7 bioprocess and metabolic engineering - 한국미생물 ... bioprocess and metabolic... ·...

1

Detection of a Ligand Using a FRET-based Molecular Sensor at Critical Temperature

Jongsik GAM, Jae-Hee MOON, Jae-Seok HA, Eugene RHA and Seung-Goo LEE*Korea Research Institute of Bioscience &Biotechnology, KRIBB.

*Corresponding author: [email protected]

Rapid molecular biosensors are important issues on both of industrial usage to produce of cellulosic ethanol and academic tool to monitor small ligands such as sugars, amino acids, nucleotides and calcium in vivo. A fluorescence resonance energy transfer (FRET)-based molecular biosensor has been developed for a reliable and rapid method to quantify multiple sugars. Ligand binding induces large conformational rearrangement of the biosensors, bringing the two fluorescent proteins into proximity so to change FRET ratio. Detection sensitivity of FRET ratio signal has been a challenge on development of a new FRET-based biosensor when using mutants of a sugar-binding protein (SBP) to detect other ligands including environmental chemicals such as trinitrotoluene. In this study, we developed a novel detection mode on the basis of our discovery on which change in FRET signal ratio was increased by differences of temperature-dependent conformational states in presence or absence of a ligand at a critical temperature. This study shows more sensitive ligand detection methods of FRET-based biosensors without optimization of linkers between SBP and fluorescent proteins, and provides background on development of a microbial whole cell senor.

*Keywords: FRET-based molecular sensor,sugar-binding protein,critical temperature

2

Schizosaccharomyces pombe genome-scale metabolic network

Chanwoo SONG and Sangyup LEE*Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Program), KAIST, Daejeon 305-701, Republic of Korea.


Yeasts have been studied extensively as a model system for higher eukaryotic organisms, including Homo sapiens. The fission yeast Schizosaccharomyces pombe, is one of two yeast systems that have been central in the study of higher eukaryotes, due to its similarity in cell division to mammalian cell, fission as opposed to budding. Additionally, S. pombe possesses genes that are similar to genes that are found in humans that are responsible for a number of genetic diseases. With the reconstruction of the genome-scale metabolic model of S. pombe, insights into the metabolic physiology of this yeast, can be examined and applied towards the improvement of human life. [This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries (NRF-2012-C1AAA001-2012M1A2A2026556) of the Ministry of Science, ICT, and Future Planning (MSIP) of Korea and the Intelligent Synthetic Biology Center (2011-0031963) through the Global Frontier Research Program of MSIP.]

*Keywords: Schizosaccharomyces pombe,genome-scale metabolic model

3

Redox Rebalancing to Optimize a Heterologous n-Butanol Pathway in Escherichia Coli

JAEHYUNG LIM1 and GyooYeol JUNG*1,2

1School of Interdisciplinary Bioscience and Bioengineering, POSTECH, Pohang 790-784, Korea. 2Department of Chemical Engineering, POSTECH, Pohang 790-784, Korea.


Advances in metabolic engineering and synthetic biology could fulfill a global demand for the production of commercially valuable chemicals. To achieve the successful design of the biological systems, however, one of the important issues to be solved is balancing the intracellular redox state that plays a governing factor for the continuation of both catabolism and anabolism. Here, we demonstrated that how the changes of intracellular redox state affect the pathway performance of n-butanol production as a model system. We built the n-butanol synthetic pathway by implementing constitutive promoters and designing synthetic 5’-untranslated regions based on predictive model, which we term “UTR engineering”, for each gene. The redox rebalancing was achieved by anaerobically activating pyruvate dehydrogenase complex and additionally tuning expression level of NAD+-dependent formate dehydrogenase through UTR engineering. As a result, we found that the optimal expression levels of fdh1 were dramatically different to efficiently produce n-butanol from glucose or galactose. This work provides intriguing insight that there is still room for strain improvement even with the same genetic contents by rebalancing intracellular redox state depending on the target products and substrates.

*Keywords: UTR engineering,Synthetic biology,Redox rebalancing

4

Role of tcs7 in Negative Regulation of FK506 Production in Streptomyces sp. Strain KCTC 11604BP

JAEYEON HWANG, Pramod B SHINDE, Dinesh KOJU, Yeon Hee BAN, Eun Young AHN, Hea Luyng SHIN, Hang Soo CHO and Yeo Joon YOON*Dept. of Chemistry and Nano Science, Ewha Womans University, Seoul 120-750, Korea.


FK506 is a clinically important 23-member polyketide macrolide with immunosuppressant activity. The sequence of the complete FK506 biosynthetic gene cluster from Streptomyces sp. Strain KCTC 11640BP was recently reported and analyzed. Based on this, LAL(large ATP-binding regulators of LuxR) family regulator was encoded by one of putative regulatory genes, tcs7. However the function of this putative regulatory gene has not been reported. In this study, we show that the function of tcs7 genes in Streptomyces sp. KCTC 11640BP by the overexpression, in-frame deletion, complementation of tcs7 deletion mutants, and transcriptional analysis of FK506 biosynthetic genes by semiquantitative reverse transcriptional-PCR(RT-PCR) in wild type and mutant strains. Above all the overexpression of tcs7 decreased the levels of FK506 production and a deletion of tcs7 lead 1.9 folds increase in FK506 production. These results imply that tcs7 plays an essential role as negative regulator of FK506 biosynthetic pathway and demonstrate the potential of manipulate regulatory gene to increase the level of FK506 biosynthesis in industrial production strains.

*Keywords: FK506, polyketide, metabolic engineering

5

A Novel RNA Scaffold System for the Enhancement of the in vivo Solubilization of Recombinant Proteins in Escherichia coli

Almando GERALDI and Sun Chang KIM*Dept. of Biological Sciences, KAIST, Daejeon 305-701, Korea.


Inclusion bodies formation of recombinant proteins is one of the major obstacles for their industrial applications. Several approaches such as the use of solubility-enhancing tags and the overexpression of folding modulators have been explored to minimize the formation of inclusion bodies. To further minimize the formation of inclusion bodies while increasing the solubility of recombinant proteins, we designed a novel RNA scaffold system in which a molecular chaperone DnaJ was fused with an RNA binding domain that specifically binds a unique RNA sequence in an engineered RNA hairpin loop structure on the 3’-UTR of the recombinant protein-encoding mRNA. Arranging molecular chaperones in proximity with the translational machinery of recombinant proteins can promote the rapid interaction between molecular chaperones and newly synthesized recombinant proteins to prevent the formation of inclusion bodies. As expected, our RNA scaffold system successfully increased the solubility of selected aggregation-prone proteins overproduced in Escherichia coli (e.g. anti p21-Ras ScFv and anti p21-Ras ScFv fused with a cell penetrating peptide). Our RNA scaffold system would provide a valuable tool for the production of recombinant proteins in soluble and active forms in E. coli.

*Keywords: RNA scaffold system,inclusion bodies,molecular chaperone

6

Improved Production of Bacterial Cellulose using Corn Steep Liquor from Gluconacetorbacter xylinus

Joon Young OH , Ji-Eun CHOI, Ji Young LEE, Hye Young NA and Jae Kwang SONG*Research Center for Bio-based Chemistry, Korea Research Institute of Chemical Technology, Daejeon, Korea.


Bacterial cellulose(BC) displays many unique properties, including higher purity, crystallinity, water absorption, tensile strength, low degree of polymerization and stronger biological adaptability compared to natural plant cellulose. This biopolymeric material have applications in different commercially important sectors such as food, textile, paper, composite membranes, medicine, biomedical material, bioadsorbent material and loud speaker diaphragms. Gluconacetobacter xylinus is one of the most extensively studied sources of BC. In this study, various media components, which were carbon sources, nitrogen sources including corn steep liquor(CSL), were optimized for BC production from Gluconacetobacter xylinus. CSL is a by-product of corn wet-milling. It is an important constituent of some growth media and an excellent source of organic nitrogen. In optimized CSL-added medium, BC production was increased excellently and BC production yield was reached up to 0.9 g of cellulose/g of consumed sugar.

*Keywords: Bacterial Cellulose,Corn Steep Liquor,Gluconacetorbacter xylinus

7

Screening whole functional sequence of metabolic enzyme

Namil LEE and Byung Gwan CHO*Dept. of Biological Science, Korea Advanced Institute of Science and Technology.


Increasing productivity by engineering metabolic enzyme is a major research stream of bio-engineering. However, method for analyzing functional sequence of enzyme is still unclear and selecting target site is time consuming step. Current researchers depend on crystal structures and homologous modeling to screen target sites for enzyme engineering. In order to understand a relationship between the sequence and function of the enzyme, we performed a high-throughput mutagenesis method that every codon of enzyme is replaced with every other amino acid. We targeted a bacterial flavin-containing monooxygenase(bFMO) which catalyzes oxidation of indole to produce bio-indigo compound. FMO is a family of various enzymes which inserts one molecule of oxygen into various substrates. A diversity of FMO substrates suggests that engineered bFMO has potential to catalyze variable profitable reactions. By using NGS, we classified whole functional sequence of bFMO and suggest appropriate target site for engineering. This strategy can be applied to other metabolic enzymes that produce phenotypically differentiable products. Even though, a crystal structure of the enzyme is not defined yet, functional domains of the metabolic enzyme can be defined by this method.

*Keywords:

8

Development of the Jet-fuel production using engineered Corynebacterium glutamicum using a CoryneBrick platform

Min-Kyoung KANG1, Youngsoon UM1, Sang Jun SIM2,3 and Han Min WOO*1,2

1Clean Energy Research Center, Korea Institute of Science and Technology, Seoul, Korea. 2Green School, Korea University, Seoul, Korea. 3Dept. of Chemical and Biological Engineering, Korea University, Seoul, Korea.


The production of alternative biofuels has been developed due to rising petroleum costs and environment concerns. Currently, biological engineering for the production of chemicals and fuels have been developed and implemented in engineered microbes. Corynebacterium glutamicum, a widely-known industrial bacterium, was engineered by introducing biofuel producing pathway constructed in the BioBrick-formatted expression vector systems. CoryneBricks, E. coli-C.glutamicum shuttle vector, were constructed with different origins of replication and controllable promoters. CoryneBrick vectors provide useful tool for users to choose the appropriate vectors in Corynebacterium and were applied to reconstruct the pinene biosynthetic pathway for the jet fuel production. A target jet-fuel precursor is further converted to high density jet-fuels via catalytic processes. So, the jet-fuel production platform by engineered C. glutamicum could be useful to biofuels production application.

*Keywords: Corynebacterium glutamicum, CoryneBrick, pinene

9

Improvement in Thin-layer Chromatography in a Quantitative Assay of Glycerolin Biodiesel

Yi-Ok KIM1, Woo-Seok CHOI2, Do-Hyung KANG3, Hyeon-Yong LEE4 and Kyung-Hwan JUNG*1

1Department of Biotechnology, Korea National University of Transportation, Jeungpyung 368-701, Korea. 2Department of Medical Biomaterials Engineering, Kangwon National University, Chuncheon 200-701, Korea. 3Korea Institute of Ocean Science and Technology, Ansan 426-744, Korea. 4Department of Food Science and Engineering, Seowon University, Cheongju, Chungbuk 361-742, Korea.


We analyzed glycerol using thin-layer chromatography (TLC) and compared the separation resolution of some mobile phases. When acetonitrile:distilled water (85:15 v/v) was used as a mobile phase, the band of glycerol on the TLC was more distinctly and rapidly separated. Using TLC analysis, we prepared a calibration curve for the glycerol concentration vs. the area of the glycerol band in which the glycerol concentration of the x-axis was converted into a log-scale ranging from 3.0 to 0.0625 (%, w/v). Based on this calibration curve, the residual glycerol concentration (0.2%, w/v) in biodiesel was determined successfully using TLC analysis. When the results of the TLC analysis were compared with those of a chemical and enzymatic assay, the results were fairly similar. We conclude that TLC without additional analytical instruments can be used as an alternative method for the quantitative analysis of the concentration of glycerol in biodiesel.

*Keywords: Biodiesel,glycerol,thin-layer chromatography

10

Investigation of Key Signaling Pathways in Chlamydomonas reinhardtii Using RNAi Strategy

Changsu LEE and Yoon-E CHOI*Department of Bioprocess Engineering, Chonbuk National Univ. Iksan Campus, Jeonbuk 570-752, Korea.


Recently, intensive global research efforts have been made with the aim of developing efficient microorganisms improving the traits for the benefit of humans. In this study, we have focused on genetic engineering of signaling transduction pathway in order to enhance the lipid content of microalgae. RNA interference(RNAi) utilized to suppress the signal transduction pathways of interest. Of the putative genes connected to microalgal signaling pathway, genes encoding G-protein(Gα, Gβ subunit), Adenylate Cyclase(AC) and phosphodiesterase(PDE) were selected. Also, three Adenylate Cyclase(AC) homologs of genes were chosen, based on the results of bioinformatics. All of RNAi constructs for Gα, Gβ, AC and PDE were made appropriately and each constructs were transformed to Chlamydomonas reinhardtii by following the protocol previously reported. The expression ratio difference of transgenic mutants were confirmed by quantitative real-time PCR(QRT-PCR). We are currently working on the characterization of each of mutant strains in order to fully understand individualsignaling pathway(s) in C. reinhardtii. The discovery in C. reinhardtii may ultimately expand to the other promising microalgal species for human benefits.

*Keywords: signaling transduction pathway,genetic engineering,microalgae

11

Development of Improved Methanol Dehydrogenase Using Directed Evolution and Biological Methanol Sensor System for the Elimination of Formaldehyde

Ji-Yeun YI1,2, Jung-Hoon SOHN1 and Bong Hyun SUNG*1

1Biochemicals and Synthetic Biology Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-806, Korea. 2Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, Republic of Korea.


Formaldehyde is an important organic precursor to many materials and chemical compounds. Despite its widespread use, exposure to formaldehyde is a significant consideration for human health due to its toxicity and volatility. Methanol dehydrogenase is an NAD+-dependent oxidoreductase that catalyzes formaldehyde to methanol reversibly. Reduction activity of MDH is noticeable in two aspects of the elimination of toxic formaldehyde and the production of methanol as an energy source. Herein, we screened improved mutants of B. methanolicus MDH in E. coli that reduce formaldehyde to methanol effectively with directed evolution and Phichia-based biological methanol sensor system. To examine the resistance to formaldehyde, E. coli strains expressing each mutant were cultured in 3mM formaldehyde, toxic concentration for E. coli. In the best mutant, three phenylalanine residues were substituted to leucine, valine and serine respectively and all the three substitutions were required to increase reduction activity for formaldehyde. Purified mutant converted formaldehyde to methanol approximately 5 times more than wild type. Based on these results, mutant MDH carry out the elimination of formaldehyde efficiently, therefore it could help solve environmental problem such as sick house syndrome.

