Earticle

Once upon binding of substrates, enzymes suffer from structural changes, i.e., from open to closed conformations. Flexible residues are closely linked to protein stability and often have an important role in conformational changes of enzymes for substrate binding. Role of flexible residues in enzyme activity could be investigated by analyzing structural differences between open and closed states. Prediction of flexible residues involved in catalytic motions is thus important not only to understand enzyme catalytic mechanism and but also to modulate activity and stability of enzymes. However, some enzymes such as xylanases do not show a distinct difference between apo and holo states. In the case of these enzymes, it is difficult to find out important flexible residues involved in structural motions with simple comparisons between two static states. In this study, we analyzed the flexibility of Bacillus circulans xylanase using molecular dynamic simulation and found out that only one surface loop (46~49) has flexible motion and this loop connects active sites pocket with second substrate binding site far from active site. However, flexible motion of this loop was constrained by cooperative interactions with other residues (cation-pi interaction (W42-R49) and electrostatic interaction (D4-R49)). This implies that flexible loop (46-49), especially, R49, seems to have important role in modulating stability and activity of xylanases. To validate this hypothesis, saturation mutagenesis of R49 was conducted and stability and kinetic parameters were determined. As expected, most mutants lost their stability due to disruption of cooperative interactions but gained higher catalytic power (up to 2 fold of Kcat /Km) due to increased flexibility. This explained typical trade-off between stability and activity of enzymes. Analysis on the flexible motion of enzyme using molecular modeling could be useful to identify important flexible residues involved in enzyme activity and stability. Further result and discussion will be presented.

The gene encoding Thermus thermophilus HJ6 DNA polymerase (Tod) was cloned and sequenced. The open reading frame (ORF) of the Tod gene was composed of 2,505 nucleotides and encoded a protein (843 amino acids) with a predicted molecular weight of 93,795 Da. The deduced amino acid sequence of Tod showed 98% and 86% identities to the Thermus thermophilus HB8 DNA pol and Thermus aquaticus DNA pol, respectively. The Tod gene was expressed under the control of the bacteriophage λ promoters PR and PL on the expression vector pJLA503 in Escherichia coli strain BL21 (DE3) codon plus. The expressed enzyme was purified by heat treatment, HiTrapTM Q column, and HiPrepTM Sephacryl S-200 HR 26/60 column chromatographies. The optimal temperature and pH for DNA polymerase activity were found to be 75~80℃ and 9.0, respectively. The optimal concentrations of Mg2+ and Mn2+ were 2.5 mM and 1 mM, respectively. The enzyme activity was activated by divalent cations, and was inhibited by monovalent cations. The result of the PCR experiment with Tod DNA polymerase indicates that this enzyme might be useful in DNA amplification and PCR-based applications. [This work was supported by the Marine and Extreme Genome Research Center Program of the Ministry of Land, Transportation and Maritime Affairs, Republic of Korea]

The recombinant epoxide hydrolase of Rhodotorula glutinis was immobilized onto magnetic nanoparticles for kinetic resolution of racemic styrene oxide. We employed a hybrid nanoparticle of Fe3O4/silica/NiO for efficient immobilization of enzyme and easy recovery of nanoparticles by magnetic separation. The isolated recombinant epoxide hydrolase was efficiently immobilized onto the magnetic nanoparticles through simple shaking of the enzyme solution and nanoparticle. More than 70% of hydrolytic activity of the free enzyme was retained. The enantiopure (S)-styrene oxide with enantiopurity higher than 98%ee could be obtained during repeatedbatch kinetic resolutions. Acknowledgment: This work was partially supported by the Marine and Extreme Genome Research Center Program, Ministry of Land, Transportation and Martime Affairs, Republic of Korea and Ministy of Knowledge Economy(MKE) and Korea Industrial Technology Foundation(KOTEF) through the Human Resource Training Project for Strategic Technology.

