- Email: [email protected]
Effect of temperature and pH on fractional precipitation for paclitaxel pre-purification
S56
Abstracts / Journal of Biotechnology 136S (2008) S22–S71
through 31st July and the plants harvested by hand at 4 steps of growth including 10 leaves plants (10 s), middle of growth (50 s), preflowering (pfs) and flowering (fs). The level of gene expression was monitored by semi quantitative RT-PCR technique. Plant growth and maturity both affected on PAL, CVOMT and EOMT gene expression and phenolic contents. References Dixon, P., Paiva, N.L., 1995. Stress-induced phenylpropanoid metabolism. Plant Cell. 7, 1085–1097. Lewinsohn, E., Ziv-Raz, I., Dudai, N., Tadmor, Y., Lastochkin, E., Larkov, O., Chaimovitsh, D., Ravid, U., Putievsky, E., Shoham, Y., 2000. Biosynthesis of estragole and methyl-eugenol in sweet basil (Ocimum basilicum L) developmental and chemotypic association of allylphenol O-methyltransferase activities. Plant Sci. 160, 27–35. Logemann, E., Parniske, M., Hahlbrock, K., 1995. Modes of expression and common structural features of the complete phenylalanine ammonia-lyase gene family in parsley. Plant Biol. 92, 5905–5909.
doi:10.1016/j.jbiotec.2008.07.118 I1-P-099 Identification and characterization of tmcC as a putative esterase of tautomycetin biosynthesis in Streptomyces sp. CK4412 Ja-Ryong Koo ∗ , Si-Sun Choi, Eung-Soo Kim Department of Biotechnology, Inha University, Incheon 402-751, Republic of Korea E-mail address: [email protected] (E.-S. Kim). Tautomycetin (TMC) is a novel activated T cell-specific immunosuppressive compound with an ester bond linkage between a terminal cyclic anhydride moiety and a linear polyketide chain bearing an unusual terminal alkene. The presence of a 1.5 kb gene of tmcC, whose deduced product was found to have some amino acid sequence homology with previously known bacterial esterase, was found just upstream of tmc PKS genes in an approximately 70-kb contiguous TMC biosynthetic gene cluster. In silico database comparisons revealed that tmcC belongs to the type B carboxylesterase. PCR-targeted disruption of the putative TMC-specific esterase gene, tmcC completely abolished TMC biosynthesis. Expectly, complementation by an integrative plasmid carrying tmcC significantly restored TMC biosynthesis. Moreover, the codon-optimzed tmcC was also over-expressed as a His-tagged fusion protein in Escherichia coli, follow by the in vitro biochemical esterase assays. References Choi, S.-S., Hur, Y.-A., Sherman, D.H., Kim, E.-S., 2007. Isolation of the biosynthetic gene cluster for tautomycetin, a linear polyketide T cell-specific immunomodulator from Streptomyces sp. CK4412. Microbiology 153, 1095–1102. Soror, S.H., Verma, V., Rao, R., Rasool, S., Koul, S., Qazi, G.N., Cullum, J., 2007. A cold-active esterase of Streptomyces coelicolor A3(2): from genome sequence to enzyme activity. J. Ind. Microbiol. Biotechnol. 34, 525–531. Tesch, C., Nikoleit, K., Gnau, V., Gotz, F., Bormann, C., 1996. Biochemical and molecular characterization of the extracellular esterase from Streptomyces diastatochromogenes. J. Bacteriol., 1858–1865.