*Keywords: Methanol dehydrogenase,Biological methanol sensor system,elimination of formaldehyde

12

Engineering of Naringenin Biosynthesis in Escherichia coli Using Unnatural Fusion Proteins

Oksik CHOI , Sun-Young KANG, Won Ho CHOI and Young-Soo HONG*KRIBB.


A phenylpropanoids, naringenin biosynthesis begins with the deamination of tyrosine by tyrosine ammonia lyase (TAL) to produce 4-coumaric acid. This product is attached to CoA by 4-coumarate:CoA ligase (CCL). Next, chalcone synthase (CHS) condenses 4-coumaroyl CoA with three molecules of malonyl CoA to form chalcone. The final naringenin structure is formed only when chalcone is spontaneously isomerized in alkaline environments. The formation of multi-enzyme complexes in biosynthetic pathways improves production yields either by colocalizing enzymes or by the channeling of substrates. For metabolic engineering purposes, linking genes together to generate a functional fusion protein offers an attractive strategy for increasing metabolic efficiency. Here we utilized the synthetic scaffolds strategy to improve naringenin production in E. coli. Thus, co-localization of the enzyme active sites provides the basis for enhanced biosynthesis of naringenin. We observed a 3.5-fold improvement over the non-scaffolded control, and a 8.1-fold increase over the previous reported with fusion silbene synthase. (Supported by grants from Basic Science Research program and Global

Frontier Project (NRF) and Next-Generation BioGreen 21 Program (RDA))

*Keywords: naringenin,biosynthesis,fusion protein

13

Monomeric sugar acid production by enzymatic degradation of alginate using recombinant alginate lyases

wang DAMAO and Kyoung Heon KIM*School of Life Sciences and Biotechnology, Korea University, Seoul 136-713, Korea.


Brown algae are considered to be promising biomass for production of biofuels and chemicals. Alginate, one of the main carbohydrates found in brown seaweeds, is composed of α-L-guluronic acid and β-D-mannuronic acid as monomeric units. Alginate lyase decomposes alginate into smaller molecules by a β-elimination reaction. Here, we cloned two alginate lyase genes alg7D and alg17C from Saccharophagus degradans 2-40T to produce 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH). Alg7D was shown to be an endo-type alginate lyase and depolymerized alginate into oligo-alginates. The optimal activity of Alg7D was shown at 50°C and pH 7. The Km, Vmax, kcat and kcat/Km of Alg7D were determined to be 3.0 mg ml-1, 6.2 U mg-1, 1.9×10-2 s-1, and 6.3×10-3 mg-1 ml-1, respectively. Alg17C was determined to be an exo-type alginate lyase, which mainly produced DEH from oligo-alginates. The optimal pH and temperature of Alg17C were found to be 6 and 40 °C. The Km and Vmax of Alg17C were 35.2 mg ml-1 and 41.7 U mg-1, respectively. By the cooperative actions of the two enzymes, 7.1 g L-1 of DEH was produced from 20 g L-1 of alginate. The enzymatic saccharification of alginate could be used to produce monomeric sugar acids which can be converted into biofuels and valuable industrial chemicals.

*Keywords: alginate lyase,enzymatic saccharification process,Saccharophagus degreadans 2-40

14

Effect of Zeta Potential of Surface Area-Increasing Materials in Fractional Precipitation Process for the Purification of Paclitaxel

Heung-Kon RYU and Jin-Hyun KIM*Dept. of Chemical Engineering, Kongju National University Cheonan 330-717, Korea.


Fractional precipitation is a simple, efficient method for purifying paclitaxel extracted from plant cell cultures. But, fractional precipitation process has been inherently problematic due to the lengthy precipitation time that is required. An improved fractional precipitation process could significantly reduce the precipitation time by increasing the surface area available for precipitation. Silica-alumina was used to increase the surface area, and the optimal surface area per working volume (i.e. volume of reaction solution) (S/V) for achieving the highest purity and yield of paclitaxel possible was found to be 1007.6 mm-1. In this study, we evaluated the effects of the zeta potential of silica-alumina on the behavior (purity, yield, fractional precipitation time, precipitate shape and size) of fractional precipitation for improving the purification efficiency of paclitaxel. As the zeta potential of surface area-increasing materials were increased, the yield of paclitaxel was increased and the precipitation time and precipitate size were decreased. Furthermore, we also found that the purity of paclitaxel was constant regardless of the zeta potential of silica-alumina. Acknowledgement: This work was performed by a research year program of the Kongju National University in 2012.

*Keywords: Paclitaxel,Fractional precipitation,Zeta potential

15

Metabolic engineering of Escherichia coli for fatty acid production using transposon mutagenesis and genome engineering methods.

Kwangsu SHIN1 and Sung Kuk LEE*1,2

1School of Nano-Bioscience and Chemical Engineering, Ulsan National Institute of Science and Technology, Ulsan 689-798, Korea. 2School of Urban and Environmental Engineering, Ulsan National Institute of Science and Technology, Ulsan 689-798, Korea.


Fatty acid-derived fuels produced by microorganisms have currently received an attention owing to serious oil crisis and global warming. Although several studies have reported improvements in fatty acid productivity from microorganisms, the yield should be higher than today to become economical and eco-friendly fuels. Given that all organisms harbor fatty acid synthetic pathway, fatty acids can offer a significant advantage as a biofuel precursor. However, there is one drawback that fatty acid synthetic pathway is tightly regulated in transcriptional and translational levels. Thus, it is inevitable to engineer microorganisms with rational or

random genetic approaches to yield higher productivity. Here, we have used transposon mutagenesis to generate a genomic mutant library of E. coli and screened mutants that showed increase in fatty acid synthesis using a fatty acid biosensor. Based on the results of library screening, the ribosome binding sites (RBSs) of identified genes will be randomly altered by multiplex automated genome engineering (MAGE). Of them, most fatty acid producers will be isolated from RBS mutant library. The resulting strain could be used for additional optimization and these screening strategies can be also applied to other metabolic pathways.

*Keywords: fatty acids,biofuel,Escherichia coli

16

Biosynthesis of Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (P(HB-co-HHx)) from butyrate using engineered Ralstonia eutropha

Jong-Min JEON1, Christopher J. BRIGHAM2, Rajesh THANGAMANI1, Yong-Hyun KIM1, Hyun-Joong KIM1, Da Hye YI1, Sang Hyun LEE1, Hyung Joo KIM1 and Yung-Hun YANG*1

1Department of Microbial Engineering, College of Engineering, Konkuk University, Seoul, South Korea. 2Department of Bioengineering, University of Massachusetts Dartmouth, 285 Old Westport Road, North Dartmouth, MA, USA.


Polyhydroxyalkanoate (PHA), a promising family of bio-based polymer products that can be synthesized by many different microorganisms and can be considered alternatives to traditional petroleum-based plastics. Among various PHAs, poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (P(HB-co-HHx)) has been shown to have favorable physical and mechanical properties, such as lower melting point, enhanced flexibility and better impact strength than PHB because of decreased crystallinity due to the presence of longer-chain 3-hydroxyhexanoate (3HHx) monomers. In this study, using engineered Ralstonia eutropha containing acetoacetyl-CoA reductase (phaB) gene deletions and transforming acetoacetyl-CoA to 3-hydroxybutyryl-CoA and replacing PHA synthase with phaC2 from Rhodococcus aetherivorans I24, we have succeeded in the production of P(HB-co-HHx) from butyrate as the sole carbon source and this is the first report for the production of P(HB-co-HHx) using butyrate in R. eutropha.

*Keywords: polyhydroxyalkanoate,Ralstonia eutropha,poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)

17

Improvement of Binding Affinity and Cellulolytic Activity towards Insoluble Cellulose by Fusion of Carbohydrate Binding Module to Family 5 Cellulase

Hyeon-dong KIM1,2, Soo-Jin YEOM1, Kil Koang KWON1, Eugene RHA1, Jae Jun SONG3 and Seung-Goo LEE*1,2

1Biochemicals &Synthetic Biology Research Center, KRIBB. 2Biosystems &Bioengineering, University of Science &Technology. 3Applied Microbiology Research Center, KRIBB.


Cellulase (CelEdx16) from an anaerobic bacterium demonstrates both endo-/exo-type cellulolytic activity. This enzyme shows low activity towards insoluble cellulose and that it lacks the carbohydrate-binding module(CBM), which is necessary for effective hydrolysis of cellulose. In order to enhance its cellulolytic activity towards insoluble cellulose, the Family III CBM and fibronectin III domain (Fn3) of unknown function, from cellobiohydrolase Cel48c Paenibacillus sp. were selected and fused to CelEdx16. All the fusion enzymes were overexpressed in E.coli, purified and characterized. The fusion enzymes, CelEdx16-CBM3 and CelEdx16-Fn3-CBM3 showed increased binding affinity and catalytic activity towards insoluble cellulose as higher than that of the native enzyme. The fusion enzyme CelEdx16-Fn3 displayed the same cellulase activity compare to that of the CelEdx16 while the solubility was increased dramatically when CBM is introduced. Thus, CBM3a and Fn3 domains play an important role in improving the cellulolytic activity towards insoluble cellulose and solubility respectively. The fusion cellulase, CelEdx16-Fn3-CBM3a that shows higher activity towards insoluble cellulose could be an attractive candidate for consolidated bioprocessing (CBP) for cellulose ethanol production.

*Keywords: Cellulase,Carbohydrate Binding Module,Fibronectin III domain,Fusion cellulase,Insoluble cellulose

18

Characterization of a novel processive cellulase from a marine bacterium Hahella chejuensis

Hee Jin LEE, In Jung KIM, In-Geol CHOI and Kyoung Heon KIM*School of Life science and Biotechnology, Korea University, Seoul 136-713, Repubilic of Korea.


The cooperative action between endoglucanase (EG), cellobiohydrolase (CBH) and ß-glucosidase are the generally approved cellulase system for the efficient degradation of crystalline cellulose. Processive endoglucanase, a cellulolytic enzyme produced by cellulose-degrading microorganisms lacking in CBH, displays a dual activity of EG and CBH. SdCel5H from Saccharophagus degradans 2-40T is a highly processive endoglucanase that generates cellobiose as a major product from cellulose. In this study, HcCel5H from a marine bacterium Hahella chejuensis was selected based on motif analysis and overexpressed in E. coli BL21 (DE3), and then its comparative study with SdCel5H was performed. HcCel5H showed a processive behavior on Avicel as shown in SdCel5H. However, HcCel5H showed an approximately 3-fold higher enzymatic activity on Avicel and was highly thermostable. Due to its thermostability and high activity, HcCel5H is expected to be industrially applicable in the enzyme hydrolysis process.

*Keywords: processive cellulase,novel enzyme

19

Metabolic Engineering of E. coli for Regiospecific Modifications of Naringenin to Produce Astragalin

Ramesh Prasad PANDEY1, Sailesh MALLA2,3, Thi Lan Huong NGUYEN1, Hem Raj RIMAL1, Byung-Gee KIM1,2 and Jae Kyung SOHNG*1

1Institute of Biomolecule Reconstruction (iBR), Department of Pharmaceutical Engineering, Sun Moon University, #100, Kalsan-ri, Tangjeon-myeon, Asan-si, Chungnam 336-708, South Korea. 2Laboratory of Molecular Biotechnology and Biomaterials, School of Chemical and Biological Engineering, Seoul National University, Seoul, South Korea. 3Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark.


Astragalin (AST) was successfully produced from regiospecific modifications of naringenin (NRN) in E. coli. The exogenously fed NRN was converted into dihydrokaempferol (DHK) and then kaempferol (KMF) in the presence of flavanone-3-hydroxylase (f3h) and flavonone synthase (fls1) from A. thaliana, respectively. KMF was further modified to produce AST by 3-O-glucosylation utilizing the endogeneous UDP-glucose in presence of UGT78K1 from G. max. The chromosomal glucose phosphate isomerase (pgi) and D-glucose-6-phosphate dehydrogenase (zwf) were deleted to channel carbon flux towards UDP-glucose. The two enzymes directly involved in the synthesis of UDP-glucose from G6P, phosphoglucomutase (nfa44530) from N. farcinia and glucose-1-phosphate uridylyltransferase (galU) from E. coli K12 were overexpressed, which diverted the carbon flow from glycolysis to the UDP-glucose. Furthermore, to prevent the dissociation of UDP-glucose into UDP and glucose, the UDP-glucose hydrolase (ushA) was deleted. The E. coli ΔpgiΔzwfΔushA mutant harboring the UDP-glucose biosynthetic pathway and the aforementioned genes for the regiospecific glucosylation produced 109.3 mg/L (244 µM) of AST representing 48.8% conversion from 500 µM of NRN in 60 h without any supplementation of extracellular UDP-glucose.

*Keywords: kaempferol-3-O-glucoside,Flavonoids,glycosylation

20

Genomic Approaches for Cloning Xylose Transporters and Its Applications in Candida tropicalis

Woo Yong SHIM and Jung Hoe KIM*Dept. of Biological Sciences, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon 305-701, Korea.


Candida tropicalis is a diploid asporogenic yeast that is widely used for industrial xylitol production because of its xylose-assimilating ability. The biomass used for xylitol production is hydrolyzed by a mineral acid to produce a hydrolysate that consists primarily of glucose, arabinose and xylose. The glucose in the hydrolysate can be used directly as a co-substrate. This approach has economic and convenience advantages. However the suppression of xylose uptake by glucose should be overcome. To overcome the suppression of xylose uptake, we cloned several xylose transporter candidates and promoters for enhanced production of xylitol. The sequences between two genes (promoter or terminator) were amplified by PCR using primers designed from the conserved sequences of neighboring genes, which were based on sequences of the closely related species P. stipites, C. albicans, D. hansseii, and C. tropicalis. In this presentation, we will introduce the bioinformatical and genomic strategies for cloning candidate region of genome and discuss on recent experimental data obtained with the genetically engineered cells.

