ist基因的DNA改组以及AHBA合酶基因的系统发生分析
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摘要
必特螺旋霉素(4″酰化螺旋霉素)是含4″-异戊酰基转移酶基因(4″-isovaleryltransferase gene，ist)的螺旋霉素产生菌的产物，即以4″-OH螺旋霉素为底物在异戊酰基转移酶催化下，在4″位加入异戊酰基侧链形成以4″位异戊酰基螺旋霉素为主成份的新抗生素。目前菌种的发酵产物是一个多组分的混合物，含有酰化的和未酰化的螺旋霉素，生产中希望尽可能提高4″酰化螺旋霉素在发酵产物中所占的比例，因此我们决定用DNA改组(DNA shuffling)的方法提高4″-异戊酰基转移酶的酰化能力，以期提高必特螺旋霉素在发酵产物中的比例。
DNA改组是一种分子水平上的定向进化(directed evolution)，它基于有性PCR原理，通过DNA碎片反复交换模板的重聚反应，能够迅速得到理想变异。
我们从6株必特螺旋霉素高产菌株中克隆得到它们的ist基因，进行改组，将最终获得的全长基因构建到链霉菌一大肠杆菌穿梭、链霉菌整合型载体pSET152上，转化到Streptomyces spiramyceticusB183中，传代后发酵，TLC检测其发酵产物，得到5株必特螺旋霉素产量比例明显增加的菌株，进一步做HPLC检测，确定了其中的4株确实产生了比例较大的4″酰化螺旋霉素，与初始基因的作用相对比，4″酰化螺旋霉素所占的比例提高了61.5％～169.7％。将这4株的ist基因送交测序，结果有2株的基因在它们的终止密码子之后发生了一些突变。
Biotechmycin(4″acylspiramycin),a mixture of spiramycin derivatives, is a new antibiotic produced by a genetic engineering strain constructed by introducing ist gene from Streptomyces thermotolerans into the chromosome of Streptomyces spiramyceticus through homologous double-crossover. The main component of the mixture is 4″ isovaleryl spiramycin, the product of isovaleryltransferase which catalyzes the addition of isovaleryl onto 4″ position of 4″-OH spiramycin. At present, there is still unacylated spiramycin in the fermentation products as well as the acylated spiramycin, and we expect that the percentage of the 4″acylspiramycin will be increased. We choose the method of DNA shuffling to enhance the acylation ability of 4″-isovalayltrasferase in order to advance the percentage of biotechmycin among the fermentation products.
DNA shuffling is a type of molecule-level directed evolution. It is based on the principle of sexual PCR, and it is easy to get the expected mutation by the reassembly of DNA fragments with the repeating template exchange.
We cloned 6 ist genes from 6 strains with high output of biotechmycin. After shuffled, the finally obtained full-length genes were cloned into pSET152, an integrating Escherichia coli-Streptomyces shuttle plasmid. The 15 recombinant plasmids were obtained and then transferred into Streptomyces spiramyceticus B183. The 15 transformants were fermented after a generation grew on SCVM medium. Five of these strains were selected because their fermentation products contained obviously increased proportion of
DNA改组是一种分子水平上的定向进化(directed evolution)，它基于有性PCR原理，通过DNA碎片反复交换模板的重聚反应，能够迅速得到理想变异。
我们从6株必特螺旋霉素高产菌株中克隆得到它们的ist基因，进行改组，将最终获得的全长基因构建到链霉菌一大肠杆菌穿梭、链霉菌整合型载体pSET152上，转化到Streptomyces spiramyceticusB183中，传代后发酵，TLC检测其发酵产物，得到5株必特螺旋霉素产量比例明显增加的菌株，进一步做HPLC检测，确定了其中的4株确实产生了比例较大的4″酰化螺旋霉素，与初始基因的作用相对比，4″酰化螺旋霉素所占的比例提高了61.5％～169.7％。将这4株的ist基因送交测序，结果有2株的基因在它们的终止密码子之后发生了一些突变。
Biotechmycin(4″acylspiramycin),a mixture of spiramycin derivatives, is a new antibiotic produced by a genetic engineering strain constructed by introducing ist gene from Streptomyces thermotolerans into the chromosome of Streptomyces spiramyceticus through homologous double-crossover. The main component of the mixture is 4″ isovaleryl spiramycin, the product of isovaleryltransferase which catalyzes the addition of isovaleryl onto 4″ position of 4″-OH spiramycin. At present, there is still unacylated spiramycin in the fermentation products as well as the acylated spiramycin, and we expect that the percentage of the 4″acylspiramycin will be increased. We choose the method of DNA shuffling to enhance the acylation ability of 4″-isovalayltrasferase in order to advance the percentage of biotechmycin among the fermentation products.
