Earticle

We investigated the relevance between the compactness of β-galactosidase inclusion bodies (β-gal IBs) and their enhanced enzymatic activity with or without the addition of D-fucose (inducer analog) or methyl α-Dglucopyranoside (α-MG, catabolite repressor,) after induction in the araBAD promoter system of Escherichia coli. Experiments conducted to evaluate the solubilization of β-gal IBs in guanidine hydrochloride, as well as their trypsin degradation and temperature stability revealed that β -gal IBs expressed in response to the addition of D-fucose or α-MG had a looser structure. Additionally, β-gal IBs expressed when D-fucose or α-MG was added were more quickly solubilized in guanidine hydrochloride or degraded by trypsin-treatment than those produced when these compounds were not added. Moreover, the specific activity of β-gal IBs expressed when D-fucose or α-MG were added was less stable at various temperatures. Consequently, we deduced that the looser structure of β-gal IBs resulted in enhanced enzymatic activity of β-gal IBs upon addition of D-fucose or -MG after induction.

In order to enhance the activity of the recombinant epoxide hydrolase of Caulobacter crescentus(CcEH), the EH gene was inserted to pColdI vector, pET21b(+) vector and pET28b vector, then expressed in the presence of various molecular chaperones and foldases. The specific activity of CcEH was the highest when the gene was expressed with pTf16 plasmid containing tig gene. We carried out enantioconvergent hydrolysis of racemic 4-chlorostyrene oxide and styrene oxide for the preparation of enantiopure (R)-4-chrolopheny-1,2-enthanediol and (R)-phenyl-1,2-ethanediol. The styrene oxide and 4-chlorostyrene oxide were analyzed by a chiral GC, and the corresponding diols were analyzed by a chiral HPLC. Chiral (R)-4-chrolopheny-1,2-enthanediol and (R)-phenyl-1,2-ethanediol with enantiopurity more than 98% was obtained with yield greater than 50%.

Homocysteine is a thiol-containing amino acid derived from the metabolism of methionine and causes increased risk for various human diseases and birth defects. Homocysteine is catalyzed by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CGL) in transsulfuration pathway. Although these two enzymes are key proteins for modulating the homocysteine concentration and also formation of hydrogen sulfide, the mutual proteins interaction is unclear. To investigate the enzyme regulation of these two enzymes by protein interaction, we preliminary studied the protein interactions for both enzymes by means of native PAGE analysis and a blot overlay analysis. In native-PAGE experiment, we could detect the new band having higher molecular weight after mixing CBS with CGL. The CBS-CGL complex exhibited strong enzyme activity for the formation of hydrogen sulfide. And in the overlays experiment, CGL with GST fusion CBS revealed higher binding affinities to compare with control protein, bovine serum albumin. Our data suggest that CBS and CGL may bind tightly maintaining enzyme activity. We are investigating the substrate specificity change of CBS-CGL complexes.

Glycerol carbonate is a key bifunctional compound employed as solvent, additive, monomer, and chemical intermediate. Candida antarctica lipase B (CalB) is an efficient biocatalyst for many organic synthesis reactions. We found that CalB catalyzed the synthesis of glycerol carbonate and biodiesel from triglyceride and dimethyl carbonate through transesterification. Various organic solvents including t-butanol and tetrahydrofuran were used as the reaction medium to enhance the formation rate of glycerol carbonate and biodiesel.

A recombinant d-lyxose isomerase from Providencia stuartii was immobilized on the of various types beads. Beads allowed the highest conversion of fructose to mannose among the various immobilization beads evaluated and then were selected for the production of mannose from fructose. The maximum activities of both the free and immobilized enzymes for fructose isomerization were observed at pH 7.5 and 45°C in the presence of 1 mM Mn2+, and enzyme half-lives were 14 and 30 h at 35°C and 3.4 and 5.1 h at 45°C, respectively. The immobilized enzyme, in a solution of 300 g/l fructose (replaced hourly), produced 75 g/l mannose at 35°C representing a conversion yield of 25% (w/w) and a productivity of 75 g l–1 h–1 mannose after 23 cycles.

