. Rapid evolution of a protein in vitro by DNA shuffling. Nature, 1994,370(4):389-391.
[47]Scheinbach S. Protoplast fusion as a means of producing new industrial yeast strains. Biotechnology Advances,1983,1:289-300.
[48]Hopwood DA, Wright HM, Bibb MJ & Cohen SN. Genetic recombination through protoplast fusion in Streptomyces. Nature,1977,268:171-174.
[49]Iwata M, Mada M & Ishiwa H. Protoplast fusion of Lactobacillus fermentum. Applied & Environmental Microbiology,1986,52:392-393.
[50]Rassoulzadegan M Binetruy B & Cuzin F. High frequency of gene transfer after fusion between bacteria and aukaryotic cells. Nature,1982,295:257-259.
[51]Zhang YX, Perry K, Vinci VA, Powell K, Stemmer WPC & del Cardayre SB. Genome shuffling leads to rapid phenotypic improvement in bacteria. Nature, 2002,8:891-898.
[52]Patnaik R, Louie S, Gavrilovic V, Perry K, Stemmer WPC, Ryan CM & del Cardayre SB. Genome shuffling of Lactobacillus for improved acid tolerance. Nature Biotechnology,2002,20:707-712.
[53]Dai MH and Copley SD. Genome Shuffling Improves Degradation of the Anthropogenic Pesticide Pentachlorophenol by Sphingobium chlorophenolicum ATCC 39723. Applied and Environmental Microbiology,2004,70(4): 2391-2397.
[54]赵凯,平文祥,张丽娜等.用Genome shuffling技术选育紫杉醇高产菌株.中国科学C辑：生命科学,2008,38(3)：221-229.
[55]徐波,王明蓉,夏永,杨奎,张春燕.应用基因组重排育种新方法筛选替考拉宁高产菌.中国抗生素杂志,2006.
[56]梁惠仪,郭勇.全基因组重排育种技术提高豆豉纤溶酶菌产酶量.中国生物工程杂志,2007.
[57]强华,黄捷,陈贻锴等.抗生素、亚硝基胍处理吸水链霉菌FC 904原生质体对雷帕霉素产量的影响.福建医科大学学报,1999,33(3)：289-293.
[58]Kojima I, et al. Carbon source nutrition of rapamycin biosynthesis in Streptomyces hgroscopicus. J Ind Micro,1995,14:436-439.
[59]Cheng YR, Fang A, Demain AL. Effect of amino acids on rapamycin biosynthesis by Streptomyces hygroscopicus. Applied microbiology and biotechnology,1995,43:1096-1098.
[60]Lee MS, et al. Effect of nitrogen source on biosynthesis of rapamycin by Streptomyces hgroscopicus. J Ind Micro&Bio,1997,19:83-86.
[61]Kim WS, et al. Methionine interference in rapamycin production involves repression of demethylrapamycin methyltransferase and S-adenosylmethionine synthetase. Antimicrobial agents and chemotherapy,2000,44(10):2908-2910
[62]Cheng YR, Hauck L, Demain AL. Phosphate,ammonium,magnetism and iron nutrition of Streptomyces hygrescopicus with respect to rapamycin biosynthesis. J Ind Microbiol,1995,14:424-427.
[63]Chen YL, et al. New process control strategy used in a rapamycin fermentation. Process Biochemistry,1999,34:383-389.
[64]Man BG, Prasad SD. A miniature bioreactor for sensing toxicity using recombinant bioluminescent E. coli cells. Biotechnol Prog,1996,12:393-397.
[65]Leuking A, Horn M, Eickhoff H, Buessow K, Lehrach H, Walter G. Protein microassays for gene expression and antibody screening. Anal Biochem,1999, 270:103-111.
[66]Kyranos JN. High-throughput analysis aids early drug discovery. Trends in Analytical Chemistry,2005,24(8):793-794.
