变构菌素生物合成相关基因功能的研究
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摘要
变构菌素(tautomycetin,TMC)是从土壤微生物灰产色链霉菌(Streptomyces griseochromogenes)的培养液中分离到的一种聚酮类化合物。TMC属于马来酸酐类化合物,其化学结构为二烷基马来酸酐与聚酮链以酯键相连。近期研究发现TMC能够特异地抑制蛋白磷酸酶PP1,具有抗真菌活性,抗肿瘤活性和免疫抑制活性。由于变构菌素的这些结构特点和其特有的生物活性,吸引了很多科研工作者对其生物合成机制开展研究,为丰富天然小分子化合物生物合成机制,并为开发新的衍生物奠定基础。
变构菌素生物合成基因簇已被成功克隆并测序,从变构菌素基因簇序列分析可以看出,变构菌素的聚酮链骨架是由模块化的Ⅰ型聚酮合酶(PKS)合成的,但是二烷基马来酸酐的合成,聚酮链的PKS后修饰机制及二烷基马来酸酐与聚酮链的酯化机制都还未经阐明。因此本文首先建立和优化了灰产色链霉菌的遗传转化体系,并且发现甘氨酸和Ca2+能够提高大肠杆菌-链霉菌属间接合转移的效率。这一发现对于链霉菌的遗传工程操作具有普遍的应用价值。
为了研究二烷基马来酸酐的生物合成机制,本研究首先选择基因簇中两个可能的候选基因ttnO和ttnK进行基因阻断失活和互补。研究结果表明ttnO参与二烷基马来酸酐的合成；而△ttnK突变株积累二烷基马来酸酐,该结果证明ttnK的失活影响二烷基马来酸酐与聚酮链酯化所涉及的酶反应。
为了阐述PKS后修饰机制,基因ttnF, ttnC, ttnD和ttnI被阻断失活。这些基因分别编码肉毒碱脱水酶(ttnF),黄素蛋白脱羧酶(ttnC),UbiD家族脱羧酶(ttnD)和P450氧化酶(ttnI)。突变株△ttnF积累的主产物为TMC-F1,与TMC相比,TMC-F1缺少C5位酮基和末端烯烃结构,多了一个3-β-羟基-丙酸。该结果表明TtnF负责TMC聚酮链的脱水反应,是PKS后修饰反应的第一步；△ttnC突变株仍然产TMC,而△ttnD主要合成TMC-D1,与TMC-F1相比,TMC-D1的C3位的取代基为3-丙烯酸,并非3-β-羟基-丙酸,该结果证明ttnD编码的脱羧酶和TtnF一起负责变构菌素合成过程中二烯烃结构的形成,TtnD负责脱羧反应,TtnC的功能有待考证;△ttnI突变株积累5-去酮基-TMC衍生物,证明TtnI在变构菌素合成过程中负责C5位的氧化,该氧化反应是变构菌素合成过程中的最后一步反应。
另外通过微生物转化实验发现氧化酶TtnI能够催化5-羟基衍生物生成5-酮基衍生物,同时也说明变构菌素的生物合成基因被阻断失活时,变构菌素的合成会表现出一定程度的非专一性(promiscuity)。
本文的研究结果使得我们对变构菌素的生物合成机制有了更深一层的认识,为利用变构菌素进行组合生物合成技术开发新的小分子候选药物奠定基础。
Tautomycetin (TMC) is a polyketide that was first isolated from the Streptomyces griseochromogenes (S. griseochromogenes). TMC is a dialkylmaleic anhydride antibiotic with an unique chemical structure of an ester bond linkage between a dialkylmaleic anhydride (DA) moiety and a linear polyketide chain. Recently it was discovered that TMC is a specific protein phosphatase I inhibitor (PP1), with antifungi, antitumor and immunosuppressive activities. The unique chemical structure and important activities of TMC draw strong attention to the biosynthesis mechanism of TMC, which is not only critical for development of novel TMC analogs through combinatorial biosynthesis, semi-synthesis or heterologous expression, but also will contribute to a better understanding of the chemistry biology of polyketide synthase (PKS).
The biosynthesis gene cluster of TMC has been cloned and fully sequenced. By analyzing the sequence of the gene cluster of TMC, it was determined that the polyketide backbone of TMC is synthesized by type I PKS. However, the mechanisms underlying the biosynthesis of DA, post-PKS tailoring and the formation of ester bond linkage between DA and the linear polyketide moiety have not been verified.
In this thesis, an intergeneric genetic transfer system for S. griseochromogenes was first established and optimized, and we demonstrated that Ca2+and especially glycin, could increase the efficiency of exconjugates, which may be very helpful for genetic manipulation of Streptomyces.
To elucidate the biosynthesis of dialkylmaleic anhydride, the two candidate genes ttnK and ttnO, which encode a carboxyl-esterase (TtnK) and a citryl-CoA lyase (TtnO) respectively, in the TMC biosynthesis gene cluster have been deleted and complemented. The results indicated that TtnO was involved in the biosynthesis of DA, whereas the mutant△ttnK accumulated dialkylmaleic anhydride, indicating a critical role for TtnK in the enzyme-dependent events related to incorporation of DA with the polyketide moiety.
To determine the post-PKS modification mechanism, ttnF, ttnC, ttnD and ttnl genes were inactivated. These genes putatively encode a L-carnitine dehydratase, a flavoprotein decarboxylase, an UbiD family decarboxylase and a P450oxidase enzymes, respectively. The mutant△ttnF accumulatetd TMC-F1as a dominant metabolite. Compared to TMC, TMC-F1lacks the C5-ketone and the terminal alkene of TMC, but with a3-β-hydroxy-propionyloxyl group. The result indicated that the TtnF is directly responsible for the dehydration of the polyketide, the first post-PKS tailoring step. The inactivation of ttnC, resulted in the production of TMC at the wild-type levels, whereas the inactivation of ttnD resulted in the accumulation of TMC-D1as a dominant metabolite. Compared to TMC-F1which has a3-β-hydroxypropanoic acid moiety, TMC-D1had an acrylic acid moiety attached to C3.The results identified TtnF and TtnD as the enzymes to form the conjugated double bonds to install the carboxyl-diene, and TtnD as the decarboxylase for the post-PKS modification steps in TMC biosynthesis, whereas the function of TtnC remains unknown. The disruption of ttnl led to the accumulation of5-des-keto-tautomycetin, revealing that TtnI was responsible for the oxidation at C5as the last step of TMC biosynthesis.
Notably, biotransformation experiments in mutants△ttnO and△ttnl suggested that the C5-hydroxyl in TMC analogs was transformed to C5-ketone via Oxidation by TtnI. Thus, there is a considerable degree of promiscuity of some of the post-PKS modification enzymes in the biosynthesis of TMC in S. griseochromogenes.
In conclusion, this thesis provided novel insights into the mechanism of the biosynthesis of TMC and the basis for developing new analogs of TMC-based small molecule drug candidates by combinatorial biosynthesis.
