재조합 대장균에서 포도당과 목당 혼합당으로부터 3-히드록시프로피온산 생산을 위한 글리세롤 대사과정 조절에 관한 연구

학위논문 (석사)-- 서울대학교 대학원 : 농생명공학부 농생명공학전공, 2016. 2. 서진호.Replacement of conventional petro-based chemicals with biomass-based substances is a central paradigm in the chemical industry. Hroxypropionic acid (3-HP) was selected as a target product, which is a precursor for various chemicals including acrylic acid, methyl acrylate and acrylamide. In previous research, accumulation of glycerol was observed during 3-HP producing culture from a mixture of glucose and xylose, which is supposed to be a result of imbalance between glycerol synthesis and consumption. It is known that accumulated glycerol inhibits glycerol dehydratase which is a key enzyme in biosynthesis of 3-HP. Therefore, it is necessary to alleviate glycerol accumulation for improving 3-HP production. For this purpose, the gpsA gene encoding glycerol-3-phosphate dehydrogenase derived from Escherichia coli K-12 which has lower activity than GPD1 from Saccharomyces cerevisiae was introduced to E. coli BL21 star (DE3). During 35 h of cultivation, the ∆gyp/pELDRR/pCPaGAR strain showed glycerol accumulation of 1.24 g/L and 3-HP concentration of 1.51 g/L, which are lower by 46% and higher by 48% than those of the ∆gyp/pELDRR/pCPaGGR strain, respectively. To achieve high concentration of 3-HP, fed-batch fermentation was carried out with feeding a mixture of glucose and xylose. After 71 h of cultivation, accumulated glycerol was transformed to 3-HP completely by using the gpsA gene with a reduced level of glycerol synthesis. Finally, 37.6 g/L of 3-HP which is 33% higher than that of the control strain was produced. Productivity of 0.63 g/L∙h and yield of 0.17 g 3-HP/g sugar were also obtained. In this study, glycerol accumulation which inhibits glycerol dehydratase was reduced by replacing the GPD1 gene by the gpsA gene. It is supposed to be a result of reduction of a carbon flux from dihydroxyacetone phosphate to glycerol-3-phosphate by GpsA which has lower activity than GPD1. As a result, enhanced production of 3-HP was possible by minimizing inactivation of glycerol dehydratase by glycerol. The result suggests that the gpsA gene overexpression would be applied to production of other chemicals which require using the glycerol dehydratase gene. In the previous research, the ∆gyp/pELDRR/pCPaGGR strain showed that xylose uptake rate decreased with culture time in glucose and xylose limited fed-batch fermentation, which resulted in a reduction of the titer and productivity of 3-HP. Therefore, xylose uptake rate needs to be increased for improving 3-HP production. A xylose transporter was introduced in order to enhance xylose uptake rate in a mixture of glucose and xylose. The 376th amino acid residue, asparagine, was changed to phenyalanine in the E. coli galactose transporter GalP for simultaneous consumption of glucose and xylose. During 16 h of cultivation, the ∆gyp/pACYCDuet-1_galPm showed an enhancement in xylose uptake rate by 34% and a decrease in glucose uptake rate by 22% compared to the control strain. Insufficient xylose uptake rate of 3-HP producing strains was improved by introducing the GalPm protein which has a high affinity with xylose. Furthermore, the xylFGH genes were deleted to increase ATP availability and to secure more space for the GalPm in the cell membrane. During 16 h of cultivation, the ∆gypx/pACYCDuet-1_galPm strain showed improved uptake rate of glucose by 20% and of xylose by 16%. To investigate 3-HP production, flask culture of the ∆gypx/pELDRR/pCPaGGRgalPm in R/5 medium containing 5 g/L glucose and 3 g/L xylose was carried out. During 30 h of cultivation, the ∆gypx/pELDRR/pCPaGGRgalPm strain showed xylose uptake rate of 0.45 g/L∙h and glucose uptake rate of 0.25 g/L∙h, which is higher by 61% and lower by 36% than those of the control strain, respectively. Also, it showed 3-HP concentration of 1.10 g/L, and that is higher by 8% than that of control strain. Further, this effect was maximized by deletion of the xylFGH genes to increase ATP availability and to secure membrane space for GalPm in the cell membrane. Improving sugar uptake rate and increasing ATP availability seem to contribute to enhanced cell growth and 3-HP production. The result suggests that the GalPm protein with point mutation and deletion of the xylFGH genes would be applied to production of other chemicals from cellulosic biomass.Chapter 1. INTRODUCTION 1 1.1 3-Hydroxypropionic acid 1 1.2 Hemicellulose and xylose 5 1.3 Metabolic pathway from glucose and xylose to 3-HP in E. coli 7 1.4 Transporter 9 1.5 Research objectives 10 Chapter 2. MATERIALS AND METHODS 11 2.1 Strains and plasmids 11 2.2 Gene deletion progress 14 2.2.1 Preparation of kanamycin resistance cassette 16 2.2.2 Expression of λ red recombinase in host strain 16 2.2.3 Kanamycin resistance cassette insert to expression strain 16 2.2.4 Recombination and adaptation 17 2.2.5 Elimination of kanamycin resistance cassette 17 2.3 E. coli DNA manipulation and transformation 18 2.3.1 Preparation of DNA 18 2.3.2 Polymerase Chain Reaction (PCR) 18 2.3.3 Point mutation by overlap extension PCR 19 2.3.4 Digestion and ligation of DNA 19 2.3.5 Transformation 20 2.4 Culture conditions 20 2.4.1 Growth media 20 2.4.2 Flask culture 21 2.4.3 Fed-batch fermentation in a bioreactor 21 2.5 Analytical methods 22 2.5.1 Dry cell weight 22 2.5.2 High performance liquid chromatography analysis 22 2.5.3 SDS-PAGE 23 Chapter 3. RESULTS AND DISCUSSION 25 3.1 Production of 3-HP from glucose and xylose 25 3.1.1 Problem of glycerol accumulation during culture 25 3.2 Manipulation of glycerol synthesis pathway 27 3.2.1 Construction of the strain without GPD1 gene 27 3.2.2 Flask culture of the strain without GPD1 gene 27 3.3 Expression of the glycerol-3-phosphate dehydrogenase derived from E. coli K-12 31 3.3.1 Sequence of the gpsA gene 31 3.3.2 Construction of the strain expressing gpsA gene 32 3.3.3 Flask culture of the strain expressing gpsA gene 33 3.3.4 Fed-batch fermentation of the strain expressing gpsA gene using constant feeding strategy 38 3.4 Expression of the galPm gene 40 3.4.1 Sequences of the galPm gene 41 3.4.2 Construction of the strain expressing galPm gene 43 3.4.3 Flask culture of the strain expressing galPm gene 45 3.5 Deletion of the xylFGH genes 48 3.5.1 Sequences of the xylFGH genes 48 3.5.2 Confirmation of deletion of the xylFGH genes 53 3.5.3 Flask culture of the xylFGH genes deleted strain 55 3.5.4 Flask culture for 3-HP production with enhanced xylose uptake rate 59 Chapter 4. CONCLUSIONS 63 REFERENCES 64 Abstract (In Korean) / 국문초록 68Maste

