Earticle

Glutamate decarboxylase (GAD) is an enzyme that catalyzes the decarboxylation of L-glutamate to γ-aminobutyrate (GABA). This reaction is of interest for its application in production of GABA as the source for manufacturing nylon-4, a biodegradable polymer. The E.coli GAD was only active under acidic environments, which severely limited the scope of the reaction. GAD undergoes a drastic conformational change in a pH-dependent manner, which eventually shuts down its activity at neutral pH. Upon the structural studies, Glu89 was shown to be one of the residues of which side chains adopt different positions in active and inactive forms of GAD. Therefore, site-directed mutagenesis of this residue was performed and the consequences were evaluated to confirm any contribution of this to the inactivation process of GAD

AbstractLactide is cyclic dimer composed by two molecules of lactate and is pivotal monomer for poly lactide (PLA) which was promising environmentally friendly polymer and applied in wide industrial field.1 Candida antarctic lipase B (CALB) catalyze hydrolysis, esterification, and transesterification in mild condition compared to chemical catalyst.1,2 Although wild-type CALB consumed methyl R-lactate as substrate completely, conversion of R-lactide reached only about 60 % and the others were converted to oligomers as by-product. Molecular dynamic simulation predicted that Asp134 residue of CALB seemed interacted with methanol, which was by-product during transesterification of methyl R-lactate and functions to degrade ring structure of R-lactide. Among D134-mutants of CALBs, D134A and D134V were selected as candidates for the improvement of R-lactide production. Interestingly, D134A CALB mutant showed 20 folds higher consumption rate of methyl R-lactate as substrate. As conclusion, D134A mutant CALB has a potential to be used as a better biocatalyst for enzymatic synthesis of R-lactide from alkyl R-lactate directly in mild condition compared to wild type CALB.

An aerobic bacteria termed AN-C16-KBRB was isolated from bovine rumen and demonstrated cellulolytic activity on a NB agar plate containing azo-carboxymethyl cellulose. The novel celEdx16 gene encoding a cellulase was cloned, sequenced, and expressed in E. coli, and the enzyme was characterized. CelEdx16 showed approximately 50% identity with known cellulolytic enzymes of various microbes including C. thermocellum, C. cellulovorans, P. rhizinflatus, N. patriciarum, R. albus, and O. joyonii. CelEdx16 was active on MeUmbG2, a fluorogenic glycoside substrate of exo-type cellulases as well as CMC. The CelEdx16 cellulase is of considerable interest because the enzyme is bifunctional, and is stable both in the presence of metal ions and under strongly alkaline conditions. (This work was supported by a high throughput screening (HTS)-based Integrated Technology Development grant from the MEST, and a basic research grant from KRIBB.)

Abstract The uncharacterized gene previously proposed as a myosin cross antigen from Macrococcus caseolyticus was cloned and expressed in Escherichia coli. The putative protein was identified as an oleate hydratase by the determination of the substrate specificity and the analysis of the amino acid sequence. The native enzyme was a 69-kDa dimer with a molecular mass of 138 kDa, as measured by gel filtration. The maximal activity of the oleate hydratase for oleate hydration was observed at pH 6.5 and 25ºC in the presence of 0.5 mM flavin adenine dinucleotide. The hydroxylated products formed from unsaturated fatty acids by the enzyme were made as the standards with a high purity (>99%) by thin layer chromatography and solvent extraction and then were identified. Using the products, the accurate specific activity and kinetic parameters for fatty acids as substrates were determined. Hydratase from Macrococcus caseolyticus exhibited hydration activity only for 9(Z)-double bonds of C16 and C18 unsaturated fatty acids and showed the highest specific activity and catalytic efficiency (kcat/Km) for oleic acid among the unsaturated fatty acid substrates. Thus, 10-hydroxystearic acid was produced from oleic acid by the recombinant enzyme and E. coli.

