Earticle

ArgR is transcriptional factor in Corynebacterium glutamicum that regulates the ornithine biosynthesis binding to the upstream region of argC, argB, argF, and argG gene. Upon proline supplementation, ArgR binding affinity was significantly reduced upstream of argB resulting in increased ornithine production. Putative transcriptional factor FarR also bound to the upstream of argB gene and it showed inhibitory effect of DNA-binding affinity of ArgR. In addition, this study reports a novel integrated metabolic pathway of amino acid production through the utilization of phenol and naphthalene in C. glutamicum. In the presence of 8.5 mM phenol and 4.2 mM naphthalene, the level of glutamate and proline production was highly increased, compared to the control condition. DNA-binding affinity of ArgR and FarR to the upstream of gltB and gdh gene, which participate glutamate biosynthesis, was reduced significantly in the presence of phenol and naphthalene. Finally, it suggests that C. glutamicum can apply to the environmentally friendly fertilization technology of aromatic compoundscontaminated soils. [This study was supported by the 21C Frontier Microbial Genomics and Applications Center Program, grant No. 11-2008- 10-002-00, Ministry of Education, Science & Technology, Republic of Korea. The authors are grateful for their support.]

In this study, we investigated the efficiencies by which the pET and pQE expression systems produce unnatural recombinant proteins by selective pressure incorporation of unnatural amino acids, a method through which it was found that type of gene expression system tremendously influences the production yield of unnatural proteins in Escherichia coli. Green fluorescent protein (GFP) and a single-chain Fv antibody against c-Met (anti-c-Met sc-Fv) were utilized as model recombinant proteins while Lhomopropargylglycine (Hpg), a methionine analogue that incorporates into the methionine residues of a recombinant protein, was used as model unnatural amino acid. The pET system produced an almost negligible amount of Hpg-incorporated unnatural protein compared to the amount of methionine-incorporated natural protein. However, comparable amounts of unnatural and natural protein were produced by the pQE expression system. The amount of unnatural GFP protein produced through pET expression was not increased despite the over-expression of methionyl tRNA synthetase, which can enhance the activation rate of methionyl-tRNA with a methionine analogue. Incorporation of Hpg decreased the productivity of active GFP by approximately 2.5 fold, possibly caused by the inefficient folding of Hpg-incorporated GFP. Conversely, the productivity of functional anti-c-Met sc-Fv was not influenced by incorporation of Hpg. We confirmed through LC-MS and LCMS/MS that Hpg was incorporated into the methionine residues of the recombinant proteins produced by the pQE expression system. In this way, this study could show the importance of expression system upon unnatural amino acids incorporation for the production of tailor-made synthetic proteins.

Expressing anti-apoptotic cloned-gene inside cells is a widely used strategy for inhibiting apoptosis. In our previous study, we reported Bombyx mori 30Kc gene could improve the production of recombinant proteins in Chinese hamster ovary (CHO) cells. In this study we reengineered the CHO cells for the production of humanized monoclonal antibodies (mAb) directed against leukemia with 30Kc6 gene and 30Kc19 gene, respectively. Over-expression of 30Kc6 in CHO cells increased the cell viability by 40.8% and mAb production by 2.31-fold after serum deprivation, but the cells over-expressing 30Kc19 varied a lot. In the case of 30Kc6 gene, the specific production rate of the cells expressing 30Kc6 was also improved. The 30Kc6 gene can increase the cell viability and productivity because it maintains the mitochondrial membrane potential (MMP) and prevents the downstream cascade responses for apoptosis. Our results indicate that 30Kc6 outperforms 30Kc19 in cell death-protective capability and it is potent to improve the productivity of monoclonal antibody in CHO cells.

Butanol production using immobilized cells of Clostridium saccharoperbutylacetonicum N1-4 (ATCC 27021T) was investigated in a semi-continuous fermentation process. Tests on the effect of glucose concentration, temperature, agitation, pH, medium modification through butyrate and/or without acetate addition, and inhibition of the hydrogenase activity of the cell have been done to optimize the yield in butanol production. Based on the results, the addition of 6.69 g/L butyrate yielded the highest butanol concentration at 18.43 g/L using 60 g/L of initial glucose. A direct relationship to the improved butanol yield was found due to capability of the cell to uptake butyrate as a precursor for butanol formation during solventogenesis. Thus, in the final run, the addition of butyrate in the Ca-alginate immobilized cell was conducted. During the semi-continuous cultivation, butanol productivity of the immobilized cell was 0.64 g/L·h compared to 0.29 g/L·h for the free-cell. In comparison, this was slightly higher compared to previous reports considering the same conditions. Butanol toxicity test was also done to compare an immobilized to nonimmobilized cell. However, no significant deviation was observed. Both were susceptible to butanol intoxication.