*Keywords:

21

Differential gene expression analysis by RNA-Seq in the Thermotolerant Yeast Kluyveromyces marxianus Transcriptome

YeonI WOO, Yoo-Bok CHO, Eun Ju LEE, Suhyung CHO and Byung-Kwan CHO*

Dept. of Biological Sciences, Korea Advanced Institute of Science and Technology,Daejeon 305-701, Korea.


The thermotolerant yeast Kluveromyces marxianus is widely used as a bioengineering platform for producing industrial, studying protein expression and secretion mechanisms, and analyzing metabolite synthesis. In spite of their great biotechnological potential, little is known for genome-wide characteristics. Although draft genomic sequence is published recently, however, actively transcribing genomic regions that reflect relevant genes for the industrial usage during development are not known. Here, we present transcriptome changes among early, mid, and late log phases. Using RNA-Seq, we catalogued gene architectures as well as differentially expressed genes. Our study should increase our understanding on the biology of this species and facilitate the potential biotechnological application. This research is funded by Bio Research Center, Samsung Advanced Institute of Technology (SAIT) and Korea Institute of Science and Technology Information (KISTI; K-13-L01-C02-S04).

*Keywords: Kluveromyces marxianus,RNA-Seq,Yeast biotechnology

22

Comparison of mineral and organic acid pretreatments for whole slurry ethanol fermentation of pretreated oil palm empty fruit bunches

Young Hoon JUNG, In Jung KIM, Hyung Kyung KIM, Hyun Min PARK and Kyoung Heon KIM*School of Life Sciences and Biotechnology, Korea University.


Oil palm empty fruit bunch (EFB) is a lignocellulosic biomass widely abundant in the world. Still, it is inherently difficult to utilize EFB for sugar production due to its recalcitrant structure. To tackle this problem, here we evaluated the hydrolysis efficacy of two differently pretreated EFB using dilute sulfuric and maleic acids, and further compared their performance on whole slurry fermentation. When EFB was pretreated by 1% (w/v) sulfuric or maleic acid at 190°C for a 3 min ramping time in a microwave digestion system and then washed by water, the respective enzymatic digestibilities were 88.5 and 60.9% of the theoretical glucose yield. The theoretical ethanol yields obtained from the simultaneous saccharification and fermentation (SSF) were 52.5 and 61.2% for sulfuric and maleic acid pretreated EFBs, respectively. The whole slurry fermentation of the pretreated EFBs, after incubation with activated carbon, resulted in the increased theoretical yield of ethanol (87.5%) for sulfuric acid-pretreated EFB. These results indicate that the sequential treatment of sulfuric acid and activated carbon adsorption warrants high ethanol yields in whole slurry fermentation of pretreated lignocellulose.

*Keywords: Sulfuric acid pretreatment,Maleic acid pretreatment,Whole slurry fermentation

23

Manipulation of LED Wavelength at Appropriate Growth Stage to Boost Biomass Productivity of Chlorella vulgaris

Dae geun KIM and Yoon E CHOI*Dept. of bioprocess engineering, Jeonbuk National University.


LEDs light offer several advantages over the conventional lamps, thereby being considered as the optimal light sources for microalgal cultivation. In addition, LEDs are suitable light source for photobioreactor(PBR) due to their compact chip size and specific narrow bands for possible manipulation of microalgal biology for human interest. In this study, various light-emitting diodes(LEDs) especially red and blue color with different light wavelengths were employed to explore the effects of light source on phototrophic cultivation of Chlorella vulgaris. Blue light illumination led to significantly increased cell size, whereas red light resulted in small-sized cell with active divisions. Based on the discovery of the effect of light wavelengths on microalgal biology, we then applied appropriate wave length at different growth stages; blue light was illuminated first and then shifted to red light. By doing so, biomass and lipid productivity of C. vulgaris could be significantly increased, compared to that in the control. Our results shed light on novel approach using LED light for microalgal biotechnology.

*Keywords: LED,Chlorella vulgaris

24

Introduction of an Engineered crp Paralog gene in Escherichia coli: A New Paradigm to Eliminate Carbon Catabolite Repression

Baeyoung CHOI and Sung Kuk LEE*

School of Nano Bioscience and Chemical Engineering, UNIST, Ulsan, Korea.


Cellulosic biomass-based industrial fermentation has been the long standing dream of scientist. However, widespread fermenting microbes have a challenge to ferment efficiently because of multiple sugars (glucose, xylose and arabinose) derived from heterogeneous nature of cellulosic biomass. Carbon catabolite repression (CCR) of most fermenting microbes, to regulate the utilization of preferred carbon sources, is a key rate-limiting step of industrial fermentation. Solving CCR to ferment multiple sugars produced by cellulosic biomass is an essential improvement for sustainable bio-production. Plenty of strategies were proposed by engineering a single orthologue crp to overcome CCR. These mutants seemed to utilize several sugars simultaneously, but they showed slow growth because of a delay in sugar uptake rate. Here, we have introduced an additional copy of crp into Escherichia coli chromosome and engineered both crp genes using multiplex automated genome engineering (MAGE). Mutants resistant to CCR will be screened based on growth rate in sugar mixture medium. Presence of one wild type crp and a mutant paralog would help in improved co-utilization of multiple sugars without delaying sugar uptake rate.

*Keywords: CCR,paralog,crp

25

Selective Production of L- and D-Lactic Acid from Jerusalem Artichoke Powder by Metabolically Engineered Kluyveromyces marxianus

Hyun-Jin KIM1, Jung-Hoon BAE1, Bong Hyun SUNG1, Chul-Ho KIM2 and Jung-Hoon SOHN*1

1Biochemicals &Synthetic Biology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Yusong, Daejeon 305-333, Korea. 2Applied Microbiology Research Center, Bio-Materials Research Institute, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Jeongeup, Jeonbuk 580-185, Korea.


Selective L- and D-lactic acid production system was constructed by metabolic engineering of Kluyveromyces marxianus. The ethanol fermentation pathway from pyruvate to ethanol was redirected to lactic acid by disruption of KmPDC1 gene and selective expression of L- and D-lactate dehydrogenase (L-and D-LDH) genes. To improve the lactic acid production yield, L-and D-LDH genes were screened from several Lactobacillus species and expressed under the control of the K. marxianus translation elongation factor 1α (TEF1) promoter, respectively. The recombinant strains expressing L- and D-LDH genes derived from Lactobacillus plantarum produced 130 g and 122g of L-lactic acid and D-lactic acid from 220 g of Jerusalem artichoke tuber powder including 140g of inulin without pretreatment and additional nutrients in batch fermentation, respectively. The conversion efficiency was more than 95% and optical purity was 99.9%.

*Keywords: L-lactic acid,D-lactic acid,Kluyveromyces marxianus

26

Efficient Recovery of Sugars from Biomass Hydrolyzate using a Lime Addition-Capacitive Deionization (CDI) Hybrid Process

Sung-Jae KIM, Jae-Hwan CHOI and Jin-Hyun KIM*Dept. of Chemical Engineering, Kongju National University, Cheonan 330-717, Korea.


In this study, we developed an efficient lime addition-capacitive deionization (CDI) hybrid process that can recover sugar from the mixture of glucose, xylose, acetic acid, and sulfuric acid, which are the major components of biomass hydrolyzate. It also optimized the key parameters of the lime addition process (type of lime, amount of lime, stirrer speed, reaction time) and of the CDI process (voltage, flow rate, feed concentration). In the lime addition process, the optimal lime type, acids (sulfuric acid and acetic acid)/lime molar ratio, stirrer speed, and reaction time for removal of sulfuric acid were CaCO3, 1:1, 500 rpm, and 8 min, respectively. For the CDI process, the optimal voltage and flow rate were 1.2 V and 20 mL/min. The efficiency of acid removal increased as the initial acetic acid concentration decreased. The developed hybrid process was able to remove 98.1% of sulfuric acid and 77.0% of acetic acid from the mixture of glucose, xylose, acetic acid, and sulfuric acid. It was able to recover most of the sugar (>99%) at high purity (97.5%).

*Keywords: Lime addition,Capacitive deionization (CDI),Removal of acid

27

Change in Amounts of Tar Compounds through the Stages of Paclitaxel Purification from Plant Cell Cultures of Taxus chinensis

Gun-joong KIM, Gyu Yeon PARK and Jin-Hyun KIM*Dept. of Chemical Engineering, Kongju National University, Cheonan 330-717, Korea.


In this study, the tar compounds derived from the plant cell cultures of Taxus chinensis were first identified and quantified via GC/MS (gas chromatography/mass spectrometry) and GC (gas chromatography). 2-Picoline, 2,5-Xylenol, Acenaphthene, 1-Methylnaphthalene and o-Xylene were found as major main tar components in biomass. These compounds were identified and confirmed by comparing their retention times with those of authentic compounds. Each compound also spiked with pure standard. The contents of 2-Picoline, 2,5-Xylenol, Acenaphthene, 1-Methylnaphthalene, and o-Xylene in biomass were 0.251, 0.159, 0.124, 0.094 wt% and 0.053 wt%, respectively. In liquid-liquid extraction, adsorbent treatment tar was removed 41.6, 89.1%, respectively. After hexane precipitation, all of tars were successfully removed. Acknowledgement: This work was supported by a grant from the National Research Foundation of Korea (NRF) funded by the Korean government (MEST) (No. 2011-0010907).

*Keywords: Paclitaxel,Tar compounds,Identification

28

Optimization of Adsorbent Treatment Conditions for the Separation and Purification of Paclitaxel from Plant Cell Cultures

Chung-Gi LEE and Jin-Hyun KIM*Dept. of Chemical Engineering, Kongju National University, Cheonan 330-717, Korea.


Biomass-derived tar and waxy compounds have a highly negative effect on the separation and purification of paclitaxel and should be removed prior to final purification. Adsorbent treatment is a simple, efficient method for removal of tar and waxy compounds from plant cell cultures. In this study, we optimized the important process parameters (solvent type, ratio, adsorption time and temperature) of adsorbent treatment to remove the tar and waxy compounds in a pre-purification step. Using the adsorbent, sylopute, we determined differences in the effectiveness of the adsorbent treatment according to changes in the solvent type, ratio (crude extract : adsorbent), adsorption time and temperature. This effect could also be confirmed by HPLC analysis of the adsorbent after treatment. In adsorbent treatment step, the purity seemed to show a small improvement but this treatment had a significant effect on convenient and feasibility of following steps by removing of tar and waxy compounds. Acknowledgement: This work was supported by a grant from the National Research Foundation of Korea (NRF) funded by the Korean government (MEST) (No. 2011-0010907).

*Keywords: Paclitaxel,Adsorbent treatment,Optimization

29

Metabolic Engineering of Escherichia coli for Co-fermentation of Hexose and Pentose Sugars Using Synthetic Constitutive Promoters

Seong Hun JEONG and Sung Kuk LEE*School of NanoBioChemical Engineering, UNIST, Ulsan 689-798, Korea.


Escherichia coli have been regarded as a valuable microorganism to produce biochemicals. However, one disadvantages of this strain is carbon catabolite repression. E. coli can utilize glucose efficiently but glucose causes the hindrance of consuming the other abundant lignocellulosic substrates such as xylose, galactose, mannose and arabinose. Wild-type E. coli cannot utilize these sugars simultaneously so causes increase of fermentation time and decreases productivity. To eliminate carbon catebolite repression in E. coli, we introduced constitutive promoters to E. coli genome to replace native sugar metabolic gene’s promoters such as araBFE, xylAF, galEP, mglB, manAX. Above all things, we focused on co-metabolism of glucose and xylose. After exchange of promoters, we tried adaptive evolution in xylose minimal media. Consequently, we found enhanced xylose consuming E. coli strains. To figure out factors affecting metabolic pattern, adapted E. coli strain was sequenced using Next-generation genome sequencing. As a result, we identified total six point mutations such as ybjG, yeiR, araE, araF coding region and thiC and wecH promoter region. To confirm accurate effects of these mutations, MAGE will be used to introduce point mutations into non-adapted strain’s genome.

*Keywords: Adaptive Evolution,Genome Sequencing,xylose

30

Efficient 2,3-Butanediol Production from Sugarcane Molasses using Metabolically Engineered Enterobacter Aerogenes

Moo Young JUNG, Hwimin JEONG, Hye-mi LEE and Min-kyu OH*Dept. of Chemical &Biological Engineering ,Korea University, 5-1 Anam-Dong, Sungbuk-Gu, Seoul.


2,3-Butanediol (2,3-BD) is a promising platform chemical due to its extensive industrial applications. Among several natural 2,3-BD producers, Enterobacter aerogenes is able to produce significant amounts of 2,3-BD from a wide range of carbon sources. In the biorefinery process, one of the biggest hindrances is the price of the carbon source. Among the inexpensive substrates, sugarcane molasses is considered to be a desired fermentative carbon source due to its low-price and high sugar content. Sugarcane molasses contains a dominant amount of sucrose. Therefore, the efficient utilization of sucrose is necessary in order to maximize the use of sugarcane molasses. To enhance sucrose utilization, a transcriptional repressor of sucrose utilization operon was disrupted. Further, we have conducted lactate dehydrogenase gene knockout. The mutant strain showed significantly enhanced the sucrose utilization when sucrose or sugarcane molasses was used as a substrate. In batch fermentation with sugarcane molasses, 2,3-BD production of mutant strain (△ldhA △scrR) increased by 60% compared to that of the wild type strain. Finally, 2,3-BD production reached 98.69 g/L at 36 h of cultivation and 269.85 g/L of sugars in sugarcane molasses was consumed in fed-batch fermentation.

*Keywords: Enterobacter aereogenes,2,3-butanediol,Sucrose regulator

31

Development of Indole-3-acetic acid-producing Eschericia coli by Expression of IpdC, AspC, and IDH

ELISA ROMASI2 and Jinho LEE*1

1Dept. of Food Science &Biotechnology, Kyungsung University, Busan 608-736, Korea. 2경성대학교.