[67]Yoo C, Cooper GG. An evaluation of a system that recommends microarray experiments to perform to discover gene regulation pathways. Artif Intel] Med, 2004,31:169-182.
[68]Kerr MK, Churchil GA. Experimental design for gene expression microassays. Trends Biotechnol,1999,17:275-281.
[69]Duetz W, Ruedi L, Hermann R, O'Conner K, Buchs J&Witholt B. Methods for intense aeration, growth, storage, and replication of bacterial strains in microtiter plates. Appl Environ Microbiol,2000,66:2641-2646.
[70]John GT, Klimant I, Wittmann C, Heinzle E. Integrated optical sensing of dissolved oxygen in microtiter plates:a novel tool for microbial cultivation. Biotechnol Bioeng,2003,81:829-836.
[71]Sathish Kumar, Christoph Wittmann, Elmar Heinzle. Minibioreactor. Biotechnology Letters,2004,26:1-10.
[72]Giege, P., Z. Konthur, Cx Walter, A. Brennicke. An ordered Arabidopsis thaliana mitochondri a) cDNA library on high-density filters allows rapid systematic analysis of plant gene expression:a pilot study. Plant J.,1999,15:721-726.
[73]Hersh BM, et al. A glutamate-dependent acid resistance gene in Escherichia coli. J. Bacteriol.,1996,178:3978-3981.
[74]Takeda H, Yamakuchi H, et al. Construction of a bovine yeast artificial chromosome (YAC) library. Anim Genet.1998,29:216-219.
[75]Minas W, Bailey JE, Duetz W. Streptomycetes in micro-cultures:Growth, production of secondary metabolites, and storage and retrieval in the 96-well format. Antoine van Leeuwenhoek,2000,78:297-305.
[76]程骥等.黑暗链霉菌与卡那链霉菌原生质体融合的研究.福州大学学报,2003,31(1)：111-115.
[77]沈齐英等.原生质体融合选育高效降酚微生物.环境污染治理技术与设备.2003,4(4)：39-42.
[78]姚曙光等.利用原生质体融合法选育林可霉素高产菌株.安徽医药,2003,7(1)：62-63.
[79]强华等.抗生素、亚硝基胍处理吸水链霉菌FC904原生质体对雷帕霉素产量的影响.福建医科大学学报,1999,33(3)：289-292.
[80]Malpartida F, Hallam SE, Kieser HM, et al. Homology between Streptomyces genes coding for synthesis of different polyketides used to clone antibiotic biosynthetic genes. Nature,1987,325:818-821.
[81]Cortes J, Haydock SF, Roberts GA, et al. An unusually large multifunctional polypeptide in the erythromycin producing polyketide synthase of Saccharopolyspora erythraea. Nature,1990,348:176-178.
[82]Baltz RH, and Seno ET. Genetics of Streptomyces fradiae and tylosin biosynthesis. Annu Rev Microbial,1988,42:547-574.
[83]Hopwood DA, Malpartida F, Kieser HM, et al. Production of'hybrid'antibiotics by genetic engineering. Nature,1985,314:642-644.
[84]Weissman KJ, Leadlay PF. Combinatorial biosynthesis of reduced polyketides. Nat Rev Microbiol,2005,3:925-936.
[85]Menzella HG, et al. Combinatorial polyketide biosynthesis by denovo design and rearrangement of modular polyketide synthase genes. Nat Biotechnol,2005,23, 1171-1176
[86]Pelzer S, et al. Novel natural compounds obtained by genomebased screening and genetic engineering. Curr Opin Drug Disc Develop,2005,8:228-238.
[87]Van Lanen SG, and Shen B. Progress in combinatorial biosynthesis for drug discovery. Drug discovery today:Technologies,2006,3:285-292.
[88]Galm U, and Shen B. Expression of biosynthetic gene clusters in heterologous hosts for natural product production and combinatorial biosynthesis. Expert Opin Drug Discov,2006,1(5):409-441.