The KRICT PX1 gene (GB: FJ380951) consisted of 996bp encoding a protein of 332 amino acids (36kDa) from the recently isolated Paenibacillus sp. strain HPL-001 (KCTC11365BP) has been cloned and expressed in Escherichia coli. The xylanase KRICT PX1 showed high activity on birchwood xylan, and was active over a pH range of 5.0 to 11.0, with two optima at pH 5.5 and 9.5 at 50°C with Km value of 2.21 and 7.70, respectively. The xylanase activity was not affected by most salts, such as NaCl, LiCl, KCl, NH4Cl, CaCl2, MgCl2, MnCl2, and CsCl2 at 1 mM except CuSO4, ZnSO4, and FeCl3. And also, 1 mM of EDTA, 2-mercaptoethanol, and PMSF were not effective on the xylanase activity. TLC analysis of the catalyzed products after reaction with birchwood xylan revealed that xylobiose was the major product with smaller amounts of xylotriose and xylose. Similarity analysis of the amino acid in KRICT PX1 resulted 72% identity with xylanase from Geobacillus stearothermophilus (GB: ZP_0304036), 70% identity with intra-cellular xylanase from an uncultured bacterium (GB: AAP51133), 68% identity with endo-1-4-xylanse from Paenibacillus sp. (GB:ZP_02847150). In addition, the amino acid alignment of KRICT PX1 with GH family 10 xylanases revealed a high degree of homology in highly conserved regions including the catalytic sites. These results imply that KRICT PX1 is a new xylanase gene, and this alkaline xylanase belongs to GH family 10.

Candida Antarctica lipase B(CALB) is an important enzyme for bioorganic synthesis. The use of E.coli for the production of CALB has faced many practical and biological problems such as insoluble aggregates and proteolytic degradation. Numerous approaches have been tried to solve these problem including optimizations of expression conditions. But the methods have been mostly specific for their targets and are not highly effective. In this work the effect of the fusion partner on the protein expression and solubility were investigate as well as the effect of different E.coil expression systems on the expression level. The attempts to increase the solubility of recombinant CALB using T7 promoter resulted in the formation of inclusion body and low expression level. Therefore, we used Skp, SurA as the N-terminus fusion partners to enhance the solubility of CALB in E.coli cytoplasm.

Recently, polylactic acid (PLA), a biodegradable polymer showing many valuable characteristics and friendly environment, has been widely used in industrial applications. Lactide formation from lactic acid has played crucial role on the PLA formation. Candida antarctica lipase B (CALB) with high enantioselectivity toward R-form of substrate was found to catalyze the lactide formation. However, the abundant source of S-lactic acid commercially produced through microbial fermentation of glucose was rarely catalyzed by CALB wild type. By using Autodock program, 2 amino acid residues Tryp104 and Thr138 on CALB displaying the key role on the enantioselectivity of CALB favouring R-form of alkyl lactate were found. CALB mutants from these sites were made and transformed successfully into Pichia pastoris X33. We are expected that these mutants will show the enantioselectivity toward S- form of lactide production.

Mannheimia succiniciproducens MBEL55E is a capnophilic gram-negative bacterium which efficiently produces succinic acid. In order to analyze metabolic pathways of M. succiniciproducens, we applied pathway analysis to the biochemical network of M. succiniciproducens, previously developed by our group. We then also analyzed the biochemical network of Escherichia coli, in the same way as above, in order to grasp the notable differences between these two organisms. In order to draw clear conclusions, we clustered the solutions of two microorganisms, and compared each other. Each of clusters showed characteristic yield of succinic acid and the number of solutions and clusters of M. succiniciproducens is greater than that of E.coli. [This work was supported by the Genome-based Integrated Bioprocess Project of the Ministry of Education, Science and Technology (No. 2005-01294). Further supports by the LG Chem Chair Professorship, IBM SUR program, and Microsoft are appreciated.

Xylose reductase (XR) is a key enzyme in D-xylose metabolism, catalyzing the reduction of D-xylose to xylitol. Expression of XR in Candida tropicalis is significantly repressed in cells grown on glucose by catabolic repression. This is one of the reasons that glucose cannot be used as a cosubstrate in xylitol production. A gene encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was isolated from the genomic DNA of C. tropicalis ATCC 20913 to construct a constitutive expression cassette for this strain. The endogenous XR gene (xyl1) was placed under the control of GAPDH promoter and integrated into the genome of C. tropicalis N43 which is xyl2-disrupted and xyl1-partially disrupted mutant. Expression of XR was confirmed by determining enzyme activity in cells grown on a medium containing glucose as a carbon source. The resulting recombinant yeast, C. tropicalis JA4, showed higher XR activity (550mU/mg of proteins) than that of the parental strain (18mU/mg of proteins). Batch culture was performed for xylitol production with JA4 using glucose as a cosubstrate. The initial xylitol production rate of JA4 was higher than that of the parental strain.