DNA shuffling is a type of molecule-level directed evolution. It is based on the principle of sexual PCR, and it is easy to get the expected mutation by the reassembly of DNA fragments with the repeating template exchange.
We cloned 6 ist genes from 6 strains with high output of biotechmycin. After shuffled, the finally obtained full-length genes were cloned into pSET152, an integrating Escherichia coli-Streptomyces shuttle plasmid. The 15 recombinant plasmids were obtained and then transferred into Streptomyces spiramyceticus B183. The 15 transformants were fermented after a generation grew on SCVM medium. Five of these strains were selected because their fermentation products contained obviously increased proportion of
引文
[1] 尚广东，戴剑漉，王以光．生技霉素稳定型基因工程菌的构建．生物工程学报．April 1999，Vol．15(2)，171—175
[2] Phillip A Patten, Russell J Howard, Willem PC Stemmer. Applications of DNA shuffling to pharmaceuticals and vaccines. Current Opinion in Biotechnology. 1997, 8:724-733
[3] 刘国奇，王海涛．DNA改组——定向进化的技术革命．军事医学科学院院刊．2000，Vol．24(2)
[4] Stemmer WP. DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution. Proc Natl Acad Sci U S A. 1994 Oct 25, Vol. 91 (22):10747-51
[5] Crameri AC, Raillard S, Stemmer WPC. DNA shuffling of a family of genes from diverse species accelerates directed evolution. Nature. 1997, Vol. 391: 288～291
[6] 张红缨，张今．蛋白质(或酶)的体外定向技术在药物和疫苗中的应用．中国生化药物杂志．2000，Vol．21(4)
[7] Leong SR, Chang JC, Ong R, etc. Optimized expression and specific activity of IL-12 by directed molecular evolution. Proc Natl Acad Sci U S A. 2003 Feb 4, Vol. 100(3):1163-8. Epub 2003 Jan 15
[8] Zhou Z, Zhang AH, Wang JR, etc. Improving the specific synthetic activity of a penicillin g acylase using DNA family shuffling. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai). 2003 Jun, Vol. 35(6):573-9.
[9] Sandmann G. Novel carotenoids genetically engineered in a heterologous host. Chem Biol. 2003 Jun, Vol. 10(6):478-9
[10] Zahnd C, Spinelli S, Luginbuhl B, etc. Directed in Vitro Evolution and Crystallographic Analysis of a Peptide-binding Single Chain Antibody Fragment (scFv) with Low Picomolar Affinity. J Biol Chem. 2004 Apr 30, Vol. 279(18):18870-7
[11] Korpimaki T, Rosenberg J, Virtanen P, etc. Further improvement of broad specificity hapten recognition with protein engineering. Protein Eng. 2003 Jan, Vol. 16(1):37-46
[12] Locher CP, Heinrichs V, Apt D, etc. Overcoming antigenic diversity and improving vaccines using DNA shuffling and screening technologies. Expert Opin Biol Ther. 2004 Apr, Vol. 4(4):589-97
[13] Locher CP, Soong NW, Whalen RG, etc. Development of novel vaccines using DNA shuffling and screening strategies. Curr Opin Mol Ther. 2004 Feb, Vol. 6(1):34-9
[14] Kim YW, Choi JH, Kim JW, etc. Directed evolution of Thermus maltogenic amylase toward enhanced thermal resistance. Appl Environ Microbiol. 2003 Aug, Vol. 69(8):4866-74
[15] 熊爱生，姚泉洪，彭日荷，等．通过体外分子进化技术获得耐高温β—葡萄糖苷酶的研究．遗传学报 2002，Vol．29(11)：1034-1040
[16] van der Veen BA, Potocki-Veronese G, Albenne C, etc. Combinatorial engineering to enhance amylosucrase performance: construction, selection, and screening of variant libraries for increased activity. FEBS Lett. 2004 Feb 27, Vol. 560(1-3):91-7
[17] Zhang J, Dawes G, Stemmer WPC. Evolution of an effective fucosidase from a galactosidase by DNA shuffling and screening. Proc Natl Acad Sci USA. 1997, Vol. 94:4504-4509
[18] http://www.maxygen.com
[19] Dr. Mac-Wan Ho reports. Death by DNA Shuffling
[20] 周佳海，陈海宝．一个简便的DNA改组(DNA shuffling)操作程序．生物化学与生物生理进展．2000，Vol．27(6)：655
[21] Bierman M, Logan R, O'Brien K, etc. Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp. Gene. 1992, Vol. 116:43—49
[22] Reetz MT. Asymmetric Catalysis Special Feature Part Ⅱ: Controlling the enantioselectivity of enzymes by directed evolution: Practical and theoretical ramifications. Proc Natl Acad Sci U S A. 2004 Apr 20, Vol. 101(16):5716-22
[1] Wehrli W. Ansarnycins: Chemistry, biosynthesis and biological activity. Top Cuur. Chem. 1977, 72:22-49.