[89]He W Q, Lei J, Liu Y Y, Wang Y G. The LuxR family members GdmRI and GdmRII are positive regulators of geldanamycin biosynthesis in Streptomyces hygroscopicus 17997. Arch Microbiol,2008,189:501-510
[90]Schwecke T, Aparicio J F, Molnar I, Konig A, Khaw L E, Haydock S F, Oliynyk M, Caffrey P, Cortes J, Lester J B, et al. The biosynthetic gene cluster for the polyketide immunosuppressant rapamycin. PNAS,1995,92(17):7839-7843.
[91]Konig A, Schwecke T, Molnar I, Bohm G A, Lowden P A, Staunton J, Leadlay P F. The pipecolate-incorporating enzyme for the biosynthesis of the immunosuppressant rapamycin--nucleotide sequence analysis, disruption and heterologous expression of rapP from Streptomyces hygroscopicus. Eur J Biochem/FEBS,1997,247:526-534
[92]Gatto G J, Jr Boyne M T,2nd, Kelleher N L, Walsh C T. Biosynthesis of pipecolic acid by RapL, a lysine cyclodeaminase encoded in the rapamycin gene cluster. J Am Chem Soc,2006,128:3838-3847
[93]Kuscer E, Coates N, Challis I, Gregory M, Wilkinson B, Sheridan R, Petkovic H. Roles of rapH and rapG in positive regulation of rapamycin biosynthesis in Streptomyces hygroscopicus. J bacteriol,2007,189:4756-4763
[94]Wendt-Pienkowski E, Huang Y, Zhang J, Li B, Jiang H, Kwon H, Hutchinson C R, Shen B. Cloning, sequencing, analysis, and heterologous expression of the fredericamycin biosynthetic gene cluster from Streptomyces griseus. J Am Chem Soc,2005,127:16442-16444.
[2]季新燕,温耀明,陈振伟,郑卫.环孢素的生物学活性及其衍生物的研究进展.国外医药：抗生素分册,2006,27(3).
[3]陈忠华.第十七届国际器官移植会议纪要(一).中华器官移植杂志,1999,1：59-60.
[4]Zyriakides G, Miller J. Use of cyclosporine in renal transplantation. Transplantation Proceedings,2004,36(2):167-172.
[5]Kino T, Hatanaka H, Hashimoto H, et al. FK506, a novel immunosuppressant isolated from a Streptomyces. I. Fermentation, isolation, and physicochemical and biological characteristic. J Antibiot,1987,40(9):1249.
[6]力弘,贾永锋.雷公藤多甙的抗炎和免疫抑制作用.上海医科大学学报,2000,27(6)：502-505.
[7]Lazary S, Stahelin H. Immumosuppressive and specific antibiotic effects of ovalicin. Experientia,1968,24:1171.
[8]Vezina C, Kudelski, Sehgal SN. Rapamycin (AY-22,989), a new antibiotic:I. Taxonomy of the producing Streptomycete and isolation of the active principle. J Antibiot,1975,28:721.
[9]Martel RR, Klicius J, Galet S. Inhibition of the immune response by rapamycin, a new antifungal antibiotic.1977. Can J Physiol Pharmacol,55(1):48-51.
[10]Goggins WC, Fisher RA, Dattilo JB, et al. Analysis of functional renal allograft tolerance with single-dose rapamycin based induction immunosuppression. Transplantatin,1997,63:310-314.
[11]Balter M. Protein matchmaker may lead new gene therapy to the alter. Nature, 1996,273:183.
[12]Cheng, Y.R., Huang, J., Qiang, H., Lin, W.L., Demain, A.L. Mutagenesis of the rapamycin producer Streptomyces hygroscopicus FC904. J Antibiot,2001,54: 967-972.
[13]Chen YL, et al. New process control strategy used in a rapamycin fermentation. Process Biochemistry,1999,34:383-389.