Biosynthesis of indole-3-acetic acid (IAA) through indole-3-pyruvic acid pathway involved three kind of enzymes, indole-3-pyruvic acid decarboxylase encoded by ipdC, aminotransferase encoded by aspC, and indole-3-acetic acid dehydrogenase encoded by idh. The ipdC from Enterobacter cloacae ATCC 13047, aspC from Escherichia coli, and idh from Ustilago maydis were cloned and expressed under the control of tac, sod, and ilvC promoters in E. coli. According to SDS-PAGE and enzyme activity, IpdC and Idh were well expressed by Ptac promoter, whereas, AspC was efficiently expressed by Psod promoter from C. glutamicum. The activities of indole-3-purivic acid decarboxylase, aminotransferase, and indole-3-acetic acid dehydrogenase from the crude extracts of recombinant E. coli DH5α showed about 92.7, 5.7, and 13.1nmol/min/mg protein, respectively. The recombinant E. coli DH5α /pCIA41/pCLTID23 and DH5α/pCIA41 expressing 3 genes (idh, aspC, and idh) and 2 genes (idh and apcC), produced about 5.0 mM IAA and 1.9 mM tryptophol, respectively, after 48 hr culture in LB medium with 10mM tryptophan.

*Keywords: Indole-3-acetic acid, aminotransferase, indole-3-purivic acid decarboxylase, indole-3-puryvic acid

32

Production of Gamma-aminobutyric acid by Metabolically-engineered Corynebacterium glutamicum

Hyukjin KWON, Kyoyoung SEO, Heeyoung KIM, Hyunghwan HYUN and Hyunehwan LEE*Dept. of Bioscience and Biotechnology, Hankuk University of Foreign Studies, Gyeonggi-do 449-791, Korea.


Gamma-aminobutyric acid (GABA) is known as an inhibitory neurotransmitter which acts on the central nerve system. Recently plenty of pharmaceutical drugs and functional foods are under development using GABA. In addition, it could be used as a monomer of biopolymers in chemical industry. In this study, metabolic engineering of Corynebacterium glutamicum HH09, the glutamate overproducer, was tried to produce GABA as high concentration.First of all, the gaba-T gene encoding GABA aminotransferase was knocked-out by Cre/loxP system to block the biosynthetic pathway from GABA to succinate semialdehyde. Secondly, the gad gene encoding glutamate decarboxylase was introduced to make GABA accumulated. In order to introduce the gad gene into C. glutamicum, the plasmid containing gad gene was constructed. Moreover, integration of gad gene using sacB system was carried out for the stable production of glutamate decarboxylase without addition of antibiotics. The disruption of gaba-T was confirmed by PCR. Western blotting and RT-PCR were used to analyze the expression of gad. As a result, the production of GABA from the metabolically-engineered C. glutamicum HH107 by fed-batch fermentation (5L) was 199 g/l by HPLC analysis.

*Keywords: GABA,C. glutamicum,Glutamate decarboxylase

33

Maleic acid pretreatment for whole slurry ethanol fermentation

Hyun-min PARK and Kyoung Heon KIM*School of Life Sciences and Biotechnology, Korea University, Seoul 136-713, Korea.


The feasibility test of whole slurry fermentation using maleic acid pretreated rice straw was conducted as an alternative bioethanol production process. Under the optimized pretreatment conditions (190°C, 3 min ramping to the set temperature with 3 min holding, 1% maleic acid) the conversion yield of glucose from glucan was 91.5% of theoretical maximum after 50 h of hydrolysis. When simultaneous saccharification and fermentation (SSF) was performed on the maleic acid-pretreated rice straw after a washing step, the theoretical ethanol yield was 62.5% based on glucan in untreated rice straw. Notably, whole slurry fermentation of pretreated rice straw without a washing step resulted in the increased theoretical yield of ethanol (90.3%). Moreover, the liquid fraction in whole slurry obtained after maleic acid pretreatment did not have a negative influence on yeast fermentation without any detoxification. Overall, the whole slurry fermentation of maleic acid-pretreated lignocellulose addressed the superior performance, implying its potentials for the industrial utilization.

*Keywords: whole slurry fermentation,acid pretreatment,lignocellulose

34

Responses of fatty acids and carbohydrate contentsin three eukaryotic microalgae to nitrogen starvation

Min-Seung HWANG1, Choul-Gyun LEE1, Dong-Il KIM1, Byung-Kwan CHO2, Seong-Joo HONG1, Yong Sung PARK1 and Choul-Gyun LEE*1

1Dept. of Biological Engineering, Inha University, Incheon 402-751, Korea. 2Dept. of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Korea.


The aim of this work is to compare lipid and carbohydrate accumulation response under nitrogen starvation of Dunaliella tertiolecta, Porphyridium cruentum and Nannochloropsis oculata. D. tertiolecta and N. oculata cells were grown photoautotrophically at 21°C under illumination from fluorescent lamps at 50 μE/m2/s in three fold f/2 medium and aeration with 2% CO2 balanced with air. P. cruentum cells were grown photoautotrophically at 20°C under illumination from fluorescent lamps at 60 μE/m2/s in Jone's medium, and aeration with 1% CO2 balanced with air. Total fatty acid contents in D. tertiolecta remained relatively the same, while total carbohydrate contents increased dramatically as the duration of nitrogen starvation increased. In P. cruentum, total carbohydrate was accumulated up to 43% of dry weight at 3.5 day and total fatty acid contents also increased slightly under nitrogen depletion. Total fatty acids content increased rapidly, reaching nearly 40% of the dry cell weight on day 3.5, from an initial content of 19% in N. oculata. These results demonstrate the possibility of couplet understanding of the fatty acid and carbohydrate biosynthesis metabolic network responding to environmental change in microalgae.

*Keywords: Dunaliella tertiolecta,Porphyridium cruentum,Nannochloropsis oculata

35

Preparation of 12-Oxo-Chenodeoxycholic Acid and 7,12-Dioxo-Lithocholic Acid from Cholic Acid by Recombinant E. coli

Dahyun HWANG1, Taewon KANG2, Jiyun LEE1, Ki-Hyun KIM2, Jeong-Keun KIM2, Seonwook HWANG1 and Young-Hee LIM*1

1Dept. of Integrated Biomedical and Life Sciences, College of Health Science, Korea University, Seoul 136-703, Korea. 2Dept. of Chemical Engineering and Biotechnology, Korea Polytechnic University, Sihung-si, Gyeonggi-do 429-793, Korea.


Ursodeoxycholic acid is known to reduce cholesterol absorption and has been used a drug to treat cholesterol gallstones. To date, ursodeoxycholic acid has been produced by seven-step chemical process from cholic acid. However, the process involved several problems such as a low yield to about 30%, the stereo- and regio-selectivity, several steps of purification and crystallization to obtain the product with a high degree of purity, and the generation of highly toxic wastes. We developed a bioconversion process using a recombinant E. coli to produce 12-oxo-chenodeoxycholic acid and 7,12-dioxo-lithocholic acid, which are critical intermediates to synthesize ursodeoxycholic acid from cholic acid. The gene encoding 12α-hydroxysteroid dehydrogenase from Clostridium chauvoei ATCC 29733 was transformed into E. coli BL21 (DE3). With whole cells of the recombinant E. coli, 12-oxo-chenodeoxycholic acid was converted from cholic acid.

However, 7,12-dioxo-lithocholic acid concentration was increased as reaction time progressed, because E. coli itself possesses 7α-hydroxysteroid dehydrogenase. The bioconversion reaction was reactivated by adding NADP+ or NAD+ cofactors. Therefore, in order to increase the number of reaction using whole cell, regeneration of NAD+ or NADP+ should be considered.

*Keywords: 12-Oxo-chenodeoxycholic acid,12alpha-Hydroxysteroid dehydrogenase,Recombination

36

Production of Taxadiene by Metabolically Engineered Escherichia coil

Gye-Hwan KIM1, Chonglong WANG1, Jung-Hun KIM1, Hui-Jeong JANG1, Eui-Sung CHOI2 and Seon-Won KIM*1

1Division of Applied Life Science (BK21 Program), PMBBRC, Gyeongsang National University, Jinju 660-701, Korea. 2Industrial Biotechnology Research Center, KRIBB, Daejeon 305-806, Korea.


Taxol is a widely used anticancer agent isolated from the bark of the pacific yew tree. At the moment, plant cell culture is another route used for its commercial supply besides bark extraction. The biosynthesis of taxadiene, the first committed intermediate of taxol biosynthesis, starts from the formation of universal precursors isopentenyl diphosphate(IPP) and dimethylallyl pyrophosphate(DMAPP) which are generated from either the mevalonate(MVA) pathway or the 2C-methyl-D-erythritol-4-phosphate(MEP) pathway. Isoprenyl pyrophosphate synthase catalyzes the condensation of IPP and DMAPP to farnesyl pyrophosphate(FPP) by FPP synthase and then to geranylgeranyl pyrophosphate(GGPP) by GGPP synthase(GGPPS). GGPP is cyclized and rearranged by taxadiene synthase(TXS) to synthesize taxadiene. In this study, we assembled TXS with various GGPPS to construct the taxadiene synthase operons. The capacities of these operons were assessed in the background of the overexpression of the MVA pathway. This work was supported by a grant (NRF-2010-C1AAA001-0029084) from the National Research Foundation, the Intelligent Synthetic Biology Center of Global Frontier Project funded by the MEST (2011-0031964), and a grant from the Next-Generation BioGreen 21 Program (SSAC, grant#: PJ009522003), RDA, Korea.

*Keywords: Taxol,Taxadiene,Escherichia coil

37

Metabolic Engineering of Pichia pastoris for Hyaluronic acid Production with High Molecular Weight

Euijoon JEONG and Jung Hoe KIM*Dept. of Biological Sciences, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon 305-701, Korea.


Hyaluronic acid (HA) is a linear high molar mass and natural polysaccharide composed of alternating glucuronic acid and N-acetyl-glucosamine. It has found a number of applications but only High MW (>1 MDa) is used in medicine and cosmetics. Extractive method using animal tissues is too costly and time consuming. Streptococcal fermentation is feasible for mass production. However, pathogen concerns and shortage of sugar precursors are disadvantages. To overcome the limits, we have firstly used yeast as a host because it has greater sugar pool that has potential to produce greater HA. Secondly, HA synthase from Xenopus laevis which has excellent polymerization rate of HA precursors has been used for HA synthesis. Finally, all genes on HA pathway are closely related to sugar precursors pool, so overexpression of these genes would be contributed to expand amounts of HA precursors. We performed that hasA and hasB from Xenopus laevis were introduced into Pichia pastoris to produce HA. Then, we constructed cassettes by using HA genes and overexpressed by various combinations of cassettes. So far, the recombinant Strains produced a maximum of 1.7 g/l HA in 1L fermenter experiments. The maximum molecular weight of the polymer from various Pichia strains is over 2.5 MDa.

*Keywords: Metabolic engineering,Pichia pastoris,Hyaluronic acid

38

A metagenome-derived enzymes for isomerization of hydroxyl groups of aromatic compounds

Ji Young LEE, Joon Young OH, Ji-Eun CHOI, Hye Young NA and Jae Kwang SONG*Dept. of Research center for bio-based chemistry, Korea Research Institute of Chemical Technology, Daejeon 305-600, Korea.


Metagenomes are genetic materials recovered directly from an environmental sample, include those of microorganisms which have not been cultured thus far. Metagenomics plays an important role in revealing novel sequences, genes and biological pathways. Metagenomic library has been used to discover novel and

potentially important enzymes for industrial applications on the basis of the functional and sequence-based search. A simple and efficient high-throughput screening system was established to identify novel isomerization activity for aromatic compounds from the metagenomic libraries. We have discovered a novel isomerase among 740,000 clones in metagenomic libraries. The gene was identified out of about 30 putative enzyme-encoding genes and confirmed through the subcloning experiment. Then we attempted to develop a novel biotransformation reaction for organic synthesis. This enzyme was overexpressed in E. coli and tested for a biotransformation reaction in various reaction conditions for an optimization. Moreover, the combination of reduction and isomerization was designed for an easy use of this reaction, and we prepared whole cell catalyst having dual activity. Using this whole cell catalyst, multi-step reaction was successfully carried out to give rise to good yields.

*Keywords: Metagenomic library,aromatic compounds,high-throughput screening

39

Enhancing 1-Butanol Tolerance of Escherichia coli by the Overexpression of the DnaJK-GrpE Chaperone System

Le Minh BUI , Almando GERALDI, Ziaur RAHMAN and Sun Chang KIM*Dept. of Biological Sciences, KAIST, Daejeon 305-701, Korea.


1-butanol has been introduced as an alternative biofuel which is superior to ethanol and has thermodynamic properties close to gasoline. Even though 1-butanol can be produced biologically, the cellular toxicity of 1-butanol is a major limit to reach the commercial production level. Among the general strategies to enhance the 1-butanol tolerance of microbial hosts, use of the molecular chaperone DnaJK-GrpE was successfully applied to improve the growth of E. coli under 1-butanol stress. Other combinatorial effects of the chaperone overexpression and physiological changes of the plasma membrane were also evaluated.

*Keywords: 1-butanol tolerance,molecular chaperone,DnaJK-GrpE

40

Two-step process using immobilized Saccharomyces cerevisiae and Pichia stipitis for ethanol production from Ulva pertusa Kjellman hydrolysate


1Department of Biotechnology, Korea National University of Transportation, Jeungpyung, Chungbuk 368-701, Republic of Korea. 2Department of Medical Biomaterials Engineering, Kangwon National University, Chuncheon 200-701, Republic of Korea. 3Korea Institute of Ocean Science and Technology, Ansan, Gyeonggi-do 426-744, Republic of Korea. 4Department of Food Science and Engineering, Seowon University, Cheongju, Chungbuk 361-742, Republic of Korea.


We established a two-step production process using immobilized S. cerevisiae and P. stipitis to produce ethanol from seaweed U. pertusa Kjellman hydrolysate. The process was designed to completely consume both glucose and xylose. In particular, the yeasts were immobilized using DEAE-corncob and DEAE-cotton, respectively. The first step of the process included a continuous column reactor using immobilized S. cerevisiae, and the second step included a repeated-batch reactor using immobilized P. stipitis. It was verified that the glucose and xylose in 20 l of medium containing the hydrolysate was converted completely to about 5.0 g/l ethanol through the two-step process, in which the overall ethanol yield from total reducing sugar was 0.37 and the volumetric ethanol productivity was 0.126 g/l/hr. This two-step process will not only contribute to the development of an integrated process for ethanol production from glucose and xylose-containing biomass hydrolysates, but could also be used as an alternative method for ethanol production.

