抗生素iso-migrastatin发酵条件优化和高产机理研究
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摘要
Iso-Migrastatin (iso-MGS)是从S. platensis NRRL18993发酵液中分离得到的12元环的大环内脂类抗生素,具有抑制肿瘤细胞转移的生物学活性。利用基因工程技术,将携带有完整iso-MGS基因簇的载体pBS11001整合到五种不同模式菌株的染色体上,成功构建五种生物合成iso-MGS的工程菌株。
本论文开展了iso-MGS工程菌的发酵培养条件优化的工作,通过培养基优化,五种工程菌异源生物合成iso-MGS的能力均得到提高,iso-MGS的产量提高了3-18倍不等,通过对比可以看出工程菌SB11001合成iso-MGS的能力明显高于其余四种工程菌,最高产量为128.6 mg/L,该菌也成为进一步组合生物合成iso-MGS的首选的工程菌。通过组合不同培养基之间的成分摸索出更适宜iso-MGS合成的培养基,摸索出一种组合B2培养基,使iso-MGS的产量进一步提高到186.7 mg/L,同时为异源生物合成其他天然产物所需培养基提供了借鉴。根据文献中报道的iso-MGS的假想生物合成路径,本实验验证了添加假想前体碳酸氢钠对iso-MGS产量的影响,添加2 g/L碳酸氢钠使iso-MGS的产量继续提高到207.8 mg/L。利用实时荧光定量RT-PCR技术研究野生菌和工程菌的mgs基因簇中每个基因在不同时期的表达丰度,从而发现与iso-MGS产量密切相关的基因,为进一步组合生物合成iso-MGS选择靶基因提供了理论基础。
Iso-migrastatin (iso-MGS), the 12-membered glutarimide-containing polyketide, was isolated from S. pLatensis NRRL18993 fermentation broth with the bioactivity against cancer cells migration. Five engineered strains were constructed upon introduction of the vector pBS11001 harboring the entire mgs biosynthetic gene cluster into five mode strains, which successfully heterologously biosynthesized iso-MGS production.
The purpose of this work is to improve iso-MGS production titers in five engineered strains by fermentation condition optimization. iso-MGS production titers were improved by 3-18 fold in five engineered strains after optimization. And SB11001 proved itself to yield the highest iso-MGS production 128.6 mg/L, which was chosen to be the candidate strain for further recombinant biosynthesized iso-MGS production. Exchanging the carbon sources between B2 medium and R2YE medium resulted in the hybrid B2 medium, in which iso-MGS production titer was further increased to 186.7 mg/L. The hybrid B2 medium provided the possibility for heterologously biosynthesizing other natural products. According to the proposed biosynthetic pathway for iso-MGS, 2 g/L NaHCO3 as the proposed precursor added to the fermentation broth further increased the iso-MGS production to 207.8 mg/L. Real Time quantitative RT-PCR demonstrated the relationship between the 11 genes in mgs cluster transcriptional level and iso-MGS production titers in wild strain and engineered strains at different phase. In order to select the targeted engineered genes for further recombinant biosynthesis iso-MGS production.
本论文开展了iso-MGS工程菌的发酵培养条件优化的工作,通过培养基优化,五种工程菌异源生物合成iso-MGS的能力均得到提高,iso-MGS的产量提高了3-18倍不等,通过对比可以看出工程菌SB11001合成iso-MGS的能力明显高于其余四种工程菌,最高产量为128.6 mg/L,该菌也成为进一步组合生物合成iso-MGS的首选的工程菌。通过组合不同培养基之间的成分摸索出更适宜iso-MGS合成的培养基,摸索出一种组合B2培养基,使iso-MGS的产量进一步提高到186.7 mg/L,同时为异源生物合成其他天然产物所需培养基提供了借鉴。根据文献中报道的iso-MGS的假想生物合成路径,本实验验证了添加假想前体碳酸氢钠对iso-MGS产量的影响,添加2 g/L碳酸氢钠使iso-MGS的产量继续提高到207.8 mg/L。利用实时荧光定量RT-PCR技术研究野生菌和工程菌的mgs基因簇中每个基因在不同时期的表达丰度,从而发现与iso-MGS产量密切相关的基因,为进一步组合生物合成iso-MGS选择靶基因提供了理论基础。
Iso-migrastatin (iso-MGS), the 12-membered glutarimide-containing polyketide, was isolated from S. pLatensis NRRL18993 fermentation broth with the bioactivity against cancer cells migration. Five engineered strains were constructed upon introduction of the vector pBS11001 harboring the entire mgs biosynthetic gene cluster into five mode strains, which successfully heterologously biosynthesized iso-MGS production.