Amino acyl tRNA synthetase (AARS) is an ATP dependent enzyme helps in charging of tRNA with cognate amino acid during protein synthesis. The accuracy of the protein translation is maintained by the proof reading activity of AARS by preventing charging of tRNA with non cognate amino acids (Unnatural amino acids). Recently researchers identified a methodology in which the endogenous tRNA synthetases can recognize and incorporate the close structural analogs of the cognate amino acid containing novel functional groups in the absence of the natural substrate called as codon reassignment. Currently, more than 15 methionine analogs were incorporated into protein in response to the sense codon with the help of methionine auxotrophic cell which provide novel application such as metabolic labeling. In this study, we have performed molecular docking analysis to predict the binding mode of the methionine analogs with the active site residues of Escherichia coli methionyl tRNA synthetase and we identified the key residues responsible for the recognition of these methionine analogs. Here, we clearly demonstrate the substrate selectivity of these methionine analogs with methionyl tRNA synthetase and offer the clear structural knowledge about the active site which will provide knowledge for rational designing of methionyl tRNA synthetase.

Proteases, playing an important role in a wide variety of physiological processes, are enzymes that selectively cleave peptide bonds in proteins and polypeptides. Because of these relationships between proteases and diseases, the need for synthesis fast, sensitive, and specific protease assay materials has increased considerably in the last few years. Dityrosine (DY), which is an unusual amino acid formed as a result of normal posttranslational processing. Many researchers have used dityrosine as an important biomarker for oxidatively modified proteins because of its fluorescence and stability. However, in this study we try to use dityrosine for another area such as protease assay. Therefore, we synthesized DBDY-(INH)2 as a simple protease assay material and experimented on assay with this material. The results of this experiment show that DBDY-(INH)2 can be used as a specific and sensitive assay material about papain.

Glutamate decarboxylase(GadB) from Escherichia coli is pyridoxal 5’phosphate-dependent enzyme which is catalyzing CO2 release from the alpha carboxyl group of L-glutamic acid to produce gamma amino butyric acid(GABA). GABA can be used as a monomer to synthesize a biobase and biodegradable polymer(polyamide 4). Gad is a key component of the most important acid resistance system, especially related to glutamic acid. GadB shows an acidic pH optimum(pH4~5) and becomes activated intracellularly because in extremely acidic environments protons leak through the bacterial cell membrane. In this research, gadB was highly expressed as soluble protein in Escherichia coli BL21(DE3) containing pET24a-gadB, which was induced by 0.5mM IPTG in LB medium. The Kcat/KM value was 24.8mM-1s-1. The highest product concentration was 102mM at pH 4.4 when we used 300mM glutamic acid as a substrate.

There has recently been much research conducted on the high temperature activity and thermal stability of reverse transcriptases. Traditional M-MLV reverse transcriptase possesses low thermal stability, allowing for reverse transcription reactions to occur only at relatively low temperatures (42℃). This limitation prevents the efficient transcription of RNA molecules with complex secondary structures. To resolve this, BIoneer has utilized synthetic biotechnology and protein engineering to develop a thermostable reverse transcriptase with activity at high temperatures of 50 °C and above, and dubbed the enzyme RocketScript. Higher temperature reverse transcription reactions allow optimization of cDNA synthesis experiments with temperature. RocketScript is Bioneer’ exclusive M-MLV-based thermostable reverse transcriptase (RTase). Because the original M-MLV RTase has maximum activity at relatively low temperatures (42 °C), it caused several problems during reverse transcription of complex secondary structure RNA species. RocketScript has thermostable activity (42~70°C), allowing efficient cDNA synthesis from complex secondary structure RNA.

Sialyltransferase(ST) is an enzyme that participates in the biosynthesis of sialyllactose. We aimed at immobilizing ST on the solid support for the repeated use of the enzyme with the ease of purification of the product. Toward this goal, we decided to install cysteinyl residue(s) in a site-specific manner. The site-specifically installed cysteine will be linked to the solid support site-specifically because of its high nucleophilicity. There are two prerequisites for the site-specific mutation of a selected residue to cysteine. First, the mutation of the selected mutation should not negatively affect the activity of ST. Secondly, newly installed cysteine should efficiently undergo a chemical reaction that eventually leads to the immobilization of ST to the solid support. Since the number of clones for assays for these requirements would be significantly large, we are aiming at the use of "totally in vitro" system. Here we report our recent progresses along with the methodology.