变构菌素生物合成基因簇已被成功克隆并测序,从变构菌素基因簇序列分析可以看出,变构菌素的聚酮链骨架是由模块化的Ⅰ型聚酮合酶(PKS)合成的,但是二烷基马来酸酐的合成,聚酮链的PKS后修饰机制及二烷基马来酸酐与聚酮链的酯化机制都还未经阐明。因此本文首先建立和优化了灰产色链霉菌的遗传转化体系,并且发现甘氨酸和Ca2+能够提高大肠杆菌-链霉菌属间接合转移的效率。这一发现对于链霉菌的遗传工程操作具有普遍的应用价值。
为了研究二烷基马来酸酐的生物合成机制,本研究首先选择基因簇中两个可能的候选基因ttnO和ttnK进行基因阻断失活和互补。研究结果表明ttnO参与二烷基马来酸酐的合成；而△ttnK突变株积累二烷基马来酸酐,该结果证明ttnK的失活影响二烷基马来酸酐与聚酮链酯化所涉及的酶反应。
为了阐述PKS后修饰机制,基因ttnF, ttnC, ttnD和ttnI被阻断失活。这些基因分别编码肉毒碱脱水酶(ttnF),黄素蛋白脱羧酶(ttnC),UbiD家族脱羧酶(ttnD)和P450氧化酶(ttnI)。突变株△ttnF积累的主产物为TMC-F1,与TMC相比,TMC-F1缺少C5位酮基和末端烯烃结构,多了一个3-β-羟基-丙酸。该结果表明TtnF负责TMC聚酮链的脱水反应,是PKS后修饰反应的第一步；△ttnC突变株仍然产TMC,而△ttnD主要合成TMC-D1,与TMC-F1相比,TMC-D1的C3位的取代基为3-丙烯酸,并非3-β-羟基-丙酸,该结果证明ttnD编码的脱羧酶和TtnF一起负责变构菌素合成过程中二烯烃结构的形成,TtnD负责脱羧反应,TtnC的功能有待考证;△ttnI突变株积累5-去酮基-TMC衍生物,证明TtnI在变构菌素合成过程中负责C5位的氧化,该氧化反应是变构菌素合成过程中的最后一步反应。
另外通过微生物转化实验发现氧化酶TtnI能够催化5-羟基衍生物生成5-酮基衍生物,同时也说明变构菌素的生物合成基因被阻断失活时,变构菌素的合成会表现出一定程度的非专一性(promiscuity)。
本文的研究结果使得我们对变构菌素的生物合成机制有了更深一层的认识,为利用变构菌素进行组合生物合成技术开发新的小分子候选药物奠定基础。
Tautomycetin (TMC) is a polyketide that was first isolated from the Streptomyces griseochromogenes (S. griseochromogenes). TMC is a dialkylmaleic anhydride antibiotic with an unique chemical structure of an ester bond linkage between a dialkylmaleic anhydride (DA) moiety and a linear polyketide chain. Recently it was discovered that TMC is a specific protein phosphatase I inhibitor (PP1), with antifungi, antitumor and immunosuppressive activities. The unique chemical structure and important activities of TMC draw strong attention to the biosynthesis mechanism of TMC, which is not only critical for development of novel TMC analogs through combinatorial biosynthesis, semi-synthesis or heterologous expression, but also will contribute to a better understanding of the chemistry biology of polyketide synthase (PKS).
The biosynthesis gene cluster of TMC has been cloned and fully sequenced. By analyzing the sequence of the gene cluster of TMC, it was determined that the polyketide backbone of TMC is synthesized by type I PKS. However, the mechanisms underlying the biosynthesis of DA, post-PKS tailoring and the formation of ester bond linkage between DA and the linear polyketide moiety have not been verified.
In this thesis, an intergeneric genetic transfer system for S. griseochromogenes was first established and optimized, and we demonstrated that Ca2+and especially glycin, could increase the efficiency of exconjugates, which may be very helpful for genetic manipulation of Streptomyces.
To elucidate the biosynthesis of dialkylmaleic anhydride, the two candidate genes ttnK and ttnO, which encode a carboxyl-esterase (TtnK) and a citryl-CoA lyase (TtnO) respectively, in the TMC biosynthesis gene cluster have been deleted and complemented. The results indicated that TtnO was involved in the biosynthesis of DA, whereas the mutant△ttnK accumulated dialkylmaleic anhydride, indicating a critical role for TtnK in the enzyme-dependent events related to incorporation of DA with the polyketide moiety.
To determine the post-PKS modification mechanism, ttnF, ttnC, ttnD and ttnl genes were inactivated. These genes putatively encode a L-carnitine dehydratase, a flavoprotein decarboxylase, an UbiD family decarboxylase and a P450oxidase enzymes, respectively. The mutant△ttnF accumulatetd TMC-F1as a dominant metabolite. Compared to TMC, TMC-F1lacks the C5-ketone and the terminal alkene of TMC, but with a3-β-hydroxy-propionyloxyl group. The result indicated that the TtnF is directly responsible for the dehydration of the polyketide, the first post-PKS tailoring step. The inactivation of ttnC, resulted in the production of TMC at the wild-type levels, whereas the inactivation of ttnD resulted in the accumulation of TMC-D1as a dominant metabolite. Compared to TMC-F1which has a3-β-hydroxypropanoic acid moiety, TMC-D1had an acrylic acid moiety attached to C3.The results identified TtnF and TtnD as the enzymes to form the conjugated double bonds to install the carboxyl-diene, and TtnD as the decarboxylase for the post-PKS modification steps in TMC biosynthesis, whereas the function of TtnC remains unknown. The disruption of ttnl led to the accumulation of5-des-keto-tautomycetin, revealing that TtnI was responsible for the oxidation at C5as the last step of TMC biosynthesis.
Notably, biotransformation experiments in mutants△ttnO and△ttnl suggested that the C5-hydroxyl in TMC analogs was transformed to C5-ketone via Oxidation by TtnI. Thus, there is a considerable degree of promiscuity of some of the post-PKS modification enzymes in the biosynthesis of TMC in S. griseochromogenes.
In conclusion, this thesis provided novel insights into the mechanism of the biosynthesis of TMC and the basis for developing new analogs of TMC-based small molecule drug candidates by combinatorial biosynthesis.
引文
[1]Sergio S. Ecology and industrial microbiology:microbial diversity-the bright and promising future of microbial manufacturing[J]. Current Opinion in Microbiology,2005, 8 (3):229-33.
[2]Newman D.J., Cragg G. M. Natural products as sources of new drugs over the last 25 years[J]. J. Nat. Prod,2007,70(3):461-477.
[3]Chin Y. W., Balunas M. J., Chai H. B., et al. Drug discovery from natural sources[J]. The AAPS Journal,2006,8(2):E239-E253.