The purpose of this work is to improve iso-MGS production titers in five engineered strains by fermentation condition optimization. iso-MGS production titers were improved by 3-18 fold in five engineered strains after optimization. And SB11001 proved itself to yield the highest iso-MGS production 128.6 mg/L, which was chosen to be the candidate strain for further recombinant biosynthesized iso-MGS production. Exchanging the carbon sources between B2 medium and R2YE medium resulted in the hybrid B2 medium, in which iso-MGS production titer was further increased to 186.7 mg/L. The hybrid B2 medium provided the possibility for heterologously biosynthesizing other natural products. According to the proposed biosynthetic pathway for iso-MGS, 2 g/L NaHCO3 as the proposed precursor added to the fermentation broth further increased the iso-MGS production to 207.8 mg/L. Real Time quantitative RT-PCR demonstrated the relationship between the 11 genes in mgs cluster transcriptional level and iso-MGS production titers in wild strain and engineered strains at different phase. In order to select the targeted engineered genes for further recombinant biosynthesis iso-MGS production.
引文
[1] Elane. J, Woo. E J, Starks. C M, Carney. J R, Arslanian. R, Cadapan. L, Zavala. S, Licari. P. Migrastatin and a new compound, isomigrastatin, from Streptomyces platensis. J Antibiot, 2002, 55: 141-146.
[2] James P. Karwowski, Marianna Jackson, Gabriela Sunga, Paul Sheldon, Jennifer B. Poddig, William L. Kohl and Sunil Kadam. Dorrigocins: novel antifungal antibiotics that change the morphology of ras-transformed NIH/3T3 cells to that of normal cells. J Antibiot,1994, 47: 862-869.
[3] Nakae, K, Yoshimoto, Y, Sawa, T, Homma, Y, Hamada, M, Takeuchi, T, Imoto, M. Migrastatin, a new inhibitor of tumor cell migration from Streptomyces sp. MK929-43F1. Taxonomy, fermentation, isolation and biological activities. J Antibiot, 2000, 53: 1130-1136.
[4] Ju J, Lim S-K, Jiang H, Shen B. Migrastatin and dorrigocins are shunt metabolites of iso-migrastain. J Am Chem Soc, 2005, 127: 1622–1623.
[5] Nakae, K, Yoshimoto, Y, Sawa, T, Homma, Y, Hamada, M, Takeuchi, T, Imoto, M. Migrastatin, a novel 14-membered lactone from Streptomyces sp.MK929-43F1. J Antibiot, 2000, 53: 1228-1230.
[6] Nakae, K, Yoshimoto, Y, Sawa, T, Homma, Y, Hamada, M, Takeuchi, T, Imoto, M. Migrastatin, a new inhibitor of tumor cell migration from Streptomyces sp. MK929-43F1. Taxonomy, fermentation, isolation and biological activities. J Antibiot, 2000, 53: 1130-1136.
[7] Reymond S, Cossy J. Migrastatin and analogues: new anti-metastatic agents. CR Chim , 2008, 11: 1447-1462.
[8] Metaferia BB, Chen L, Baker HL, Huang X-Y & Bewley CA. Synthetic macrolides that inhibit breast cancer cell migration in vitro. J Am Chem Soc , 2007, 129: 2434-2435.
[9] Shan D, Chen L, Njardarson JT, Gaul C, Ma X, Danishefsky SJ & Huang X-Y. Synthetic analogues of migrastatin that inhibit mammary tumor metastasis inmice. Proc Natl Acad USA, 2005, 102: 3772-3776.
[10] Nakae K, Nishimura Y, Ohba S, Akamatsu Y. Migrastatin acts as a muscarinic acetylcholine receptor antagonist. J Antibiot, 2006, 59: 685-692.
[11] Takemoto Y, Tashiro E, Imoto M. Suppression of multidrug resistance by migrastatin. J Antibiot , 2006, 59: 435-438.