In this study, we investigated toxic effects of two pharmaceuticals (i.e., verapamil, tramadol) and two commonly used pesticides (i.e., glyphosate, methidathion) on physiological responses such as viability, growth, and reproduction as well as molecular responses such as gene and protein expression in water flea, Daphnia magna. The expression of Arnt, Vtg, CYP4, CYP314 and Dhb was analyzed as representative biomarkers after exposing D. magna to hazardous materials. Our results showed that the expression of these genes in D. magna were significantly down-regulated in response to pesticides. Among five investigated genes, Cyp4 and Cyp314 genes, expressed differently in exposure of glyphosate and methidathion, respectively. This indicated that these two genes could be considered as potential biomarkers. However, in case of pharmaceuticals, although they did not give high affects on gene expression in D. magna, the chronic toxicity tests indicated that both verapamil and tramadol are harmful to the tested organism with respect to reproduction and growth rate. The results suggest that these pharmaceuticals and pesticides have distinguishable toxic modes between them. While the pesticides led to molecular damage such as significantly reduced of five genes, the pharmaceuticals led to a chronic toxicity, not the effect of gene expression caused by acute exposure.

A single chain Fv (scFv) fragment is a versatile antibody format that is applicable in therapy, diagnostics and so on. In many cases, the scFvs are labeled by fluorescent probes, conjugated by synthetic polymers and immobilized on chip/arrays. These modifications are facilitated using the site-specific addition of bio-orthogonal reactive groups to the scFv proteins. Especially, the N-terminus of scFv is an attractive target site for the addition of reactive groups in manufacturing scFv-based herapeutics, chips and biosensors. This work describes a strategy for producing a scFv that bears a bio-orthogonal group that was selectively added onto its N-terminus in the cytoplasm of Escherichia coli. To achieve the production of N-terminal modified scFvs we employed the successfully demonstrated in vivo based methionineresidue substitution method. The major problem of using the methionine-residue substitution method for N-terminal modification is that all the methionine residues in the target protein are globally replaced by methionine surrogates, which prevents the N-terminal specific modification. In this study, an approach for engineering a target scFv sequence into a sequence that is methionine-free except for at N-terminus is proposed, and the N-terminal specific incorporation of a methionine analogue into the engineered scFv is demonstrated using the methionine-residue substitution method.

Sm23, a novel SGNH esterase from Sinorhizobium meliloti 1021 (Sm23), which has conserved amino acid sequences of Gly8(G), Asp9(D), Ser10(S), and Leu11(L) in N-terminal region, is identified and characterized. Enzymatic functions of the enzyme are investigated using activity staining and enantioselective assay with (R,S)-methyl-β- hydroxyisobutyrate, which showed that this enzyme is specific for short chain ester with (R)-specificity. We also studied oligomeric state of Sm23 using chemical cross-linking, high performance liquid chromatography, and mass-tof mass spectrometry which has trimer in native state.

Cytochrome P450 isolated from Streptomyces peucetius (CYP107AJ1; molecular mass 45kDa) showing high homology with peroxygenases is believed to catalyze variety of biological reactions which uses peroxides to provide electrons, protons and oxygen, and further evading requirement for a protein partner. Cytochrome P450 used in this study was cloned into pET28(a) to express in E. coli. Soluble protein was subjected to column-chromatographic purification for carrying out enzyme assay. As an attempt to prove its catalytic function, dealkylation of 7-Ethoxycoumarin was carried out. HPLC analysis of the extracted product corresponding to its LC-MS analysis showed dealkylated 7-ethoxycoumarin which was further established by subsequent GC mass spectra.

Nitrogen oxides (NOx) accumulation in the atmosphere, owing principally to the combustion of fossil fuels, could contribute to the problem of global warming. Removal of NOx using microalgae system emerged as a promising technology because it can easily grow in altered conditions and its biomass can be used as source of biodiesel. Scenedesmus sp. was grown in a photobioreactor using carbon dioxide (CO2) as a carbon source and nitric oxide (NO) as a nitrogen source with or without iron-EDTA complex. The effect of the addition of iron chelated EDTA in the cultivation of microalgae was analyzed. The Scenedesmus cell concentration increased significantly as the NO was removed efficiently upon addition of Fe(II)EDTA or Fe(III)EDTA. For the lipid accumulation, the system with Fe(II)EDTA showed higher total lipid % than that of the system with Fe(III)EDTA. This indicates that the algal system with iron-EDTA for NO removal is a possible process to be used as a biomass production system. To optimize further the lipid production, the effect of different NO concentrations and harvesting time were investigated. The lipid content increased as the NO concentration depleted under which the cell mass was much lower. Furthermore, the lipid content of the cells obtained after 6 days is higher than that obtained after 2 days. The longer incubation time led to less nitrogen concentration in the medium and resulted to higher accumulation of lipid inside the cells. Compromising between increasing lipid content, microalgal cultivation and harvesting time was necessary to obtain higher values of both the lipid and biomass productivity.