[4]Oberlies N. H., Kroll D.J. Camptothecin and taxol:historic achievements in natural products research[J]. J Nat Prod,2004,67(2):129-135.
[5]Chiu H. T., Hubbard B. K., Shah A. N., et al. Molecular cloning and sequence analysis of the complestatin biosynthetic gene cluster[J]. PNAS,2001,98(15):8548-8553.
[6]Koglin A., Walsh C. T. Structural insights into nonribosomal peptide enzymatic assembly lines[J].Natural product reports,2009,26(8):987-1000.
[7]Motamedi H., Shaf iee A. The biosynthetic gene cluster for the macrolactone ring of the immunosuppressant FK506[J]. Eur. J.Biochem,1998,256(3):528-534.
[8]Staunton J. Biosynthesis of erythromycin and rapamycin[J]. Chem. Rev,1997,97(7):2611-2629.
[9]Keating T. A., Walsh C. T. Initiation, elongation, and termination strategies in polyketide and polypeptide antibiotic biosynthesis[J]. Current Opinion in Chemical Biology,1999,3(5):598-606.
[10]Fischbach M. A.,Walsh C.T. Assembly-line enzymology for polyketide and nonribosomal peptide antibiotics:logic, machinery and mechanisms[J]. Chem. Rev,2006,106(8):3468-3496.
[11]Chan Y. A., Podevels A. M., Kevany B. M., et al. Biosynthesis of polyketide synthase extender units [J]. Nat. Prod. Rep,2009,26(1):90-114.
[12]Ben S. Polyketide biosynthesis beyond the type Ⅰ, Ⅱ and Ⅲ polyketide synthase paradigms[J], Current Opinion in Chemical Biology,2003,7(2):285-295.
[13]Chen H., Tseng C. C., Hubbard B. K., et al. Glycopeptide antibiotic biosynthesis: Enzymatic assembly of the dedicated amino acid monomer (S)-3,5-dihydroxyphenyl glycine[J]. PNAS,2001,98(26):14901-14906.
[14]Dirk S., Robert F., Marahiel M. A. Nonribosomal peptides:from genes to products[J]. Nat Prod Rep,2003,20(3):275-287.
[15]Byford M. F., Baldwin J.E., Shiau C. Y., et al. The mechanism of ACV synthetase. Chem Rev[J],1997,97(7):2631-2650.
[16]Weber G., Schorgendorfer K., Schneider-Scherzer E., et al.The peptide synthetase catalyzing cyclosporine production in Tolypocladium niveum is encoded by a giant 45.8-kilobase open reading frame. Curr Genet[J],1994,26(2):120-125.
[17]Kni jnenburg A. D., Kapoerchan V. V., Grotenbreg G. M., et al. Synthesis and evaluation of strand and turn modified ring-extended gramicidin S derivatives. Bioorg Med Chem[J],2011,19(11):3402-3409.
[18]Pfeifer B. A., Wang C. C.C.Walsh C.T.,et al. Biosynthesis of yersiniabactin, a complex polyketide-nonribosomal peptide, using Escherichia coli as a heterologous host[J]. Applied and environmental microbiology,2003,69(11):6698-6702.
[19]Hofle G., Bedorf N., Steinmetz H., et al. Epothilone A and B-novel 16-membered macrolides with cytotoxic activity:isolation, crystal structure, and conformation in solution [J].Angewandte Chemie,1996,35(1):1567-1569.
[20]Nicolaou K.C., Roschangar F.,Vourloumis D.Chemical biology of epothilones[J]. Angew. Chem. Int. Ed,1998,37 (2):2014-2045.
[21]Bollag D. M.,McQueney P. A., Zhu J., et al. A new class of microtubule-stabilizing agents with a taxol-like mechanism of action[J]. Cancer Res,1995,55(11):2325-33.
[22]Molnar I., Schupp T., Ono M.,Zirkle R.,et al. The biosynthetic gene cluster for the microtubule-stabilizing agents epothilones A and B from Sorangium cellulosum So ce90[J]. Chem Biol,2000,7(2):97-109.
[23]Tang L., Shah S., Chung L., et al. Cloning and Heterologous Expression of the Epothilone Gene Cluster[J]. Science,2000,287(5453):639-642.
[24]Walsh C.T., Connor S. E., Schneider T.L. Polyketide-nonribosomal peptide epothilone antitumor agents:the EpoA, B, C subunits[J] J. Ind.Micobiol. Biotechnol,2003,30(8): 448-455.
[25]王锋鹏.现代天然产物化学[M].北京：科学出版社,2009：940-956.
[26]McDaniel R, Ebert-Khosla S, Hopwood DA, et al. Engineered biosynthesis of novel polyketides[J]. Science.1993,262(5139):1546-50.
[27]Kao C.M., Luo G., Katz L., et al. Engineered biosynthesis of a triketide lactone from an incomplete modular polyketide synthase[J]. J. Am. Chem. Soc,1994,16(25):11612-11613.
[28]Khosla C., Gokhale R. S., Jacobsen J. R., et al. Tolerance and specificity of polyketide synthases[J]. Annu. Rev. Biochem,1999,68(0):219-253.
[29]Mcdaniel R., Thamchaipenet A., Gustafsson C., et al. Multiple genetic modification of the erythromycin polyketide synthase to produce a library of novel unnatural natural products[J]. Proc. Natl. Acad. Sci.,1999,96(5):1846-1851.
[30]Kao C.M., Luo G., Katz L., et al.Manipulation of macrolide ring size by directed mutagenesis of a modular polyketide synthase[J]. J. Am. Chem. Soc.,1995,117(35): 9105-9106.
[31]Stefano D., Staver M. J., McAlpine J. B., et al. Modular organization of genes required for complex polyketide biosynthesis [J]. Science,1991,252(5006):675-679.
[32]Oliynyk M., Brown M. JB, Cortes J., et al. A hybrid modular polyketide synthase obtained by domain swapping[J].1996, Chemistry & Biology,3(10):833-839.
[33]Tang L., Fu H., McDaniel R. Formation of functional heterologous complexes using subunits from the picromycin, erythromycin and oleandomycin polyketied synthase [J], Chem &Bio,2000,7(2):77-84.
[34]Khosla C. Harnessing the biosynthetic potential of modular polyketide synthases[J]. Chem. Rev.1997,97(7):2577-2590.
[35]Menzella H.G., Reid R., Carney J. R, et al. Combinatorial polyketide biosynthesis by de novo design and rearrangement of modular polyketide synthase genes[J]. Nature Biotechnology.2005,23(9):1171-1176.
[36]Khosla C. Structures and Mechanisms of Polyketide Synthases[J]. J. Org. Chem.2009, 74(17):6416-6420.
[37]Cortes J., Haydock S. F., Roberts G.A., et al. An unusually large multifunctional polypeptide in the erythromycin-producing polyketide synthase of Saccharopolyspora erythraea[J]. Nature 1990,348(6297):176-178.
