变构菌素及衍生物的制备与生物合成机制探讨
摘要
变构菌素(Tautomycetin, TMC)是一种具有抗真菌、抗癌和免疫抑制剂作用的抗生素。研究表明,该抗生素是具有特异性的蛋白磷酸酶(PP1)抑制剂,但其生物合成及调控机制并未完全研究清楚。因此,本课题研究的重点主要是通过分离鉴定TMC及相关类似物,推测变构菌素的生物合成机制。
变构菌素具有马来酸酐结构,极易水解、醇解且没有标准对照品,分离纯化过程困难。本课题采用分析、纯化过程直接加酸的方法,将变构菌素的纯度由文献报道的96%提高到98%以上,同时提高了产品的收率及分离纯化速度。针对变构菌素发酵收率低的问题,通过对发酵培养时间、发酵液体积等发酵条件进行初步优化,大大提高了目标化合物的产量。目标产物的发酵优化条件如下：
冻存的孢子悬液以合适的比例接种至种子培养基中,培养1-1.5天。然后将培养好的种子液按合适比例转接至发酵培养基中,继续培养3天,培养过程中对TMC的产量进行HPLC分析。将发酵液用乙酸乙酯萃取得到粗提油状物。获得的粗提物通过正相硅胶层析、反相半制备色谱进行精制,浓缩、干燥得到目标产物。获得的产品进行UV、MS、NMR等分析测定,对得到的图谱进行解析,确定其化学结构。
本研究从野生菌发酵液中分离得到TMC、重要衍生物3′-脱羟基-TMC、7-乙基-TMC及TMC合成前体物3′-脱羟基-C8环,化学裂解法得到C8环、TMC-PKS及TMC-post-PKS链。其中3′-脱羟基-TMC、7-乙基-TMC、3′-脱羟基-C8环、C8环均为首次分离并进行了结构确证。这些化合物的分离鉴定为TMC生物合成机制的阐明提供了不可或缺的物质基础,具有重要的理论价值。通过对野生菌发酵液及酯酶基因敲除菌发酵液进行HPLC分析,确定两者发酵液均存在3′-脱羟基-C8环而不存在C8环,得出3′-脱羟基-C8环作为起始物进行TMC的生物合成。本课题研究结果为提出TMC独特新颖的生物合成机制奠定了基础。
Tautomycetin (TMC) is an antibiotic having antibacterial, immune suppression and anticancer activities. The antibiotic is a specific protein phosphatase (PP1) inhibitors, but its biosynthesis and regulation mechanism are not entirely clear. Therefore, the focus of this research is to isolate TMC and novel analogues and confirm their structures, so that the biosynthesis mechanism of TMC can be speculated from the molecular level.
TMC is easy to hydrolyze and alcoholysis because of its maleic anhydride structure, and currently no standard is available, which makes it hard to be analysed. In this study, addition of acid during analysing and preparing of TMC was found to improve its purity from 96% to 98%, at the same time the product yield and purification speed were also improving. To solve the problem of the low fermentation yield of TMC, fermentation process of TMC was optimized, which results in the significant yield improvement. The optimized fermentation procedure was described as following:
The frozen spore suspension is inoculated to seed medium by appropriate ratio for 1-1.5 days, after which the seed culture is inoculated to fermentation medium by appropriate ratio, and is cultured for 3-4 days under the same conditions. During the culture process, the production of TMC is monitored by HPLC analysis. The fermentation broth is extracted with EtOAc to get the oily residue, which was applied onto a silica gel column and semi-prepared chromatography before being concentrated and dried to get the pure compounds. Through UV, MS, and NMR analysis, the chemical structures of the analogs are determined.
In this study, several important analogues including 3'-dehydroxyl-TMC,7-ethyl-TMC and the synthetic starting compound 3'-dehydroxyl-C8 Ring, as well as C8 Ring,TMC-PKS ring and TMC-post-PKS ring by degradation methods were obtained.3'-dehydroxyl-TMC, 7-ethyl-TMC,3'-dehydroxyl-C8 Ring and C8 Ring are first to be separated and provide essential material foundation for TMC biosynthesis Through analyzing the fermentation broth of the wild type and the knock out mutant of the ester enzyme gene by HPLC, it was comfirmed that the 3'-dehydroxyl-C8 Ring, instead of C8 Ring existed in the fermentation broth of both strains, which indicates that 3'-dehydroxyl-C8 Ring is the starting compound for the biosynthesis of TMC. The results from this study set basis for proposing a distinct pathway for TMC biosynthesis.