*Keywords: Saccharomyces cerevisiae,Pichia stipitis,Ulva pertusa Kjellman

41

RecA Mediated SOS Response Providing Tolerance Against Geraniol in Escherichia coli

Asad Ali SHAH1, Chonglong WANG1, Sang-Hwal YOON1, Eui-Sung CHOI2 and Seon-Won KIM*1

1Division of Applied Life Science (BK 21 Program), PMBBRC, Gyeongsang National University, 501 Jinju Dae-ro , Jinju 660-701, Korea. 2Industrial Biotechnology Research Center, Korean Research Institute of Bioscience and Biotechnology, 125 Gwahak-ro, Yuseong-gu, Daejeon 305-806, Korea.


Geraniol is an important commodity chemical and also considered as candidate for advanced biofuel among

isoprenoids. However, Escherichia coli DH5α known to an optimal host for isoprenoids production have little tolerance to geraniol, restraining the economic viability of this biofuel candidate. Rational engineering for higher geraniol production relies upon identification of genes conferring tolerance and understanding the mechanism of tolerance. To date, the current knowledge of essential genes involved in geraniol tolerance is limited. RecA gene enhancing the geraniol tolerance was identified through screening of shot-gun library clones constructed from E. coli chromosomal DNA. It was deciphered that recA provided tolerance against geraniol through SOS response network. The SOS induced tolerance is a novel finding, by which cells can counteract toxicity of geraniol. This SOS based mechanism of geraniol tolerance is a significant addition in overcoming of biofuel toxicity to a production host. This work was funded by a grant (NRF-2010-C1AAA001-0029084) from the National Research Foundation, the Intelligent Synthetic Biology Center of Global Frontier Project funded by the MEST (2011-0031964) and a grant from the Next-Generation BioGreen 21 Program (SSAC, PJ009522003) RDA, Korea.

*Keywords: RecA,Geraniol tolerance,SOS response

42

Metabolic engineering of Escherichia coli for the production of fumaric acid

chanwoo SONG, dongin KIM, sol CHOI, jaewon JANG and sangyup LEE*Dept. of chemical and biomolecular engineering, KAIST, Daejeon 305-701, Korea.


In this study, Escherichia coli was metabolically engineered for the production of fumaric acid. To design optimal production pathway to fumaric acid, firstly the iclR gene was deleted to redirect the carbon flux through the glyoxylate shunt. In addition, the three known fumarase genes (fumA, fumB and fumC) were also deleted. The resulting strain was able to produce 1.45 g/L of fumaric acid from 15 g/L of glucose in flask culture. This base strain was further engineered by overexpression of the native ppc gene, encoding phosphoenolpyruvate carboxylase (PPC), based on in-silico aided prediction strategy, which resulted in the production of 4.09 g/L of fumaric acid. And then, the arcA and ptsG genes were sequentially deleted to reinforce the oxidative TCA cycle flux, and the aspA gene was deleted to block the conversion of fumaric acid into L-aspartic acid. The native promoter of the galP gene was replaced with the strong trc promoter to increase glucose uptake rate and fumaric acid productivity. Fed-batch culture of the final strain CWF812 allowed production of 28.2 g/L fumaric acid in 63 h. (Development of systems metabolic engineering platform technologies for biorefineries; NRF-2012-C1AAA001-2012M1A2A2026556) funded by the Ministry of Education, Science and Technology)

*Keywords: Fumaric acid,Escherichia coli,Metabolic engineering

43

Efficient Transcription of Meta-genomic Genes by Random Insertion of T7 Promoter in Escherichia coli

Haseong KIM*2, Haseong KIM3, Su-Lim CHOI3, Eugene RHA3, Seo-Hyun KIM3, Seung-Goo LEE3

1KRIBB. 2한국생명공학연구원 바이오화학연구센터. 3Biochemicals and Synthetic Biology Research Center.


Meta-genome is an unlimited resource for novel biocatalysts that contribute cost-effective industrial biorefinery processes. Although there are various well-designed methods to identify novel enzymes from meta-genome libraries, most are suffered from a problem that metagenomic genes are not expressed well in heterologous surrogate hosts. In order to improve the expression efficiency of the metagenomic libraries, we constructed synthetic transposons containing bidirectional T7 promoters, and randomly inserted them into metagenomic DNAs in fosmid vectors in E. coli. When the modified library were investigated for halo-forming colonies on tributyrin agar plates, they showed dramatic increases in both the appearance time (2-fold) and the size of haloes (~10-fold), compared to the original library. Therefore, the insertion of synthetic transposons containing bidirectional T7 promoters increases the possibility that one can identify useful genes having weak activity, which will be practically useful for the rapid screening from in large-scale genomic resources.

*Keywords: Meta-genome,Gene expression

44

Secretion of cellulases from Escherichia coli for consolidated bioprocessing

Seohee KANG1, Sang-Hwal YOON1, Sunil S. GHATGE1, Hyun-Dong SHIN2, Mi-Kyoung KIM3, Young Soon UM4 and Seon-Won KIM*1

1Division of Applied Life Sciences (BK21), PMBBRC, Gyeongsang National University, Jinju, 660-701, Korea. 2School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, Atlanta,. 3Naturence Corp., Yeongi-gun, Chungnam, 339-824, Korea. 4Korean Institute Of Science and Technology (KIST), Daejeon,

305-806, South Korea.


In an effort to develop a low-cost fermentation using biomass, consolidated bio-processing (CBP) is emerging as an important issue. By carrying out saccharification and fermentation in one pot, this process would cut down an expense used in protein purification and separation of reducing sugar, the end-product of saccharification. For the development of tractable CBP host strain, E. coli was engineered to produce cellulases, hence it can grow on cellulose. Cellulases have been known to have high optimum reaction temperature above 50℃. However, cellulases for CBP are required to show high activity at a growth temperature of host strain. Thus, we searched candidates for the CBP enzyme from marine bacteria such as Saccharophagus degradans and Hahella chejuensis which live at low temperature. Cellulases were expressed and released from E. coli. After incubation, the enzymes in culture broth were used to determine their activity and synergism on insoluble cellulose. The work was supported by a grant from the International Collaborative R&D Program of Knowledge Economy Technology Innovation Program, MKE, a grant (NRF-2010-C1AAA001-0029084) from the National Research Foundation, MEST of Korea. The financial support from the KIST through Future Key Technology Program.

*Keywords: CBP,marine bacteria,cellulase

45

Enhanced bioethanol production from seaweed, Gelidium amansii, using yeasts acclimated to high osmotic pressure

Hye young CHO and Sung-Koo KIM*Department of Biotechnology, Pukyong National University, Busan 608-737, Korea.


Gelidium amansii, red seaweed, has advantage of high carbohydrate contents converted to fermentable sugar such as glucose and galactose. In spite of those advantages, seaweed has problem with high salinity over 10 %. High salt concentration in the hydrolysate from G. amansii repressed fermentation process of yeasts. Therefore, acclimation of high salt concentration to yeasts is required to resolve the problem.In this study, pretreatment for the saccharification of Gelidium amansii was performed with 16 % (w/v) seaweed slurry, 1 % (v/v) H2SO4 and autoclaved at 121℃ for 60 min. To obtain glucose, enzymatic saccharification was carried out using 8.4 EGU/ml of Celluclast 1.5 L and 1.2 FBG/ml of Viscozyme L for 2 days. As a result of saccharification, the galactose of 40 g/l and the glucose concentration of 23 g/l were obtained. Fermentation was performed with Candida tropicalis KCTC 7212, Kluyveromyces marxianus KCCM 1129 and Saccharomyces cerevisiae KCCM 1129 acclimated to high osmotic pressure, respectively. Using acclimated C. tropicalis, K. marxianus and S. cerevisiae, ethanol concentrations of 27.6 g/l, 32.2 g/l and 22.1 g/l were produced, respectively.

*Keywords: Gelidium amansii,Thermal acid hydrolysis,Enzymatic saccharification

46

D-Lactic acid Production from Xylose to D-lactic acid by Recombinant Saccharomyces cerevisiae

Ja-Ryong KOO, Se Kyung KIM, Yong Ho NOH, Hye Min PARK and Hyun Shik YUN*Department of Biotechnology, Inha University, Nam-Gu, Incheon 402-751, Korea.


The yeast Saccharomyces cerevisiae efficiently ferments glucose for useful products. However, it is unable to utilize xylose in lignocellulosic materials. Since yeasts are more tolerant to low pH than E. coli, high density cultivation for the production of lactic acid is possible. In this study, recombinant Saccharomyces cerevisiae was constructed to convert xylose to D-lactic acid. A recombinant Saccharomyces cerevisiae containing D-xylose isomerase from Lactobacillus hilgardii strain ATCC 8290 and D-lactate dehydrogenase from Lactobacillus rhamnosus ATCC 53103 gene was constructed. The recombinant cells could uptake xylose and produce D-lactic acid. The expression of xylA and ldhD in Saccharomyces cerevisiae was confirmed. The D-lactic acid was very high optical purity of 98% or higher.

*Keywords: Saccharomyces cerevisiae,xylose isomerase,lactate dehydrogenase

47

Heterologous Production of 4‑O‑Demethylbarbamide, a new barbamide derivative, which is a Marine Cyanobacterial Natural Product

hea luyng SHIN, Eun ji KIM, Young Ji YOO, Eunji KIM, Hwa Gyung CHOI, Myoun Su KIM, Ji Yoon BEOM and Yeo Joon Yoon YOON*

Dept. of Chemistry and Nano Science, Ewha Womans University, Seoul 120-750, Korea.


This paper show that Heterologous expression of the barbamide biosynthetic gene cluster, obtained from the marine cyanobacterium Moorea producens, in the Streptomyces venezuelae, As a resulted in the production of a new barbamide congener 4-O-demethylbarbamide, demonstrating the potential of this approach for investigating the assembly and tailoring of complex marine natural products.Here, we demonstrate a new barbamide derivative that only lacks an O-methyl group relative to the parent structure but shows several-fold improved molluscicidal activity compared to barbamide. Most important thing in this paper is the first successful functional heterologous expression of a marine cyanobacterial NRPS/PKS gene cluster in a genetically amenable terrestrial host. The structure of newly produced barbamid derivative was confirmed through mass spectroscopy anlysis and NMR spectroscopy and then Molluscicidal activity of 4-O-demethylbarbamide.The results presented herein provide an important precedent and approach for the biosynthetic study of marine natural products derived from genetically intractable organisms. In this work, we demonstrated heterologous production and identification of a novel barbamide derivative, which had not been previously isolated from the natural producer.

*Keywords: marine cyanobacterium Moorea producens,heterologous expression in the Streptomyces venezuelae,novel barbamide derivative

48

Butyrate Production in Engineered Escherichia coli with Synthetic Scaffolds

Sang-Woo LEE , Jang-Mi BAEK, Suman MAZUMDAR, Moo-Young JUNG and Min-Kyu OH*Dept. of Chemical and Biological Engineering, Korea University, Seoul, Korea.


Butyrate pathway was constructed in recombinant Escherichia coli using the genes from Clostridium acetobutylicum and Treponema denticola. However, the pathway constructed from exogenous enzymes did not efficiently convert carbon flux to butyrate. Three steps of the productivity enhancement were attempted in this study. First, pathway engineering to delete metabolic pathways to by-products successfully improved the butyrate production. Second, synthetic scaffold protein that spatially co-localizes enzymes was introduced to improve the efficiency of the heterologous pathway enzymes, resulting in threefold improvement in butyrate production. Finally, further optimizations of inducer concentrations and pH adjustment were tried. The final titer of butyrate was 4.3 and 7.2 g/L under batch and fed-batch cultivation, respectively. This study demonstrated the importance of synthetic scaffold protein as a useful tool for optimization of heterologous butyrate pathway in E. coli.

*Keywords: Scaffold,Synthetic biology,Butyrate

49

Bioethanol Production from Euchema spinosum and Reduction of Toxicity using Active Charcoal

Min-ji KIM, Jung-Soo KIM and Sung-Koo KIM*Dept. of Biotechnology, Pukyong National University, Busan 608-737, Korea.


Seaweed is a third-generation biomass that can be used in bioenergy production and has many advantages. The seaweed grows quickly, is lignin-free and is not used as a food crop. The red seaweed, Euchema spinosum, is cultivated in Indonesia. Seaweed slurry was pretreated by thermal acid hydrolysis and enzyme treatment. Optimal pretreatment condition was determined with 11% (w/v) solid content of seaweed and 150 mM sulfuric acid at 121℃ for 40 min. Enzymatic saccharification was carried out using Celluclast 1.5L and Viscozyme L at pH 4.5, 40℃. However, E. spinosum produced 5-HMF of 6 g/L after thermal acid hydrolysis. To removed 5-HMF, the addition of active charcoal was carried out in the range of 3-6% at room temperature for 2 hour. 5-HMF was reduced from 5.7 g/L to 0.8 g/L on 5% active charcoal. The ethanol fermentation was carried out using SHF with Kluyveromyces marxianus KCTC 7150. The ethanol fermentation from 5-HMF removed E. sponosum hydrolysate produced 18 g/L using K. marxianus for 48 h. Ethanol was produced 10 g/L during 36 h when 5-HMF existed. HMF was affected on yeast growth and ethanol production.

*Keywords: Euchema spinosum,Bioethanol,Seaweed

50

Identification of Protein-Protein Interaction in Bacterial Cells by Active Particles Display

Yu Jung KIM1, Jongsik GAM1, Su-Lim CHOI1, Hak-Sung KIM2 and Seung-Goo LEE*1

1Biochemicals and Synthetic Biology Research Center, KRIBB. 2Department of Biological Sciences, KAIST.


The active particles, non-classical inclusion bodies (ncIBs), are composed from significant amounts of properly folded and biologically active proteins, trapped into the network of misfolded proteins. Recently, active particles have been focused on a new immobilized enzyme carrier. Here we developed a protein-protein interaction (PPI) system using the active particles as interaction trappers in a bacterial cell. A cellulose-binding domain (CBD) from Cellulomonas fimi was introduced as a fusion partner which induces the formation of active particles. We clearly demonstrated that a fluorescence labeled prey protein with high binding affinity was co-localized onto active particles displaying bait proteins, while a prey with no affinity was not. These results indicate that our active particle display technique would provide a powerful method to detect protein-protein or protein-ligand interactions by observing microscopic co-localization. Also this method along with flow cytometry providing enables us to identify the interacted proteins and their characteristics rapidly

*Keywords: protein-protein interaction (PPI), active particle, flow cytometry, fluorescence microscopy

51

Accurate and High-throughput Detection of Increased Bacterial Poly-3-Hydroxybutyrate Using New Fluorescent Probes.