[38]Tang L., Fu H., Betlach M. C., McDaniel R. Elucidating the mechanism of chain termination switching in the picromycin/methymycin polyketide synthase[J]. Chem Biol.1999,6(8):553-558.
[39]Walsh C. T, Chen H., Keating T. A., et al. Tailoring enzymes that modify nonribosomal peptides during and after chain elongation on NRPS assembly lines[J]. Current Opinion in Chemical Biology,2001,5(5):525-534.
[40]Hopwood D. A. Genetic contributions to understanding polyketide synthases[J]. Chem. Rev. 1997,97(7):2465-2497.
[41]PetkovicH, Sandmann A, ChallisIR, et al. Substrate specif icity of the acyl transferase domains of EpoC from the epothilone polyketide synthase.Org Biomol Chem,2008,6(3): 500-506.
[42]Aparicio J. F..Molnar I., Schwecke T., et al. Organization of the biosynthetic gene cluster for rapamycin in Streptomyces hygroscopicus:analysis of the enzymatic domains in the modular polyketide synthase[J]. Gene,1996,169(1):9-16.
[43]Bevitt D.J., Cortes J.,Haydock S. F.,et al.6-Deoxyerythronolide-B synthase 2 from Saccharopolyspora erythraea:cloning of the structural gene, sequence analysis and inferred domain structure of the multifunctional enzyme[J]. Eur. J. Biochem,1992, 204(1):39-49.
[44]Zhang Y., Rao M. S., Heath R. J., et al. Identification and Analysis of the Acyl Carrier Protein (ACP)Docking Site on β-Ketoacyl-ACP Synthase Ⅲ[J]. J Biol Chem,2001,276 (11):8231-8.
[45]Alekseyev V.Y., Liu C. W., Cane D.E., et al. Solution structure and proposed domain-domain recognition interface of an acyl carrier protein domain from a modular polyketide synthase[J]. Protein Science,2007,16(10):2093-2107.
[46]Tang Y., Kim C. Y.,Mathews I. I., et al. The 2.7A Structure of a 194-kDa Homodimeric Fragment of the 6-deoxyerythronolide B Synthase[J]. PNAS,2006,103(30),11124-11129.
[47]Gokhale R. S., Hunziker D., Cane D. E. et al. Mechanism and specificity of the terminal thioesterase domain from the erythromycin polyketide synthase [J]. Chem. Bio,1999,6(2): 117-125.
[48]Tsai S., Miercke L. J. W.,Krucinski J., et al. Crystal structure of the macrocycle-forming thioesterase domain of the erythromycin polyketide synthase versatility from a unique substrate channel[J]. PNAS.2001,98(26):14808-14813.
[49]Tsai S., Lu H., Cane D. E., et al. Insights into channel architecture and substrate specificity from crystal structures of two macrocycle-forming thioesterases of modular polyketide synthases[J]. Biochemistry,2002,41 (42):12598-12606.
[50]Gu L. C., Wang B.,Kulkarni A., et al. Polyketide decarboxylative chain termination preceded by O-sulfonation in curacin a biosynthesis[J]. J Am Chem Soc,2009,131(44): 16033-16035.
[51]Gokhale R. S.,Tsuji S.Y.,Cane D.E., et al. Dissecting and Exploiting Intermodular Communication in Polyketide Synthases[J]. Science,1999,284(5413):482-485.
[52]Broadhurst R. W., Nietlispach D., Wheatcrof t M. P., et al. The structure of docking domains in modular polyketide synthases[J]. J. Chem. Biol,2003,10(8):723-731.
[53]Pfeifer B. A., Admiraal S. J., Gramajo H., et al. Biosynthesis of complex polyketides in a metabolically engineered strain of E.coli[J]. Science,2001,291(5509):1790-1792.
[54]Pieper R., Luo G., Cane D. E. Cell-free synthesis of polyketides by recombinant erythromycin polyketide synthases[J]. Nature,1995,378(16):263-266.
[55]Xue Q..Ashley G.,Hutchinson C. R.et al.A multiplasmid approach to preparing large libraries of polyketides[J]. PNAS,1999,96(21):11740-11745.
[56]Kieser T., Bibb M. J., Buttner M. J. L., et al. Practical streptomyces genetics. John Innes Foundation[M]. Norwich, UK.2000:311-318.
[57]Gust B.,Challis G. L., Fowler K., et al. PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin[J]. Proc. Natl. Acad. Sci.,2003,100(4):1541-1546.
[58]崔洪霞.微生物转化法在药物芳环羟基化代谢研究中的应用[D].沈阳：沈阳药科大学,2001.
[59]谢丽萍.阿霉素的微生物转化及dauU基因的阻断突变研究[D].上海：上海医药工业研究院,2006.
[60]Tang L.,Qiu R. G., Li Y., et al. Generation of novel epothilone analogs with cytotoxic activity by biotransformation[J].2003,56(1):16-23.
[61]Chen X., Zheng Y., Shen Y. Natural products with maleic anhydride structure:nonadrides, tautumycin, chaetomellic anhydride and other compounds[J]. Chem. Rev,2007,107(5): 1777-1830.
[62]McCluskey A., Sim A. T. R., Sakoff J. A. Serine-threonine protein phosphatase inhibitors: development of potential therapeutic strategies[J].Journal of medicinal chemistry, 2002,45(6):1151-1169.
[63]Cheng X..Kihara T., Kusakabe H., et al. A new antibiotic, tautomycin[J]. Journal of Antibiotics,1987,40(6):907-909.
[64]Cheng X., Ubukata M., Isono K.The structure of tautomycin, a dialkylmaleic anhydride antibiotic[J]. Journal of Antibiotics,1990,43(7):809-819.
[65]Liu W., Sheppeck J. E., Colby D. A., et al. The selective inhibition of phosphatases by natural toxins:the anhydride domain of tautomycin is not a primary factor in controlling PP1/PP2A selectivity[J]. Bioorganic & Medicinal Chemistry Letters,2003, 13(9):1597-1600.
[66]Sugiyama Y.,.Ohtani II., Isobe M., et al. Molecular shape analysis and activity of tautomycin, a protein phosphatase inhibitor[J]. Bioorganic & Medicinal Chemistry Letters,1996,6(1):3-8.
[67]Adler J. T., Cook M., Luo Y., et al. Tautomycetin and tautomycin suppress the growth of medullary thyroid cancer cells via inhibition of glycogen synthase kinase-3β [J].Mol Cancer Ther,2009,8(4):914-920.
[68]Kelker M. S., Page R.,Peti W. Crystal structures of protein phosphatase-1 bound to nodularin-R and tautomycin:a novel scaffold for structure-based drug design of serine/threonine phosphatase inhibitors [J]. J. Mol. Biol,2009,385(1):11-21.
[69]Oikawa H. Synthesis of specific protein phosphatase in hibitors, tautomycin and tautomycetin toward structure-activity relationship study[J]. Current Medicinal Chemistry,2002,9(22):2033-2054.
