G_ Enzyme, Protein Engineering, and Metabolic Engineering

Please download to get full document.

View again

of 8
All materials on our website are shared by users. If you have any questions about copyright issues, please report us to resolve them. We are always happy to assist you.
Information Report
Category:

Retail

Published:

Views: 4 | Pages: 8

Extension: PDF | Download: 0

Share
Description
44 th Annual Meeting & International Symposium F-14 Comparative Growth Kinetics of CHO Cells by Virus Contamination Young CHOI 1, Woon Young KO 2, Jae Il LEE 2, Jae-Eun PARK 3 and In Seop KIM* 1 1 Department
Transcript
44 th Annual Meeting & International Symposium F-14 Comparative Growth Kinetics of CHO Cells by Virus Contamination Young CHOI 1, Woon Young KO 2, Jae Il LEE 2, Jae-Eun PARK 3 and In Seop KIM* 1 1 Department of Biological Sciences and Biotechnology, Hannam University, Daejon 34054, Republic of Korea. 2 Center for Biopharmaceuticals Safety Validation, BioPS Co., Ltd. Daedeuk Valley Campus, Hannam University, Daejon 34054, Republic of Korea. 3 Y-Biologics, N416, 11-3, Techno-1-Ro, Youseung, Daejon 34015, Republic of Korea. *Corresponding author: Chinese hamster ovary (CHO) cells have been widely used to manufacture recombinant proteins for human therapeutic use. During the manufacturing process of biopharmaceuticals using CHO cells, virus contaminations of CHO-cell derived bulk harvest are rare, but have occurred. The contaminating viruses were identified as Mice minute virus, Reovirus, Cache Valley virus, Epizootic hemorrhagic disease virus, and Vesivirus Since all these viruses induced cytopathic effect(cpe) in CHO cells, they can be easily detected. However, some viruses such as parainfluenza virus type 3, pseudorabies virus, or porcine parvovirus cannot induce CPE, but replicate in CHO cells. Therefore it is very important to detect such virus contaminations during CHO cell cultures for the assurance of virus safety of biopharmaceuticals. For the rapid detection of such viruses at the QC level, it is necessary to find a key parameter that can determine the sign of virus contamination during culture. In this study we conducted experiments for comparative growth kinetics of CHO cells by virus contamination. The comparative growth kinetics of CHO cells by virus contamination will be presented. F-15 Electric Treatment Toward Increasing Growth Rate of Microalga, Haematococcus pluvialis Jee Young KIM 1, Changsu LEE 2 and Yoon-E CHOI* 1 1 Division of Environmental Science and Ecological Engineering, Korea University, Seoul, Korea. 2 Microbiology and Functionality Research Group, World Institute of Kimchi, 86 Kimchi-ro, Nam-gu, Gwangju 61755, Korea. *Corresponding author: Haematococcus pluvialis has been reported as nature source of high valuable material, astaxanthin. However, it requires a long time to accumulate an astaxanthin since H. pluvialis grows slowly in the green growth stage. Therefore, to enhance cell growth rate of H. pluvialis in the green growth stage, the possible application of electric treatment on H. pluvialis was investigated in this study. During the cultivation of H. pluvialis at the green growth stage, H. pluvialis was treated electrically with various voltages. Subsequently, cell density, cell size, dry weight, and the amount of total chlorophyll and astaxanthin were evaluated during cultivation. Interestingly, with the electric treatment on green staged H. pluvialis, cell growth was enhanced. However, the electric treatment wad not effective on the brown staged H. pluvialis. These results indicate that the effectiveness of electrical treatment is evident to green growth stage of H. pluvialis decreasing the required timeframe for microalgal cultivation. Since the green growth is universal across different microalga species, we expect that our novel approach can provide a feasible way to enhance not only H. pluvialis growth, but also other microalgal productivity. Keywords: Treatment, Haematococcus pluvialis, Astaxanthin G_ Enzyme, Protein Engineering, and Metabolic Engineering G-1 Biosynthesis of Poly(lactate-co-glycolate) Based Copolymers as Novel Biomaterials in Escherichia coli So Young CHOI 1, Si Jae PARK 2, Won Jun KIM 1, Jung Eun YANG 1, Hyuk LEE 3, Jihoon SHIN 4 and Sang Yup LEE* 1 1 Dept. of Chemical and Biomolecular Engineering, KAIST. 