doi:10.1016/j.jbiotec.2008.07.119
I1-P-100 Universal model for designing 5 -UTRs for precise gene expression in Escherichia coli Sang Woo Seo 1,∗ , Jin A. Yang 1 , Gyoo Yeol Jung 1,2 1
Department of Chemical Engineering, Pohang University of Science and Technology, San 31, Hyoja-dong, Nam-gu, Pohang, Gyeongbuk 790-784, Republic of Korea 2 School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, San 31, Hyoja-dong, Nam-gu, Pohang, Gyeongbuk 790-784, Republic of Korea A large number of natural and unnatural products from microorganisms such as biologically active compounds and commodity chemicals have attracted huge attention in the various industries including pharmaceutical and chemical industries. However, functional improvement of microorganisms meeting with commercial need is limited by the biological constraints developed during natural evolutionary process. “Metabolic Engineering” aims the purposeful redesign of the biological systems and requires the accurate information of the cellular metabolic networks and proper tools for the reconstruction of the biological systems such as regulatory elements and engineered proteins. The goal of “Synthetic Biology” is to synthesize whole biological system or its subsystem intentionally, and designing parts such as the regulatory elements and functional gene should be necessarily predictable. Numerous regulatory elements such as promoter libraries and tunable intergenic regions (TIGRs) can be applied for the modulation of gene expression. However, it is difficult to detect the proper variants required for balanced expression of multiple genes. In this study, we constructed a universal model for designing 5 -UTRs for precise gene expression in Escherichia coli integrating randomized 5 -UTRs, followed by superfolder GFP as a reporting system into the chromosome. Mathematical model was constructed by mapping between the secondary structures of 5 -UTRs and the expression level of superfolder GFP. Examples using the other genes will show the potentials of this model to predict the precise expression level and consequently, will provide a valuable tool for “Synthetic Biology”. doi:10.1016/j.jbiotec.2008.07.120 I1-P-101 Effect of temperature and pH on fractional precipitation for paclitaxel pre-purification Jin-Hyun Kim ∗ , Seul-Gi Kim, Sun Im Jeon Department of Chemical Engineering, Kongju National University, Kongju 314-701, Republic of Korea E-mail address: [email protected] (J.-H. Kim). Fractional precipitation is a simple, efficient method for prepurifying paclitaxel from plant cell cultures. The storage temperature of fractional precipitation was optimized in terms of the yield and purity of paclitaxel with a fixed methanol concentration in water (61.5%, v/v), paclitaxel content in the crude extract (0.5%, w/v), and storage time (3 days). The greatest yield (∼84%) was obtained with storage at a constant temperature (0 ◦ C) for 3 days. Conversely, the highest purity (∼79%) was obtained with stepwise reduction in temperature over 3 days. For a constant storage temperature, the maximum purity (∼67%) was obtained at 0 ◦ C. There was little difference in the yield of paclitaxel between −20 and 12 ◦ C. At a constant storage temperature (0 ◦ C), both the precipitate yield and purity increased rapidly up to 18 h, and then
Abstracts / Journal of Biotechnology 136S (2008) S22–S71
increased more slowly. The maximum yield and purity of paclitaxel were obtained at pH 8.0 with potassium phosphate buffer. This prepurification process serves to minimize solvent usage, and the size and complexity of the high-performance liquid chromatography (HPLC) operation required for paclitaxel purification.
S57
I1-P-104 Isolation of the biosynthetic gene cluster for tautomycetin, a linear polyketide T cell-specific immunomodulator from Streptomyces sp. CK4412 Si-Sun Choi ∗ , Eung-Soo Kim
References Jeon, K.Y., Kim, J.H., 2007. Optimization of micellar extraction for the pre-purification of paclitaxel from Taxus chinensis. Biotechnol. Bioprocess Eng. 12, 354–358. Kim, J.H., 2006. Paclitaxel: recovery and purification in commercialization step. Kor. J. Biotechnol. Bioeng. 21, 1–10. Kim, J.H., Kang, I.S., Choi, H.K., Hong, S.S., Lee, H.S., 2002. A novel prepurification for paclitaxel from plant cell cultures. Process Biochem. 37, 679–682. Pyo, S.H., Park, H.B., Song, B.K., Han, B.H., Kim, J.H., 2004. A large-scale purification of paclitaxel from cell cultures of Taxus chinensis. Process Biochem. 39, 1985–1991.