[70]Li W, Ju J, Osada H., et al. Utilization of the methoxymalonyl-acyl carrier protein biosynthesis locus for cloning of the tautomycin biosynthetic gene cluster from Streptomyces spiroverticillatus[J]. Journal of Bacteriology,2006,188(11):4148-4152.
[71]Li W., Ju J., Rajski S.R.,et al. Characterization of the tautomycin biosynthetic gene cluster from streptomyces spiroverticillatus unveiling new insights into dialkylmaleic anhydride and polyketide biosynthesis[J]. The Journal of Biological Chemistry,2008, 283(42):28607-28617.
[72]Ju J., Li W., Yuan Q., et al. Functional characterization of ttmMuniveils new tautomycin analogs and insight into tautomycin biosynthesis and acitivity[J]. Organic letters 2009, 11(7):1639-1642.
[73]Li W., Luo Y., Ju J., et al. Characterization of the tautomycetin biosynthetic gene cluster from Streptomyces grisechromogenes provides new insight into dialkylmaleic anhydride biosynthesis[J]. J. Nat. Prod,2009,72(3):450-459.
[74]Cheng X. C., Kihara T., Ying X., et al.A new antibiotic, tautomycetin[J]. J. Antibiot, 1989,42(1):141-144.
[75]Cheng X. C., Makoto U., Kiyoshi I. The structure of tautomycetin, a dialkylmaleic anydride antibiotic[J]. J. Antibiot,1990,43(7):890-896.
[76]Mitsuhashi S..Matsuura N., Ubukata M.,et al. Tautomycetin is a Novel and Specific Inhibitor of Serine/threonine Protein Phosphatase Type 1, PP1[J]. Biochem. Biophys. Res. Commun,2001,287(2):328-331.
[77]Woo S. M., Min K. J., Kwon T. K. Calyculin a causes sensitization to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis by ROS-mediated down-regulation of cellular FLICE-inhibiting protein (c-FLIP) and by enhancing death receptor 4 mRNA stabilization[J]. Apoptosis,2012,17(11):1223-34.
[78]Matsushima R., Yoshizawa S., Watanabe M. F., et al. In vitro and in vivo effects of protein phosphatase inhibitors, microcystins and nodularin, on mouse skin and f ibroblasts[J]. Biochem Biophys Res Commun,1990,171(2):867-74.
[79]Takeuchi T..Takahashi N., Ishi K., etal. Antitumor antibiotic fostriecin covalently binds to cysteine-269 residue of protein phosphatase 2A catalytic subunit in mammalian cells[J]. Bioorg Med Chem,2009,17(23):8113-8122.
[80]Fujiki H., Suganuma M.Unique features of the okadaic acid activity class of tumor promoters[J].J. Cancer Res. Clin. Oncol,1999,125(34):150-155.
[81]McConnell J.L., Wadzinski B. E. Targeting protein serine/threonine phosphatases for drug development[J]. Molecular Pharmacology,2009,75(6):1249-1261.
[82]Dounay A. B.,Forsyth C. J. Okadaic acid:the archetypal serine/threonine protein phosphatase inhibitor[J]. Curr Med Chem,2002,9 (22):1939-80.
[83]Honkanen R. E., Golden T. Regulators of serine/threonine protein phosphatases at the dawn of a clinical era[J]. Curr.Med. Chem,2002,9(22):2055-2075.
[84]Kim J. H., Lee T. Y..Park J., et al. Effects of tautomycetin on proliferation and fibronectin secretion in vascular smooth muscle cells and glomerular mesangial cells[J]. Transplant Proceedings,2005,37(4):1959-1961.
[85]Han D.J.,Jeong Y.L,Wee Y. M., et al. Tautomycetin as a novel immunosuppressant in transplantation [J].Transplantation Proceedings,2003,35 (1):547.
[86]Mitsuhashi S., Shima H., Tanuma N., et al. Usage of tautomycetin, a novel inhibitor of protein phosphatase 1(PP1), reveals that PP1 is a positive regulator of Raf-1 in vivo[J]. The Journal of Biological Chemistry,2003,278 (1):82-88.
[87]Shim J. H., Lee H. K., Chang E. J., et al. Immunosuppressive effects of tautomycetin in vivo and in vitro via T cell-specific apoptosis induction[J]. Proceedings of Naional Academy of Sciences,2002,99 (16):10617-10622.
[88]Ubukata M., Cheng X. C., Uzawa J.,et al. Biosynthesis of the dialkylmaleic anhydride-containing antibiotics, tautomycin and tautomycetin[J]. J. Chem. Soc., Perkin Trans,1995, 1(0):2399-2404.
[89]Reid R., Tang L. Biosynthetic gene cluster for tautomycetin[P].2005. WO/2005/118797.
[90]Choi S. S., HurY. A., Sherman D. H., et al. Isolation of the biosynthetic gene cluster for tautomycetin, a linear polyketide T cell-specific immunomodulator from Streptomyces sp. CK4412[J].Microbiol,2007,153 (PT4):1095-1102.
[91]Scaglione J. B., Akey D. L., Sullivan R. et al. Biochemical and structural characterization of the tautomycetin thioesterase:analysis of a stereoselective polyketide hydrolase[J]. Angew. Chem. Int. Ed.2010,49(33):5726-5730.
[92]HurY., Choi S. Sherman D. H. et al. Identification of TmcN as a pathway-specific positive regulator of tautomycetin biosynthesis in Streptomyces sp. CK4412[J]. Microbiology, 2008,154(Pt10):2912-2919.
[93]吴胜,夏焕章.链霉菌基因转移的方法[J].生物工程进展,2002,22(1)：91-95.
[94]Sambrook J, Russell DW.黄培堂,译.分子克隆实验指南[M].3版.北京：科学出版社,2002.
[95]Natsume M.,Marumo S.Calcium signal modulators inhibit aerial mycelium formation in Streptomyces alboniger[J]. The Journal of Antibiotics (Tokyo),1992,45(6):1026-1028.
[96]Gust B., Kieser T., Chater K. REDIRECT technology:PCR-targeting system in Streptomyces coelicolor. John Innes Centre,2002. http://wenku.baidu.com/view/84f9cb2cb4daa58 da0114ad8.html.
[97]Pettit, R. K. Mixed fermentation for natural product drug discovery [J]. Appl. Microbiol. Biotechnol,2009,83(1):19-25.
[98]Luo Y., Li W., Ju J., et al. Functional characterization of TtnD and TtnF, unveiling new insights into tautomycetin biosynthesis [J]. J. Am. Chem. Soc,2010,132(19):6663-6671.
[99]刘波Tautomycetin新型衍生物的分离鉴定及其生物合成机制的初步探讨[D].大连：大连理工大学,2011.
[2]Newman D.J., Cragg G. M. Natural products as sources of new drugs over the last 25 years[J]. J. Nat. Prod,2007,70(3):461-477.