[2] Lancini G. Ansamycins. In: Vining LC (ed) Biochemistry and genetic regulation of commercially important antibiotics. Addision-Wesley, Reading, Mall., USA, 1983, pp231-254.
[3] Rinehart K. L., Shield L.S. Chemistry of the ansamycin antibiotics. In: Herz W., Grisbach H., Kirby G.W. (eds) Progress in the chemistry of organic natural products, vol 33, Springer, Vienna New York, 1976, pp. 232-307
[4] Sasaki K, Rinehart KL. Jr., Slomp G, et al. Geldanamycin. 1: Structure assignment. J. AM. Chem. 1970, Soc. 92: 7591-7593.
[5] Redeuilh G. J Biol Chem, 1997, 262: 6969-6975.
[6] 陶佩珍，章天．抗病毒抗生素17997体外联合用药研究．中国抗生素杂志，2001，26：292．
[7] August P. R., Tang L., Yoon Y.J., etc. Biosynthesis of the ansamycin antibiotic rifamycin: deductions from the molecular analysis of the rif biosynthetic gene cluster of Amycolatopsis mediterranei S699. Chemistry&Biology, 1998, 5:69-79.
[8] Chen S., Bamberg D. V., Hale V., etc. Biosynthesis of ansatrienin (mycotrienin) and naphthomycin Identification and analysis of two separate biosynthetic gene clusters in Streptomyces collinus Tu 1892. Eur. J. Biochem., 1999, 261: 98-107.
[9] Yu T. W., Bai L. Q., Clade D., etc. The biosynthetic gene cluster of the maytansinoid antitumor agent ansamitocin from Actinosynnema pretiosum. PNAS. 2002, 99: 7968-7973.
[10] http://gaia.biotech.vt.edu/calendar/lectures/309,1, What is phylogenetic analysis?
[11] Caro-Beth Stewart, Ph. D. Phylogenetic Analysis. http://www.genome.gov/Pages/Hyperion/COURSE2000/Pdf/Phylogenetic_Analysis.pdf
[12] Chun-Gyu Kim, Tin-Wein Yu, Craig B. Fryhle, etc. 3-Amino-5-hydroxybenzoic Acid Synthase, the Terminal Enzyme in the Formation of the Precursor of mC7N Units in Rifamycin and Related Antibiotics. The Journal of Biological Chemistry. 1998, Vol. 273(11): 6030-6040
[13] Fiona S. Lawson, Robert L, Charlebois, and Jo-Anne R. Dillon. Phylogenetic Analysis of Carbamoylphosphate Synthetase Genes: Complex Evolutionary History Includes an Internal Duplication Within a Gene Which Can Root the Tree of Life. Mol. Bid. Ed. 1996, Vol. 13(7):970-977.
[14] Wen Liu, Joachim Ahlert, Qunjie Gao, ect. Rapid PCR amplification of minimal enediyne polyketide synthase cassettes leads to a predictive familial classification model. PNAS October 14, 2003, vol. 100(21): 11959-11963
[15] Chen S, Bamberg DV, Hale V etc. Eur J Biochem. 1999, 261, 98-107.
[16] 3. Yu TW, Bai LQ, Clade D etc. PNAS, 2002, 99(12), 7968-7973
[17] Anderson M. G., Kibby J. J., Rickards. R. W., etc. J. Chem. Soc. Chem. Commun. 1980, 1227-1278.
[18] Ghisalba O. & Nuesch J. J. Antibiot. (Tokyo)., 1981, 34:64-71.