doi:10.1016/j.jbiotec.2008.07.121 I1-P-102 Spore inoculum optimization to maximize cyclosporin A production in Tolypocladium niveum Mi-Jin Lee ∗ , Eung-Soo Kim Department of Biological Engineering, Inha University, Incheon 402751, Republic of Korea E-mail address: [email protected] (E.-S. Kim). The cyclic undecapeptide, cyclosporin A (CyA), is one of the most commonly prescribed immunosuppressive drugs (Borel, 1983). It is generated non-ribosomally from a multi-functional cyclosporin synthetase enzyme complex by the filamentous fungus, Tolypocladium niveum. In order to maximize the production of CyA by wild-type T. niveum (ATCC 34921), each of three culture stages (sporulation culture, growth culture, and production culture) were sequentially optimized (Chun and Agathos, 1991). Among the three potential sporulation media, the SSMA medium generated the highest numbers of T. niveum spores. The SSM and SM media were then selected as the optimal growth and production culture media, respectively. The addition of valine and fructose to the SM production medium was also determined to be crucial for CyA biosynthesis. In this optimized three-stage culture system, 3% of the spore inoculum generated the highest level of CyA productivity in a 15-day T. niveum production culture, thereby implying that the determination of an appropriate size of T. niveum spore inoculum plays a critical role in the maximization of CyA production.
Department of Biological Engineering, Inha University, Incheon 402751, Republic of Korea E-mail address: [email protected] (E.-S. Kim). The bacterial genus Streptomyces has long been appreciated for its ability to produce various kinds of medically important secondary metabolites, such as antibiotics, anti-tumour agents, immunosuppressants and enzyme inhibitors. Tautomycetin (TMC), which is produced by Streptomyces sp. CK4412, is a novel activated T cellspecific immunosuppressive compound with an ester bond linkage between a terminal cyclic anhydride moiety and a linear polyketide chain bearing an unusual terminal alkene1. Using a Streptomyces polyketide methylmalonyl-CoA acyltransferase gene as a probe, three overlapping cosmids were isolated from the genomic library of TMC-producing Streptomyces sp. CK4412. Sequence information of an approximately 70 kb contiguous DNA region revealed two multi-modular type I polyketide synthases (PKSs), and 12 additional gene products presumably involved in TMC biosynthesis. The deduced roles for most of the TMC PKS catalytic domains were consistent with the expected functions necessary for TMC chain elongation and processing. In addition, disruption of regions included TMC biosynthetic genes, located upstream of the PKS gene locus, completely abolished TMC biosynthesis. Taken together, these data provide strong supporting evidence that the cloned gene cluster identified in this study is responsible for TMC biosynthesis in Streptomyces sp. CK4412, and set the stage for detailed genetic and biochemical studies of the biosynthesis of this important metabolite. The more detailed results of genetic characterization and manipulation of this TMC gene cluster will be further discussed. References Choi, S.S., Hur, Y.A., Sherman, D.H., Kim, E.S., 2007. Isolation of the biosynthetic gene cluster for tautomycetin, a linear polyketide T cell-specific immunomodulator from Streptomyces sp. CK4412. Microbiology 153, 1095–1102. Shim, J.H., Lee, H.K., Chang, E.J., Chae, W.J., Han, J.H., Han, D.J., Morio, T., Yang, J.J., Bothwell, A., Lee, S.K., 2002. Immunosuppressive effects of tautomycetin in vivo and in vitro via T cell-specific apoptosis induction. Proc. Natl. Acad. Sci. U.S.A. 99, 10617–10622.
doi:10.1016/j.jbiotec.2008.07.123 References Borel, J.F., 1983. Cyclosporin: Historical perspectives. Transplant. Proc. 15 (1), 2219. Chun, G.-T., Agathos, S.N., 1991. Comparative studies of physiological and environmental effects on the production of cyclosporine A in suspended and immobilized cells of Tolypocladium inflatum. Biotechnol. Bioeng. 37, 256–265.