变构菌素具有马来酸酐结构,极易水解、醇解且没有标准对照品,分离纯化过程困难。本课题采用分析、纯化过程直接加酸的方法,将变构菌素的纯度由文献报道的96%提高到98%以上,同时提高了产品的收率及分离纯化速度。针对变构菌素发酵收率低的问题,通过对发酵培养时间、发酵液体积等发酵条件进行初步优化,大大提高了目标化合物的产量。目标产物的发酵优化条件如下：
冻存的孢子悬液以合适的比例接种至种子培养基中,培养1-1.5天。然后将培养好的种子液按合适比例转接至发酵培养基中,继续培养3天,培养过程中对TMC的产量进行HPLC分析。将发酵液用乙酸乙酯萃取得到粗提油状物。获得的粗提物通过正相硅胶层析、反相半制备色谱进行精制,浓缩、干燥得到目标产物。获得的产品进行UV、MS、NMR等分析测定,对得到的图谱进行解析,确定其化学结构。
本研究从野生菌发酵液中分离得到TMC、重要衍生物3′-脱羟基-TMC、7-乙基-TMC及TMC合成前体物3′-脱羟基-C8环,化学裂解法得到C8环、TMC-PKS及TMC-post-PKS链。其中3′-脱羟基-TMC、7-乙基-TMC、3′-脱羟基-C8环、C8环均为首次分离并进行了结构确证。这些化合物的分离鉴定为TMC生物合成机制的阐明提供了不可或缺的物质基础,具有重要的理论价值。通过对野生菌发酵液及酯酶基因敲除菌发酵液进行HPLC分析,确定两者发酵液均存在3′-脱羟基-C8环而不存在C8环,得出3′-脱羟基-C8环作为起始物进行TMC的生物合成。本课题研究结果为提出TMC独特新颖的生物合成机制奠定了基础。
Tautomycetin (TMC) is an antibiotic having antibacterial, immune suppression and anticancer activities. The antibiotic is a specific protein phosphatase (PP1) inhibitors, but its biosynthesis and regulation mechanism are not entirely clear. Therefore, the focus of this research is to isolate TMC and novel analogues and confirm their structures, so that the biosynthesis mechanism of TMC can be speculated from the molecular level.
TMC is easy to hydrolyze and alcoholysis because of its maleic anhydride structure, and currently no standard is available, which makes it hard to be analysed. In this study, addition of acid during analysing and preparing of TMC was found to improve its purity from 96% to 98%, at the same time the product yield and purification speed were also improving. To solve the problem of the low fermentation yield of TMC, fermentation process of TMC was optimized, which results in the significant yield improvement. The optimized fermentation procedure was described as following:
The frozen spore suspension is inoculated to seed medium by appropriate ratio for 1-1.5 days, after which the seed culture is inoculated to fermentation medium by appropriate ratio, and is cultured for 3-4 days under the same conditions. During the culture process, the production of TMC is monitored by HPLC analysis. The fermentation broth is extracted with EtOAc to get the oily residue, which was applied onto a silica gel column and semi-prepared chromatography before being concentrated and dried to get the pure compounds. Through UV, MS, and NMR analysis, the chemical structures of the analogs are determined.
In this study, several important analogues including 3'-dehydroxyl-TMC,7-ethyl-TMC and the synthetic starting compound 3'-dehydroxyl-C8 Ring, as well as C8 Ring,TMC-PKS ring and TMC-post-PKS ring by degradation methods were obtained.3'-dehydroxyl-TMC, 7-ethyl-TMC,3'-dehydroxyl-C8 Ring and C8 Ring are first to be separated and provide essential material foundation for TMC biosynthesis Through analyzing the fermentation broth of the wild type and the knock out mutant of the ester enzyme gene by HPLC, it was comfirmed that the 3'-dehydroxyl-C8 Ring, instead of C8 Ring existed in the fermentation broth of both strains, which indicates that 3'-dehydroxyl-C8 Ring is the starting compound for the biosynthesis of TMC. The results from this study set basis for proposing a distinct pathway for TMC biosynthesis.