[3]Chin Y. W., Balunas M. J., Chai H. B., et al. Drug discovery from natural sources[J]. The AAPS Journal,2006,8(2):E239-E253.
[4]Oberlies N. H., Kroll D.J. Camptothecin and taxol:historic achievements in natural products research[J]. J Nat Prod,2004,67(2):129-135.
[5]Chiu H. T., Hubbard B. K., Shah A. N., et al. Molecular cloning and sequence analysis of the complestatin biosynthetic gene cluster[J]. PNAS,2001,98(15):8548-8553.
[6]Koglin A., Walsh C. T. Structural insights into nonribosomal peptide enzymatic assembly lines[J].Natural product reports,2009,26(8):987-1000.
[7]Motamedi H., Shaf iee A. The biosynthetic gene cluster for the macrolactone ring of the immunosuppressant FK506[J]. Eur. J.Biochem,1998,256(3):528-534.
[8]Staunton J. Biosynthesis of erythromycin and rapamycin[J]. Chem. Rev,1997,97(7):2611-2629.
[9]Keating T. A., Walsh C. T. Initiation, elongation, and termination strategies in polyketide and polypeptide antibiotic biosynthesis[J]. Current Opinion in Chemical Biology,1999,3(5):598-606.
[10]Fischbach M. A.,Walsh C.T. Assembly-line enzymology for polyketide and nonribosomal peptide antibiotics:logic, machinery and mechanisms[J]. Chem. Rev,2006,106(8):3468-3496.
[11]Chan Y. A., Podevels A. M., Kevany B. M., et al. Biosynthesis of polyketide synthase extender units [J]. Nat. Prod. Rep,2009,26(1):90-114.
[12]Ben S. Polyketide biosynthesis beyond the type Ⅰ, Ⅱ and Ⅲ polyketide synthase paradigms[J], Current Opinion in Chemical Biology,2003,7(2):285-295.
[13]Chen H., Tseng C. C., Hubbard B. K., et al. Glycopeptide antibiotic biosynthesis: Enzymatic assembly of the dedicated amino acid monomer (S)-3,5-dihydroxyphenyl glycine[J]. PNAS,2001,98(26):14901-14906.
[14]Dirk S., Robert F., Marahiel M. A. Nonribosomal peptides:from genes to products[J]. Nat Prod Rep,2003,20(3):275-287.
[15]Byford M. F., Baldwin J.E., Shiau C. Y., et al. The mechanism of ACV synthetase. Chem Rev[J],1997,97(7):2631-2650.
[16]Weber G., Schorgendorfer K., Schneider-Scherzer E., et al.The peptide synthetase catalyzing cyclosporine production in Tolypocladium niveum is encoded by a giant 45.8-kilobase open reading frame. Curr Genet[J],1994,26(2):120-125.
[17]Kni jnenburg A. D., Kapoerchan V. V., Grotenbreg G. M., et al. Synthesis and evaluation of strand and turn modified ring-extended gramicidin S derivatives. Bioorg Med Chem[J],2011,19(11):3402-3409.
[18]Pfeifer B. A., Wang C. C.C.Walsh C.T.,et al. Biosynthesis of yersiniabactin, a complex polyketide-nonribosomal peptide, using Escherichia coli as a heterologous host[J]. Applied and environmental microbiology,2003,69(11):6698-6702.
[19]Hofle G., Bedorf N., Steinmetz H., et al. Epothilone A and B-novel 16-membered macrolides with cytotoxic activity:isolation, crystal structure, and conformation in solution [J].Angewandte Chemie,1996,35(1):1567-1569.
[20]Nicolaou K.C., Roschangar F.,Vourloumis D.Chemical biology of epothilones[J]. Angew. Chem. Int. Ed,1998,37 (2):2014-2045.
[21]Bollag D. M.,McQueney P. A., Zhu J., et al. A new class of microtubule-stabilizing agents with a taxol-like mechanism of action[J]. Cancer Res,1995,55(11):2325-33.
[22]Molnar I., Schupp T., Ono M.,Zirkle R.,et al. The biosynthetic gene cluster for the microtubule-stabilizing agents epothilones A and B from Sorangium cellulosum So ce90[J]. Chem Biol,2000,7(2):97-109.
[23]Tang L., Shah S., Chung L., et al. Cloning and Heterologous Expression of the Epothilone Gene Cluster[J]. Science,2000,287(5453):639-642.
[24]Walsh C.T., Connor S. E., Schneider T.L. Polyketide-nonribosomal peptide epothilone antitumor agents:the EpoA, B, C subunits[J] J. Ind.Micobiol. Biotechnol,2003,30(8): 448-455.
[25]王锋鹏.现代天然产物化学[M].北京：科学出版社,2009：940-956.
[26]McDaniel R, Ebert-Khosla S, Hopwood DA, et al. Engineered biosynthesis of novel polyketides[J]. Science.1993,262(5139):1546-50.
[27]Kao C.M., Luo G., Katz L., et al. Engineered biosynthesis of a triketide lactone from an incomplete modular polyketide synthase[J]. J. Am. Chem. Soc,1994,16(25):11612-11613.
[28]Khosla C., Gokhale R. S., Jacobsen J. R., et al. Tolerance and specificity of polyketide synthases[J]. Annu. Rev. Biochem,1999,68(0):219-253.
[29]Mcdaniel R., Thamchaipenet A., Gustafsson C., et al. Multiple genetic modification of the erythromycin polyketide synthase to produce a library of novel unnatural natural products[J]. Proc. Natl. Acad. Sci.,1999,96(5):1846-1851.
[30]Kao C.M., Luo G., Katz L., et al.Manipulation of macrolide ring size by directed mutagenesis of a modular polyketide synthase[J]. J. Am. Chem. Soc.,1995,117(35): 9105-9106.
[31]Stefano D., Staver M. J., McAlpine J. B., et al. Modular organization of genes required for complex polyketide biosynthesis [J]. Science,1991,252(5006):675-679.
[32]Oliynyk M., Brown M. JB, Cortes J., et al. A hybrid modular polyketide synthase obtained by domain swapping[J].1996, Chemistry & Biology,3(10):833-839.
[33]Tang L., Fu H., McDaniel R. Formation of functional heterologous complexes using subunits from the picromycin, erythromycin and oleandomycin polyketied synthase [J], Chem &Bio,2000,7(2):77-84.
[34]Khosla C. Harnessing the biosynthetic potential of modular polyketide synthases[J]. Chem. Rev.1997,97(7):2577-2590.
[35]Menzella H.G., Reid R., Carney J. R, et al. Combinatorial polyketide biosynthesis by de novo design and rearrangement of modular polyketide synthase genes[J]. Nature Biotechnology.2005,23(9):1171-1176.
[36]Khosla C. Structures and Mechanisms of Polyketide Synthases[J]. J. Org. Chem.2009, 74(17):6416-6420.