doi:10.1016/j.jbiotec.2008.07.122
I1-P-105 Multiple knock-out system of Mannheimia succiniciproducens using mutant Lox sequences Ji Mahn Kim 1,∗ , Sang Yup Lee 1,2 1
Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical & Biomolecular Engineering and BioProcess Engineering Research Center, KAIST, 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, Republic of Korea 2 Department of BioSystems and Bioinformatics Research Center, KAIST, 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, Republic of Korea A multiple gene deletion method was developed to generate genetically modified Mannheimia succiniciproducens, which is a rumen bacterium producing succinic acid as a major metabolite. A temperature sensitive plasmid was generated using pMVSCS1, a native
Abstracts / Journal of Biotechnology 136S (2008) S22–S71
through 31st July and the plants harvested by hand at 4 steps of growth including 10 leaves plants (10 s), middle of growth (50 s), preflowering (pfs) and flowering (fs). The level of gene expression was monitored by semi quantitative RT-PCR technique. Plant growth and maturity both affected on PAL, CVOMT and EOMT gene expression and phenolic contents. References Dixon, P., Paiva, N.L., 1995. Stress-induced phenylpropanoid metabolism. Plant Cell. 7, 1085–1097. Lewinsohn, E., Ziv-Raz, I., Dudai, N., Tadmor, Y., Lastochkin, E., Larkov, O., Chaimovitsh, D., Ravid, U., Putievsky, E., Shoham, Y., 2000. Biosynthesis of estragole and methyl-eugenol in sweet basil (Ocimum basilicum L) developmental and chemotypic association of allylphenol O-methyltransferase activities. Plant Sci. 160, 27–35. Logemann, E., Parniske, M., Hahlbrock, K., 1995. Modes of expression and common structural features of the complete phenylalanine ammonia-lyase gene family in parsley. Plant Biol. 92, 5905–5909.
doi:10.1016/j.jbiotec.2008.07.118 I1-P-099 Identification and characterization of tmcC as a putative esterase of tautomycetin biosynthesis in Streptomyces sp. CK4412 Ja-Ryong Koo ∗ , Si-Sun Choi, Eung-Soo Kim Department of Biotechnology, Inha University, Incheon 402-751, Republic of Korea E-mail address: [email protected] (E.-S. Kim). Tautomycetin (TMC) is a novel activated T cell-specific immunosuppressive compound with an ester bond linkage between a terminal cyclic anhydride moiety and a linear polyketide chain bearing an unusual terminal alkene. The presence of a 1.5 kb gene of tmcC, whose deduced product was found to have some amino acid sequence homology with previously known bacterial esterase, was found just upstream of tmc PKS genes in an approximately 70-kb contiguous TMC biosynthetic gene cluster. In silico database comparisons revealed that tmcC belongs to the type B carboxylesterase. PCR-targeted disruption of the putative TMC-specific esterase gene, tmcC completely abolished TMC biosynthesis. Expectly, complementation by an integrative plasmid carrying tmcC significantly restored TMC biosynthesis. Moreover, the codon-optimzed tmcC was also over-expressed as a His-tagged fusion protein in Escherichia coli, follow by the in vitro biochemical esterase assays. References Choi, S.-S., Hur, Y.-A., Sherman, D.H., Kim, E.-S., 2007. Isolation of the biosynthetic gene cluster for tautomycetin, a linear polyketide T cell-specific immunomodulator from Streptomyces sp. CK4412. Microbiology 153, 1095–1102. Soror, S.H., Verma, V., Rao, R., Rasool, S., Koul, S., Qazi, G.N., Cullum, J., 2007. A cold-active esterase of Streptomyces coelicolor A3(2): from genome sequence to enzyme activity. J. Ind. Microbiol. Biotechnol. 34, 525–531. Tesch, C., Nikoleit, K., Gnau, V., Gotz, F., Bormann, C., 1996. Biochemical and molecular characterization of the extracellular esterase from Streptomyces diastatochromogenes. J. Bacteriol., 1858–1865.