引文
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[2]马田田.纤维素酶用于中药提取的初步研究[J].中草药,1994,25(3)：123.
[3]江泽飞,宋三泰,冯奉仪.中国肿瘤临床与康复,[J].2000,7(2)：66-67.
[4]袁金辉,谢弘,等.紫杉醇抗肿瘤药物的新用途.[P].CN99116954.9,1999-10-11.
[5]纪元,毕建男,严冰,等.[J].生物工程学报,2006,22(1)：1～6.
[6]K. Gerth, N. Bedorf, G. Hfle, et al.Epothilone A and B:antifungal and cytotoxic compounds from Sorangium cellulosum (Myxobacteria)production, physico chemical and biological properties. [J]. J.Antibiot.1996:560-563.
[7]Hofle G, N. Bedorf, K. Gerth & H. Epothilons, process for preparing the same and their use as medicaments and as plant protecting agents. [P]. Wo 93/10121,published May,27,1993.
[8]Bollag D. M, P. A. McQuency, J. Zhu, et al.Epothilones, a newclass of microtubule-stabilizing agents with a taxol-like mechanism of action. [J]. Cancer Res.55: 2325-2333,1995.
[9]Gerth K, D. Schummer, G. Hofle, et al.A new antifungal compound from Sorangium cellulosum (Myxobacteria). Production, physico-chemical and biological properties .[J].J. Antibiotics.
[10]Altmann, K. H. Mini-Rev. [J]. Med. Chem.2003,3,149.
[11]Altaha R, Fojo T, Reed E, et al. [J]. Pharm. Des.2002,8,1707.
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[13]Nicolau k. c, winssinger n, pastor j, et al. [J]. Nature1997,387,268.
[14]Buey R.M,Diaz J. F,Andreu J.M, et al. [J]. Chem. Biol.2004,11,225.
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[16]icolaou K.C,Vallberg H,King N. P, et al. [J]. Chem. Eur. J.1997,3,1957.
[17]Goodin G, Kane M.P, Rubin E. H. J. Clin. [J]. Oncol.2004,22,2015.
[18]孙宇辉,邓子新.中国抗生素杂志,[J].2006,31(1)：6～18.
[19]McDaniel R, EbertKhosla S, Hopwood D A, et al. Engineered biosynthesis of novel polyketides [J].Science,1993,262:1546.
[20]Shen B, Liu W, Nonaka K. Enediyne natural products:biosynthesis and prospect toward engineering novel antitumor agent. [J]. Curr Med Chem,2003,10:2317-2325.
[21]Cheng X. C, et al. A new antibiotic, tautomycetin. [J]. J Antibiotic(1989) 42,141-144.
[22]Ubukata, M. et al. Biosynthesis of the dialkylmaleic anhydride-containing antibiotics, Tautomycin and tautomycetin. [J]. J. Chem. Soc. Perkin Trans.1995, 1:2439-2404.
[23]Mitsuhashi S, et al. Tautomycetin is a novel and specific Inhibitor of serine/ threonine protein phosphatase typel,PPl. [J].Biochem. Biophy. Res.2001,287: 328-331.
[24]Li W. et al. Utilization of the methoxylmalonyl-Acyl carrier protein biosynthesis locus for cloning of the tautomycin biosynthetic gene cluster from Streptomyces piroverticillatus[J]. J. Bacteri.2006,188:4148-4152.
[25]Li et al. Characterization of the Tautomycin Biosynthetic Gene Cluster from Streptomyces spiroverticillatus. [J]. J. Biol. Chem..2008; 283:28607-28617.
[26]Ju J et al. Functional Characterization of ttmM Unveils New Tautomycin Analogs and Insight into Tautomycin Biosynthesis and Activity. [J]. Org Lett.2009 Apr 2;11(7):1639-42.