[37]Cortes J., Haydock S. F., Roberts G.A., et al. An unusually large multifunctional polypeptide in the erythromycin-producing polyketide synthase of Saccharopolyspora erythraea[J]. Nature 1990,348(6297):176-178.
[38]Tang L., Fu H., Betlach M. C., McDaniel R. Elucidating the mechanism of chain termination switching in the picromycin/methymycin polyketide synthase[J]. Chem Biol.1999,6(8):553-558.
[39]Walsh C. T, Chen H., Keating T. A., et al. Tailoring enzymes that modify nonribosomal peptides during and after chain elongation on NRPS assembly lines[J]. Current Opinion in Chemical Biology,2001,5(5):525-534.
[40]Hopwood D. A. Genetic contributions to understanding polyketide synthases[J]. Chem. Rev. 1997,97(7):2465-2497.
[41]PetkovicH, Sandmann A, ChallisIR, et al. Substrate specif icity of the acyl transferase domains of EpoC from the epothilone polyketide synthase.Org Biomol Chem,2008,6(3): 500-506.
[42]Aparicio J. F..Molnar I., Schwecke T., et al. Organization of the biosynthetic gene cluster for rapamycin in Streptomyces hygroscopicus:analysis of the enzymatic domains in the modular polyketide synthase[J]. Gene,1996,169(1):9-16.
[43]Bevitt D.J., Cortes J.,Haydock S. F.,et al.6-Deoxyerythronolide-B synthase 2 from Saccharopolyspora erythraea:cloning of the structural gene, sequence analysis and inferred domain structure of the multifunctional enzyme[J]. Eur. J. Biochem,1992, 204(1):39-49.
[44]Zhang Y., Rao M. S., Heath R. J., et al. Identification and Analysis of the Acyl Carrier Protein (ACP)Docking Site on β-Ketoacyl-ACP Synthase Ⅲ[J]. J Biol Chem,2001,276 (11):8231-8.
[45]Alekseyev V.Y., Liu C. W., Cane D.E., et al. Solution structure and proposed domain-domain recognition interface of an acyl carrier protein domain from a modular polyketide synthase[J]. Protein Science,2007,16(10):2093-2107.
[46]Tang Y., Kim C. Y.,Mathews I. I., et al. The 2.7A Structure of a 194-kDa Homodimeric Fragment of the 6-deoxyerythronolide B Synthase[J]. PNAS,2006,103(30),11124-11129.
[47]Gokhale R. S., Hunziker D., Cane D. E. et al. Mechanism and specificity of the terminal thioesterase domain from the erythromycin polyketide synthase [J]. Chem. Bio,1999,6(2): 117-125.
[48]Tsai S., Miercke L. J. W.,Krucinski J., et al. Crystal structure of the macrocycle-forming thioesterase domain of the erythromycin polyketide synthase versatility from a unique substrate channel[J]. PNAS.2001,98(26):14808-14813.
[49]Tsai S., Lu H., Cane D. E., et al. Insights into channel architecture and substrate specificity from crystal structures of two macrocycle-forming thioesterases of modular polyketide synthases[J]. Biochemistry,2002,41 (42):12598-12606.
[50]Gu L. C., Wang B.,Kulkarni A., et al. Polyketide decarboxylative chain termination preceded by O-sulfonation in curacin a biosynthesis[J]. J Am Chem Soc,2009,131(44): 16033-16035.
[51]Gokhale R. S.,Tsuji S.Y.,Cane D.E., et al. Dissecting and Exploiting Intermodular Communication in Polyketide Synthases[J]. Science,1999,284(5413):482-485.
[52]Broadhurst R. W., Nietlispach D., Wheatcrof t M. P., et al. The structure of docking domains in modular polyketide synthases[J]. J. Chem. Biol,2003,10(8):723-731.
[53]Pfeifer B. A., Admiraal S. J., Gramajo H., et al. Biosynthesis of complex polyketides in a metabolically engineered strain of E.coli[J]. Science,2001,291(5509):1790-1792.
[54]Pieper R., Luo G., Cane D. E. Cell-free synthesis of polyketides by recombinant erythromycin polyketide synthases[J]. Nature,1995,378(16):263-266.
[55]Xue Q..Ashley G.,Hutchinson C. R.et al.A multiplasmid approach to preparing large libraries of polyketides[J]. PNAS,1999,96(21):11740-11745.
[56]Kieser T., Bibb M. J., Buttner M. J. L., et al. Practical streptomyces genetics. John Innes Foundation[M]. Norwich, UK.2000:311-318.
[57]Gust B.,Challis G. L., Fowler K., et al. PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin[J]. Proc. Natl. Acad. Sci.,2003,100(4):1541-1546.
[58]崔洪霞.微生物转化法在药物芳环羟基化代谢研究中的应用[D].沈阳：沈阳药科大学,2001.
[59]谢丽萍.阿霉素的微生物转化及dauU基因的阻断突变研究[D].上海：上海医药工业研究院,2006.
[60]Tang L.,Qiu R. G., Li Y., et al. Generation of novel epothilone analogs with cytotoxic activity by biotransformation[J].2003,56(1):16-23.
[61]Chen X., Zheng Y., Shen Y. Natural products with maleic anhydride structure:nonadrides, tautumycin, chaetomellic anhydride and other compounds[J]. Chem. Rev,2007,107(5): 1777-1830.
[62]McCluskey A., Sim A. T. R., Sakoff J. A. Serine-threonine protein phosphatase inhibitors: development of potential therapeutic strategies[J].Journal of medicinal chemistry, 2002,45(6):1151-1169.
[63]Cheng X..Kihara T., Kusakabe H., et al. A new antibiotic, tautomycin[J]. Journal of Antibiotics,1987,40(6):907-909.
[64]Cheng X., Ubukata M., Isono K.The structure of tautomycin, a dialkylmaleic anhydride antibiotic[J]. Journal of Antibiotics,1990,43(7):809-819.
[65]Liu W., Sheppeck J. E., Colby D. A., et al. The selective inhibition of phosphatases by natural toxins:the anhydride domain of tautomycin is not a primary factor in controlling PP1/PP2A selectivity[J]. Bioorganic & Medicinal Chemistry Letters,2003, 13(9):1597-1600.
[66]Sugiyama Y.,.Ohtani II., Isobe M., et al. Molecular shape analysis and activity of tautomycin, a protein phosphatase inhibitor[J]. Bioorganic & Medicinal Chemistry Letters,1996,6(1):3-8.
[67]Adler J. T., Cook M., Luo Y., et al. Tautomycetin and tautomycin suppress the growth of medullary thyroid cancer cells via inhibition of glycogen synthase kinase-3β [J].Mol Cancer Ther,2009,8(4):914-920.
[68]Kelker M. S., Page R.,Peti W. Crystal structures of protein phosphatase-1 bound to nodularin-R and tautomycin:a novel scaffold for structure-based drug design of serine/threonine phosphatase inhibitors [J]. J. Mol. Biol,2009,385(1):11-21.