[12] Christoph Gaul, Jo′n T. Njardarson, Dandan Shan, David C. Dorn, Kai-Da Wu, William P. Tong, Xin-Yun Huang, Malcolm A. S. Moore, Samuel J. Danishefsky. The Migrastatin Family: Discovery of Potent Cell Migration Inhibitors by Chemical Synthesis. J Am Chem Soc, 2004, 126: 11326-11337.
[13] Christoph Gaul, Jo′n T. Njardarson, Dandan Shan, David C. Dorn, Kai-Da Wu, William P. Tong,Xin-Yun Huang, Malcolm A. S. Moore, Samuel J. Danishefsky. The Migrastatin Family: Discovery of Potent Cell Migration Inhibitors by Chemical Synthesis. J Am Chem Soc, 2004, 126: 11326-11337.
[14] V. Sai Baba, Parthasarathi Das, K. Mukkanti, Javed Iqbal. A convergent synthesis of the macrolide core of migrastatin. Tetrahedrom Letters, 2006, 47:6083-6086.
[15] Sebastien Reymond, Janine Cossy. Synthesis of migrastatin and its macrolide core. Tetrahedron, 2007, 63: 5918-5929.
[16] Dandan Shan, Lin Chen, Jon T. Njardarson, Christoph Gaul, Xiaojing Ma?, Samuel J. Danishefsky, Xin-Yun Huang. Synthetic analogues of migrastatin that inhibit mammary tumor metastasis in mice. Proc Natl Acad Sci USA, 2005, 102: 3772-3776.
[17] Rude MA, Khosla C. Engineered biosynthesis of polyketides in heterologous hosts. Chem Eng Sci, 2004, 59: 4693-4701.
[18] McDaniel R, Ebert-Khosla, Hopwood DA, et al Engineered biosynthesis of novel polyketides. Science, 1993, 262: 1546.
[19] Gerth K, Bedorf N, Hofle G, et al.Epothilons A and B: antifungal and cytotoxic compounds from Sorangium cellulosum. Production, physico-chemical and biological properties. J Antibiot, 1996, 49: 560-563.
[20] Piel J. A polyketide synthase-peptide synthetase gene cluster from an unculturedbacterial symbiont of Paederus beetles. Proc Natl Acad Sci USA, 2002, 99: 14002-14007.
[21] Jacobsen JR, Hutchinson CR, Cane DE, et al. Precursordirected biosynthesis of erythromycin analogs by an engineered polyketide synthase. Science, 1997, 277:367-369.
[22] Shen B. Polyketide biosynthesis beyond the type I, II and III polyketide synthase paradigms. Curr Opion Chem BioL, 2003, 7: 285-295.
[23] Yi-Qiang Cheng, Gong-Li Tang, Ben Shen. Type I polyketide synthase requiring a discrete acyltransferase for polyketide biosynthesis. Proc Natl Acad Sci USA, 2003, 100: 3149-3154.
[24] Heathcote, ML, Staunton, J, Leadlay, PF.. Chem. Biol, 2001, 8: 207-220.
[25] Lim, S-K., J. Ju, E. Zazopoulos, H. Jiang, J-W. Seo, Y. Chen, Z. Feng, S. R. Rajski, C. M. Farnet, and B. Shen (iso-Migrastatin, Migrastatin, and Dorrigocin Production in Streptomyces platensis NRRL 18993 Is Governed by a Single Biosynthetic Machinery Featuring an Acyltransferase-less Type I Polyketide Synthase. J B C, 2009, 284: 29746–29756.
[26] Mcdaniel R, Ebert2Khosla S, Hopwood DA, et al . Engineered biosynthesis of novel polyketides . Science, 1993, 26: 15462-15550 .
[27] Newman, D. J., G. M.Cragg, and K. M. Snader. Natural products as sources of new drugs over the period 1981-2002. J Nat Prod, 2003, 66: 1022-1037.
[28] Miao V, Coeffet-legal, MF, Brian P, Brost R, Ford R, Parr I, Bouchard M, Silva CJ,Wrigley SK, Baltz RH. Microbiology , 2005, 151: 1507-1523.
[29] Feng, Z., L. Wang, S.R. Rajski, Z. Xu, M.F. Coeffet-LeGal, and B. Shen. Engineered production of iso-migrastatin in heterologous Streptomyces hosts. Bioorgan Med Chem, 2009, 17: 2147-2153.