[27]McCluskey A,Sim A. T. R, Sakoff J. A. Serine-Threonine Protein Phosphatase Inhibitors:Development of Potential Therapeutic Strategies. [J]. J. Med. Chem, (Perspective),2002,45(6),1151-1175.
[28]Oikawa, H. Synthesis of specific protein phosphatase inhibitors, tautomycin and tautomycetin toward structure-activity relationship study. [J]. Curr. Med. Chem. 2002,9:2033-2054.
[29]Oikawa, H. [J]. Curr. Med. Chem.2002,9,2033-2054.
[30]Sugiyama Y, Ohtani I.I, Isobe M, et al. [J]. Med. Chem. Lett.1996,6,3-8.
[31]Pager M. S,Chamberlin L,Atrih A, et al. [J]. J. Bacteriol.1999,181,204-211.
[32]Bialojan C,Takai A. [J]. Biochem. J.1988,256,283-290.
[33]Reid R, Tang, L. [P] 2005, WO/2005/118797.
[34]Choi S. S, Hur Y. 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]. Microbiology.2007.153.1095-1102.
[35]D. J. Douglas, U. P. Ramsey, J. A. Walter and J. L. C. Wright. [J]. J. Chem. Soc., Chrm. Commun., I 992,7 14.
[36]M. Ubukata, X.-C. Cheng, M. Isobe and K. Isono. [J].J. Chem. Soc., Perkin Trans. I,1993,617.
[37]MacKintosh C, Klumpp S. [J].FEBS Lett.1990,277,137-140.
[38]Li W. L, Luo Y. G. Ju, J. H. Rajski, et al. Characterization of the Tautomycetin Biosynthetic Gene Cluster from Streptomyces griseochromogenes Provides New Insight into Dialkylmaleic Anhydride Biosynthesis[J]. J. Nat. Prod.2009,72,
450-459.
[39]Staunton J, Weissman K. J. [J]. Nat. Prod. Rep.2001,18,380-416.
[40]Gust B, Challis G. L, Fowler K, et al. [J]. Proc. Natl. Acad. Sci. U.S.A.2003,100, 1541-1546.
[2]马田田.纤维素酶用于中药提取的初步研究[J].中草药,1994,25(3)：123.
[3]江泽飞,宋三泰,冯奉仪.中国肿瘤临床与康复,[J].2000,7(2)：66-67.
[4]袁金辉,谢弘,等.紫杉醇抗肿瘤药物的新用途.[P].CN99116954.9,1999-10-11.
[5]纪元,毕建男,严冰,等.[J].生物工程学报,2006,22(1)：1～6.
[6]K. Gerth, N. Bedorf, G. Hfle, et al.Epothilone A and B:antifungal and cytotoxic compounds from Sorangium cellulosum (Myxobacteria)production, physico chemical and biological properties. [J]. J.Antibiot.1996:560-563.
[7]Hofle G, N. Bedorf, K. Gerth & H. Epothilons, process for preparing the same and their use as medicaments and as plant protecting agents. [P]. Wo 93/10121,published May,27,1993.
[8]Bollag D. M, P. A. McQuency, J. Zhu, et al.Epothilones, a newclass of microtubule-stabilizing agents with a taxol-like mechanism of action. [J]. Cancer Res.55: 2325-2333,1995.
[9]Gerth K, D. Schummer, G. Hofle, et al.A new antifungal compound from Sorangium cellulosum (Myxobacteria). Production, physico-chemical and biological properties .[J].J. Antibiotics.
[10]Altmann, K. H. Mini-Rev. [J]. Med. Chem.2003,3,149.
[11]Altaha R, Fojo T, Reed E, et al. [J]. Pharm. Des.2002,8,1707.
[12]Nicolau K. C, Ritzen A, Namoto K. [J]. Chem. Commun. (Cambridge) 2001,1523-1535.
[13]Nicolau k. c, winssinger n, pastor j, et al. [J]. Nature1997,387,268.
[14]Buey R.M,Diaz J. F,Andreu J.M, et al. [J]. Chem. Biol.2004,11,225.
[15]Nicolaou K. C, Vourloumis D,Li,T. et al. [J]. Int. Ed. Engl.1997,36,2097.