Jieun CHOI, Hye Young NA, Ji Young LEE, Joon young OH and Jae Kwang SONG*Research Center for Bio-based Chemistry, Korea Research Institute of Chemical Technology, Daejeon, Korea.


Polyhydroxyalkanoates (PHA) are commercially interesting thermoplastic because they are produced from renewable resources and are completely biodegradable. Poly-3-hydroxybutyrate (PHB) is the most abundant occurring PHA and can be synthesized in a variety of microorganisms. The Recombinant E.coli cloned with PHB biosynthesis genes has been well studies and is considered a primary candidate for industrial PHB production based on its high accumulation of PHB. Therefore, needs for simultaneous monitoring of the produced PHB concentration during cultivation is strongly demanded. Many molecular fluorescent probes including Nile red and BODIPY for detecting accumulating PHB in vivo system have been recovered. Recently, LipidGreen as a new small molecule probe which stains accumulated lipids in animal cell and zebrafish was reported. In this study we used LipidGreen to visualize bacterial PHB generation and to quantify. From the series of experiments, we confirmed that it could penetrated cell wall and efficiently bound to polyesters with fluorescence microscopic images. Furthermore, fluorescence intensity of LipidGreen combined with PHB was successfully measured through fluorescence spectrometer, and the amount of accumulated PHB had linear correlation with fluorescence intensity.

*Keywords: Poly-3-hydroxybutyrate,High-throughput Screening,LipidGreen

52

Ethanol production from glycerol using Pachysolen tannophilus in surface-aerated fermentor


1Department of Biotechnology, Korea National University of Transportation, Jeungpyung, Chungbuk 368-701, Republic of Korea. 2Department of Medical Biomaterials Engineering, Kangwon National University, Chuncheon 200-701, Republic of Korea. 3Korea Institute of Ocean Science and Technology, Ansan, Gyeonggi-do 426-744, Republic of Korea. 4Department of Food Science and Engineering, Seowon University, Cheongju, Chungbuk 361-742, Republic of Korea.


We investigated on the condition for ethanol production from glycerol after screening of a yeast P. tannophilus ATCC 32691. When the yeast produced ethanol form glycerol, it was important that the aeration was finely controlled. Therefore, we attempted to produce ethanol using surface-aerated fermentor. When we produced ethanol using 880 ml of YPG medium (1% yeast extract, 2% peptone, 2% glycerol), the optimal aeration condition for ethanol production was that surface aeration rate and agitation speed were 500 ml/min and 300 rpm, respectively. Finally, we accomplished the fed-batch culture for ethanol production form glycerol using the surface-aerated fermentor, in which the maximum ethanol production and the ethanol yield from glycerol (Ye/g) were 5.74 g/l and 0.166, respectively, at the elapsed time of the culture.

*Keywords: Ethanol,Fed-batch culture,Glycerol

53

Engineering MVA pathway for production of santalene in E.coli

LIYANG YANG, Chonglong WANG, Jia ZHOU, Jung-Hun KIM, Hui-Jeong JANG, Gye-Hwan KIM, Guang-Bin LI, Sang-Hwal YOON and Seon-Won KIM*Division of Applied Life Science , Gyeongsang National University, Jinju 660-701, Korea.

*Corresponding author: [email protected]([email protected])

Santalene is the major olefinic sesquiterpenes responsible for the fragrance of sandalwood oil and derived from the precursor of FPP which can be synthesized from IPP and its isomer DMAPP. In this study, we generated a library with different orders of the enzymes involved in the bottom portion of MVA pathway to differentiate their expression level. Santalene synthase was co-transformed to assess their performance of IPP and DMAPP synthesis. The efficient bottom MVA pathway plasmid selected from the library was further optimized by the sequential replacement of the pathway enzymes with their homologs from different species. By this approach, we successfully produced santalene of 146.7 mg/L from E.coli harboring the well-performing bottom portion of MVA pathway with addition of 5mM MVA. This study shows the highest production of santalene reported up to date and provides a new platform for isoprenoids production. This work was supported by a grant (NRF-2010-C1AAA001-0029084) from the National Research Foundation, the Intelligent Synthetic Biology Center of Global Frontier Project funded by the MEST (2011-0031964), and a grant from the Next-Generation BioGreen 21 Program (SSAC, grant#: PJ009522003), RDA, Korea.

*Keywords: MVA pathway,santalene,E.coli

54

Programmable Synthetic Pathway: Fine-tuned by Inverting Promoter

Donghui CHOE1,2, Soo-in LEE1,2, Suhyung CHO1,2, Sun Chang KIM1,2 and Byung-Kwan CHO*1,2

1Department of Biological Sciences and KIB, KAIST, Daejeon, South Korea. 2Intelligent Synthetic Biology Center, South Korea.


Since synthetic biology has been emerged, biological research which just studied about how to enhance cell’s productivity has shifted its paradigm toward re-programming of organism considering physiology and systems of the cell. With this trend, genetic circuit such as logic gates has been embodied in cellular system to control metabolic pathways. However, many of these systems work in irreversible manner. To construct a programmable and reversible synthetic system, we designed a promoter, positioned between the specific binding sites of Mycobacterium phage Bxb1 integrase/excisionase, which can be inverted by site-specific DNA recombination. This invertible promoter can initiate transcription selectively according to the cellular signal. In this research, bio-indigo pathway was a target synthetic pathway and, also, quorum-sensing system was adopted as an indicator of level of metabolites. We demonstrated that pathway can be turned ON and OFF according to the amount of quorum molecules, respectively. This programmable synthetic system provides an efficient tool needed to advance fine-tuned controllable metabolic processes for industrial applications.(Supported by ISBC 2011-0031957)

*Keywords:

55

Engineering of Escherichia coli for the optimal expression of plant genes

Junhyeong LEE1 and Sung Kuk LEE*1,2

1School of Nano-Biosicence and Chemical Engineering, Ulsan National Institute of Science and Technology, Ulsan 689-798, Korea. 2School of Urban and Environmental Engineering, Ulsan National Institute of Science and Technology, Ulsan 689-798, Korea.


Useful products from plants have been used as drugs, dyes, perfumes and resins. However industrial production of these materials from plants has many limitations. So microorganisms are attractive for heterologous production of many useful compounds derived from plants. Escherichia coli is especially valuable as an industry microorganism because its growth rate is high and many genetic tools are available compared to other microbes. But functional expression of plant proteins in E. coli has not been satisfactory, probably because they have different codon usage bias. Genes involved in amino acid biosynthesis directly related to cell growth tested through complementary experiments, where E. coli genes were replaced with the corresponding plant genes. The expression of target genes involved in growth was analyzed by their growth rate. We have chosen four target plant genes that may not be expressed well in E. coli. For further study, rare tRNA promoters will be simultaneously engineered by multiplex automated genome engineering (MAGE) strategy and fast-growing cells will be selected. In addition to the promoter engineering, the expression of E. coli chaperones will be engineered to enhance plant protein folding by changing expression levels and time points of chaperone proteins.

*Keywords: Echerichia coli,tRNA/Chaperone,MAGE

56

Enhanced tyrosine and cadaverin production by Escherichia coli using synthetic regulatory small RNAs

Chanwoo SONG, Seungmin YOO, Dokyun NA, Hannah CHUNG, Hyegwon PARK and Sangyup LEE*Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Program), KAIST, Daejeon 305-701, Republic of Korea.


Small regulatory RNAs (sRNAs) are short non-coding RNAs that can finely control the expression of target genes in trans at post-transcriptional level in prokaryotes. An engineered E. coli strain (tyrR csrA repressed S17-1 strain) capable of producing 21.9 g/L of tyrosine was developed by combinatorial knockdown experiments on various candidate genes in 14 different strains using respective synthetic sRNAs. Also, sRNAs was applied to an already metabolically engineered strain producing cadaverine by applying a library of 130 synthetic sRNAs and repression of murE allowed 55% increase in cadaverine production. This study demonstrates the possibility for the efficient production of tyrosine and cadaverine by metabolically engineered E. coli using synthetic sRNAs. [This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries (NRF-2012-C1AAA001-2012M1A2A2026556) and the Intelligent Synthetic Biology Center through the Global Frontier Project (2011-0031963) of the Ministry of Science, ICT, and Future Planning (MSIP) through the National Research Foundation of Korea.]

*Keywords: synthetic sRNA,tyrosine,cadaverine

57

Retinoids Production from Recombinant E. coli Harnessing Retinoids Modification Enzymes

Hui-Jeong JANG1, Bo-Kyung HA1, Sang-Hwal YOON1, Chong-Long WANG1, Zhou JIA1, Deok-Kun OH2 and Seon-Won KIM*1

1Division of Applied Life Science (BK21), PMBBRC,Gyeongsang National University, Jinju 660-701, Korea. 2Department of Bioscience and Biotechnology, KonkukUniversity, Seoul 143-503, Korea.


Retinoids include retinol, retinal, retinoic acid, and retinyl esters, and also exist in other various structures differentiated by their functional groups. Retinal is converted to retinol and retinoic acid by retinol dehydrogenase and retinal dehydrogenase/oxidase, respectively. Retinol is esterified to retinyl esters by retinol acyltransferase. The formation of other retinoids derived from retinal by promiscuous enzymes in E. coli was considered. Because retinal could be converted into retinol, retinoic acid, and retinyl ester by cellular enzymatic reactions, these retinal derivatives were analyzed in the E. coli cultures. Based on this result, the retinoid synthesis pathway of E. coli was reengineered to produce a specific retinoid through elaborative genetic manipulations. Therefore, a composition of retinoids was differentiated by controlled expression of retinoids modification enzymes. This work was supported by a grant (NRF-2010-C1AAA001-0029084) from the National Rewearch Foundation, the Intelligent Synthetic Biology Center of Global Frontier Project funded by the MEST (2011-0031964), and a grant from the Next-Generation BioGreen 21 Program (SSAC,grant#:PJ009522003),RDA,Korea.

*Keywords: Recombinant E.coli,Retinoids

58

Long-term Fed-batch Fermentation with In Situ Gas-stripping of Alcohol Mixture Using Engineered Clostridium acetobutylicum Strains

Joungmin LEE, Yu-Sin JANG, Sung Jun CHOI and Sang Yup LEE*Dept. of Chem. Biomol. Eng., KAIST, Daejeon 305-701, Korea.


Solventogenic clostridia have been grabbing attention of many researchers since they are capable of producing n-butanol, a promising advanced biofuel. Unfortunately, many clostridial species produce acetone along with butanol, which is considered not appropriate as biofuel. Since genetic engineering of clostridia is still difficult compared to other familiar microbial hosts, we previously suggested rather a simple strategy, conversion of acetone into isopropanol, which are used as a gasoline additive for its high octane-rating. However, mass production of mixed alcohols is limited by high toxicity of butanol. In this study, we applied in situ gas-stripping to recover solvents from the fermentation broth of an engineered Clostridium acetobutylicum strain and thus to reduce butanol titer in the reactor. With in situ recovery, fermentation time could be successfully extended to ca. 300 hours without any notable symptoms of strain degeneration. [This work was supported by the Advanced Biomass R&D Center of Korea (ABC-2010-0029799) through the Global Frontier

Research Program of the Ministry of Education, Science and Technology (MEST). Further supports by BioFuelChem, EEWS program of KAIST, and the World Class University program (R32-2008-000-10142-0) of the MEST are appreciated.]

*Keywords: Metabolic Engineering,Butanol,In situ recovery

59

Regulation of Ttranscriptional Regulator Controlling the cbb Pathway in Rhodobacter sphaeroides

Hyun Jeong LEE2, Yang-Hoon KIM3 and Jiho MIN*1

1Dept. of Semiconductor and Chemical Engineering, Jeonju 561-756 South Korea. 2Dept. of Semiconductor and Chemical Engineering, Jeonju 561-756, South Korea. 3Department of Microbiology, Chungbuk National University, Cheongju 361-763, South Korea.


Rhodobacter sphaeroides is a nonsulfur photosynthetic bacterium that possesses two cbb operons, cbbI and cbbII, encoding exzymes involved in the Calvin-Bensom-Bassham (CBB) reductive pentose phosphate pathway of carbon dioxide fixation. In this study, the CbbR-binding sites on the cbb operon R. sphaeroides were characterized by chromatin immunoprecipitation (ChIP). The ChIP assay indicated that the CbbR protein binds to the upstream regions cbbF in cbbI operon and cfxB in cbbII operon. Various metabolites were used as activator or repressor. CbbR binding affinity upstream of cbbF and cfxB was enhanced in the presence of RuBP. In contrast, fructose 1,6-bisphosphate have different pattern by culture conditions. Also, we have used electrophoretic mobility shift assays (EMSAs) for major points to bind CbbR to the cbb genes. Our data demonstrated that the interaction between CbbR and cbb genes may represent a novel mechanism for transcriptional regulation of CbbR.

*Keywords: Rhodobacter sphaeroides,Transcriptional regulator,CbbR

60

Production of Recombinant Proteins in an Insertion Sequence-free Escherichia coli MS56

Myung Keun PARK, Jun Hyoung LEE, Kyung Seok YANG, Suk Chae JUNG and Sun Chang KIM*DepT. of Biological Sciences, KAIST, Daejeon 305-701, Republic of Korea.


The genomic stability and integrity of host strains are critical for the production of recombinant proteins in biotechnology because genomes of microorganisms contain numerous insertion sequences (ISs) that cause a variety of genetic rearrangements, resulting in adverse effects such as instability of genomes and recombinant clones. To minimize the harmful effects of ISs on the expression of recombinant proteins in Escherichia coli, we recently developed an IS-free E. coli strain (MS56) in which all ISs and unnecessary genes were removed from the E. coli MG1655 genome. Here, we compared the expression profiles of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and bone morphogenetic protein-2 (BMP2) in MG1655 and MS56. The hopping of ISs into the TRAIL and BMP2 genes occurred at the rate of ~10-8/gene/h in MG1655 whereas no IS hopping was observed in MS56. Even though IS hopping occurred very rarely, the IS-inserted TRAIL and BMP2 clones became dominant after fermentation because of its rapid growth, significantly reducing recombinant proteins production in batch fermentation. Our findings clearly indicate that IS hopping is detrimental to the industrial production of recombinant proteins, emphasizing the importance of the development of IS-free host strains.