Osteoblastic bone formation (bone remodeling) is a coupled process where bone resorption is normally followed by new bone formation. During some pathological processes in osteoporotic patients, bone remodeling is uncoupled, and bone resorption is not followed by new bone formation. In this study, naringin and lipoic acid was enzymatically esterified by catalyzing with lipase (Novozym 435) in 2-methyl-2-buthanol, to get naringin derivative having lipoic acid at 6-position of its glucose moiety. The chemical structure was characterized by NMR and Mass spectroscopy. The synthesized naringin lipoate significantly enhanced alkaline phosphatase activity, osteocalcin level and matrix protein synthesis in osteoblast like cells. Moreover, naringin lipoate increased bone morphogenic protein synthesis in a dose-dependant manner. Therefore, this result suggests that naringin lipoatecan be used as a nutraceutical and a drug for osteoporosis and related symptoms.

Among 27 cytochrome P450s (CYPs) of Nocardia farcinica IFM10152, three CYPs were identified to have O-dealkylation catalytic activity. Between two of them encoding CYP154 subfamilies, CYP154 encoded by nfa22930 gene showed distinct O-dealkylation and subsequent hydroxylation activities for formononetin. Firstly, formononetin was O-dealkylated into daidzein, and daidzein was subsequently mono-hydroxylated at 3’-position of B-ring into ortho-dihydroxy-isoflavone. Apparent kcat/Km values of CYP154 for formononetin O-dealkylation and daidzein hydroxylation reaction were 3.57 and 1.84 μM-1min-1, respectively. The dissociation constants based on spectral changes for binding with each substrate were 5.16 and 3.11 μM, respectively. Homology modeling and docking simulation predicted that Thr247 is a key residue responsible for the 3’-position hydroxylation reaction by forming hydrogen bond with 4’-hydroxyl group of daidzein and making the proton atom at 3’-position face to heme center. And site-directed mutagenesis of Thr247 into alanine led to a drastic decrease in the binding affinity for daidzein (9.73 μM) and 3’-position hydroxylation catalytic activity down to 3 folds (0.48 μM-1min-1).

Alginate lyase from a novel marine microorganism, Streptomyces sp. ALG-5, was expressed in the presence of various molecular chaperones in Escheichia coli BL21(DE3). The hydrolytic degradation activity toward 0.4 %(w/v) alginate solution was two times enhanced when the alginate lyase gene was expressed in pColdI plasmid than pET-21b(+) at low temperature, 15oC. The reaction condition of alginate lyase-catalyzed hydrolysis of alginate was optimized. The purified enzyme was immobilized onto the Fe3O4/silica/NiO magnetic nanoparticles efficiently, and re-used five times with retention of more than 50% of initial activity in repeated batch operations.

Site-directed mutagenesis (SDM) is one of the common methods used in studying the structure-function relationships of proteins. Mutations at the level of nucleic acids are related to altered properties such as the level of expression, the stability, and/or the change in the catalytic activity of a target protein. To this end, nucleotide-directed mutagenesis methods based on polymerase chain reaction (PCR) is widely used. Even though mutagenesis methods are efficient, the accompanying steps such as transformation, colony peaking, and in vivo expression of the mutant proteins are very time consuming. Thus, we integrated the PCR-based mutagenesis method in the up-stream steps of cell-free protein synthesis to build an efficient system to carry out scanning mutations in parallel. The mutagenic system is devised to enrich mutated DNA while completely removing the residual wild-type template DNA using combination of DNA ligase, polynucleotide kinase, Dpn I, and lambda exonuclease. Results from the proof-of-concept experiment will be presented.