2 Dept. of Chemical Engineering and Materials Science, Ewha Womans University. 3 Division of Drug Discovery Research, Korea Research Institute of Chemical Technology. 4 Center for Bio-based Chemistry, Green Chemistry & Engineering Division, Korea Research Institute of Chemical Technology. *Corresponding author: We previously reported biosynthesis of PLGA in engineered Escherichia coli harboring an evolved polyhydroxyalkanoate (PHA) synthase and propionyl-coa transferase. Here, using the PLGA producing E. coli, 3-hydroxybutyrate or 4-hydroxybutyrate were copolymerized to enhance polymer properties in terms of elasticity, suitable degradation rate and strength as novel biomaterials. First, for in vivo synthesis of 3HB-CoA, R. eutropha phaa and phab genes encoding β-ketothiolase and acetoacetyl-coa reductase were introduced. Second, in order to produce 4-hydroxybutyrate containing polymers, the Clostridium kluyveri sucd and 4hbD genes encoding CoA-dependent succinate semialdehyde dehydrogenase and 4HB dehydrogenase, respectively, were introduced. Then, the metabolic flux for 4HB was concentrated by inactivating the competing pathways. The produced terpolymers showed different mechanical properties especially, significant increase of elasticity, to lead various applications over PLGA. [This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries from the Ministry of Science, ICT and Future Planning (MSIP) through the National Research Foundation (NRF) of Korea (NRF-2012M1A2A and NRF-2012M1A2A )]. Keywords: Biopolymer, poly (lactate-co-glycolate), Systems metabolic engineering G-2 Metabolic Engineering of Corynebacterium glutamicum to Improve Xylose Utilization and Succinate Production Jinkyung YOON, Jaehyun PARK and Han Min WOO* Department of Food Science and Biotechnology, Sungkyunkwan University (SKKU), 2066 Seobu-ro, Jangan-gu, Suwon 16419, Republic of Korea. *Corresponding author: The nonpathogenic microorganism Corynebacterium glutamicum was metabolically engineered to broaden its substrate utilization range to include the pentose sugar xylose, which is the second most abundant sugar-derived agricultural residues. We introduced xylose isomerase (XI) pathway to xylose-negative C. glutamicum and analyzed that two functionally-redundant transcriptional regulators (GntR1 and GntR2) repress the pentose phosphate (PPP) pathway genes in presence of xylose as sole carbon. Also, we improved xylose utilization of C. glutamicum harboring XI pathway by overexpressing the PPP pathway gene (gnd or tal) and the heterologous phosphoketolase (PHK) pathway gene (xpka and fpka). Subsequently, the engineered strain produced succinate of which production rate by 2.5-fold compared to wild type with XI pathway alone. Thus, this study could be useful for amino acid and other value-added chemical production from xylose as the sole carbon source. This work was supported by a Golden Seed Project grant funded by the Ministry of Agriculture, Ministry of Oceans and Fisheries. Keywords: Corynebacterium glutamicum, Xylose, Metabolic engineering The Korean Society for Microbiology and Biotechnology 57 G-3 Crystal Structure of Ralstonia eutropha Polyhydroxyalkanoate Synthase C-terminal Domain and Reaction Mechanisms So Young CHOI 2, Jieun KIM 1, Yeo-Jin KIM 1, Sang Yup LEE 2 and Kyung-Jin KIM* 1 1 School of Life Sciences, KNU Creative BioResearch Group, Kyungpook National University. 