[14]Sehgal SN. Rapamycin (Sirolimus):An overview and mechanism of action. Ther Drug Monil,1995,17:660.
[15]Betetta L, Gingras AC, Svitkin YV et al. Rapamycin blocks the phosphorylation of 4E-BP1 and inhibits cap-dependent iniation of translation. EMBO J.1995, 15:658.
[16]Sabatini DM, Pierchala BA, Barrow RK et al. The Rapamycin and FKBP12 traget (RAFT) displays phospatidylinositol 4-kinase activity. J Biol Chern, 1995,270:20875.
[17]夏国伟,夏穗生,张元芳.雷帕霉素研究进展.中华器官移植杂志,2002,23(1)：60-62.
[18]董济民,慕为民.西罗莫司抗肿瘤研究的进展.湖南中医药大学学报,2009,29(8)：65-67.
[19]Harrison DE, et al. Rapamycin fed late in life extends lifespan in genetically heterogeneous mice. NATURE,2009,460(16):392-396.
[20]涂响安.雷帕霉素及其在肾移植中的应用.国外医学泌尿系统分册,2003,23(1)：14-16.
[21]赵武述.免疫抑制剂的研究进展及在器官移植中的应用.中国处方药,2003.4：66-69.
[22]陈纪林,高润霖,杨跃进等.雷帕霉素药物洗脱支架在冠心病复杂病变中应用的近远期疗效.中华心血管病杂志,2004,32(4)：867-869.
[23]杨培增,张震.葡萄膜炎免疫治疗的进展与展望.中华眼科杂志,2002,38(9)：574-576.
[24]杨震坤,沈卫峰,张瑞岩等.雷帕霉素洗脱支架治疗冠状动脉慢性完全性闭塞病变.中国循环杂志,2006.
[25]Yu K, Toral-Barza L, Discafani C, et al. mTOR, a novel target in breast cancer: the efect of CCI-779, an mTOR inhibitor, in preclinical models of breast cancer. Endocr Relat Cancer,2001,8(3):249-258.
[26]Hidalgo M, Buckner JC, Erlichman C, et al. A phase Ⅰ and pharmacokinetic study of temsirolimus(CCI-779) administered intravenously daily for 5 days every 2 weeks to patients with advanced cancer. Clin Cancer Res,2006,12(19): 5755-5763.
[27]Oza AM, Elit L, Provencher D, et al. A phase Ⅱ study of Temsirolimus(CCI-779) in patients with metastatic and/or recurrent endometrial cancer. J Clin Oncol,2008,26(15):5516.
[28]Fasolo A, Sessa C. mTOR inhibitors in the treatment of cancer. Expert Opin Investig Drugs,2008,17(11):1717-1734.
[29]Mabuchi S, Altomare DA, Cheung M, et al. RAD001 inhibits human ovarian cancer cell proliferation, enhances cisplatin-induced apoptosis and prolongs survival in an ovarian cancer model. Clin Cancer Res,2007,13(14): 4261-4270.
[30]Lane HA, Wood JM, McSheehy PM. mTOR inhibitor RAD00 1(Everolimus) has antiangiogenic/vascular properties distinct from a VEGFR tyrosine kinase jnhibitor. Clin Cancer Res,2009,15(5):1612-1622.
[31]李秀营,李东吉.免疫抑制剂-艾罗莫司.齐鲁药事,2007,26(12)：759-760.
[32]Hartfdrd CM, Desai AA, Janisch L. A phase I trial to determine the safety, tolerability, and maximum tolerated dose of deforolimus in patients with advanced malignan cies. Clin Cancer Res,2009,15(4):1428-1434.
[33]Meyer SD, Miwa T, Nakatsuka M, Schreiber SL. Synthetic investigations of rapamycin.1. Synthesis of a C10-C21 fragment. J Org Chem,1992,57: 5058-5060.