We previously reported that the systematically mined β-glucosidase from Shinorhizobium meliloti efficiently converted the indican or plant extracts (containing the natural indican) of Polygonum tinctorium into a bright blue pigment indigo. The β-glucosidase was determined to be a monomer with a calculated molecular mass of 52 kDa, and also expressed at high level in soluble fraction under the control of novel expression system equipped with a selected promoter from metagenome. Subsequently, we develop a sensitive reporter system for gene cloning, which was suitable for discriminating the recombinant clones from negative ones in a high throughput manner. Using the novel chromogenic substrate indican, we analyzed the reporter system in terms of sensitivity, reliability and reproducibility. In addition, the Shinorhizobium β-glucosidase was also readily hydrolyzed the fluoregenic substrate MUG(4-Methylumbeliferyl-β-D-glucopyranoside) and a typical chromogenic X-gal, thereby providing a possibility that our system was broadly applied to various fields of molecular biology and bioengineering.

Oxaloacetate decarboxylase complex, encoded by oadGAB operon in M. succiniciproducens, is predicted to convert oxaloacetate into pyruvate, while competing with malate dehydrogenase leading to succinic acid production. As a verification of the homology analysis, oadGAB operon of M. succiniciproducens was deleted using a markerless gene deletion method. The fermentation of oadGAB deletion mutant verified that oxaloacetate decarboxylase did not affect the succinic acid production of wild type M. succiniciproducens.

As a reporter gene for gene expression and protein localization in a variety of cells and organisms, enhanced green fluorescent protein (EGFP) has been developed. However, N-terminal minimal domain has not been precisely studied for prokaryotes although several investigations have been reported for prokaryotic and eukaryotic cells. In this study, we studied N-terminal minimal domain and the Nterminal minimal domain of EGFP was determined to start after the fifth amino acid. Additionally, it was found that each amino acid from 5 to 11 was essential for the maintenance of the fluorescence of EGFP in E. coli.

In this study, a methodology for the development of a chemically defined medium (CDM) using genome-scale metabolic network and flux balance analysis and its improvement by metabolic engineering technology are demonstrated. Two amino acids, cysteine and methionine, and four vitamins, nicotinic acid, pyridoxine HCl, thiamine, and Ca-pantothenate, were identified as non-substitutable essential compounds to be supplemented to a minimal medium for the sustainable growth of Mannheimia succiniciproducens by the genome-based in silico analysis. The in silico predictions were confirmed by cultivating the Mannheimia succiniciproducens on a CDM containing the six nonsubstitutable essential compounds, and it was further verified by observing no cell growth on the CDM lacking any one of the non-substitutable essentials. To enhance the cell growth and the succinic acid production, as a target product, an optimal CDM was developed with a single-addition technique. The fermentation on the optimal CDM increased the succinic acid productivity by 36%, the final succinic acid concentration by 17%, and the succinic acid yield on glucose by 15% compared to the cultivation using a complex medium. Moreover, metabolic engineering technologies were applied to improve the Mannheimia succiniciproducens for the further enhancement of CDM in an economical aspect.

SSR markers had transferability and availability in similar species. The sea-buckthorns (Hippophae rhamnoides) are deciduous shrubs in the genus Hippophae, family Elaeagnaceae, with a range extending from the Atlantic coasts of Europe right across to northwestern China. More than 90 percent or about 1.5 million hectares of the world's sea buckthorn resources can be found in China where the plant is exploited for soil and water conservation purposes. While Grape and Hippophae belonged to the same item, but not the same faculty, SSR markers adapting to Hippophae rhamnoides were screened using reported Grape’s SSR primers. Two varieties (Zhongguo Shaji and Liaofu No.1) were evaluated for the detection of polymorphism. Among 107 primers, 16 primers showed polymorphism. Total 38 alleles were gotten according to their amplification, including 24 aberrant alleles (accounting for 36.16%). Allele number of each primer ranged from 1~4 with a mean of 2.375. The genetic similarity coefficient between two varieties was 0.570, and the genetic distance was 0.430.