[69]Oikawa H. Synthesis of specific protein phosphatase in hibitors, tautomycin and tautomycetin toward structure-activity relationship study[J]. Current Medicinal Chemistry,2002,9(22):2033-2054.
[70]Li W, Ju J, Osada H., et al. Utilization of the methoxymalonyl-acyl carrier protein biosynthesis locus for cloning of the tautomycin biosynthetic gene cluster from Streptomyces spiroverticillatus[J]. Journal of Bacteriology,2006,188(11):4148-4152.
[71]Li W., Ju J., Rajski S.R.,et al. Characterization of the tautomycin biosynthetic gene cluster from streptomyces spiroverticillatus unveiling new insights into dialkylmaleic anhydride and polyketide biosynthesis[J]. The Journal of Biological Chemistry,2008, 283(42):28607-28617.
[72]Ju J., Li W., Yuan Q., et al. Functional characterization of ttmMuniveils new tautomycin analogs and insight into tautomycin biosynthesis and acitivity[J]. Organic letters 2009, 11(7):1639-1642.
[73]Li W., Luo Y., Ju J., et al. Characterization of the tautomycetin biosynthetic gene cluster from Streptomyces grisechromogenes provides new insight into dialkylmaleic anhydride biosynthesis[J]. J. Nat. Prod,2009,72(3):450-459.
[74]Cheng X. C., Kihara T., Ying X., et al.A new antibiotic, tautomycetin[J]. J. Antibiot, 1989,42(1):141-144.
[75]Cheng X. C., Makoto U., Kiyoshi I. The structure of tautomycetin, a dialkylmaleic anydride antibiotic[J]. J. Antibiot,1990,43(7):890-896.
[76]Mitsuhashi S..Matsuura N., Ubukata M.,et al. Tautomycetin is a Novel and Specific Inhibitor of Serine/threonine Protein Phosphatase Type 1, PP1[J]. Biochem. Biophys. Res. Commun,2001,287(2):328-331.
[77]Woo S. M., Min K. J., Kwon T. K. Calyculin a causes sensitization to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis by ROS-mediated down-regulation of cellular FLICE-inhibiting protein (c-FLIP) and by enhancing death receptor 4 mRNA stabilization[J]. Apoptosis,2012,17(11):1223-34.
[78]Matsushima R., Yoshizawa S., Watanabe M. F., et al. In vitro and in vivo effects of protein phosphatase inhibitors, microcystins and nodularin, on mouse skin and f ibroblasts[J]. Biochem Biophys Res Commun,1990,171(2):867-74.
[79]Takeuchi T..Takahashi N., Ishi K., etal. Antitumor antibiotic fostriecin covalently binds to cysteine-269 residue of protein phosphatase 2A catalytic subunit in mammalian cells[J]. Bioorg Med Chem,2009,17(23):8113-8122.
[80]Fujiki H., Suganuma M.Unique features of the okadaic acid activity class of tumor promoters[J].J. Cancer Res. Clin. Oncol,1999,125(34):150-155.
[81]McConnell J.L., Wadzinski B. E. Targeting protein serine/threonine phosphatases for drug development[J]. Molecular Pharmacology,2009,75(6):1249-1261.
[82]Dounay A. B.,Forsyth C. J. Okadaic acid:the archetypal serine/threonine protein phosphatase inhibitor[J]. Curr Med Chem,2002,9 (22):1939-80.
[83]Honkanen R. E., Golden T. Regulators of serine/threonine protein phosphatases at the dawn of a clinical era[J]. Curr.Med. Chem,2002,9(22):2055-2075.
[84]Kim J. H., Lee T. Y..Park J., et al. Effects of tautomycetin on proliferation and fibronectin secretion in vascular smooth muscle cells and glomerular mesangial cells[J]. Transplant Proceedings,2005,37(4):1959-1961.
[85]Han D.J.,Jeong Y.L,Wee Y. M., et al. Tautomycetin as a novel immunosuppressant in transplantation [J].Transplantation Proceedings,2003,35 (1):547.
[86]Mitsuhashi S., Shima H., Tanuma N., et al. Usage of tautomycetin, a novel inhibitor of protein phosphatase 1(PP1), reveals that PP1 is a positive regulator of Raf-1 in vivo[J]. The Journal of Biological Chemistry,2003,278 (1):82-88.
[87]Shim J. H., Lee H. K., Chang E. J., et al. Immunosuppressive effects of tautomycetin in vivo and in vitro via T cell-specific apoptosis induction[J]. Proceedings of Naional Academy of Sciences,2002,99 (16):10617-10622.
[88]Ubukata M., Cheng X. C., Uzawa J.,et al. Biosynthesis of the dialkylmaleic anhydride-containing antibiotics, tautomycin and tautomycetin[J]. J. Chem. Soc., Perkin Trans,1995, 1(0):2399-2404.
[89]Reid R., Tang L. Biosynthetic gene cluster for tautomycetin[P].2005. WO/2005/118797.
[90]Choi S. S., HurY. A., Sherman D. H., et al. Isolation of the biosynthetic gene cluster for tautomycetin, a linear polyketide T cell-specific immunomodulator from Streptomyces sp. CK4412[J].Microbiol,2007,153 (PT4):1095-1102.
[91]Scaglione J. B., Akey D. L., Sullivan R. et al. Biochemical and structural characterization of the tautomycetin thioesterase:analysis of a stereoselective polyketide hydrolase[J]. Angew. Chem. Int. Ed.2010,49(33):5726-5730.
[92]HurY., Choi S. Sherman D. H. et al. Identification of TmcN as a pathway-specific positive regulator of tautomycetin biosynthesis in Streptomyces sp. CK4412[J]. Microbiology, 2008,154(Pt10):2912-2919.
[93]吴胜,夏焕章.链霉菌基因转移的方法[J].生物工程进展,2002,22(1)：91-95.
[94]Sambrook J, Russell DW.黄培堂,译.分子克隆实验指南[M].3版.北京：科学出版社,2002.
[95]Natsume M.,Marumo S.Calcium signal modulators inhibit aerial mycelium formation in Streptomyces alboniger[J]. The Journal of Antibiotics (Tokyo),1992,45(6):1026-1028.
[96]Gust B., Kieser T., Chater K. REDIRECT technology:PCR-targeting system in Streptomyces coelicolor. John Innes Centre,2002. http://wenku.baidu.com/view/84f9cb2cb4daa58 da0114ad8.html.
[97]Pettit, R. K. Mixed fermentation for natural product drug discovery [J]. Appl. Microbiol. Biotechnol,2009,83(1):19-25.
[98]Luo Y., Li W., Ju J., et al. Functional characterization of TtnD and TtnF, unveiling new insights into tautomycetin biosynthesis [J]. J. Am. Chem. Soc,2010,132(19):6663-6671.
[99]刘波Tautomycetin新型衍生物的分离鉴定及其生物合成机制的初步探讨[D].大连：大连理工大学,2011.