Cholesterol esterase (CHE; EC 3.1.1.13) hydrolyzes fatty acid esters of cholesterols and is widespread in nature. Mammalian CHEs have been extensively studied, because they have importance in cholesterol absorption and metabolism. Some microbial CHEs are used as diagnostic reagents for measuring total serum cholesterol in blood(1). And also the enzyme is responsible for the digestion and absorption of lipid nutrients similarly to triglyceride lipase and phospholipase, therefore extensive research has been devoted to the structure-function relationship of this protein as well as its physiological roles in lipid metabolism. Using enzyme immobilization technology we can extensively utilize the enzyme as a biosensor. A novel CHE genes from Pseudomonas aeruginosa, Vibrio parahaemolyticus, Streptomyces avermitilis are cloned and expressed by E.coli. Using PCR(Polymerase Chain Reaction)technology we cloned target sequence and also Using PQE30 verctor we transformed it into a competent cell. Protein expression experiment was carried out by IPTG induction and expression level was confirmed by SDS-PAGE. Their cholesterol activity was investigated by Cholesterol/Cholesteryl Ester Quantitation Kit (BioVision03-100). In further study we will investigate the substrate specificity of the enzyme and immobilization possibility.

A fungal peroxidase from Coprinus cinereus (CiP) was successfully being used for the oxidative polymerization of phenolic compounds. During polymerization reaction, CiP showed low stability in organic solvent especially water miscible solvent such as 2-propanol. Generally speaking, the thermostability of enzyme can be related with the stability in organic solvent. CiP variants were created by site directed mutagenesis and their enzymatic activities in organic solvent and the thermostability were studied. In various CiP variants, the C50 values were remarkably higher for the thermostable variants than for the other mutated variants. The stability in organic solvent is proportionate to thermal stability for CiP variants. Furthermore, the strongly stable CiP variant against thermal inactivation also showed the resistance against to organic solvent, which suggests that the mechanism of irreversible thermal and solvent-induced inactivation seem to be similar.

A metagenomic approach is an efficient way for discovery of novel and useful genes from uncultured environmental microorganisms. In this study, we constructed metagenomic libraries from cow rumen fluids. Screenings of the libraries revealed a clone showing carboxymethyl cellulolytic activity. Further analysis of the insert DNA showed an ORF encoding cellulase. A multiple sequence analysis with the deduced amino acid sequences of engR1 showed the highest identity with endoglycosidase from uncultured bacterium (62%) followed by beta-1,4-endoglucanase from Prevotella bryantii (49%). The recombinant EngR1 protein was not only highly active against soluble cellulose containing β-1,4 linkages, such as barley β-glucan but also active against hemicellulose such as xylan. The highest enzymatic activity was observed at pH 4.0 and 43℃with recombinant EngR1. The enzyme was highly stable at 50℃ for 120 min.

Xylanase (EC 3.2.1.8) is the name given to a class of enzymes which degrade the linear polysaccharide beta-1,4-xylan into xylose, thus breaking down hemicellulose, which is a major component of the cell wall of plants. Previously, a new xylanase gene, KRICT-PX1 isolated from Paenibacillus sp. HPL-001 was expressed in E. coli, and the biochemical properties of the enzyme was examined. Error-Prone PCR conducted on the xylanase gene KRICT-PX1, and produced a mutation library. Enhanced xylanase activity from mutant clones were effectively identified using DNS assay in 96-well micro-plate after sonic cell disruption. From 272 mutant clones, a P1H1 clone showing strong xylanase activity was selected. The xylanase activity of the P1H1 was 53.5 U/mg protein at pH7 and 50°C, which is approximately 5 time-increased comparing to the original KRICT-PX1 xylanase with the activity of 10.25 U/mg protein at the same condition. The TLC analysis of the products of birchwood xylan catalyzed by two xylanases revealed that the hydrolysis by P1H1 xylanase was significantly faster than that of KRICT-PX1. DNA sequencing of the P1H1 clone showed that it had one amino acid substitution (168Gly/Gln). Further research on the biochemical property and the structural analysis of the point-mutated xylanase will be necessary for the catalysis mechanism of xylanase.