3-Hydroxypropionic acid (3-HP) is a potential feedstock for producing many industrially important chemicals. Previously, we reported the production of 3-HP from glycerol by recombinant E. coli SH-BGK1 with a highest titer of 2.8 g/L in shake-flask experiments and 38.7 g /L in bioreactor experiments. The yield was appeared to be about 35%. Most carbons in glycerol were diverted to growth and the production of other metabolites like lactic acid, acetic acid etc. To improve the yield, glycerol kinase (glpFKX) gene cluster which includes glycerol transport protein were disrupted. Then, for the glycerol transport, glpF gene was cloned and overexpressed under a constitutive tac promoter in a low copy pACYCDuet vector. The resulting construct was grown in a rich medium and the cells were resuspended and used for resting cell experiment for the production of 3-HP. With the new construct, the yield on glycerol improved to the theoretical maximum of 100%. However, it was necessary to supplement glucose as a carbon source for maintaining cell viability and enzymatic activities for the 3-HP synthesis from glycerol.

The analysis of microbial structure and dynamics in the methanogenesis, associated with performance pattern, were investigated in two anaerobic batch digesters using swine wastewater at the same process condition. All two bioreactors were initially seeded with anaerobic sludge originating from a local municipal wastewater treatment plant, based on 1% of TSS (total suspended solids), and processed in the batch mode for 80 days. The analysis of process performance of methanogenic digesters called M1, M2 revealed different process pattern in the same condition of performance. The accumulated amounts of methane production in the M1 and M2 were 7.9 L/L, and 4.5 L/L, respectively; in addition, the starting points of acetate reduction were 37th day, and 47th day; the initial points of propionate consumption were 51st day, 61st day, respectively. For the determination of causes to arouse differences between both digesters, denaturing gradient gel electrophoresis (DGGE) was carried out, and the populations of bacteria and archaea were monitored, at order levels with real-time PCR based on the 16S rRNA gene. The bacterial population in M1 was higher than that in M2 throughout batch process, and in case of Archaea, Methanobacteriales (MBT) and Methanomicrobiales (MMB) were predominated in both of digesters among archaea. The initially bacterial concentration in M1 was somewhat higher than in M2; in contrast, the archaeal concentration in M1 was a little lower than in M2. In case of M1, MBT and MMB showed the outstanding increase of their populations at the reduction points of acetate and propionate. However, in case of M2, the population increase of MBT was not so high at the reduction points of acetate and propionate; moreover, it was not observed for the population of MMB at the propionate reduction period to increase. For the correlation analysis with redundancy analysis (RDA), MMB was more deeply associated to acetate and propionate concentrations than MBT was. Based on these results, we concluded and that MMB increase in the propionate reduction stage must be important to the abundant methane production, even though both MBT and MBT were predominated in all digesters, and that the higher initial bacterial concentration was, the better methanogenesis of MMB, MBT was due to the increase of acidogenic activity of bacteria. Through this finding, it is possible to investigate why the uplicated trials of methano-process at the same condition lead to different results, which can be basically applied to the optimization of methanogenesis in the future. Acknowledgements This work was financially supported in part by the Korea Ministry of Environment (MOE) as Human Resource Development Project for Waste to Energy, Manpower Development Program for Energy & Resources by the Ministry of Knowledge and Economy (MKE), Korea Energy Management Corporation (2006-N-BI-02-P-09-0-000-2007) program and by the BK-21 program.