Since many medical devices are implanted in the human body, it should be supplied with constant voltage by a long-life battery. So, we are developing a bio-anode that uses blood glucose as an energy source. It converts blood glucose to electric current by using enzyme oxidizing glucose. Although glucose oxidase (GOX) has been widely used so far, we selected glucose dehydrogenase (GDH) as an alternative enzyme source for the reason, their no toxic byproduct formation and low Km value. In order to get long-life GDHs, we cloned GDH genes from thermophiles. The aldose sugar dehydrogenase and membrane-bound glucose dehydrogenase from Escherichia coli were used as reference enzymes. Most of them require pyrroloquinoline quinine (PQQ) for their activity. PQQ is a redox cofactor with higher reduction potential than others(NAD, FAD). It is expected to transfer electrons from glucose to electrode more efficiently. GDH using FAD as a cofactor has a cytochrome c subunit to compensate its low conductivity. PAN nanofiber and carbon nanotube are used to immobilize the GDHs. This work was supported by the Basic Research Program(Grant No. 2009-0082812) of MEST and BK21 program of Korea.

2,3-butanediol (2,3-BDO) can be chemically changed into butadiene which is an important precursor of synthetic rubber, thus the production of 2,3-BDO have been attracting attention continuously in scholar circle. It can be produced by fermenting several bacteria such as Enterobacter asburiae. However, different culture conditions will affect the production and productivity of 2,3-BDO. For example, under different pH values, the productivity of 2,3-BDO from glucose vary a lot. As well, different substrate concentrations will also influence the final results of 2,3-BDO production due to the potential possibility of substrate inhibition effect. In this work, fermentation experiments by a new Enterobacter asburiae strain under different pH control conditions from 5 to 8 have been conducted to examine the influence on 2,3-BDO productivity. Also, different initial glucose concentrations have been also examined in the experiment to observe the substrate shortage condition and substrate inhibition condition and the optimized substrate concentration for producing 2,3-BDO by Enterobacter asburiae was determined by the experimental results.

Saccharomyces cerevisiae is widely known and used as an industrial strain especially for the production of ethanol. Ethanol is the most important biofuel these days, while a lot of longer chain alcohols are being developed as commodity chemicals. 2,3-butanediol, as one of the longer chain alcohols, is a chemical with multiple practical applications. It is naturally produced by microorganisms such as Klebsiella pneumonia, Klebsiella oxytoca and Enterobacter aerogenes. (1) We attempted to engineer S. cerevisiae for 2,3-butanediol production. In an attempt to redirect carbon flux towards 2,3-butanediol production, we have devised two metabolic engineering strategies. For each strategy, we have constructed a variety of strains by applying loxP/Cre mediated PCR-based gene deletion method and also gene overexpression technique. In the first strategy, pyruvate decarboxylase genes (PDC1, PDC5, PDC6) were deleted. In the second strategy, ADH1 gene (encoding alcohol dehydrogenase) and ALD6 gene (encoding major cytosolic acetaldehyde dehydrogenase) are deleted from the wild type strain. Then, acetolactate synthase from Bacillus subtilis (AlsS) and/or butanediol dehydrogenase (BDH1) gene were overexpressed. We compare the production of 2,3-butanediol from both strategies.