[19] Becker a. M., Herlt a. J., Hilton G.L., etc. J. Antibiot. (Tokyo)., 1983, 36:1323-1328.
[20] Kibby J. J., McDonald I. A., Rickards R.W.J. Chem. Spc. Chem. Commun. 1980, 768-769.
[21] 高群杰 Geldanamycin产生菌吸水链霉菌17997中安莎类抗生素生物合成基因簇的克隆与研究 [博士学位论文] 北京 中国医学科学院中国协和医科大学医药生物技术研究所，2002
[2] Phillip A Patten, Russell J Howard, Willem PC Stemmer. Applications of DNA shuffling to pharmaceuticals and vaccines. Current Opinion in Biotechnology. 1997, 8:724-733
[3] 刘国奇，王海涛．DNA改组——定向进化的技术革命．军事医学科学院院刊．2000，Vol．24(2)
[4] Stemmer WP. DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution. Proc Natl Acad Sci U S A. 1994 Oct 25, Vol. 91 (22):10747-51
[5] Crameri AC, Raillard S, Stemmer WPC. DNA shuffling of a family of genes from diverse species accelerates directed evolution. Nature. 1997, Vol. 391: 288～291
[6] 张红缨，张今．蛋白质(或酶)的体外定向技术在药物和疫苗中的应用．中国生化药物杂志．2000，Vol．21(4)
[7] Leong SR, Chang JC, Ong R, etc. Optimized expression and specific activity of IL-12 by directed molecular evolution. Proc Natl Acad Sci U S A. 2003 Feb 4, Vol. 100(3):1163-8. Epub 2003 Jan 15
[8] Zhou Z, Zhang AH, Wang JR, etc. Improving the specific synthetic activity of a penicillin g acylase using DNA family shuffling. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai). 2003 Jun, Vol. 35(6):573-9.
[9] Sandmann G. Novel carotenoids genetically engineered in a heterologous host. Chem Biol. 2003 Jun, Vol. 10(6):478-9
[10] Zahnd C, Spinelli S, Luginbuhl B, etc. Directed in Vitro Evolution and Crystallographic Analysis of a Peptide-binding Single Chain Antibody Fragment (scFv) with Low Picomolar Affinity. J Biol Chem. 2004 Apr 30, Vol. 279(18):18870-7
[11] Korpimaki T, Rosenberg J, Virtanen P, etc. Further improvement of broad specificity hapten recognition with protein engineering. Protein Eng. 2003 Jan, Vol. 16(1):37-46
[12] Locher CP, Heinrichs V, Apt D, etc. Overcoming antigenic diversity and improving vaccines using DNA shuffling and screening technologies. Expert Opin Biol Ther. 2004 Apr, Vol. 4(4):589-97
[13] Locher CP, Soong NW, Whalen RG, etc. Development of novel vaccines using DNA shuffling and screening strategies. Curr Opin Mol Ther. 2004 Feb, Vol. 6(1):34-9
[14] Kim YW, Choi JH, Kim JW, etc. Directed evolution of Thermus maltogenic amylase toward enhanced thermal resistance. Appl Environ Microbiol. 2003 Aug, Vol. 69(8):4866-74
[15] 熊爱生，姚泉洪，彭日荷，等．通过体外分子进化技术获得耐高温β—葡萄糖苷酶的研究．遗传学报 2002，Vol．29(11)：1034-1040
[16] van der Veen BA, Potocki-Veronese G, Albenne C, etc. Combinatorial engineering to enhance amylosucrase performance: construction, selection, and screening of variant libraries for increased activity. FEBS Lett. 2004 Feb 27, Vol. 560(1-3):91-7
[17] Zhang J, Dawes G, Stemmer WPC. Evolution of an effective fucosidase from a galactosidase by DNA shuffling and screening. Proc Natl Acad Sci USA. 1997, Vol. 94:4504-4509
[18] http://www.maxygen.com
[19] Dr. Mac-Wan Ho reports. Death by DNA Shuffling
[20] 周佳海，陈海宝．一个简便的DNA改组(DNA shuffling)操作程序．生物化学与生物生理进展．2000，Vol．27(6)：655
[21] Bierman M, Logan R, O'Brien K, etc. Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp. Gene. 1992, Vol. 116:43—49
[22] Reetz MT. Asymmetric Catalysis Special Feature Part Ⅱ: Controlling the enantioselectivity of enzymes by directed evolution: Practical and theoretical ramifications. Proc Natl Acad Sci U S A. 2004 Apr 20, Vol. 101(16):5716-22
[1] Wehrli W. Ansarnycins: Chemistry, biosynthesis and biological activity. Top Cuur. Chem. 1977, 72:22-49.