고속로 노심해석을 위한 몬테칼로-결정론적 하이브리드 방법론 연구

학위논문(석사) - 한국과학기술원 : 원자력및양자공학과, 2014.2, [ v, 43 p. ]This thesis investigates a hybrid Monte Carlo-Deterministic method for the analysis of fast reactors. In this method, the effective multi-group cross sections and high-order Legendre scattering cross sections are generated by using the collision estimator in the MCNP5 code in combination with an added data generation module. In both the new hybrid and conventional methods, the cross section data is based on a simple RZ homogenous core model. As a computational model, a 300MWe SFR (sodium-cooled fast reactor) TRU burner core has been introduced. The generated multi-group cross sections are used by a 3-D diffusion core analyzer to calculate the resulting k-eff and assembly power distribution. A whole core calculation of the heterogeneous core model is performed using MCNP5 in order to determine the reference solution for the evaluation of the analysis methods. For an in-depth verification of the hybrid method in fast reactors, the new method was also applied to both unrodded and heavily rodded cores. In the case of rodded core, a new core modeling named RRZ was proposed for a better modeling of the self-shielding effect of the control assembly in the fast reactor. Additionally, the nodal equivalence theory was successfully applied to fast reactor analysis for the first time in this work. To apply the nodal equivalence theory, a simple 1-D spectral geometry was developed to determine the flux discontinuity factor of a control assembly region on the interface between fuel and control assembly regions. The generated group-wise DF values were used to correct the cross sections of the control assembly region. Moreover, the sensitivity of the DF to the 1-D spectral geometry model was also evaluated in this work. It is concluded that the hybrid method works well for the analysis of the SFR core. Particularly, the special application of the nodal equivalence theory greatly improves the accuracy of the new hybrid method for fast reactor analysis.한국과학기술원 : 원자력및양자공학과

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Ocean Color Algorithm Development Environment for High-Speed Data Processing of GOCI-II

Geostationary Ocean Color Imager&#8211 II (GOCI-II), the follow-up satellite sensor platform of GOCI, will be launched in Mar. 2019. For estimating ocean color information from this new sensor data, the dedicated ocean color algorithms will be newly developed. However, the existing ocean color algorithm development methodology without consideration for the high-speed data processing will not be employed because the amount of GOCI-II data will be significantly large and theirdata will have to be processed and distributed in real time. In this study, therefore, we designed the ocean color algorithm development environment (OCADE) to support high-performance computing and storage using the parallelism. Also, OCADE enables the source code projects for GOCI-II ocean color products to be automatically built and deployed. We expect that GOCI-II ocean color algorithm developers will easily create and manage their parallelized source code projects at any time and any place through the OCADE cloud service. Additionally, they will be able to validate the output and evaluate the processing performance under OCADE in real-time. We believe that OCADE will contribute to the field of massive satellite data processing.ll be newly developed. However, the existing ocean color algorithm development methodology without consideration for the high-speed data processing will not be employed because the amount of GOCI-II data will be significantly large and theirdata will have to be processed and distributed in real time. In this study, therefore, we designed the ocean color algorithm development environment (OCADE) to support high-performance computing and storage using the parallelism. Also, OCADE enables the source code projects for GOCI-II ocean color products to be automatically built and deployed. We expect that GOCI-II ocean color algorithm developers will easily create and manage their parallelized source code projects at any time and any place through the OCADE cloud service. Additionally, they will be able to validate the output and evaluate the processing performance under OCADE in real-time. We believe that OCADE will contribute to the field of massive satellite data processing.1