The high osmolarity glycerol (HOG) mitogen-activated protein kinase pathway plays important role in osmoadaptation in yeasts. We characterized the HOG1 homologue of the methylotrophic yeast Hansenula polymorpha. The amino acid sequence of HpHOG1 shows 70% of identity and 75% of similarity to that of S. cerevisiae Hog1. Deletion of Hphog1 gene caused the cell sensitive to high concentrations of NaCl and sorbitol. To understand the regulatory role of HpHOG1, transcriptome profiles of wild-type and Δhog1 mutant H. polymorpha strains challenged with 0.5 M NaCl for 30 min were analyzed. About 400 genes were differentially regulated in wild-type and Δhog1 mutant strain and the majority of them was involved in metabolism. Interestingly, many genes involved in the osmotic stress response, general stress response, and cell surface and cell wall formation were specifically up-regulated in wild-type strain. On the other hand, numerous genes involved in ribosomal protein synthesis were specifically down-regulated in Δhog1 mutant strain. Our results indicate that the osmotic stress response in H. polymorpha is modulated by the HOG1 MAP kinase pathway and other signal transduction mechanism.

Although 1.42 million single-nucleotide polymorphisms (SNPs) were identified in human genome, the relationships between diseases and SNPs have not been clarified. For this reason, high-throughput genotyping methods are needed for analyzing human genes and many researchers are searching for various and effective detection methods. In this study, we prepared repetitive polypeptides for SNP detection using capillary electrophoresis in free-solution. We demonstrate a method for multiplexed single-base extension (SBE) genotyping that takes advantage of the unique separation modalities made possible via end-labeled free-solution electrophoresis (ELFSE). A series of “rag-tags”was created using biological synthesis and used to achieve DNA separation by microchannel electrophoresis without a polymeric sieving matrix. This method is shown to be applicable for SNP genotyping and expected to search for the various genetic diseases..

Bombyx mori genome has three ferritins, iron storage proteins, Fer1HCH, Fer2LCH, and Fer3HCH. Of them, only Fer2LCH mRNA was found in our EST library constructed from the day 1 of silkworm eggs and had a putative N-glycosylation site. Temporal, spatial, and inducible expression analyses during embryogenesis revealed that the silkworm Fer2LCH mRNA was abundant from 6 h to 6 days after oviposition. Transcription of Fer2LCH was not affected by immune challenge, oxidative, and iron stress. To understand the difference in folding and assembly of recombinant protein secreted in insect cell or silkworm, Fer2LCH purified in both production systems was analyzed. The Fer2LCH proteins produced by both systems were N-glycosylated but not O-glycosylated. Lectin blot analysis indicated that only Fer2LCH produced by silkworm was terminated by mannose, GlcNAc, and Fucose residues. The association of ERp57 with Fer2LCH glycoprotein from hemolymph was stronger than with it from cell, and the interaction with PDI, Bip, calnexin, and calreticulin were very similar in both systems. Our data suggest that Fer2LCHs synthesized in cells and silkworm interact probably differently with the ER chaperones.

Recently, Chlorotyrosine containing proteins has the attention of protein engineers for developing novel biomarker as well as in preparing pharmacologically active substances. Similarly, fluorination of recombinant protein is significant for analyzing the structural, biological and physical property of protein. Due to its significance and requirements, in vivo incorporation of Chloro and Fluoro amino acids into the target protein is essential. Recently genetic incorporation of novel functionality embedded within the non canonical amino acids (NCAA) became an indispensable quest to design and manipulate target proteins with new and enhanced properties. The protein modification can be achieved by two different methods 1. "Genetic code engineering" is based on the reassignment of the sense codon through the selective pressure incorporation method (SPI). 2. “ite specific incorporation”methodology use DNA mutagenesis to introduce in-frame termination triplets (e.g. the amber stop codon) that are considered as blank codons for the expansion of the cellular genetic code. In general, genetic code engineering will utilize the host translational machinery (tRNA/tRNA synthetase system), whereas the site specific incorporation requires 21st tRNA/synthetase pair (orthogonal) for NCAA incorporation into protein. Both methods are successfully utilized to incorporate different NCAA into protein for improving its biophysical and functional properties. Recently, we have developed an interesting GFP for NCAA incorporation with improved stability and folding efficiency. Here, the promise of this new GFP is utilized to exemplify the introduction of a halogen group containing aromatic NCAA into Tyr residues of GFP through SPI method. We observed the high level expression of F-Tyr containing GFP (38mg/L), when compared to that of the parent GFP (34mg/L) and medium supplemented with Cl-Tyr (18mg/L). Due to the bulky nature, we expected that Cl-Tyr incorporation might affect the GFP expression level by forming protein aggregates. The dichroic profiles confirmed the overall secondary structural characteristic of the protein remains same even after incorporation of Cl-Tyr and F-Tyr into the protein. The refolding kinetic parameters of chloro variant was almost similar to that of the parent GFP but the fluorine atom incorporation into protein stabilizes protein folding efficiency this result supports the earlier report. As expected the incorporation of halogen group into GFP, alters its spectral properties such as emission maxima and relative fluorescence intensity. Here we report, the successfully incorporation of Cl-Tyr into GFP without altering the host translational machinery. This demonstrates that protein with a NCAA opens new strategies for the design of tailor made proteins with superior to those of the parent protein.