[16]icolaou K.C,Vallberg H,King N. P, et al. [J]. Chem. Eur. J.1997,3,1957.
[17]Goodin G, Kane M.P, Rubin E. H. J. Clin. [J]. Oncol.2004,22,2015.
[18]孙宇辉,邓子新.中国抗生素杂志,[J].2006,31(1)：6～18.
[19]McDaniel R, EbertKhosla S, Hopwood D A, et al. Engineered biosynthesis of novel polyketides [J].Science,1993,262:1546.
[20]Shen B, Liu W, Nonaka K. Enediyne natural products:biosynthesis and prospect toward engineering novel antitumor agent. [J]. Curr Med Chem,2003,10:2317-2325.
[21]Cheng X. C, et al. A new antibiotic, tautomycetin. [J]. J Antibiotic(1989) 42,141-144.
[22]Ubukata, M. et al. Biosynthesis of the dialkylmaleic anhydride-containing antibiotics, Tautomycin and tautomycetin. [J]. J. Chem. Soc. Perkin Trans.1995, 1:2439-2404.
[23]Mitsuhashi S, et al. Tautomycetin is a novel and specific Inhibitor of serine/ threonine protein phosphatase typel,PPl. [J].Biochem. Biophy. Res.2001,287: 328-331.
[24]Li W. et al. Utilization of the methoxylmalonyl-Acyl carrier protein biosynthesis locus for cloning of the tautomycin biosynthetic gene cluster from Streptomyces piroverticillatus[J]. J. Bacteri.2006,188:4148-4152.
[25]Li et al. Characterization of the Tautomycin Biosynthetic Gene Cluster from Streptomyces spiroverticillatus. [J]. J. Biol. Chem..2008; 283:28607-28617.
[26]Ju J et al. Functional Characterization of ttmM Unveils New Tautomycin Analogs and Insight into Tautomycin Biosynthesis and Activity. [J]. Org Lett.2009 Apr 2;11(7):1639-42.
[27]McCluskey A,Sim A. T. R, Sakoff J. A. Serine-Threonine Protein Phosphatase Inhibitors:Development of Potential Therapeutic Strategies. [J]. J. Med. Chem, (Perspective),2002,45(6),1151-1175.
[28]Oikawa, H. Synthesis of specific protein phosphatase inhibitors, tautomycin and tautomycetin toward structure-activity relationship study. [J]. Curr. Med. Chem. 2002,9:2033-2054.
[29]Oikawa, H. [J]. Curr. Med. Chem.2002,9,2033-2054.
[30]Sugiyama Y, Ohtani I.I, Isobe M, et al. [J]. Med. Chem. Lett.1996,6,3-8.
[31]Pager M. S,Chamberlin L,Atrih A, et al. [J]. J. Bacteriol.1999,181,204-211.
[32]Bialojan C,Takai A. [J]. Biochem. J.1988,256,283-290.
[33]Reid R, Tang, L. [P] 2005, WO/2005/118797.
[34]Choi S. S, Hur Y. 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]. Microbiology.2007.153.1095-1102.
[35]D. J. Douglas, U. P. Ramsey, J. A. Walter and J. L. C. Wright. [J]. J. Chem. Soc., Chrm. Commun., I 992,7 14.
[36]M. Ubukata, X.-C. Cheng, M. Isobe and K. Isono. [J].J. Chem. Soc., Perkin Trans. I,1993,617.
[37]MacKintosh C, Klumpp S. [J].FEBS Lett.1990,277,137-140.
[38]Li W. L, Luo Y. G. Ju, J. H. Rajski, et al. Characterization of the Tautomycetin Biosynthetic Gene Cluster from Streptomyces griseochromogenes Provides New Insight into Dialkylmaleic Anhydride Biosynthesis[J]. J. Nat. Prod.2009,72,
450-459.
[39]Staunton J, Weissman K. J. [J]. Nat. Prod. Rep.2001,18,380-416.
[40]Gust B, Challis G. L, Fowler K, et al. [J]. Proc. Natl. Acad. Sci. U.S.A.2003,100, 1541-1546.