A novel enzyme support derived from cellulose and chitosan was prepared under an environment-friendly preparation processes using ionic liquids. Cellulose and chitosan were codissolved in [Emim] [acetate]. The obtained clear and viscous solution was introduced dropwise into water bath using a syringe needle. Then the hydrogel beads were collected and extensively washed with deionized water. Prepared cellulose-chitsoan composite bead was treated with glutaraldehyde and then used as the support to immobilize formate dehydrogenase. Formate dehydrogenase was successfully cross-linked on the surface of cellulose-chitosan bead. Immobilized enzyme was easily reused and remained activity after 5 times reuse was over 95% of initial activity. Thermostability and pH stability of formate dehydrogenase immobilized on cellulose-chitosan bead were also highly enhanced. Half-life times of immobilized enzymes were 1.8 and 7.1 times increased at 52℃ and pH 4.3, respectively.

Despite the promising potential of cell-free protein synthesis, the high cost of the required reagents has plagued its practical application. In particular, most of the reagent cost is attributed to the expenses for conventional energy sources such as phophsoenol pyruvate (PEP) and creatine phopshate (CP). In this study, for the preparative expression of recombinant proteins at a reduced energy cost, we attempted to use the glycolytic intermediates to drive the cell-free protein synthesis in a continuous-exchange cell-free protein synthesis (CECF) reaction. Among the glycolytic intermediates examined, use of fructose 1,6-bisphosphate (FBP) gave the highest yield of protein synthesis, producing approximately 7 mg/ml of a chloramphenicol acetyl transferase (CAT) protein. We also found that protein synthesis in the CECF reaction was able to be directed by PCR-amplified linear DNA templates by employing the measures to stabilize mRNA in the reaction mixture.

Transaminase (TA) catalyzes transfer of an amino group between amino acids and α-keto acids. The TA can be divided into two classes depending on the position of the amino group in an amino donor relative to the carboxyl group, i.e. α-TA and ω-TA. The TA requires pyridoxal 5'-phosphate (PLP) as a cofactor. The TA has gained increasing attention owing to high turnover rate, precise enantioselectivity and no cofactor requirement. To develop novel biocatalytic processes for production of unnatural amino acids, we cloned three bacterial ω-TAs selected by amino acid sequence homology using the well-studied w-TA from Vibrio fluvialis JS17. The three ω-TAs from Paracoccus denitrificans, Ochrobactrum anthropi and Pseudomonas stutzeri were cloned into pET28 expression vector. Despite of high sequence homology, the three ω-TAs showed very similar enzymatic properties including substrate specificity, optimal pH and inhibitions by substrates and products. Benzylamine and (S)-a-ethylbenzylamine ((S)-a-EBA) are the best amino donors among the ones tested. In the case of amino acceptor specificity, the ω-TAs show high activities towards 2-oxobutyricacid, pyruvate, butanal and benzaldehyde. Strong substrate inhibitions by pyruvate and butanal were observed whereas substrate inhibition by amino donors including (S)-a-EBA and benzylamine was moderate. In the case of product inhibition, benzaldehyde and propiophenone exerted high enzyme inhibition. The optimum pH and temperature for the enzymes were 9 and 37 oC, respectively. We carried out asymmetric synthesis of L-2-aminobutyric acid using 2-oxobutyricacid (10 mM) and benzylamine (20 mM) as substrates and the conversion yield reached 79, 69 and 76 % using the ω-TAs from P. denitrificans, O. anthropi and P. stutzeri, respectively, after 5 h reaction.

Enzymes catalyze industrially important reactions in organic solvents. But their activity in anhydrous media is extremely low compared with its activity in water media. It is due to decrease of enzyme motion in the absence of lubricant water followed by enzyme flexibility decrease. Enzyme flexibility has relation to enzyme activity because enzyme does flexible motion for catalysis. Thus, enzyme flexibility modulation can lead enzyme activity enhancement. In this study, Candida antarctica lipase B(CalB) was selected as a model enzyme for mutations to change enzyme flexibility. From the analysis using spring model, it was detected that forced and twisted regions of CalB were located in residues surrounding active site. The edges of these helices were selected as target sites aiming at increasing flexibility. The details will be presented and discussed.