Recently, great efforts have been devoted to expanding the genetic code for the in vivo biological incorporation of non-canonical amino acids (NCAA) into proteins. Current approaches such as reassignment of sense (residue specific incorporation) and non sense codon (site specific incorporation) provide non natural amino acid as a new set of building blocks for the Eukaryotic and prokaryotic translational machinery. Even though, these methods successfully alter the protein functionality, the limitations of above methods is that they allow only incorporation of a single unnatural amino acid into the recombinant protein. This single protein exhibited two different novel functionalities acquired from the genetically incorporated NCAA, which is an interesting and not an inherent property of the protein. To incorporate two different NCAA, we developed a promising alternative approach by coupling both the residue-specific and site-specific incorporation of NCAA into a single protein. To explore the feasibility for MUAA incorporation, a model protein, green fluorescent protein (GFP), was selected. GFP has five methionine (Met) residues, which will be reassigned with Met surrogate L-homopropargylglycine (L-Hpg). Simultaneously, the tyrosine (Tyr) analogue, 3,4-dihydroxy-L-phenylalanine (L-Dopa), will be site specifically incorporated into K15 by evolved Methanococcus jannaschii tRNA/synthetase pairs (mutant TyrRS). The successful incorporation of L-DOPA further facilitates the selective chemical cross-linking of protein-polysaccharide interaction in vitro. GFP protein has Dopa residue were oxidized upon sodium periodate (NaIO4) which lead to the formation an orthoquinone intermediate that is subsequently attacked by nucleophilic side chains of amine group interacting polysaccharide, through either Michael addition or Schiff base formation. Further the Met analogue L-Hpg whose functionality can be chemoselectively modified with specific alkyne bearing reagent by means of a copper mediated azide-alkyne cycloaddition. This is the first study to demonstrate that the protein has multi-functional property through combination of expanding and engineering the genetic code of amino acids. This approach will drive us toward a post proteomics era as well as it will also open a new door for synthetic biologist to generate multi characteristic proteins.

Translation, process that synthesizes the protein, is related with the cell growth because proteins which synthesized through translation play the major role in the biological system. The modulation of translation elongation rate could affect protein synthesis rate and change the growth rate. Among the translation elongation factors, EF-G is a key factor in translation elongation since it determines how fast translocation of ribosome occurs. To modulate the growth rate, a genetic circuit was constructed and applied to on/off switch. fusA gene coding EF-G is regulated by lambda promoter which is repressed by cI repressor which is expressed by IPTG induction. The regulation system of fusA by cI repressor was validated by measuring the growth rate of BL21 (DE3) deleted chromosomal fusA in the various IPTG induction time and concentration. In this research, growth rate was decreased after 2hr from IPTG induction generally and more decreased as IPTG concentration was increased. It is shown that this genetic circuit could control the growth of cell.