2 Dept. of Chemical and Biomolecular Engineering, KAIST. *Corresponding author: Polyhydroxyalkanoates (PHAs) are natural polyesters synthesized by microorganisms and have attracted much attention as substitutes for petroleum-based plastics. Here, we first report the crystal structure of PHA synthase from Ralstonia eutropha (RePhaC1) and structure-based mechanisms for PHA biosynthesis (Other group also reported xxx). RePhaC1 contains the N-terminal and C-terminal (RePhaC1CD) domains, and exists as a dimer. RePhaC1CD catalyzes polymerization via non-processive ping-pong mechanism using a Cys-His-Asp catalytic triad. Molecular docking simulation of 3-hydroxybutyrylCoA reveals residues involved in the formation of substrate binding pocket and binding tunnel. Comparative analysis with other PHA synthase elucidates how different classes enzymes show different substrate specificities. Furthermore, we attempted structure-based protein engineering and developed a mutant with enhanced activity. [This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries from the Ministry of Science, ICT and Future Planning (MSIP) through the National Research Foundation (NRF) of Korea (NRF- 2012M1A2A and NRF-2012M1A2A ).] Keywords: Polyhydroxyalkanoate synthase, Crystal structure, Biopolymer G-5 In Vitro Characterization of Glycoengineered Interferon beta 1a Ju Young LEE 1, Hyun Mi LEE 2, Jin Chul YOON 2, Eun Young SHIM 2, Chang Ho JANG 2, Yeung Chul KIM 2, Gi Sun BAEK 2, Young Gyu CHO 2 and In Seop KIM* 1 1 1Department of Biological Sciences & Biotechnology, Hannam University, Daejion 34054, Korea. 2 Y-Bioloics, Daejion 34015, Korea. *Corresponding author: Type I IFN-β(Interferon beta) is a well-known cytokine having anti-viral and anti-proliferative activity, and divers immune-modulatory effects. Recombinant IFN-β has been proven to be effective in diminishing relapsing-remitting multiple sclerosis, and used more than 20 years as a first line therapy. However, low producivity and short half-life in vivo need to be improved. To improve these issues, PEGylation, albuminfusion, Fc protein fusion, and introduction of new glycosylation have been used. Especially, N-Glycosylation has been known to increase the half-life and decrease the immunogenicity without affecting on the activity of protein clinically. Also it showed the increaed productivity. In this study, several glycosylation site were introduced in IFN-β and tested their property, producivity and activity with various experimental methods. The characteristics were analyzed by SDS-PAGE, Western blotting, and PNGase treatment. The activity was measured by cell proliferation, MHC-1 expression, RT-qPCR, and reporter assay for promoter. The results showed introduction of new glycosylation are affecting the properties, productivity, and activity. Some of these glycosylation will be explored further. Keywords: N-GlycoEngineering, Interferon beta, In vitro characterization G-4 Biosynthesis of Non-natural Medical Polymer, Poly (lactate-co-glycolate) by Systems Metabolically Engineered Bacteria So Young CHOI 1, Si Jae PARK 2, Won Jun KIM 1, Jung Eun YANG 1, Hyuk LEE 3, Jihoon SHIN 4 and Sang Yup LEE* 1 1 Dept. of Chemical and Biomolecular Engineering, KAIST. 2 Dept. of Chemical Engineering and Materials Science, Ewha Womans University. 3 Division of Drug Discovery Research, Korea Research Institute of Chemical Technology. 4 Center for Bio-based Chemistry, Green Chemistry & Engineering Division, Korea Research Institute of Chemical Technology. *Corresponding author: The biorefinery technologies which transform biomass into fuel, power, and chemicals have received a great deal of attention as a sustainable alternative to fossil fuels. Here, we developed the microbial production system of non-natural polymer, poly(lactate-co-glycolate) (PLGA) from renewable biomass by adapting the bacterial polyester biosynthesis system. PLGA is biodegradable, biocompatible, and has been widely used in biomedical applications such as drug delivery and tissue engineering. To produce PLGA, we engineered Escherichia coli to produce lactate and glycolate by constructing and optimizing the Dahms pathway of Caulobacter in E. coli based on the genome-wide simulation. Then, propionyl-coa transferase and polyhydroxyalkanoate synthase were co-expressed to convert lactate and glycolate to lactyl-coa and glycolyl-coa, respectively and PLGA. The unwanted 2-hydroxybutyrate incorporation was removed by inactivating isoleucine pathway, finally PLGA was successfully produced. [This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries from the Ministry of Science, ICT and Future Planning (MSIP) through the National Research Foundation (NRF) of Korea (NRF-2012M1A2A and NRF-2012M1A2A )]. Keywords: poly(lactate-co-glycolate), Systems metabolic engineering, Polyhydroxyalkanoate G-6 Regulation of Glycogen Metabolism and Pathogenicity of Vibrio vulnificus MO6-24/O Modulated by the Concentration of MgCl 2 So-Ra PARK 1, Ji-Yeong KIM 2, Eui-Yeong HWANG 2 and Jung-Wan KIM* 1,2 1 Dept. of Life Science, Graduate School of Incheon National University, Incheon, , Korea. 