Peroxidase catalyzes the oxidation by hydrogen peroxide of a number of substates, which has important roles in plants under oxidative stress. Leyumus chinensis (Chinese leymus) is become as a soil-binding plant to protect soil from desertification. In this study, we analyzed PRX gene of certain model plants (including rice, Arabidopsis thaliana, wheat, and bread wheat), and discussed the homology in term of plant genetics. We found that the diversity among sequences is great, expect of two sets of sequences from wheat and bread wheat (100% and 95%, respectively). Others had less than 33% of homology, even from the same species (HV-AF411228 and HV-AJ003141). So it is said that PRX gene of Chinese leymus which is closely relative with wheat according to genetics, was difficult to orientate in the homology tree written by 20 sequences of model plants. Compared with Chinese leymus EST peroxidase data and HV-DL138603 and TA-AY506502, results suggested that defence-relative EST sequences had higher homology (61%) with wheat, but not CD809225 and other metabolism relative EST sequences. The results are suitable to further study of cloning, analysis and orientation of PRX gene of L. chinensis.

Identification and determination of intracellular metabolites are the most important elements in metabolic engineering. Mostly metabolic flux analysis (MFA), metabolic control analysis (MCA) needs information about intracellular metabolites. Recent studies show that analytical instruments which have high sensitivity and high resolution power are quite useful for intracellular metabolites detection research. Gas chromatography and mass spectrometry (GC/MS) are powerful methods for detection and identification of various metabolites1). Though there are many attempts to analysis intracellular metabolites of various cells, it’s mainly focused on vitamins or amino acids1-2). In this study, intracellular metabolites that mainly related with glycolysis are identified using GC/MS. Detected intracellular metabolites are including glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-bisphosphate. Main parameters for optimizing detection condition were split ratio, injection volume and oven temperature. Also, sample preparation methods, including cell extraction methods, are tested for optimal detection.

1,2-Propanediol (1,2-PD) has plenty of applications, unsaturated polyester resins, antifreeze, food, and drug, etc [1]. 1,2-PD was produced by using renewable resource as commercially produced 1,2-PD has a variety of problems [2]. Saccharomyces cerevisiae was genetically modified and fermentation conditions were optimized through the regulation of variable factors. mgs and gldA genes which are related to 1,2-PD pathway were extracted from E. coli K-12 MG 1655. After cloning the genes in pYES2/CT and pYES3/CT, transformation was carried out. Subsequently, recombinant S. cerevisiae, S. cerevisiae::pYES2/CT/mgs + pYES3/CT/gldA, were obtained. After seed culture, Cells were inoculated at an OD600 of 0.1 (1% v/v) and induced using galactose when glucose was consumed, totally. 0.5% galactose was added in YPD medium at an interval of 4 hours. 1,2-PD was analyzed by HPLC with RI detector (flow rate : 0.8ml/min, oven temperature : 40℃).

1,2-Propanediol, also known as propylene glycol, is a major commodity chemical with world market of $ 123.6 millions/yr. In our previous research, we engineered S. cerevisiae strain to produce 1,2-propanediol with tpi1(triosephosphate isomerase) deleted mutant. By introduction of E. coli mgs(methylglyoxal synthase) and gldA(glycerol dehydrogenase) using a multicopy plasmid, 1.11 g/l 1,2-propanediol was achieved with 2%(v/v) ethanol as a major carbon source. In this study, glycerol was used as a sole carbon source under microaerobic condition, much higher 1,2-propanediol productivity was achieved with overexpression of mgs and gldA. The productivity was enhanced when fps1 gene encoding glycerol transporter was overexpressed in S. cerevisiae. As result, we got 3.23 g/l 1,2-propanediol concentration during 144h flask cultivation with 1%(v/v) glycerol as a carbon source.