doi:10.1016/j.jbiotec.2008.07.119
I1-P-100 Universal model for designing 5 -UTRs for precise gene expression in Escherichia coli Sang Woo Seo 1,∗ , Jin A. Yang 1 , Gyoo Yeol Jung 1,2 1
Department of Chemical Engineering, Pohang University of Science and Technology, San 31, Hyoja-dong, Nam-gu, Pohang, Gyeongbuk 790-784, Republic of Korea 2 School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, San 31, Hyoja-dong, Nam-gu, Pohang, Gyeongbuk 790-784, Republic of Korea A large number of natural and unnatural products from microorganisms such as biologically active compounds and commodity chemicals have attracted huge attention in the various industries including pharmaceutical and chemical industries. However, functional improvement of microorganisms meeting with commercial need is limited by the biological constraints developed during natural evolutionary process. “Metabolic Engineering” aims the purposeful redesign of the biological systems and requires the accurate information of the cellular metabolic networks and proper tools for the reconstruction of the biological systems such as regulatory elements and engineered proteins. The goal of “Synthetic Biology” is to synthesize whole biological system or its subsystem intentionally, and designing parts such as the regulatory elements and functional gene should be necessarily predictable. Numerous regulatory elements such as promoter libraries and tunable intergenic regions (TIGRs) can be applied for the modulation of gene expression. However, it is difficult to detect the proper variants required for balanced expression of multiple genes. In this study, we constructed a universal model for designing 5 -UTRs for precise gene expression in Escherichia coli integrating randomized 5 -UTRs, followed by superfolder GFP as a reporting system into the chromosome. Mathematical model was constructed by mapping between the secondary structures of 5 -UTRs and the expression level of superfolder GFP. Examples using the other genes will show the potentials of this model to predict the precise expression level and consequently, will provide a valuable tool for “Synthetic Biology”. doi:10.1016/j.jbiotec.2008.07.120 I1-P-101 Effect of temperature and pH on fractional precipitation for paclitaxel pre-purification Jin-Hyun Kim ∗ , Seul-Gi Kim, Sun Im Jeon Department of Chemical Engineering, Kongju National University, Kongju 314-701, Republic of Korea E-mail address: [email protected] (J.-H. Kim). Fractional precipitation is a simple, efficient method for prepurifying paclitaxel from plant cell cultures. The storage temperature of fractional precipitation was optimized in terms of the yield and purity of paclitaxel with a fixed methanol concentration in water (61.5%, v/v), paclitaxel content in the crude extract (0.5%, w/v), and storage time (3 days). The greatest yield (∼84%) was obtained with storage at a constant temperature (0 ◦ C) for 3 days. Conversely, the highest purity (∼79%) was obtained with stepwise reduction in temperature over 3 days. For a constant storage temperature, the maximum purity (∼67%) was obtained at 0 ◦ C. There was little difference in the yield of paclitaxel between −20 and 12 ◦ C. At a constant storage temperature (0 ◦ C), both the precipitate yield and purity increased rapidly up to 18 h, and then
Abstracts / Journal of Biotechnology 136S (2008) S22–S71
increased more slowly. The maximum yield and purity of paclitaxel were obtained at pH 8.0 with potassium phosphate buffer. This prepurification process serves to minimize solvent usage, and the size and complexity of the high-performance liquid chromatography (HPLC) operation required for paclitaxel purification.
S57
I1-P-104 Isolation of the biosynthetic gene cluster for tautomycetin, a linear polyketide T cell-specific immunomodulator from Streptomyces sp. CK4412 Si-Sun Choi ∗ , Eung-Soo Kim
References Jeon, K.Y., Kim, J.H., 2007. Optimization of micellar extraction for the pre-purification of paclitaxel from Taxus chinensis. Biotechnol. Bioprocess Eng. 12, 354–358. Kim, J.H., 2006. Paclitaxel: recovery and purification in commercialization step. Kor. J. Biotechnol. Bioeng. 21, 1–10. Kim, J.H., Kang, I.S., Choi, H.K., Hong, S.S., Lee, H.S., 2002. A novel prepurification for paclitaxel from plant cell cultures. Process Biochem. 37, 679–682. Pyo, S.H., Park, H.B., Song, B.K., Han, B.H., Kim, J.H., 2004. A large-scale purification of paclitaxel from cell cultures of Taxus chinensis. Process Biochem. 39, 1985–1991.