Production of bioethanol for use as an alternative fuel has been extensively growing due to the increasing demand for energy resources. Distillery wastewater refers to the effluent generated from alcohol distilleries and its volume is about 10 times of produced ethanol. This effluent is in the temperature range of 70-80 0C and extremely high concentration of organic materials. A thermophilic mediator-less microbial fuel cell (MFC) was developed for continuous electricity production while treating this waste water. The power density of about 70 mW/m2 was generated continuously while about 60% if COD was removal. Several limiting factors which involve in cathodic reaction, anodic reaction and membrane fouling were examined. Using buffer as catholyte and increasing flowrate increased the current generation. The strength and feeding rate of distillery wastewater to anode strongly affected to the efficiency of reactor. The addition of methanogen inhibitor into the wastewater inhibited the methane fermentation which is popular in thermophilic reactor and consequently increasing the coloumbic efficiency of MFC. These results showed that distillery wastewater can be effectively treated using thermophilic MFCs.

3-Hydroxypropionic acid (3-HP), an important platform chemical for several commodity and speciality chemicals was produced in E. coli by over expressing dhaB of Klebsiella pneumoniae DSM 2026 encoding glycerol dehydratase and aldH of E. coli K-12 MG1655 encoding aldehyde dehydrogenase. Adenosylcobalamin, an essential and expensive coenzyme should be provided extrinsically for the activity of DhaB in production of 3-HP in E. coli. To overcome the limitation of coenzyme B12 in E. coli, the K. pneumoniae strain, which naturally synthesizes 3-hydroxypropionaldehyde (3-HPA), an intermediary compound of glycerol metabolism and serves as the precursor for 3-HP production, was studied. An efficient production of 3-HP in K. pneumoniae required expression of aldH encoding aldehyde dehydrogenase which converts 3-HPA to 3-HP. By over expressing an aldH genes in K. pneumoniae under various cultural conditions a reasonable titer of 3-HP was achieved. Under microaerobic condition, a maximum titre of 1.44 ± 0.2 gram per litre was successfully produced.

Neocarzinostatin is an endiyene antibiotic with potent antitumor activity. Aponeocarzinostatin (apo-NCS)-component of Neocarzinostatin (NCS), is biologically inactive but is essential to stabilize and regulate the availability of labile chromophore. Here, we studied the effect of apoprotein on NCS production by overexpression of apoprotein, ncsA in neocarzinostatin producer strain, S. carzinostaticus. The results revealed that the overexpression of apoprotein enhanced the production of neocarzinostatin by stabilizing the NCS-chromophore. Moreover, apoprotein overexpressed strain showed enhanced growth rate revealing the expression of apoprotein not only protects labile NCS-chromophore but also protects the host strain from NCS toxicity.

The aim of this study was to investigate the microbial population dynamics in the salted Chinese cabbage and the recycled brine solution. For this, we performed the polymerase chain reactiondenaturing gradient gel electrophoresis (PCR-DGGE) analysis using a universal primer pair of 338f-GC and 518r, with the genomic DNAs of bacterial cells from salted Chinese cabbage and the brine solution after repeated salting processes in 13% salt solution for 15hr at 10℃. As results, the bacterial species identified were Pseudoalteromonas sp. Halomonas sp. Cobetia sp. Bacillus sp. and Lactobacillus sakei. Particularly, while Pseudoalteromonas sp. and Halomonas sp. were found in the brine solution, Lb. sakei was only detected in Chinese cabbage samples. Along with the repeated use of brine solution, the halotolerant bacteria were accumulated to become a dominant group in the brine solution and Lb. sakei disappeared in the salted Chinese cabbage. This result reveals that repeated use of brine solution to produce the salted Chinese cabbage result in unsafe and unfavorable effects on kimchi fermentation. From this experiment, PCR-DGGE analysis was proven useful for investigating microbial dynamics in foods.

The aim of this study was to isolate and characterize biogenic amines (BAs)-producing bacteria in fermented soybean paste. For this, an improved decarboxylating agar medium was used as a culture dependant method and the multiplex PCR analysis was used as culture independant method. As a result, two BAs-producing bacteria were isolated from traditional type soybean pastes. One was a histamineproducing strain which identified as Clostridium perfringens and another was a tyramine-producing strain identified as Pseudomonas entomophila. The multiplex PCR analysis showed the existance of histidine decarboxylase (hdc) gene and tyrosine decarboxylase (tydc) gene in C. perfringens and P. entomophila, respectively. At the same time, their abilities to produce BAs in histidine and tyrosinecontaining media were confirmed by HPLC analysis.