Lactic acid is a very important chemical having various applications in the field of pharmaceutical, cosmetics, textile, leather and food industries. Pure sources of the D(-) and L(+) enantiomers can be blended to vary the physical properties and biodegradation rates of polylactides. Lactic acid is able to be produced by chemical synthesis or bacterial fermentation, but none of chemical production routes shows technical and economical viability. Significant advantage of biotechnological production of lactic acid by fermentation over chemical synthesis is that can sue cheap raw materials. In this presentation, we developed several metabollically engineered strain for the production of pure L- or Denantiomer and will show the production of lactic acid using these lactic acid bacteria.. Detailed results will be presented.

β-1,3-glucanase was widely used in various biotechnological processes and overproduction was required for versatile industrial utilization. We subcloned β-1,3-glucanase gene (exgA) from Aspergillus oryzae and examined overexpression of β-1,3-glucanase by using δ -targeted multiple integration. We constructed two plasmids for multiple integration of exgA, pRSδ-exgA and pRSδH-exgA for first and second round integration, respectively. These plasmids contain ADH1 promoter and exoinulinase signal sequence (Inus.s) for secretion of β-1,3-glucanase. The pRSδ-exgA plasmid was transformed into S. cerevisiae BY4742△ exg1 strain. Subsequently, the pRSδH-exgA plasmid was transformed into S. cerevisiae BY4742△exg1/pRSδ-exgA strain. In the BY4742△exg1/pRSδ-exgA and BY4742△ exg1/pRSδ-exgA/pRSδH-exgA strains, β-1,3-glucanase activity was 0.94 and 1.4 unit/㎖�, respectively. Copy numbers of exgA gene were 6.7 and 10-fold increased by first and second round integration, respectively. These results suggested that exgA gene was successfully secreted and overexpressed by using δ-sequence integration. We will further increase of copy number by using repeated δ-integration.

Erythrose reductase (ER) is a key enzyme in biosynthesis of erythritol, which catalyzes the reduction of erythrose to erythritol in the presence of reducing cofactor NAD(P)H. The osmotolerant yeast, Candida magnoliae produces a functional sweetener, erythritol, from glucose, fructose and sucrose. Expression of erythrose reductase gene in C. magnoliae was examined by enzyme activity assay and reverse transcription polymerase chain reaction (RT-PCR) in a variety of stress conditions. Expression level of the ER in C. magnoliae significantly increased in the medium containing KCl and NaCl, indicating that expression of ER might be closely related to stress response pathways in C. magnoliae.

We identified two novel fungal exo-cellobiohydrolase (CBH) genes, PeCBH1 and PeCBH2 from Penicillium sp. PeCBH1 (1,641 bp without intron), showed 84% similarity to the Penicillium oxalicum CBH1, encoded 547 amino acid residues (aa) including 26 aa signal peptide and a cellulose binding module (CBM). PeCBH2 showed a 78.4% similarity to the gene from the Penicillium decumbens CBH and has 452 aa including two introns and 17 aa signal peptide. Its internal amino acid sequence showed a significant homology with glycoside hydrolase family 7. CBM of PeCBH1 was classified as carbohydrate binding module family 1. Both PeCBH1 and PeCBH2 were over-expressed using several translational fusion partners in yeast Saccharomyces cerevisiae and purified for further characterization. The optimum pH and temperature of PeCBH1 were pH5 and 50 ℃ and the optimum pH and temperature of PeCBH2 were pH5 and 55 ℃, respectively. The para-nitrophenyl-beta-D-cellobiotetraoside cleavage patterns showed that both PeCBHs preferred the reducing end cleavage. Two novel exo-cellobiohydrolases, PeCBH1 and PeCBH2 will be useful for the enzymatic saccharification of cellulosic biomass for the production of bioethanol.