doi:10.1016/j.jbiotec.2008.07.121 I1-P-102 Spore inoculum optimization to maximize cyclosporin A production in Tolypocladium niveum Mi-Jin Lee ∗ , Eung-Soo Kim Department of Biological Engineering, Inha University, Incheon 402751, Republic of Korea E-mail address: [email protected] (E.-S. Kim). The cyclic undecapeptide, cyclosporin A (CyA), is one of the most commonly prescribed immunosuppressive drugs (Borel, 1983). It is generated non-ribosomally from a multi-functional cyclosporin synthetase enzyme complex by the filamentous fungus, Tolypocladium niveum. In order to maximize the production of CyA by wild-type T. niveum (ATCC 34921), each of three culture stages (sporulation culture, growth culture, and production culture) were sequentially optimized (Chun and Agathos, 1991). Among the three potential sporulation media, the SSMA medium generated the highest numbers of T. niveum spores. The SSM and SM media were then selected as the optimal growth and production culture media, respectively. The addition of valine and fructose to the SM production medium was also determined to be crucial for CyA biosynthesis. In this optimized three-stage culture system, 3% of the spore inoculum generated the highest level of CyA productivity in a 15-day T. niveum production culture, thereby implying that the determination of an appropriate size of T. niveum spore inoculum plays a critical role in the maximization of CyA production.
Department of Biological Engineering, Inha University, Incheon 402751, Republic of Korea E-mail address: [email protected] (E.-S. Kim). The bacterial genus Streptomyces has long been appreciated for its ability to produce various kinds of medically important secondary metabolites, such as antibiotics, anti-tumour agents, immunosuppressants and enzyme inhibitors. Tautomycetin (TMC), which is produced by Streptomyces sp. CK4412, is a novel activated T cellspecific immunosuppressive compound with an ester bond linkage between a terminal cyclic anhydride moiety and a linear polyketide chain bearing an unusual terminal alkene1. Using a Streptomyces polyketide methylmalonyl-CoA acyltransferase gene as a probe, three overlapping cosmids were isolated from the genomic library of TMC-producing Streptomyces sp. CK4412. Sequence information of an approximately 70 kb contiguous DNA region revealed two multi-modular type I polyketide synthases (PKSs), and 12 additional gene products presumably involved in TMC biosynthesis. The deduced roles for most of the TMC PKS catalytic domains were consistent with the expected functions necessary for TMC chain elongation and processing. In addition, disruption of regions included TMC biosynthetic genes, located upstream of the PKS gene locus, completely abolished TMC biosynthesis. Taken together, these data provide strong supporting evidence that the cloned gene cluster identified in this study is responsible for TMC biosynthesis in Streptomyces sp. CK4412, and set the stage for detailed genetic and biochemical studies of the biosynthesis of this important metabolite. The more detailed results of genetic characterization and manipulation of this TMC gene cluster will be further discussed. References Choi, S.S., Hur, Y.A., Sherman, D.H., Kim, E.S., 2007. Isolation of the biosynthetic gene cluster for tautomycetin, a linear polyketide T cell-specific immunomodulator from Streptomyces sp. CK4412. Microbiology 153, 1095–1102. Shim, J.H., Lee, H.K., Chang, E.J., Chae, W.J., Han, J.H., Han, D.J., Morio, T., Yang, J.J., Bothwell, A., Lee, S.K., 2002. Immunosuppressive effects of tautomycetin in vivo and in vitro via T cell-specific apoptosis induction. Proc. Natl. Acad. Sci. U.S.A. 99, 10617–10622.
doi:10.1016/j.jbiotec.2008.07.123 References Borel, J.F., 1983. Cyclosporin: Historical perspectives. Transplant. Proc. 15 (1), 2219. Chun, G.-T., Agathos, S.N., 1991. Comparative studies of physiological and environmental effects on the production of cyclosporine A in suspended and immobilized cells of Tolypocladium inflatum. Biotechnol. Bioeng. 37, 256–265.
doi:10.1016/j.jbiotec.2008.07.122
I1-P-105 Multiple knock-out system of Mannheimia succiniciproducens using mutant Lox sequences Ji Mahn Kim 1,∗ , Sang Yup Lee 1,2 1
Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical & Biomolecular Engineering and BioProcess Engineering Research Center, KAIST, 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, Republic of Korea 2 Department of BioSystems and Bioinformatics Research Center, KAIST, 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, Republic of Korea A multiple gene deletion method was developed to generate genetically modified Mannheimia succiniciproducens, which is a rumen bacterium producing succinic acid as a major metabolite. A temperature sensitive plasmid was generated using pMVSCS1, a native