Glutamic acid decarboxylase (GAD) catalyzes the decarboxylation of L-glutamate to gamma-amino butyric acid (GABA), which is not only a chief inhibitory neurotransmitter in human nervous system but also a monomer for polyamide 4, a new attractive biodegradable material. Therefore, GAD is of interest in large-scale production of GABA and polyamide 4. In this approach, a 6xHis tag was fused to GAD at the N-terminus and overexpressed in E. coli. However, overexpression of GAD resulted in the formation of inclusion body. Efforts were endeavored to increase the amount of soluble enzyme, 0.1 g/l pyridoxine in the culture proved to be the most efficient for the purpose. On the other hand, the activity of GAD was only detected at acidic pH, which limits the reaction at relatively low concentration of the substrate. Since the C-terminal amino acid residues are considered to be responsible for the inactivation of this enzyme at neutral pH, C-terminal residues were truncated for wider pH range of GAD.

The production of biodiesel by enzymatic transesterification has attracted much attention for high purity product and enables easy separation. Candida antarctica lipase B is one of the most important enzyme in the biodiesel reaction. In reaction, CALB shows low organic solvent stability while having relation with methanol. In this study, we concerned about enhancing organic solvent stability in hydrophilic solvents. In watermiscible solvents, water molecule seems to play an important role in biological structure and function. Water molecules in the enzyme are stripped away or replaced with hydrophilic solvent molecules and this causes deformation and denaturation of the enzyme. Molecular dynamic simulation were proceed to search how enzyme actually motion in organic solvents. RMSD value of each residue were calculated for distinguish having highly alteration amino acids, which are determined as weak spots. For further analysis, secondary structure, solvent accessible area, intra molecule interaction and water molecule movements were identified and calculated.

Candida Antarctica lipase B(CALB) is an important enzyme for biodiesel reaction. The use of E.coli for the production of CALB has faced many practical and biological problems such as insoluble aggregates and proteolytic degradation. And CALB shows low organic solvent stability in methanol. Numerous approaches have been tried to solve these problems 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 investigated ad well as the effect of different E.coli expression system on the expression level. Skp, pelB, GST were used as the N-terminus fusion partners to enhance the solubility of CALB in cytoplasm for the direct fusion expression of CALB. To compare expression level, Skp was co-expressed. Consequently, the study led to the conclusion that Skp was a powerful solubility enhancer.

Lipase B from Candida antarctica (CalB) showing several excellent characteristics such as high stereo-selectivity and stability in organic solvent has attracted much attention for various enzymatic bioconversion reactions [1]. To apply lipase to commercial process, the thermal stability is one of the prerequisites. In this study, we improved the thermal stability of CalB by increasing the number of disulfide bridges based on B-factor. MODIP program was used to determine the probability of a disulfide bridge formation between two adjacent amino acid residues. As a result, 21 amino acid couples with the highest possibility (grade A) were firstly chosen for further investigation. Among those couples in grade A, we selected four candidates (S50-A273, Q156-L163, A162-K308, N169-F304) based on B-factor of its residues. Four double CalB mutants (S50C-A273C. Q156C-L163C, A162C-K308C and N169C-F304C) were constructed and investigated to determine the thermal stability. Two mutants A162C-K308C and N169C-F304C showed improved thermal stability compared to its wild type. We anticipate that those mutants will be one of the good candidates for further study to improve the thermal stability of CalB.

Corynebacterium glutamicum, a Grame-positive soli bacterium, is a fast-growing, nonmotile with a long history in the microbial fermentation industry for amino acids and nucleic acid. In experiment performed to identify transcriptional regulators of the TCA cycle and engineered for production of succinic acid of Corynebacterium glutamicum, the regulator of DeoR-type SucR was isolated and verified by DNA affinity chromatography and electrophoretic mobility shift assays with the promoter region of the sucCD operon encoding succinyl-CoA synthetase. The regulatory effects of this protein on sucCD were tested by promoter activity assays and gene expression level measurement. The sucCD expression of sucR deletion mutant was 25 times higher than those of wild type in cells grown on acetate. Transcriptional lacZ fusion result indicated that SucR repressed the expression of the sucCD. Evidence was obtained that SucR functions as a repressor of sucCD expression on acetate metabolism. This work was supported by National Research Foundation of Korea Grant funded by the Korean Government (2009-0073856)