[34]Romo D, Johnson D, Plamondon L, Miwa T, Schreiber SL. Synthetic investigations of rapamycin.2. Synthesis of a C(22)-C(42) fragment. J Org Chem,1992,57:5060-5063.
[35]Meyer SD, Romo D, Johnson DD, Schreiber SL. Total synthesis of (-)-rapamycin using an Evans-Tishchenko fragment coupling. J Am Chem Soc, 1993,115:7906-7907.
[36]Kojima N, et al. NOVEL RAPAMYCIN PRODUCER. PA/US 1993/001534.
[37]林文良,吴晓华等.新型强效免疫抑制剂F904的结构测定.中国抗生素杂志2002,27(12)：713-716.
[38]Istvan Mlonar, Jesus F.Apraicio, Stephen. Organisation of the biosynthetic gene cluser for rapamycin in Streptomyces hgroscopicus:analysis of genes flanking the polyketide synthase. Gene,1996,169:1-7.
[39]Jesus F, et al. Organisation of the biosynthetic gene cluser for rapamycin in Streptomyces hgroscopicus:analysis of the enzymatic domains in the modular polyketide synthase.Gene,1996,169:9-16.
[40]Staunton J, Wilkinson B. Biosynthesis of Erythromycin and Rapamycin. Chem Rev,1997,97:2611-2629.
[41]Cheng YR, et al. Mutagenesis of the Rapamycin Producer Streptomyces hygroscopicus FC904. J Antibiotics,2001,54 (11):967-972.
[42]Tsuchida T, et al. Production of L-Leucine by a Mutant of Brevibacterium lactofermentum 2256. Agricultural and Biological Chemistry,1974,38(10): 1907-1911.
[43]李寅,陈坚,陈燕,黄敏,芮新生,潘春.丙酮酸高产芮株的选育及中试研究.工业微生物,2001,31(2).
[447 http://www.ncbi.nlm.nih.gov/genomes.
[45]Stephanopoulos G. Metabolic engineering:perspective of a chemical engineer. AIChE Journal.2002,48(5):920-926.
[46]Stemmer WPC. Rapid evolution of a protein in vitro by DNA shuffling. Nature, 1994,370(4):389-391.
[47]Scheinbach S. Protoplast fusion as a means of producing new industrial yeast strains. Biotechnology Advances,1983,1:289-300.
[48]Hopwood DA, Wright HM, Bibb MJ & Cohen SN. Genetic recombination through protoplast fusion in Streptomyces. Nature,1977,268:171-174.
[49]Iwata M, Mada M & Ishiwa H. Protoplast fusion of Lactobacillus fermentum. Applied & Environmental Microbiology,1986,52:392-393.
[50]Rassoulzadegan M Binetruy B & Cuzin F. High frequency of gene transfer after fusion between bacteria and aukaryotic cells. Nature,1982,295:257-259.
[51]Zhang YX, Perry K, Vinci VA, Powell K, Stemmer WPC & del Cardayre SB. Genome shuffling leads to rapid phenotypic improvement in bacteria. Nature, 2002,8:891-898.
[52]Patnaik R, Louie S, Gavrilovic V, Perry K, Stemmer WPC, Ryan CM & del Cardayre SB. Genome shuffling of Lactobacillus for improved acid tolerance. Nature Biotechnology,2002,20:707-712.
[53]Dai MH and Copley SD. Genome Shuffling Improves Degradation of the Anthropogenic Pesticide Pentachlorophenol by Sphingobium chlorophenolicum ATCC 39723. Applied and Environmental Microbiology,2004,70(4): 2391-2397.
[54]赵凯,平文祥,张丽娜等.用Genome shuffling技术选育紫杉醇高产菌株.中国科学C辑：生命科学,2008,38(3)：221-229.
[55]徐波,王明蓉,夏永,杨奎,张春燕.应用基因组重排育种新方法筛选替考拉宁高产菌.中国抗生素杂志,2006.