[30] Chelly , J. Kahn. RT-PCR and mRNA quantitative. Polymerase Chain React, 1994, 269: 101-109.
[31] Chomczynski P, Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem, 1987, 162: 156-159.
[32]周德庆《微生物学教程》高等教育出版社1993.
[33] Wegmann K. Osmotic regulation of photosynthetic glycerol production in DunaLieLLa. Biochim. Biophys .Acta, 1971, 234: 317-323.
[34]沈同,王镜岩《生物化学》高等教育出版社,1981.
[35] Eng LF, Lee YL, Murphy GM, Yu AC. A RT-PCR study of gene expression in a mechanical injury model. Prog Brain Res, 1995, 105: 219-229.
[36] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real time quantitative PCR and the 2-ΔΔCt method. Methods, 2001, 25: 402-408.
[2] James P. Karwowski, Marianna Jackson, Gabriela Sunga, Paul Sheldon, Jennifer B. Poddig, William L. Kohl and Sunil Kadam. Dorrigocins: novel antifungal antibiotics that change the morphology of ras-transformed NIH/3T3 cells to that of normal cells. J Antibiot,1994, 47: 862-869.
[3] Nakae, K, Yoshimoto, Y, Sawa, T, Homma, Y, Hamada, M, Takeuchi, T, Imoto, M. Migrastatin, a new inhibitor of tumor cell migration from Streptomyces sp. MK929-43F1. Taxonomy, fermentation, isolation and biological activities. J Antibiot, 2000, 53: 1130-1136.
[4] Ju J, Lim S-K, Jiang H, Shen B. Migrastatin and dorrigocins are shunt metabolites of iso-migrastain. J Am Chem Soc, 2005, 127: 1622–1623.
[5] Nakae, K, Yoshimoto, Y, Sawa, T, Homma, Y, Hamada, M, Takeuchi, T, Imoto, M. Migrastatin, a novel 14-membered lactone from Streptomyces sp.MK929-43F1. J Antibiot, 2000, 53: 1228-1230.
[6] Nakae, K, Yoshimoto, Y, Sawa, T, Homma, Y, Hamada, M, Takeuchi, T, Imoto, M. Migrastatin, a new inhibitor of tumor cell migration from Streptomyces sp. MK929-43F1. Taxonomy, fermentation, isolation and biological activities. J Antibiot, 2000, 53: 1130-1136.
[7] Reymond S, Cossy J. Migrastatin and analogues: new anti-metastatic agents. CR Chim , 2008, 11: 1447-1462.
[8] Metaferia BB, Chen L, Baker HL, Huang X-Y & Bewley CA. Synthetic macrolides that inhibit breast cancer cell migration in vitro. J Am Chem Soc , 2007, 129: 2434-2435.
[9] Shan D, Chen L, Njardarson JT, Gaul C, Ma X, Danishefsky SJ & Huang X-Y. Synthetic analogues of migrastatin that inhibit mammary tumor metastasis inmice. Proc Natl Acad USA, 2005, 102: 3772-3776.
[10] Nakae K, Nishimura Y, Ohba S, Akamatsu Y. Migrastatin acts as a muscarinic acetylcholine receptor antagonist. J Antibiot, 2006, 59: 685-692.
[11] Takemoto Y, Tashiro E, Imoto M. Suppression of multidrug resistance by migrastatin. J Antibiot , 2006, 59: 435-438.
[12] Christoph Gaul, Jo′n T. Njardarson, Dandan Shan, David C. Dorn, Kai-Da Wu, William P. Tong, Xin-Yun Huang, Malcolm A. S. Moore, Samuel J. Danishefsky. The Migrastatin Family: Discovery of Potent Cell Migration Inhibitors by Chemical Synthesis. J Am Chem Soc, 2004, 126: 11326-11337.
[13] Christoph Gaul, Jo′n T. Njardarson, Dandan Shan, David C. Dorn, Kai-Da Wu, William P. Tong,Xin-Yun Huang, Malcolm A. S. Moore, Samuel J. Danishefsky. The Migrastatin Family: Discovery of Potent Cell Migration Inhibitors by Chemical Synthesis. J Am Chem Soc, 2004, 126: 11326-11337.