Gamma aminobutyric acid (GABA) is the non-essential amino acid not found in natural proteins and is found in the human brain and eyes. It serves as a major inhibitory neurotransmitter with hypertensive and diuretic effects in animals. GABA has been applied in health functional foods, which may promote relaxation and relieve nervous tension. Recently, bio-based fuels and polymers have become important due to the increase of oil price and concerns about environmental pollution. Because GABA can be used for the possible monomer of nylon 4, which is the biodegradable heat-resistant polymer, production of GABA from cheap carbon source such as L-glutamate (MSG) become important in an industrial application. In this presentation, we report bioconversion of L-glutamate to GABA by recombinant Escherichia coli expressing glutamate decarboxylase. Temperature, pH, and initial L-glutamate concentration during reaction were examined for the production of GABA.

Clopidogrel is a platelet aggregation inhibitor widely administered to atherosclerotic patients with the risk of a heart attack or stroke that are caused by the formation of a clot in the blood. Plavix (clopidogrel bisulfate) is marketed worldwide in nearly 110 countries, with sales of $6.4 billion per year. It had been the second top selling drug in the world for a few years and was still growing by over 20%. Methyl (R)-2-chloromandelate is key intermediate of clopidogrel and should be enantiopure. Considering the enantioselective nature of enzyme, biological transformation of ketones into optically active alcohols could be an efficient strategy. In the present study, we investigate the factors that influence the reduction of methyl-2-chlorobenzoylformate to find optimum conditions enabling the efficient production of methyl (R)-2-chloromandelate. [This work was supported by the Small & Medium Business Administration].

Yeast has been studied extensively as a model system for higher eukaryotic organisms, including Homo sapiens and has contributed greatly towards a better understanding of higher eukaryotic cellular functions. The fission yeast Schizosaccharomyces pombe, is an attractive system as a model due to the similarity in cell division with mammals, fission as opposed to budding, which is used in the more characterized yeast Saccharomyces cerevisiae. Additionally, S. pombe possesses genes that are similar to genes that are found in humans that are responsible for a number of genetic diseases. Here we have reconstructed the genome-scale metabolic model of the fission yeast S. pombe to serve as a platform for better understanding the metabolic phenotype of S. pombe, and other eukaryotic systems. [This work was supported by the Korean Systems Biology Research Project (20090065571) of the Ministry of Education, Science and Technology (MEST) through the National Research Foundation of Korea (NRF). Further supports by the World Class University Program (R32-2008-000-10142-0) of the MEST, LG Chem Chair Professorship, IBM SUR program, and Microsoft are appreciated.]

Previous studies suggested that p38 MAP kinase inhibition can suppress the glial cell activation. This study demonstrated that NADPH oxidase activity is essential for inflammogen-mediated induction of reactive oxygen species (ROS) and iNOS expression in murine N9 microglial cells. The NADPH oxidase inhibitor, diphenyleneiodonium chloride, blocked lipopolysaccharide (LPS)-induced nitric oxide (NO) production in parallel with inhibition of NADPH oxidase activity. Of interest, p38 MAP kinase inhibition suppressed both NO production and NADPH oxidase activity which were induced by LPS. In addition, this study revealed that quercetin, a bioactive phenolic compound known to inhibit oxidative stress and inflammation in macrophage, significantly attenuates microglial production of NO and ROS, a product of NADPH oxidase in response to LPS treatment. Furthermore, quercetin inhibited p38 MAP kinase activation induced by LPS in microglia. These results suggest that quercetin may inhibit LPS-induced microglia activation through inhibition of p38 MAP kinase.