[56]梁惠仪,郭勇.全基因组重排育种技术提高豆豉纤溶酶菌产酶量.中国生物工程杂志,2007.
[57]强华,黄捷,陈贻锴等.抗生素、亚硝基胍处理吸水链霉菌FC 904原生质体对雷帕霉素产量的影响.福建医科大学学报,1999,33(3)：289-293.
[58]Kojima I, et al. Carbon source nutrition of rapamycin biosynthesis in Streptomyces hgroscopicus. J Ind Micro,1995,14:436-439.
[59]Cheng YR, Fang A, Demain AL. Effect of amino acids on rapamycin biosynthesis by Streptomyces hygroscopicus. Applied microbiology and biotechnology,1995,43:1096-1098.
[60]Lee MS, et al. Effect of nitrogen source on biosynthesis of rapamycin by Streptomyces hgroscopicus. J Ind Micro&Bio,1997,19:83-86.
[61]Kim WS, et al. Methionine interference in rapamycin production involves repression of demethylrapamycin methyltransferase and S-adenosylmethionine synthetase. Antimicrobial agents and chemotherapy,2000,44(10):2908-2910
[62]Cheng YR, Hauck L, Demain AL. Phosphate,ammonium,magnetism and iron nutrition of Streptomyces hygrescopicus with respect to rapamycin biosynthesis. J Ind Microbiol,1995,14:424-427.
[63]Chen YL, et al. New process control strategy used in a rapamycin fermentation. Process Biochemistry,1999,34:383-389.
[64]Man BG, Prasad SD. A miniature bioreactor for sensing toxicity using recombinant bioluminescent E. coli cells. Biotechnol Prog,1996,12:393-397.
[65]Leuking A, Horn M, Eickhoff H, Buessow K, Lehrach H, Walter G. Protein microassays for gene expression and antibody screening. Anal Biochem,1999, 270:103-111.
[66]Kyranos JN. High-throughput analysis aids early drug discovery. Trends in Analytical Chemistry,2005,24(8):793-794.
[67]Yoo C, Cooper GG. An evaluation of a system that recommends microarray experiments to perform to discover gene regulation pathways. Artif Intel] Med, 2004,31:169-182.
[68]Kerr MK, Churchil GA. Experimental design for gene expression microassays. Trends Biotechnol,1999,17:275-281.
[69]Duetz W, Ruedi L, Hermann R, O'Conner K, Buchs J&Witholt B. Methods for intense aeration, growth, storage, and replication of bacterial strains in microtiter plates. Appl Environ Microbiol,2000,66:2641-2646.
[70]John GT, Klimant I, Wittmann C, Heinzle E. Integrated optical sensing of dissolved oxygen in microtiter plates:a novel tool for microbial cultivation. Biotechnol Bioeng,2003,81:829-836.
[71]Sathish Kumar, Christoph Wittmann, Elmar Heinzle. Minibioreactor. Biotechnology Letters,2004,26:1-10.
[72]Giege, P., Z. Konthur, Cx Walter, A. Brennicke. An ordered Arabidopsis thaliana mitochondri a) cDNA library on high-density filters allows rapid systematic analysis of plant gene expression:a pilot study. Plant J.,1999,15:721-726.
[73]Hersh BM, et al. A glutamate-dependent acid resistance gene in Escherichia coli. J. Bacteriol.,1996,178:3978-3981.
[74]Takeda H, Yamakuchi H, et al. Construction of a bovine yeast artificial chromosome (YAC) library. Anim Genet.1998,29:216-219.
[75]Minas W, Bailey JE, Duetz W. Streptomycetes in micro-cultures:Growth, production of secondary metabolites, and storage and retrieval in the 96-well format. Antoine van Leeuwenhoek,2000,78:297-305.
[76]程骥等.黑暗链霉菌与卡那链霉菌原生质体融合的研究.福州大学学报,2003,31(1)：111-115.