2 Division of Bioengineering, Incheon National University, Incheon, , Korea. *Corresponding author: Glycogen is the major energy reservoir in microorganisms for long-term survival under various growth conditions. Especially, for pathogens with alternative habitat such as V. vulnificus, modulation of glycogen metabolism according to various environmental factors would be very critical in their successful propagation during their life cycle between marine environment and their hosts. Previous study has reported that malt played as a positive regulator in glycogen metabolism of V. vulnificus in complex medium when maltodextrin was the excessive carbon source. In this study, both growth and glycogen accumulation of the malt mutant were similar to those of wild type when 1% glucose was added to M9 medium. Moreover, V. vulnificus accumulated more than two fold glycogen in the cell as the concentration of Mg 2+ decreased to 0.25 mm in M9 medium supplemented with 1% maltodextrin. TD 50 of V. vulnificus was the shortest in M9 medium with 0.25 mm MgCl 2 and maltodextrin. However, the effects of MalT and the concentration of Mg 2+ was relieved and reversed, respectively, in M9 medium supplemented with 1% glucose. This suggested that there might be multiple pathways for glycogen metabolism regulated by various factors other than MalT. Keywords: Vibrio vulnificus, Glycogen metabolism, MgCl 2 58 44 th Annual Meeting & International Symposium G-7 Enhancing Functional Expression of CadA Through Random Substitution of Genetic Codes in the 5 coding Region Binna LEE, YunJon HAN, Jae Jun SONG and Jong Hyun CHOI* Industrial Microbial Biotechnology research Center, Integrated Biorefinery Research Institute, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 181 Ipsin-gil, Jeongeup-si, Jeonbuk , Republic of Korea. *Corresponding author: To date, although the microbial fermentation process has been considered as the sustainable technology to produce valuable chemicals, it is economically unfavorable than the petrochemical process in the production costs and yields. Accordingly, the increase of the productivity achieved by the biological process is to be the first priority problem. Itaconic acid (IA) is an unsaturated dicarboxylic acid which utilized as biochemical building block for production of resins, plastics, paints and synthetic fiber. Itaconic acid (IA) is considered as future bio-based platform chemical. However, IA hasn t been improved during the past 40 years owing to physiological and genetical disadvantages of Aspergillus terreus. In our study, we readily produced about 7.23 g/l itaconic acid from glycerol in E. coli by introducing a single cada mutant gene encoded by the first 10 synonymous codons without any metabolic engineering approach. A single mutant cada gene was improved to mainly be expressed as a soluble form. Although this titer is still inferior to those obtained using A. terreus, the application of improved scvcada_no8 together with host improvements through metabolic engineering approaches might enable generation of an E. coli strain as an industrial IA producer. Keywords: CadA, Itaconic acid, IA G-9 Screening, Cloning, Purification, and Characterization of Novel Lipases from Marine Microorganisms Isolated from Antarctic Ocean Se Hyeon PARK, Seuk Jae WON, Chae Gyeong PARK and Hyung Kwoun KIM* Dept. of Biotechnology, The Catholic University of Korea, Gyeonggi Korea. *Corresponding author: Antarctic marine environment provides good source of novel lipases possessing unusual properties, i.e. resistance to harsh physical and chemical conditions. Especially, marine lipase enzymes can be used in many industrial applications due to their useful features, i.e. high stability in organic solvents and unique substrate specificity. In this