*Keywords: insertion sequences, genomic stability, E. coli

61

Development of Phyto-sensor: A Synthetic Biology approach

sujin KIM, Myung Hee KIM, Sang Jun LEE, Ohsuk KWON, Choong-Min RYU, Eugene RHA and Seung-Goo LEE*Korea Research Institute of Bioscience Bioscience &Biotechnology(KRIBB),Daejeon, 305-806, Korea.


Synthetic biology is a new area of biological engineering that synthesizes new biological molecules, pathways, and networks to rewire and reprogram organisms.The engineering rules are expected to lead our lives closer to new chemicals, green fuels, and targeted therapies for diseases. A particular interest has been given to the development of sentinel plants or phyto-sensors to provide a rapid, low-cost, in-situ monitoring of environmental hazards and plant diseases. The sensors are useful for the ecological risk assessment of agricultural and industrial chemicals. In this regard, we are interested in developing phyto-sensors that sense

hazardous aromatics such as explosive trinitrotoluenes (TNTs). However, because no sufficient knowledge on how plants respond to aromatics is known, we focus on getting proper Bio-Bricks from bacterial systems, altering the specificity and sensitivity, and finally implementing them into plant chassis. In this study, we employed the bacterial two-component system (bacterial TCS) that is responsive to aromatic hazards such as toluene, dinitrotoluene, trinitrotoluene (TNT). The bacterial TCS was constructed by a fusion with VP16 transcription activator. Parts for interchangeable sensors and reporters were developed successfully.

*Keywords: Bio-Bricks,environmental hazards,phyto-sensors

62

Free fatty acid generation for biodiesel conversion in Rhodococcus opacus PD630

Chanwoo SONG, Hyemi KIM and Sangyup LEE*Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Program), KAIST, Daejeon 305-701, Republic of Korea.


One of the Rhodococcus species, R. opacus PD630 was isolated from a soil sample collected in Germany. It is well known to accumulate incredible amounts of triacylglycerols (TAGs) which can be isolated and chemically converted to fatty acid methyl esters (FAMEs). FAMEs generated from TAGs can be used for a diesel alternative. In this study, we overexpressed the heterologous TAG lipase from a few of organisms R. opacus PD630 using the R. opacus-Escherichia coli shuttle vectors to generate free fatty acids from TAGs. Fatty acids generated from TAGs in fed-batch fermentation were chemically converted to FAME with 30 g/L in titers. We also overexpressed Acinetovactor baylyi ADP1 TAG lipase in R. opacus PD630 to enhance the production of fatty acids which is glycerol free form. Overexpression of A. baylyi lipase in R. opacus PD630 resulted in increased free fatty acids in the flask cultivation. [This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries (NRF-2012-C1AAA001-2012M1A2A2026556) of the Ministry of Science, ICT, and Future Planning (MSIP) of Korea and the Advanced Biomass R&D Center of Korea (ABC-2011–0028386) through the Global Frontier Research Program of MSIP.]

*Keywords: R. opacus PD630,fatty acid methyl esters

63

A chimeric FPP Synthase Mediates the Synthesis of Unusual Z,E-FPP in E. coli

Chonglong WANG, Jia ZHOU, Jung-Hun KIM, Hui-Jeong JANG, Liyang YANG, Gye-Hwan KIM, Guang-Bin LI, Sang-Hwal YOON and Seon-Won KIM*Division of Applied Life Science (BK21 Program), PMBBRC, Gyeongsang National University, Jinju 660-701, Korea.


Production of Z-type farnesyl diphosphate (FPP) has not been reported in E. coli. Here we present the fusion enzyme (ILRv) of E. coli E,E-FPP synthase (IspA) and Mycobacterium tuberculosis Z,E-FPP synthase (Rv1086), which can produce primarily Z,E-FPP rather than E,E-FPP, the predominant stereoisomer found in most organisms. Z,E-farnesol (FOH) was produced from E. coli harboring the bottom portion of the MVA pathway and the fusion FPP synthase (ILRv) at a titer of 115.6 mg/L in 2YT medium containing 1% (v/v) glycerol as a carbon source and 5 mM mevalonate. The Z,E-FOH production was improved by 15-fold, compared with 7.7 mg/L obtained from the co-overexpression of separate IspA and Rv1086. The Z,E-FPP was not metabolized in native metabolic pathways of E. coli. It would be of interest to produce Z,E-FPP derived sesquiterpenes from recombinant E. coli due to no loss of Z,E-FPP substrate in endogenous metabolism of the host strain. This work was supported by a grant (NRF-2010-C1AAA001-0029084) from the National Research Foundation, the Intelligent Synthetic Biology Center of Global Frontier Project funded by the MEST (2011-0031964), and a grant from the Next-Generation BioGreen 21 Program (SSAC, grant#: PJ009522003), RDA, Korea.

*Keywords: Z,E-FPP,FPP synthase,Protein fusion

64

Development of a High-Throughput Screening Method for the Selection of High Alkane-Producing Escherichia coli Strains

ziaur RAHMAN, Le Minh BUI and Sun Chang KIM*Dept. of Biological Sciences. KAIST, Daejeon 305-701, Korea.


Alkanes are the promising replica of gasoline and jet fuel and the demand for alkanes as biofuels is ever increasing. To meet the growing demand for alkanes, efforts are made to engineer microbial systems. Nevertheless, the microbial productions of alkanes are far below our satisfaction. To screen and select the high alkane-producing (HAP) strains with rational engineering and/or directed evolution, a high-throughput strain selection method is needed. Here, we construct an artificial circuit for the selection of HAP strains. An alkane sensor plasmid (pALK) was constructed, consists of an alkane responsive promoter, PalkB and a green fluorescence protein (GFP) as a reporter. In the presence of alkanes, the circuit turned on and GFP was expressed. To observe the response of the PalkB promoter in E. coli harboring pALK, alkanes in several concentrations were added. The promoter responded in a concentration-dependent manner. For the selection of HAP strains, the pALK was co-transformed with alkane-producing plasmid in E. coli. With our artificial circuit, HAP strains could be screened successfully. Our results suggest that pALK could be used as a reliable tool for the rapid screening of HAP hosts and to facilitate the process of rapid strain development.

*Keywords: Alkanes,Biofuels,Synthetic biology

65

Agitation Programmable Picoliter Droplet Arrays for HTS of Escherichia Coli

Ji Won LIM1, Taesung KIM2 and Sung Kuk LEE*1

1Ulsan National Institute of Science and Technology, School of Nano Bio-Chemical engineering. 2Ulsan National Institute of Science and Technology, School of Mechanical and Advanced Material Engineering.


We report an integrated, versatile microfluidic system that produces picoliter droplets, traps them on a microcavity array located at a downstream area and then agitates them in a programmable format. Water-in-oil based droplets generated at an upstream region are individually, sequentially captured by the microcavities located at a downstream region by using a specific gravity difference between water (nutrient media) and oil (hexadecane). E. coli cells encapsulated within medium droplets in the microcavities are grown both in a static and dynamic manner by manipulating flow rates of oil over the droplet array. Therefore, it is possible to program an agitation cycle of the droplets for the entire period of growth time, which is one of the common and conventional methods for the better growth of bacterial cells. In addition, we demonstrate that the same microfluidic platform can be applied to high-throughput screening (HTS) of metabolites produced by recombinant E. coli cells

*Keywords: high throughput screening,microdroplet,agitation

66

Generation of Novel Compound by Enzymatic Modification of 7,8-Dihydroxyflavone by Methylation and Glycosylation

KOIRALA NIRANJAN, Ramesh Prasad PANDEY, Biplav SHRESTHA, Joo-Ho LEE and Jae Kyung SOHNG*Department of Pharmaceutical Engineering, Institute of Biomolecule Reconstruction, Sun Moon University, Asansi, Chungnam, 336-708, Republic of Korea.


In this study, an O-methyltransferase gene, SpOMT-2884, was identified from Streptomyces peucetius ATCC27952. Among the several substrates tested for SpOMT-2884 catalyzed O-methylation of flavonoids, 7,8-dihydroxyflavone (7, 8-DHF), quercetin, luteolin, fisetin and rutin. 7,8-DHF was found to be the best substrates. We further proceeded for in-vivo biotransformation of 7,8-DHF where we used E. coli BL21 (DE3) expressing SpOMT-2884 as a biocatalyst for the production of methylated derivative of 7,8-DHF. The supplementation of 0.2 mM of 7, 8-DHF in the growing induced culture of E. coli BL21 (DE3) harboring pET28-SpOMT-2884 recombinant resulted in the production of 7-methyl-8-hydroxyflavone which was confirmed by HPLC (Rt:17 min), high resolution LC-QTOF-ESI/MS (m/z+ 269.08) and NMR spectroscopy. Further, this enzymatically synthesized methylated derivative of 7, 8-DHF was used as a substrate invitro for glycosylation by Yjic, a glycosyltransferase from Bacillus licheniformis DSM13. This in-vitro reaction mixture analysis revealed the presence of glycosylated product which was confirmed by HPLC and LC-QTOF-ESI/MS (m/z+ 431.13). The glycosylation of the target was further supported by our findings from insilico docking analysis.

*Keywords: 7,8-dihydroxyflavone,Methylation,Glycosylation

67

Artificial Biosynthesis of Coumaric Acid, Caffeic Acid and Ferulic Acid in Escherichia coli Strain

Sun-young KANG1,2, Oksik CHOI1, Bang Yeon HWANG2 and Young-Soo HONG*1

1Chemical Biology Research Cente, Korea Research Institute of Bioscience and Biotechnology. 2Department

of Pharmacy Graduate School, Chungbuk National University, Cheongju.


The phenylpropanoid metabolites are an extremely diverse group of natural products biosynthesizedby plants, fungi, and bacteria. Although these compounds are widely used in human health care and nutrition services, their availability is limited by regional variations, and isolation of single compounds from plants is often difficult. Recent advances in synthetic biology and metabolic engineering have enabled artificial production of plant secondary metabolites in microorganisms. We develop an Escherichia coli system containing an artificial biosynthetic pathway that yields phenylpropanoic acids, such as 4-coumaric acid, caffeic acid, and ferulic acid, from simple carbon sources. These artificial biosynthetic pathways contained a codon-optimized tal gene that improved the productivity of 4-coumaric acid and ferulic acid, but not caffeic acid in a minimal salt medium. These heterologous pathways extended in E. coli that had biosynthesis machinery overproducing tyrosine. Finally, the titers of 4-coumaric acid, caffeic acid, and ferulic acid reached 974 mg/L, 150 mg/L, and 196 mg/L, respectively, in shake flasks after 36-hour cultivation.

*Keywords: Phenylpropanoic acid,Biosynthesis

68

Metabolic profiling of central carbon metabolites and its 13C-isotope dynamic study in Kluyveromyces marxianus

joonyoung JUNG, tae yeon KIM, dae kyun IM and min kyu OH*Dept of chemical and biological eng, Korea Univ.


Calculation of fluxes in the central metabolic pathways has improved our understanding about microbial physiology of metabolically engineered strains. For this purpose, accurate, reliable and reproducible measurement of intracellular metabolites is prerequisite. The critical step is the establishment of proper quenching, extraction and sampling protocol, which ensures rapid arrest of all metabolic activity, effective extraction of target metabolites from the cells. Up to now, flux analysis using computational calculation tracing site-specific 13C pattern has been widely used. But new technology, metabolome-based 13C flux analysis, has been recently introduced, which directly measures isotope tracer conversion rates of target metabolites for flux analysisIn this study, we performed qualitative and quantitative analysis of intermediate metabolites on glycolysis, pentose phosphate pathway and TCA cycle with GC-MS. Base on the metabolite profiling method, isotope-based dynamic studies were performed with uniformly labeled 13C glucose. From time series isotopic studies, conversion rates of glucose into the central metabolic pathway were well demonstrated, which showed pattern of glucose utilization in K. marxinus strain.

*Keywords: 13C isotope,GC/MS,non-stationary

69

Engineering Escherichia coli towards an L-isoleucine Producer.

Joungmin LEE1, Ji Young KIM1, Jin Hwan PARK2 and Sang Yup LEE*1

1Dept. of Chem. and Biomol. Eng., KAIST, Daejeon 305-701, Korea. 2Samsung Advanced Institute of Technology, Yongin 446-712, Korea.


L-isoleucine is one of the essential amino acids in mammals, and it is used to manufacture beverages and animal feedstuffs. In this study, L-isoleucine overproducing E. coli strain was developed by rational metabolic engineering. L-threonine overproducing strain of E. coli TH20, a previously reported threonine-producing strain, was used as the starting strain, as L-isoleucine is synthesized from L-threonine through five enzymatic steps. The thrABC, ilvA, ilvIH, and ygaZH genes were amplified by plasmid-born overexpression. The ilvCED and lrp genes were also amplified by replacing the promoter in the chromosome to further increase the flux toward L-isoleucine. The final engineered E. coli strain was able to produce 9.46g/L of L-isoleucine with a yield of 0.14g/g of glucose by fed-batch culture. [This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries (NRF-2012-C1AAA001-2012M1A2A2026556) of the Ministry of Science, ICT, and Future Planning (MSIP) of Korea and the Intelligent Synthetic Biology Center (2011-0031963) through the Global Frontier Research Program of MSIP.]

*Keywords: L-isoleucine,Metabolic Engineering,Amino acids

70

Genetic Engineering of Escherichia coli to Improve Biofuel Tolerance

Jae Ok LEE, Hye Yun OH and Ok Bin KIM*Dept. of Life science, Ewha Womans University, 120-750 Seoul, Korea.


Biofuels like ethanol, butanol and diverse organic hydrocarbons are toxic to bacteria. Genetic engineering to improve tolerance against solvent is therefore a prerequisite for microbial production of biofuel. The organic solvent tolerances (OSTs) of gram-negative bacteria are related with the rigidity of cell membrane, composition of outer membrane, solvent efflux systems, osmoprotectants and other resistance systems. For targeted engineering to improve tolerance, we investigated genes that participate in relevant mechanisms to tolerance. Among the genes that affect the bacterial resistance, manipulations of three regulator genes had the best impact to improve tolerance. The enhanced solvent-tolerance of the strains was shown by spot assay and growth test. Finally the evidences of improved tolerance were analyzed by membrane composition and intracellular accumulation of solvent.