Due to global warming and rising price of fossil fuel, microbial production of bio-fuel from organic byproducts has acquired significance in recent years. Over the few years ethanol has been trusted as an alternate fuel for the future. The ethanologenic pathway in Z. mobilis (Zymomonas mobilis), like that of Saccharomyces cerevisiae, consist of two essential activities, pyruvate decarboxylase (pdc) and alcohol dehydrogenase (adh). E. coli was able to ferment sugars into ethanol by inserting Z. mobilis genes encoding pyruvate decarboxylase and alcohol dehydrogenase II. These two enzymes produces 30 to 50% of the soluble proteins which activates the glycolysis pathway in Z. mobilis. In this study expression vector carrying pdc and adhB genes were constructed by using pSTV28 vector. Ethanol productivity of E. coli strains were thoroughly affected by the expression of pdc gene along with adhB genes. By successful gene mutation we will establish in constructing new E. coli strains that produces ethanol efficiently. Also to confirm the production of ethanol, fermentation experiment of recombinant ethanolic E. coli MG1655 and E. coli W3110 are going to be performed in aerobic conditions. Also to increase the ethanol productivity, galP (D-galactose transporter) gene and glk (glucokinase) gene in both E. coli strains were over-expressed by the expression vector ptrc99a. To confirm the production of ethanol in these new recombinant E. coli strains, fermentation experiment in aerobic conditions are also going to be performed. Pretreatment of marine algae were also going to be used as the culture medium of the aerobic fermentation experiments preformed by mutated E. coli strains as above.

Decursin is a pyranocoumarin isolated from the root of Anglica gigas Nakai and has been reported to have anti-inflammatory and anticancerous (1, 2). Recently, it has been reported that Decursin stimulates glucose uptake by activating AMP-activated protein kinase (AMPK) (3). However, the mechanism by which Decursin activates AMPK remains undefined. So, the mechanism is investigated through which decursin activates AMPK in skeletal muscle cells by comparing insulin and metformin signal (4, 5). Activation of AMPK requires the phosphorylation of Thr172 α-subunit by upstream AMPK kinase (AMPKK) (6). Presently, two upstream AMPKKs have been discovered: LKB1 and Ca2+/calmodulin-dependent protein kinase kinase (CaMKK). Phosphorylation of AMPK by LKB1 requires an increase in AMP or AMP mimetic agents (7). However, CaMKK activates AMPK by a rise in the intracellular Ca2+ concentration in an AMP independent manner (8, 9). The aim of this study is to investigate the mechanism leading to AMPK activation by Decursin in muscle cells. The results reveal that Decursin activates AMPK by Ca2+-independent manner. However, insulin and metformin activates AMPK by Ca2+-dependent manner.

The cyclic undecapeptide, cyclosporin A (CyA), one of the most valuable immunosuppressive drugs, is produced non-ribosomally by a multi-functional cyclosporin synthetase enzyme complex by the filamentous fungus Tolypocladium niveum. The various structural modifications of cyclosporins such as a regiospecific hydroxylation at N-methyl leucines lead to a dramatic changes of their bioactive spectra. A Rare Actinomycetes named Sebekia benihana was identified to be able to hydroxylate CyA at the position of 4th Nmethyl leucine. Previously, six different novel Cytochrome P450 hydroxylase (CYP) genes have been isolated from S. benihana, followed by the complete sequencing and characterization1). Since a rare actinomycetes speices including, S. benihana is not currently feasible for genetic manipulation, we tried to establish genome engineering techniques for S. benihana in order to maximize its potential as a bioconversion host of various valuable metabolites including CyA in vivo. Detailed results including interspecies conjugation, foreign gene-integration, and a targeted-gene-disruption in S. benihana will be discussed.

The advent of in silico genome-scale model developed various algorithms for the gene targeting. Flux balance analysis (FBA) optimizes a specific objective function by linear programming under pseudo-steady state based on the stoichiometry of metabolic reactions. . However, single objective algorithms optimize only the limited objective function. To improve a strain for biochemical production, the organism should be investigated from diverse sides simultaneously: for instance, biomass formation, biochemical production, and waste formation In this respect, we propose a new approach called the flux scanning with compromised objective fluxes (FSCOF), multi-objective algorithm. [This work was supported by Korean Systems Biology Research program (M10309020000-03B5002-00000) of the Ministry of Education, Science and Technology. Further supports by LG Chem Chair Professorship, Microsoft and IBM SUR program are appreciated.]