Recently small regulatory RNAs have been discovered in prokaryotic cells to regulate intracellular processes. They repress translation by preventing ribosome binding to mRNA. The regulatory small RNAs could provide a new level of regulation in synthetic biology to control synthetic circuits in concert with transcriptional regulations. We developed synthetic small regulatory RNAs repressing the translation of DsRed2 mRNA and also constructed three different sRNAs for the mRNAs of LuxR, AraC, and KanR without cross-reactivity. The results suggest that gene expression can be fine-tuned by designed artificial small RNAs. The possibility of designing small regulatory RNAs may facilitate the development of precisely regulated synthetic circuits. [This work was supported by the Korean Systems Biology Research Project (20100002164) of the Ministry of Education, Science and Technology (MEST) through the National Research Foundation of Korea. Further support by the World Class University Program (R32-2008-000-10142-0) through the National Research Foundation of Korea funded by the MEST is appreciated.]

Polylactic acid (PLA) is a promising biomass-derived polymer, but is currently synthesized by a two-step process: fermentative production of lactic acid followed by chemical polymerization. Here we report production of PLA homopolymer and its copolymer, poly(3-hydroxybutyrate-co-lactate), by direct fermentation of metabolically engineered E. coli. Introduction of the heterologous metabolic pathways involving engineered propionate CoA-transferase and polyhydroxyalkanoate synthase allowed synthesis of PLA and P(3HB-co-LA) in E. coli, but at relatively low efficiency. In this study, the metabolic pathways of E. coli were further engineered based on in silico genome-scale metabolic flux analysis. Using this engineered strain, PLA homopolymer and P(3HB-co-LA) copolymers containing up to 70 mol% lactate could be produced up to 11 wt% and 46 wt% from glucose, respectively. Thus, the strategy of combined systems-level metabolic engineering and enzyme engineering allowed efficient bio-based one-step production of PLA and its copolymers. [This work was supported by the Korean Systems Biology Research Project (20100002164) of the Ministry of Education, Science and Technology (MEST) through the National Research Foundation of Korea. Further support by the World Class University Program (R32-2008-000-10142-0) through the National Research Foundation of Korea funded by the MEST is appreciated.]

A great deal of genomic knowledge has been leaped from a-decade-long efforts, but such achievement has not been transformed into full fruition yet in drug discovery field. With this motivation, we report a systems biological approach for drug targeting and discovery against an opportunistic human pathogen Vibrio vulnificus CMCP6 as an example. This systems biological approach utilizes newly updated genomic information to reconstruct the genome-scale metabolic network of this organism, named VvuMBEL943. Drug targets are selected by metabolite-centric drug targeting method. This metabolite-centric approach for drug targeting is performed by implementing constraints-based flux analysis, which is an optimization-based simulation technique, and predicts so called essential metabolites whose absence disrupts cell growth. Initial set of the predicted essential metabolites was further filtered with additional criteria based on organism specificity and maximal disruptive damage to the cell. Essentiality of final five essential metabolites was experimentally validated by gene knockout experiments. Finally, structural analogs of final five essential metabolites were selected out of large chemical compound library for subsequent high-throughput screening. This systematic approach should facilitate initial stage of drug discovery as well as other human-related diseases. [This work was supported by the Korean Systems Biology Research Project (20100002164) of the Ministry of Education, Science and Technology (MEST) through the National Research Foundation of Korea. Further support by the World Class University Program (R32-2008-000-10142-0) through the National Research Foundation of Korea funded by the MEST is appreciated.]

Isoprene (2-methyl-1,3-butadiene) is a volatile C5 terpenoid released mainly from leaves of many deciduous trees and some bacteria. Isoprene is present as a colorless liquid under standard conditions. Also, it is the monomer of natural rubber and a precursor to an immense variety of naturally occurring terpenoids. Isoprene 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 made from dimethylallyl diphosphate (DMAPP) by isoprene synthase via methyl erythritol 1-phosphate pathway (MEP) in plants. In this study, we constructed plasmid containing isoprene synthase (ispS) from three kinds of plant, Populus alba, Populus trichocarpa, and Pueraria Montana (kudzu) and introduced into E. coli to produce isoprene. By GC analysis, we confirmed isoprene production in E. coli. To produce more isoprene, we introduced pSNA plasmid containing whole mevalonate pathway for DMAPP synthesis and codon optimized ispS gene into E. coli. This work was supported by a grant (NRF-2010-C1AAA001-0029084) from the National Research Foundation (MEST), the KRIBB Research Initiative Program, and BK21 program of Korea.