[14] V. Sai Baba, Parthasarathi Das, K. Mukkanti, Javed Iqbal. A convergent synthesis of the macrolide core of migrastatin. Tetrahedrom Letters, 2006, 47:6083-6086.
[15] Sebastien Reymond, Janine Cossy. Synthesis of migrastatin and its macrolide core. Tetrahedron, 2007, 63: 5918-5929.
[16] Dandan Shan, Lin Chen, Jon T. Njardarson, Christoph Gaul, Xiaojing Ma?, Samuel J. Danishefsky, Xin-Yun Huang. Synthetic analogues of migrastatin that inhibit mammary tumor metastasis in mice. Proc Natl Acad Sci USA, 2005, 102: 3772-3776.
[17] Rude MA, Khosla C. Engineered biosynthesis of polyketides in heterologous hosts. Chem Eng Sci, 2004, 59: 4693-4701.
[18] McDaniel R, Ebert-Khosla, Hopwood DA, et al Engineered biosynthesis of novel polyketides. Science, 1993, 262: 1546.
[19] Gerth K, Bedorf N, Hofle G, et al.Epothilons A and B: antifungal and cytotoxic compounds from Sorangium cellulosum. Production, physico-chemical and biological properties. J Antibiot, 1996, 49: 560-563.
[20] Piel J. A polyketide synthase-peptide synthetase gene cluster from an unculturedbacterial symbiont of Paederus beetles. Proc Natl Acad Sci USA, 2002, 99: 14002-14007.
[21] Jacobsen JR, Hutchinson CR, Cane DE, et al. Precursordirected biosynthesis of erythromycin analogs by an engineered polyketide synthase. Science, 1997, 277:367-369.
[22] Shen B. Polyketide biosynthesis beyond the type I, II and III polyketide synthase paradigms. Curr Opion Chem BioL, 2003, 7: 285-295.
[23] Yi-Qiang Cheng, Gong-Li Tang, Ben Shen. Type I polyketide synthase requiring a discrete acyltransferase for polyketide biosynthesis. Proc Natl Acad Sci USA, 2003, 100: 3149-3154.
[24] Heathcote, ML, Staunton, J, Leadlay, PF.. Chem. Biol, 2001, 8: 207-220.
[25] Lim, S-K., J. Ju, E. Zazopoulos, H. Jiang, J-W. Seo, Y. Chen, Z. Feng, S. R. Rajski, C. M. Farnet, and B. Shen (iso-Migrastatin, Migrastatin, and Dorrigocin Production in Streptomyces platensis NRRL 18993 Is Governed by a Single Biosynthetic Machinery Featuring an Acyltransferase-less Type I Polyketide Synthase. J B C, 2009, 284: 29746–29756.
[26] Mcdaniel R, Ebert2Khosla S, Hopwood DA, et al . Engineered biosynthesis of novel polyketides . Science, 1993, 26: 15462-15550 .
[27] Newman, D. J., G. M.Cragg, and K. M. Snader. Natural products as sources of new drugs over the period 1981-2002. J Nat Prod, 2003, 66: 1022-1037.
[28] Miao V, Coeffet-legal, MF, Brian P, Brost R, Ford R, Parr I, Bouchard M, Silva CJ,Wrigley SK, Baltz RH. Microbiology , 2005, 151: 1507-1523.
[29] Feng, Z., L. Wang, S.R. Rajski, Z. Xu, M.F. Coeffet-LeGal, and B. Shen. Engineered production of iso-migrastatin in heterologous Streptomyces hosts. Bioorgan Med Chem, 2009, 17: 2147-2153.
[30] Chelly , J. Kahn. RT-PCR and mRNA quantitative. Polymerase Chain React, 1994, 269: 101-109.
[31] Chomczynski P, Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem, 1987, 162: 156-159.
[32]周德庆《微生物学教程》高等教育出版社1993.
[33] Wegmann K. Osmotic regulation of photosynthetic glycerol production in DunaLieLLa. Biochim. Biophys .Acta, 1971, 234: 317-323.
[34]沈同,王镜岩《生物化学》高等教育出版社,1981.
[35] Eng LF, Lee YL, Murphy GM, Yu AC. A RT-PCR study of gene expression in a mechanical injury model. Prog Brain Res, 1995, 105: 219-229.
[36] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real time quantitative PCR and the 2-ΔΔCt method. Methods, 2001, 25: 402-408.