work, we purified chromosomal DNAs from many marine lipolytic bacteria isolated from Antarctic ocean and constructed shotgun library in Escherichia coli using puc19 vector. We found some lipolytic E. coli colonies transformed using various genomic DNAs from Marinobacter lipolyticus, Marinobacter maritimus, Psychrobacter nivimaris and Pseudoalteromonas atlantica. DNA sequence analysis revealed surprisingly that all lipolytic clones contained the same lipase gene (lipbl). Subsequently, LipBL was over expressed in E. coli BL21 (DE3) cells and purified by an anion exchange chromatography and gel filtration chromatography. For industrial application, we characterized some useful enzymatic properties including optimal temperature and ph and substrate specificity. Keywords: Lipase, Marinobacter lipolyticus, Antarctic bacteria G-8 Design of Bio-Inspired Silica-Encapsulated Protein A for Improved Immunoprecipitation Assays Ki Sung PARK, Mi Ran KI and Seung Pil PACK* Department of Biotechnology and Bioinformatics, Korea University, 2511 Sejong-Ro, Sejong 30019, Republic of Korea. *Corresponding author: Since Staphylococcus aureus Protein A (SpA) has a high affinity to the Fc region of immunoglobulins, SpA-immobilized matrices are widely used for antibody (Ab) purification or immunoprecipitation (IP) assays. Here, we developed novel stragtgy that a bio-inspired silica-encapsulation method to improve the immunoprecipitation assay using an silicaencapsulated SpA. Two kinds of novel silica-forming peptides (SFPs), namely R5 and EctP1, were separately introduced at the C-terminus of SpA to generate two recombinant fusion proteins (SpA-SFPs) with auto-silicifying abilities. When SpA-SFPs were employed as Ab-binding partner on a 96-well microtiter plate, they showed an effective Ab-binding ability and performance than intact SpA. A high binding ability was observed even when an auto-encapsulated SpA silica matrix was prepared. When we compared the performance of IP using a commercial SpA-agarose, showed a higher performance than SpA-Agarose particles and no loss of particles. Moreover, in IP assays, showed an approximately 300% higher precipitation of target protein than the SpA-Agarose when a small amount of cell lysate was used. These findings demonstrated that SpA-SFP could be useful for the development of an efficient immunoassay system. Keywords: Silica forming peptide, Biosilica, Protein A G-10 Production of 20(S)-Protopanaxatriol from Protopanaxatriol-type Ginsenosides in Ginseng Leaf Extract by Beta-Glycosidase from Dictyoglomus turgidum Combined with Beta-Glycosidase Pyrococcus furiosus Eun-Joo YANG, Kyung-Chul SHIN and Deok-Kun OH* Dept. of Bioscience and Biotechnology, Konkuk University, Seoul 05029, Korea. *Corresponding author: Ginsenosides have excellent pharmacological efficacies. Among them, 20(S)-protopanaxatriol has anti-tumor and memory enhancement activities. Beta-glycosidase from Pyrococcus furiosus hydrolyzes the outer glycoside at C-6 but not the inner glucose at C-6 and the glucose at C-20 linked to APPT. Dictyoglomus turgidum hydrolyzes the glucose residues in PPT-type ginsenosides but not other glycosides. Thus, the combined use of DT-bgl and PF-bgl is expected to increase the conversion of PPT-type ginsenosides of APPT. For the increased production of ginsnosides Re and PPT-type ginsenosides in Panax ginseng leaf extract to APPT, the ratio of DT-bgl and PF-bgl, concentrations of enzyme and substrate, and reaction time were optimized. DT-bgl combined with PF-bgl converted 1.0 mg/ml PPT-type ginsenoside in P. ginseng leaf extract to 0.58 mg/ml APPT without other ginsenosides, with a molar con
We Need Your Support
Thank you for visiting our website and your interest in our free products and services. We are nonprofit website to share and download documents. To the running of this website, we need your help to support us.

Thanks to everyone for your continued support.

No, Thanks
SAVE OUR EARTH

We need your sign to support Project to invent "SMART AND CONTROLLABLE REFLECTIVE BALLOONS" to cover the Sun and Save Our Earth.

More details...

Sign Now!

We are very appreciated for your Prompt Action!

x