[2] Lancini G. Ansamycins. In: Vining LC (ed) Biochemistry and genetic regulation of commercially important antibiotics. Addision-Wesley, Reading, Mall., USA, 1983, pp231-254.
[3] Rinehart K. L., Shield L.S. Chemistry of the ansamycin antibiotics. In: Herz W., Grisbach H., Kirby G.W. (eds) Progress in the chemistry of organic natural products, vol 33, Springer, Vienna New York, 1976, pp. 232-307
[4] Sasaki K, Rinehart KL. Jr., Slomp G, et al. Geldanamycin. 1: Structure assignment. J. AM. Chem. 1970, Soc. 92: 7591-7593.
[5] Redeuilh G. J Biol Chem, 1997, 262: 6969-6975.
[6] 陶佩珍，章天．抗病毒抗生素17997体外联合用药研究．中国抗生素杂志，2001，26：292．
[7] August P. R., Tang L., Yoon Y.J., etc. Biosynthesis of the ansamycin antibiotic rifamycin: deductions from the molecular analysis of the rif biosynthetic gene cluster of Amycolatopsis mediterranei S699. Chemistry&Biology, 1998, 5:69-79.
[8] Chen S., Bamberg D. V., Hale V., etc. Biosynthesis of ansatrienin (mycotrienin) and naphthomycin Identification and analysis of two separate biosynthetic gene clusters in Streptomyces collinus Tu 1892. Eur. J. Biochem., 1999, 261: 98-107.
[9] Yu T. W., Bai L. Q., Clade D., etc. The biosynthetic gene cluster of the maytansinoid antitumor agent ansamitocin from Actinosynnema pretiosum. PNAS. 2002, 99: 7968-7973.
[10] http://gaia.biotech.vt.edu/calendar/lectures/309,1, What is phylogenetic analysis?
[11] Caro-Beth Stewart, Ph. D. Phylogenetic Analysis. http://www.genome.gov/Pages/Hyperion/COURSE2000/Pdf/Phylogenetic_Analysis.pdf
[12] Chun-Gyu Kim, Tin-Wein Yu, Craig B. Fryhle, etc. 3-Amino-5-hydroxybenzoic Acid Synthase, the Terminal Enzyme in the Formation of the Precursor of mC7N Units in Rifamycin and Related Antibiotics. The Journal of Biological Chemistry. 1998, Vol. 273(11): 6030-6040
[13] Fiona S. Lawson, Robert L, Charlebois, and Jo-Anne R. Dillon. Phylogenetic Analysis of Carbamoylphosphate Synthetase Genes: Complex Evolutionary History Includes an Internal Duplication Within a Gene Which Can Root the Tree of Life. Mol. Bid. Ed. 1996, Vol. 13(7):970-977.
[14] Wen Liu, Joachim Ahlert, Qunjie Gao, ect. Rapid PCR amplification of minimal enediyne polyketide synthase cassettes leads to a predictive familial classification model. PNAS October 14, 2003, vol. 100(21): 11959-11963
[15] Chen S, Bamberg DV, Hale V etc. Eur J Biochem. 1999, 261, 98-107.
[16] 3. Yu TW, Bai LQ, Clade D etc. PNAS, 2002, 99(12), 7968-7973
[17] Anderson M. G., Kibby J. J., Rickards. R. W., etc. J. Chem. Soc. Chem. Commun. 1980, 1227-1278.
[18] Ghisalba O. & Nuesch J. J. Antibiot. (Tokyo)., 1981, 34:64-71.
[19] Becker a. M., Herlt a. J., Hilton G.L., etc. J. Antibiot. (Tokyo)., 1983, 36:1323-1328.
[20] Kibby J. J., McDonald I. A., Rickards R.W.J. Chem. Spc. Chem. Commun. 1980, 768-769.
[21] 高群杰 Geldanamycin产生菌吸水链霉菌17997中安莎类抗生素生物合成基因簇的克隆与研究 [博士学位论文] 北京 中国医学科学院中国协和医科大学医药生物技术研究所，2002