Branched chain amino acids, including L-valine, L-leucine, and L-isoleucine, have gained commercial attention due to their important applications in feed additives, pharmaceuticals as well as cosmetics. We focused our research on the microbial production of L-isoleucine, one of essential amino acids, which has been utilized as a constituent of infusions and dietary products. Biosynthetic pathway of L-isoleucine in Escherichia coli consists of five enzymatic reactions from L-threonine as a starting metabolite. Along this pathway, E. coli possesses complex regulatory mechanism by feedback inhibition and transcription regulation. In this study, we attempted to remove metabolic regulations that hamper the production of L-isoleucine, and then to overexpress genes encoding enzymatic reactions and exporters that facilitate production of L-isoleucine. L-isoleucine-producing E. coli strain developed in this study demonstrates that metabolic engineering approaches could contribute to the construction of industrial strains in the field of amino acids. [This work was supported by the Korean Systems Biology Research Project (20090065571) of the Ministry of Education, Science and Technology (MEST) through the National Research Foundation. Further supports by the World Class University Program (R32-2008-000- 10142-0) of the MEST, LG Chem Chair Professorship, IBM SUR program, and Microsoft are appreciated.]

3-hydroxypropionic acid (3-HP) is expected to build up huge market in biomass derived valuable platform chemicals recently and its commercial mass production will led to bio-refinery industrial development. In glycerol metabolism of bacteria, 3-HP is generally known as converting from 3-hydroxypropionaldehyde (3-HPA) by aldehyde dehydrogenase. To find out the appropriate enzyme for achieving the effective catalysis, 32 kinds of gene encoding each of the dehydrogenase from Lactobacillus reuteri were cloned with dhaT which encoding 1,3-propandiol oxidoreductase, and expressed in Klebsiella pneumoniae AK strain (1). Among 32 recombinants, a half of them illustrated enhancement aspects comparing with the control while eight showed lower 3-HP production. The highest 3-HP production was investigated in the recombinant strain which containing a gene encoding propanediol utilization protein. The enzyme will be useful for improvement of the 3-HP production.

Hepatitis B virus causes the acute and chronic liver diseases. The gene coding for the S domain of hepatitis B virus surface antigen (sHBsAg) fused with the mating factor α (MFα) signal sequence was introduced by the chromosomal δ-integration to make S. cerevisiae 2805/pδMFα_sHBsAg strain. The sHBsAg gene was expressed under the control of the galactoseinducible GAL1 promoter. In this study, protein folding accessory proteins such as PDI1, SEC23, TRX2 and SCJ1 were coexpressed individually in recombinant S. cerevisiae 2805/pδMFα_sHBsAg to enhance the production of sHBsAg. Their coexpression led to a 2-fold increase each in the expression level of sHBsAg, compared to that of the control strain. The combinatorial expression of PDI1 and SEC23 gave a synergistic effect on the sHBsAg titer and productivity. Also KEX2 protease was coexpressed to obtain intact sHBsAg. In a glucose-limited fed-batch fermentation with YPDG medium, 15.2 g/L of dry cell mass and 5.81 mg/g cell of specific sHBsAg content were obtained in recombinant S. cerevisiae 2805/ pδMFα_sHBsAg +pPDI1+ pSEC23+pKEX2, which were approximately 11 and 12 times higher than those of the control strain, respectively.

Taurine, a free amino acid, has been shown to be essential in various ways; mammalian development and clinical treatment of serious diseases. Mammals are able to synthesize taurine but most mammals are more dependent on dietary supplementation of taurine. Due to the dependency of taurine uptake on dietary sources, five to six thousands of tons of taurine are produced annually. However, its production has relied on chemical synthesis, which led us to develop a biotechnological method for taurine production. Toward this goal, novel synthetic pathway for taurine production was established in Escherichia coli. It is the first approach for taurine production using unique synthetic engineering and it gives a good example of metabolic engineering for the production of valuable metabolites. [This work was supported by the Korean Systems Biology Research Project (20090065571) of the Ministry of Education, Science and Technology (MEST) through the National Research Foundation of Korea (NRF). Further supports by the World Class University Program (R32-2008-000-10142-0) of the MEST, LG Chem Chair Professorship, IBM SUR program, and Microsoft are appreciated.]