*Keywords: Organic Solvent Tolerance,Fatty acid composition,Intracellular accumulation

71

The Half-Adder circuit with Boolean logic computation using trans-splicing by a split DnaE intein

Dae Eun CHEONG, Yunjon HAN, Kyong-Cheol KO, Jong Hyun CHOI and Jae Jun SONG*Korea Research Institute of Bioscience and Biotechnology.


In general, published logic gates such as AND, OR, NOR and NAND are to be layered by sets consisted of transcription and translation processes more than three or require specialized hosts. These could not only have been made the design of synthetic networks to be complicated, but stimulate stochastic properties of biological systems occurring unpredictable errors of genetic programs in vivo. To generate the intuitive circuit in a single layer, we applied a self-splicing intein to effector proteins for the output and function. A natural split intein, DnaE Npu intein from Nostoc punctiforme, was incorporated into circular permissive sites of GFPuv and CFP derived from GFPuv by inducing point mutations at F64L, S65T, Y66H and Y145F using PCR. Resulting split the N-terminus region with a fluorophore of GFPuv or CFP and common C-terminal region was inserted downstream PBAD, PRham and PTrc promoter, respectively. Subsequently, the AND logic gate in which two-color fluorescence was regulated by three input molecules, L-arabinose, L-Rhamnose and IPTG, was successfully designed and observed to be functional in vivo. To our knowledge, this is the first example of the inter-domain swapping by an intein, as well as the first Half-Adder AND gate in the biological system.

*Keywords: synthetic biology, AND gate, intein, fluorescence protein

72

Potential of Microalgal Bio-char as a Solid Acid Catalyst for Biodiesel Production

SEULGI LEE, SEULGI LEE, Young-Jin RYU, Dong Ho SEONG, Sang-Min LIM, Choul-Gyun LEE and Choul-Gyun LEE*Marine Bioenergy Research Center &Department of Biological Engineering, Inha University, Incheon, Korea.


Biodiesel is an attractive alternative to fossil fuel because of its similar characteristics to petro-diesel. Its manufacturing processes generally contain a step carried out by either alkaline or acid homogeneous catalyst. However, the homogeneous liquid catalyst must be removed by a rather complex purification process. Solid heterogeneous catalysts have a potential to overcome these problems, strong acidic ion exchange resins and inorganic-oxide solid acids were studied but those have some undesirable characteristics, such as high cost, low activity, etc. In this study, bio-char from microalgae was tested as a potential matrix to be used for a solid acid catalyst. The bio-char based catalyst was prepared after reacting bio-char with sulfuric acid and tested as a catalyst for trans-esterification of triolein and esterification of oleic acid using methanol. The potential of bio-char as a heterogeneous acid catalyst for biodiesel production was found to be higher than that of Nafion, a representative strong acidic ion exchange resin.

*Keywords: microalgae,biodiesel,solid catalyst

73

Bio-isoprene Production in Metabolically Engineered E. coli

Jung-Hun KIM1, Chong-Long WANG1, Sang-Hwal YOON1, Seo-Hee KANG1, Gye-Hwan KIM1, Jia ZHOU1, Eui-Sung CHOI2 and Seon-Won KIM*1

1Division of Applied Life Science, Gyeongsang National University, Jinju 660-701, Korea. 2Korea Research

Institute of Bioscience &Biotechnology, Daejeon, Korea.

*Corresponding author: [email protected] ([email protected])

Isoprene (2-methyl-1,3-butadiene) is a volatile C5 terpenoid released from leaves of many deciduous trees and some bacteria. It is the monomer of natural rubber and a precursor to an immense variety of naturally occurring terpenoids. Isoprene derived from oil refinery is an important chemical feedstock used in the synthetic rubber industry. About 800,000 tons per year of cis-polyisoprene are produced from the polymerization of isoprene, and most of this polyisoprene is used in the tire and rubber industry. Isoprene is biologically made from methyl erythritol 1-phosphate pathway in plants. In this study, we constructed plasmids containing isoprene synthases (ispS) of Populus alba, Populus trichocarpa, and Pueraria Montana trees and introduced them into E. coli to produce isoprene. By GC analysis, we confirmed isoprene production from E. coli. To produce more isoprene, we introduced pSNA plasmid containing whole mevalonate pathway and a codon optimized ispS gene into E. coli. This work was supported by a grant (NRF-2010-C1AAA001-0029084) from the National Research Foundation, the Intelligent Synthetic Biology Center of Global Frontier Project funded by the MEST (2011-0031964), and a grant from the Next-Generation BioGreen 21 Program (SSAC, grant#: PJ009522003), RDA, Korea.

*Keywords: Bio-isoprene,Escherichia coli,Metabolic engineering

74

Bioconversion Process for Production of 7-Ketolithocholic Acid from Chenodeoxycholic Acid by E. coli

jiyun LEE2, Naeun PARK1, Seonwook HWANG2, Young-Hee LIM2 and Jeong-Keun KIM*1

1Department of Chemical Engineering and Biotechnology, Korea Polytechnic University, Sihung-si, Gyeonggi-do 429-793, Korea. 2Department of Integrated Biomedical and Life Sciences, College of Health Science, Korea University, Seoul 136-703, Korea.


Bioconversion is very effective process to reduce process time and cost to carry out a chemical reaction, and offers minimal generation of hazardous wastes. This research developed a bioconversion process to convert chenodeoxycholic acid (CDCA) to 7-ketolithocholic acid (7-KLCA) using 7α-hydroxysteroid dehydrogenase (7α-HSDH) produced from E. coli ATCC 25922. The bioconversion reaction was carried out with CDCA in 0.1 M phosphate buffer (pH 8.0) at 35ºC for 48 h with 120 rpm shaking. The bioconversion was performed with E. coli whole cell and the cells were reused four times. The conversion rate was 98%, 32.4%, 27.6%, 20.1%, and 0% in the first, the second, the third, the fourth, and the fifth time use, respectively. However, once 8,000 μg/ml of NAD+ was added in the reaction after the fifth reaction, 7-KLCA was produced from CDCA with 19% of conversion rate. It means that the activity of 7α-HSDH in E. coli depends on NAD+

. The

products were purified by TLC and HPLC and were confirmed the structures using 1H NMR and 13C NMR. In this study, we established an effective bioconversion process to produce 7-KLCA from CDCA, which reduces the process steps to produce ursodeoxycholic acid from cholic acid compared with the chemical process currently used.

*Keywords: 7-Ketolithocholic Acid,7alpha-Hydroxysteroid dehydrogenase,E. coli

75

Metabolic engineering of Escherichia coli for geraniol production

jia ZHOU1, Chonglong WANG1, Sang-Hwal YOON1, Hui-Jeong JANG1, Eui-Sung CHOI2, Seon-Won KIM1 and Seon-Won KIM*1

1Division of Applied Life Science, Gyeongsang National University, Jinju 660-701, Korea. 2Systems Microbiology Research Center, KRIBB, Daejeon 305-806, Korea.


Monoterpene alcohol geraniol has versatile applications in fragrance industries, pharmacy and agrochemistry. Moreover, geraniol can be an ideal gasoline alternative. Thus, Escherichia coli was metabolically engineered to produce geraniol. GPP was produced in E. coli MG1655 by overexpression of GPP synthase and the MVA bottom pathway. Direct dephosphorylation of GPP by endogenous promiscuous phosphatases enabled geraniol formation at a level of 14 mg/L in 48 h. Enhancement in dephosphorylation of GPP by employing geraniol synthase from O. basilicum further improved geraniol production to 111 mg/L in 48 h. However, geraniol was observed to be dehydrogenized and isomerized into other geranoids (nerol, neral and geranial) by endogenous promiscuous dehydrogenases, among which YjgB was shown with major responsibility for geraniol dehydrogenation. Deletion of gene yjgB improved geraniol purity and updated geraniol production to 137 mg/L. Replacement of the bottom MVA pathway with the whole MVA pathway further increased geraniol production up to a level of 193 mg/L, which was 39-fold and 1,043-fold of the highest geraniol productions from yeast and E. coli, respectively.

*Keywords: MVA pathway,Geraniol,E. coli

76

Oil production from four microalgae under various culture conditions

Chang-Han KANG, Yeaung-Bin HYEAN, Jin-Ho CHOI and Sung-Koo KIM*Dept. of Biotechnology, Pukyoung National University, Busan 608-737, Korea.


Microalgae, such as marine microalgae and cyanobacteria, produce long-chain polyunsaturated fatty acids. This study was carried out using four microalgae strains for high biomass and oil productions. The maximum biomass of Dunaliella tertiolecta was 0.30 g dcw/L with a nitrate concentration of 24.74 mg/L, and the oil content increased from 18% to 54.54% in 2 days at 0 psu in two-stage culture with salt stress. The maximum biomass of Dunaliella salina was 0.29 g dcw/L with 18.55 mg/L nitrate, and the oil content increased from 16.32% to 47.30% in 1 day at 10 psu in two-stage culture with salt stress. The maximum biomass of Isochrysis galbana was 0.82 g dcw/L with 24.74 mg/L nitrate, and the oil content increased from 23.15% to 57.03% in 2 days at 10 psu in two-stage culture with salt stress. The maximum biomass of Nannochloropsis oculata was 0.51 g dcw/L with 24.74 mg/L nitrate, and the oil content increased from 8.23% to 28.57% in 4 days at 0 psu in two-stage culture with salt stress

*Keywords: Nitrate concentration,Salinity,Oil production

77

gTME-driven Development of Recombinant Saccharomyces cerevisiae with Enhanced Ethanol Production Ability

Yeong-Je SEONG1, Haeseong PARK1, Hye-Jin LEE1, Mikyung PARK1, Kyu-Sang PARK1, Soo young JUN1, Jung-Eun LEE1, Kyoung Heon KIM2 and Yong-Cheol PARK*1

1Dept. of Bio and Fermentation Convergence, Kookmin University, Seoul 136-702, Korea. 2School of Life Science and Biotechnology, Korea University, Seoul 136-713, Korea.


Global transcription machinery engineering (gTME) is a high-throughput technology to screen a robust yeast strain by generating various mutations of the TATA-binding protein encoded by SPT15 in Saccharomyces cerevisiae. In this study, gTME was used to construct recombinant S. cerevisiae ETS2 and ETS3 strains with an enhanced ethanol production ability. In micro-aerobic batch cultures using a chemically defined medium with 470 g/L glucose, the ETS2 and ETS3 strains showed 2.5, 3.2 and 1.3 fold increases in glucose consumption rate, ethanol production rate and ethanol yield, compared with those of the BY4741 host strain, respectively. A batch culture of ETS3 in a bioreactor containing complex medium with 300 g/L glucose resulted in 22.2 g/L dry cell mass, 98.1 g/L ethanol concentration and 1.82 g/L-hr ethanol productivity, which were about 13-16% increases relative to those of BY4741 strain. As a result, recombinant S. cerevisiae strains constructed through gTME possessed the improved fermentation properties of cell growth and ethanol production for fuel ethanol manufacturing.

*Keywords: bioethanol,Saccharomyces cerevisiae,global transcription machinery engineering

78

Gene expression and enzymatic characterization of enoate reductases for reduction of cyclic α,β-unsaturated carbonyl compound

Hye Young NA, Ji Eun CHOI, Ji Young LEE, Joon Young OH and Jae Kwnag SONG*Research center for bio-based chemistry, Korea Research Institute of Chemical Technology, Daejeon, Korea.


Carbon-carbon double bonds in α,β-unsaturated carbonyl compounds can be selectively reduced by enoate reductases. Enoate reductase-catalyzed reduction of different compounds such as conjugated enals, enones, imides and nitroalkenes has been also reported. We tried to use the catalytic action of enoate reductases in converting a biomass-derived chemical to a useful chemical feedstock. First, five enoate reductase-encoding genes of XenA from Pseudomonas putida, KYE1 from Kluyveromyces lactis, Yers-ER from Yersinia bercovieri, PETN from Enterobacter cloacae, and YqjM from Trypanosoma cruzi were cloned into pET-21a(+) plasmid and expressed in Escherichia coli BL21(DE3). Catalytic activities in resting whole cells, crude cell extracts and purified enzymes were tested on the α,β-unsaturated carbon bond of various compounds such as (Z)-6,7-dihydro-1H-azepin-2(5H)-one, 3-buten-2-one, 2-cyclohexen-1-one, 3-methyl-2-cyclohexenone, 2,5-pyrroledione. We selected the enoate reductase with relatively high activity for production of our desired product and also investigated some biochemical properties of the enzyme.

*Keywords: Enoate reductase

79

Genetic Transformation of Gram-Positive, CO- Utilizing Acetogenic Bacterium, Eubacterium limosum KIST612

Ji Yeong JEONG1, Jihee YOON2, Donggeon CHOI2, Shinyoung PARK2 and Jiyeong JEONG*1

1School of Environmental Science and Engineering, Gwangju Institute of Science and Technology (GIST), 123 Cheomdan-gwagiro, Buk-gu, Gwangju 500-712, Republic of Korea. 2School of Environmental Science and Engineering, Gwangju Institute of Science and Technology (GIST),123 Cheomdan-gwagiro, Buk-gu, Gwangju 500-712, Republic of Korea.


Renewable energy production using bio-process has been considered to be a promising alternative for chemical conversion processes due to the depletion of fossil fuels. Genetic manipulation for yield enhancement and metabolite diversity also has been tried in the bio-process. Gram-positive bacteria such as acetogens which utilize synthesis gas have been used as biocatalysts; however, it is difficult to apply genetic tools for Gram-positive bacteria. The aim of this research is to construct genetic tools for further research to increase the production of specific biochemicals. In this study, we applied the gene transformation technique into an acetogenic bacterium (Eubacterium limosum KIST612) isolated from an anaerobic digestor. Three plasmids were used for transformation of the bacterium; pKD46 is designed for lambda recombinase, pBR322 and pJIR418 are shuttle vectors. We adapted conjugation, electroporation, and sonoporation to transform E. limosum KIST612. As a result, two plasmids pBR322 and pJIR418 could not be transferred into E. limosum KIST612 because restriction endonuclease might eliminate two plasmids. Here, we applied in-vivo methylation in Escherichia coli strains in order to avoid the effect of endonusclease on two plasmids, but could not obtain stable transformants. Consequently, only pKD46 was successfully transferred to E. limosum KIST612 by electroporation, and sonoporation.

*Keywords: Genetic transformation,Gram-positive,Acetogenic bacteria

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