Microbial pathogens are constantly developing resistance to antibiotics, giving impetus for the development of efficient methods for drug targeting and discovery. Genome-scale biological network is expected to play a significant role, as it has succeeded in capturing the physiological characteristics of various organisms. We herein employed two systematic approaches. One of them is constraints-based flux analysis, an optimization-based simulation technique that calculates metabolic fluxes (1). Another important work is chokepoint analysis, which is network-topology-based method that selects enzymes or metabolites as a target that has a single ingoing and/or outgoing reaction (2). We combined these methods to generate novel drug targets in several emerging drug-resistant pathogens, including Escherichia coli, Helicobacter pylori, Mycobacterium tuberculosis and Staphylococcus aureus. This study demonstrates that drug targeting using in silico approaches enables a rational design of experiments applicable to biomedical science. [This work was supported by the Korean Systems Biology Research Project (M10309020000–03B5002–00000) of the Ministry of Education, Science and Technology. Further supports by the LG Chem Chair Professorship, Microsoft, and IBM SUR program are appreciated.

than those of daidzein. P450s have the ability of converting daidzein to hydroxydaidzein i.e. 3΄-hydroxy, 6-hydroxy, and 8-hydroxydaidzein. We already found that CYP105D7 (SAV7469) has the specific activity of converting daidzein to hydroxydaidzein. We compared the various strains for the biosynthesis of hydroxydaidzeins. Among strains examined, Streptomyces avermitilis MA4680 highly converted daidzein to orthospecific dihydroxyisoflavones. We investigated recombinant Streptomyces avermitilis strain found to have 3.6 times higher ability than wild type to convert 3΄-hydroxydaidzein. In this report, we found a new pathway-specific regulator which binds to the promoter region of CYP105D7 (SAV7469) in Streptomyces avermitilis by the DNA affinity capture assay method. Since this regulator is well conserved throughout the Streptomyces species, it is expected to have an important role in Streptomyces avermitilis. We made an overexpression mutant, deletion mutant of this regulator and checked the activity of converting daidzein to hydroxydaidzein compared to the wild type strain.

Elevated total homocysteine (Hcy) levels have been associated with an increased risk of atherosclerotic disease, coronary heart disease, and clinical stroke. Cysteine (Cys) can be obtained as the final product of the transsulfuration pathway through Hcy metabolism. It is essential and important to develop some pre-column fluorescent derivatizing reagents for the detection of the Hcy and Cys with ease, quick, accuracy and specificity. 4-maleimide-7-nitro-2,1,3-benzoxadiazole (NBD-M), a novel pre-column fluorescent derivatizing reagent was designed and synthesized. NBD-M was synthesized on the base of bezofurazan skeleton. Maleimide and 4-chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl) were dissolved in the acetonitrile solution, respectively. NBD-Cl solution was added drop-wise to the maleimide solution containing sodium carbonate and was reacted at room temperature for 30 minutes. And then the product was separated by silica gel chromatography and determined by mass spectroscopy. NBD-M has relative low molecular weight and reacted with Hcy and Cys selectively. The high stabilities of the derivatives of Hcy and Cys with NBD-M are suitable for the HPLC analysis.

1,2-Propanediol (1,2-PD: Propylene glycol) is a major commodity chemical that is currently derived from propylene, a non-renewable resource. 1,2-PD is three-carbon diol with a stereogenic center at the central carbon atom. The Saccharomyces cerevisiae is a successful industrial microorganism and it has been characterized fully. For 1,2- PD production, 2 key genes (mgs and gldA) from Escherichia coli were placed under the control of the CUP1 promoter. The induction time control led to the increase in 1, 2-PD production. The effect of inducer concentration was studied. Also, the effect of temperature on the production of 1, 2-PD was examined from 28°C to 30°C. By using the data obtained from shake flask culture, cells were grown in 5L fermenter.

The Gram-positive soil bacterium, S. coelicolor A3(2) produces various antibiotics. There are various approaches to improve antibiotics production and we focused transcriptional regulators. By controlling not only pathway-specific transcriptional regulators but also novel regulators, we can control antibiotics production. To find novel regulators, we used DACA method developed in our lab, and the method to analyse microarray data. It is possible to get the raw array data from the NCBI’s GEO. We obtained 11 Streptomyces GEO datasets, which are the mRNA expression data from various conditions. After combining every dataset, we performed normalization. And we compared every normalized profile with interesting antibiotics cluster profiles, and in this case, we used RED cluster and ACT cluster. By using Pearson product-moment correlation, we selected 119 genes using RED cluster and 85 genes using ACT cluster. Our hypothesis is that genes which show similar expression profiles with antibiotics cluster have possibility to affect antibiotics production. In conclusion, we found 15 regulator genes, some are already reported and the others are not yet reported, but studied in our lab.