α-agarase hydrolyzes α-1,3 linkage of agarose, yielding agarooligosaccharides and is poorly known compared with β-agarase. The α-agarase gene (AgaA, 2.3kb ORF) from Thalassomonas JAMB A33 was subcloned into each expression vector (pPIC9 [AOX1p for Pichia pastoris expression], pVT103U and pYInu [ADH1p and GAL10p for Saccharomyces cerevisiae expression, respectively]). The constructed plasmid pPIC9-AgaA was integrated into HIS4 gene of P. pastoris genome and pVT-AgaA and pYInu-AgaA were transformed in S. cerevisiae FY833 and SEY2102, respectively. Firstly, in each transformant, recombinant α-agarase activity was detected by using iodine method (active staining) that red halos around α-agarase expressing cells were showed. Subsequently, transformed cells were cultured on each medium (BMMY[methanol 0.5%], YPD, YPDGal) and α-agarase activity was analyzed. The recombinant α -agarases were successfully overexpressed with activity range of 0.27 ~ 1.34 unit/ml. In FY833/pYInu-AgaA (GAL10p-Inu s.s) strain, α-agarase activity showed maximum unit, 1.34 unit/ml, that is optimal host-vector system for secretory overexpression of α-agarase. On the other hand, Pichia host-vector system is not suitable for expression of α-agarase.

In this study, we developed an efficient process for 1,3-PD production from glycerol by genetic engineering of K. pneumoniae AK mutant strains, in which by-products formation was eliminated but IPTG induction was necessary for maximal production of 1,3-PD. A series of recombinant strain was designed to constitutively express dhaB and/or dhaT gene using bacteriophage T5 PDE20 and rho-independent transcription termination signal of Rahnella aquatilis levansurcrase gene. Among them, AK/pConT expressing dhaT alone was the most proper for 1,3-PD production. Fed-batch fermentation showed efficient production of 1,3-PD from both pure or crude glycerol without by-products formation. Further engineering to enhance production level would provide an economical biological process for 1,3-PD production.

The thermotolerant methylotrophic yeast Hansenula polymorpha has been shown to metabolize and ferment ethanol from glucose, xylose, cellobiose, starch, and xylan substrates which makes it an ideal candidate for lignocellulosic biomass-based ethanol fermentation. In addition to biomass, crude glycerol, a co-product of biodiesel transesterification, has become a valuable substance for ethanol production due to its high abundance, low cost, and high reducibility. However, ethanol production from glycerol by H. polymorpha has not been investigated thoroughly. In this study, we compared the transcriptome profiles of H. polymorpha grown on glycerol with those of glucose-grown cells both under aerobic and microaerobic conditions. About two percent of the 5.848 H. polymorpha genes were either up- or down-regulated more than two-fold during growth on glycerol. As expected the majority of the up-regulated genes was involved in metabolism including some of glycerol metabolic genes. On the other hand, the majority of the down-regulated genes was involved in central metabolism and cellular transport. Our results will allow us to understand and engineer the glycerol metabolism in H. polymorpha.

“etabolic Engineering”aims the purposeful redesign of the biological systems and requires the accurate information of the cellular metabolic networks and proper tools for the reconstruction of the biological. Numerous regulatory elements such as promoter libraries and RBS calculator can be applied for the modulation of gene expression. However, without carefully considering the structural information of 5’unstranslated region (5’UTR) sequence, it is insufficient to precisely design and modulate the gene expression level. To address this issue, in this study, we randomized 5’UTRs maintaining ribosome binding affinity using superfolder GFP as a reporting system in Escherichia coli. A mathematical model was constructed by mapping between the secondary structures of 5’UTRs and the expression level of superfolder GFP. Examples using the other genetic contexts will show the potentials of this model to predict the precise expression level based on the structural information and consequently will provide a valuable tool for “etabolic Engineering”