[77]沈齐英等.原生质体融合选育高效降酚微生物.环境污染治理技术与设备.2003,4(4)：39-42.
[78]姚曙光等.利用原生质体融合法选育林可霉素高产菌株.安徽医药,2003,7(1)：62-63.
[79]强华等.抗生素、亚硝基胍处理吸水链霉菌FC904原生质体对雷帕霉素产量的影响.福建医科大学学报,1999,33(3)：289-292.
[80]Malpartida F, Hallam SE, Kieser HM, et al. Homology between Streptomyces genes coding for synthesis of different polyketides used to clone antibiotic biosynthetic genes. Nature,1987,325:818-821.
[81]Cortes J, Haydock SF, Roberts GA, et al. An unusually large multifunctional polypeptide in the erythromycin producing polyketide synthase of Saccharopolyspora erythraea. Nature,1990,348:176-178.
[82]Baltz RH, and Seno ET. Genetics of Streptomyces fradiae and tylosin biosynthesis. Annu Rev Microbial,1988,42:547-574.
[83]Hopwood DA, Malpartida F, Kieser HM, et al. Production of'hybrid'antibiotics by genetic engineering. Nature,1985,314:642-644.
[84]Weissman KJ, Leadlay PF. Combinatorial biosynthesis of reduced polyketides. Nat Rev Microbiol,2005,3:925-936.
[85]Menzella HG, et al. Combinatorial polyketide biosynthesis by denovo design and rearrangement of modular polyketide synthase genes. Nat Biotechnol,2005,23, 1171-1176
[86]Pelzer S, et al. Novel natural compounds obtained by genomebased screening and genetic engineering. Curr Opin Drug Disc Develop,2005,8:228-238.
[87]Van Lanen SG, and Shen B. Progress in combinatorial biosynthesis for drug discovery. Drug discovery today:Technologies,2006,3:285-292.
[88]Galm U, and Shen B. Expression of biosynthetic gene clusters in heterologous hosts for natural product production and combinatorial biosynthesis. Expert Opin Drug Discov,2006,1(5):409-441.
[89]He W Q, Lei J, Liu Y Y, Wang Y G. The LuxR family members GdmRI and GdmRII are positive regulators of geldanamycin biosynthesis in Streptomyces hygroscopicus 17997. Arch Microbiol,2008,189:501-510
[90]Schwecke T, Aparicio J F, Molnar I, Konig A, Khaw L E, Haydock S F, Oliynyk M, Caffrey P, Cortes J, Lester J B, et al. The biosynthetic gene cluster for the polyketide immunosuppressant rapamycin. PNAS,1995,92(17):7839-7843.
[91]Konig A, Schwecke T, Molnar I, Bohm G A, Lowden P A, Staunton J, Leadlay P F. The pipecolate-incorporating enzyme for the biosynthesis of the immunosuppressant rapamycin--nucleotide sequence analysis, disruption and heterologous expression of rapP from Streptomyces hygroscopicus. Eur J Biochem/FEBS,1997,247:526-534
[92]Gatto G J, Jr Boyne M T,2nd, Kelleher N L, Walsh C T. Biosynthesis of pipecolic acid by RapL, a lysine cyclodeaminase encoded in the rapamycin gene cluster. J Am Chem Soc,2006,128:3838-3847
[93]Kuscer E, Coates N, Challis I, Gregory M, Wilkinson B, Sheridan R, Petkovic H. Roles of rapH and rapG in positive regulation of rapamycin biosynthesis in Streptomyces hygroscopicus. J bacteriol,2007,189:4756-4763
[94]Wendt-Pienkowski E, Huang Y, Zhang J, Li B, Jiang H, Kwon H, Hutchinson C R, Shen B. Cloning, sequencing, analysis, and heterologous expression of the fredericamycin biosynthetic gene cluster from Streptomyces griseus. J Am Chem Soc,2005,127:16442-16444.