Tautomycetin (TMC), which is produced by Streptomyces sp. CK4412, is a novel activated T cell-specific immunosuppressive compound with an ester bond linkage between a terminal cyclic anhydride moiety and a linear polyketide chain bearing an unusual terminal alkene. Previously, we isolated and characterized the entire TMC biosynthetic gene cluster from Streptomyces sp. CK44121). And also, TMC pathway-specific gene, tmcN was successfully utilized to increase TMC productivity by introducing its extra copies, implying the importance of the regulatory network optimization for strain improvement2). It was previously reported that a 64-amino-acid encoding wblA inhibited the biosynthesis of doxorubicin in S. peucetius as well as the antibiotics production in S. coelicolor, suggesting that wblA and its homologs act globally among streptomycetes as putative down-regulators of antibiotic biosynthesis3). We identified a positive cosmid clone containing wblA-tmc via genomic DNA library screening using the degenerated primers based on wblA sequence from S. coelicolor A3(2). A 0.39kb gene, wblA-tmc showed a high amino acid similarity compared to a previously-known S. coelicolor wblA. Gene disruption of wblA-tmc from the Streptomyces sp. CK4412 chromosome resulted in significantly different morphological differentiation pattern as well as TMC productivity. The more detailed results including complementation data will be discussed

In our previous study, the genes encoding the benzoate metabolism of Chromohalobacter sp. HS-2 were cloned into a cosmid vector, sequenced, and then analyzed to reveal the m-hydroxybenzoate hydroxylase gene (mobA), p-hydroxybenzoate hydroxylase gene (pobA). And two putative regulatory genes (pcaQ and pobR) were also detected. The HS-2 genes involved in benzoate and m-, p-hydroxybenzoate degradation were clustered within approximately 40 kb region, and showed quite a different genetic organization from those of other benzoate catabolic genes. In this study, mobA and pobA gene were ligated with the pHCEIIB vector and the constructed plasmid were transformed into E. coli DH5α. After 16 hour, each recombinant E. coli was constitutive expressed MobA (72 kDa) and PobA (23.3 kDa) without IPTG induction. The HPLC and LC/MS analysis revealed that resting cells of E. coli DH5α harboring mobA oxidized mhydroxybenzoate to protocatechuate and those harboring pobA converted p-hydroxybenzoate to protocatechuate, respectively, at 37℃.

The number of patent applications containing gene sequences increase each year, with gene sequences appearing in patents that may be broadly applied to many aspects of biotechnology and drug development. Nonetheless, relatively few reports have focused on gene-related patents, and there has been no prior attempt to study patent-related sequences from a biological perspective. We herein used bioinformatics analysis to identify patent-associated genes. The biological sequences disclosed in published patents were compared with RefSeq database, and the patent-associated hits were mapped to Entrez Gene, OMIM and GO databases. Mapping analysis revealed that 24,273 (61%) of known human genes were related to patenting, and asthma was the frequently patent-associated disease appearing in 264 patents. We have consolidated the association results and the patent sequence data to a relational database and implemented a web-based user interface to provide search service. We also provide web-based BLAST facility, allowing users to compare their sequences against patent sequences.

The individual enantiomers (R-1,2-PD and S-1,2-PD) have potential uses as chiral synthons for the production of pharmaceuticals and novel polymers. S. cerevisiae which is able to produce 1,2-propandiol was developed by inserting two important genes (mgs and dhaD genes). As a result, the batch fermentation of S. cerevisiae YPH500, harboring pJES27 vector inserted a mgs gene, resulted in a yield of 0.17g/L. In order to achieve higher yield of 1,2-PD we selected the pESC-TRP vector which is able to co-express the dhaD gene with the pJES27 vector. In prior studies, the maximal yield of 1,2-PD achieved in the 1% galactose batch fermentation by pJES27 and pJES30 harboring S. cerevisiae was 0.45g/L. Recently, our numbers were improved the maximal yield of 1,2-PD into 0.51g/L by optimizing fermentation.