Nystatin-like Pseudonocardia Polyene (NPP) is believed to be a typical tetraene macrolide antifungal antibiotic synthesized by the action of socalled type I modular polyketide synthases. The sequence information of an approximately 125.7 kb contiguous DNA region was completely characterized and revealed to contain a total 23 open reading frames involved in a nystatin-like NPP biosynthesis[1]. Although NPP might be a valuable candidate for novel antifungal drug-lead compound, the complete structural elucidation of NPP is still hampered by its extremely low productivity as well as mixture production. A detailed genetic analysis of nysR genes, the positive regulators for the nystatin biosynthetic gene cluster in Streptomyces norsei, has been reported [2]. Pseudonocardia autotrophica also has six putative regulatory genes that are located at the flanking region of the NPP biosynthetic gene cluster. To increase our nderstanding about P. autotrophica NPP regulatory system as well as its application for NPP overproduction, genetic analysis of pathway-specific regulatory genes located within the NPP biosynthetic gene clusters should be necessary for designing molecular genetic strategies to maximize NPP productivity. In this study, we describe the characterization and application of six pathway-specific NPP regulators in P. autotrophica, whose detailed results will be further discussed.

The aim of this study was to investigate the microbial diversity and their changes during the commercial manufacturing process of kimchi. For this, we chose a kimchi-manufacturing company and measured the viable counts of total cells, lactic acid bacteria (LAB), leuconostocs, bacillus, and yeasts of various raw materials, ingredients, and samples obtained from kimchi manufacturing processes. These samples were diluted with physiological saline, spread on 5 selective media, incubated under the aerobic conditions and the isolates were characterized by cell morphology and 16S rDNA sequencing analysis. Briefly, this study showed that various microbial biota including LAB and leuconostocs were evenly detected from most of the raw materials (cabbage and ingredients like pepper, garlic, ginger, salts) and their counts were not significantly reduced along with the washing processes. Therefore, for the long time preservation and quality control of commercial kimchi products, broad sterilization of kimchi ingredients and starter addition were recommended.

Cheongju is a traditional Korean refined rice wine brewed by a complex of microorganisms involving mold, yeast and bacteria. We monitored the microbial changes during Cheongju fermentation in a commercial brewing company using polymerase chain reactiondenaturing gradient gel electrophoresis (PCR-DGGE) method. As results, the dominant yeast and mold ecology along with brewing process was identified as Saccharomyces cerevisiae, Pichia species, Candida magnolia and Aspergillus niger. Also, the bacterial ecology through the fermentation period was mainly identified as Lactobacillus species, Leuconostoc species and Weissella confusa. Cluster analysis of the yeast and bacterial DGGE profiles highlighted the uniqueness of the Cheongju fermentation processes studied. PCR-DGGE method provided an overview of the microbial dynamics from substrates (rice and koji) to pre- and main-culture.

E. tarda ghost vaccines may provide immune efficacy against edwardsiellosis in farmed fish. However, the leakage of antibioticresistance gene from cloned cells leads to occurrence of antibiotic resistant bacteria and negative effect on environment. Thus, plasmid based on an aspartate-semialdehyde-dehydrogenase (asd) gene was constructed for the E. tarda ghost vaccine to prevent the spread of the antibiotic resistance gene in environment. Target gene for the construction of auxotrophic E. tarda was asd gene, which is an essential gene for the construction of cell wall. The auxotrophic strain was generated by allelic exchange using a suicidal vector harboring DNA segments for asd gene deletion. A pBR origin, asd gene from pYA3342 and E lysis gene controlled by lambda PR/cI regulatory system from pGhost were transfered into the recombinant plasmid. Efficient promoter and signal peptide systems on the vector will be studied for better antigen expression and delivery of the antigen to outer membrane. The auxotrophic E.tarda ghost vaccine with antigen delivery system can lead to safe environment with as antibiotic-free system and increase the protection efficacy against dwardsiellosis.

L-6-Hydroxynorleucine is a chiral intermediate useful for the synthesis of a asopeptidase inhibitor in clinical trials and for the synthesis of C-7 substituted azepinones as potential intermediates for other antihypertensive metalloprotease inhibitors. It has also been used for the synthesis of siderophores, indospicine, and peptide hormone analogues. Previous synthetically useful methods for obtaining this intermediate have involved synthesis of the racemic compound followed by enzymatic resolution. D-Amino acid oxidase has been used to convert the Damino acid to the ketoacid leaving the L-enantiomer. In a second approach, racemic N-acetylhydroxynorleucine has been treated with L-amino acid acylase to give the L-enantiomer. Transaminases have good characteristics required for industrial biocatalysts such as broad substrate specificity, high enantioselectivity, high turnover number, and no requirement for external cofactor regeneration. In the present study, branched chain aminotransfese from E. coli has been successfully used to synthesize enantiopure L-6-Hydroxynorleucine from corresponding keto acid in quantitative yields and high enantiomeric purity (e.e. > 99 %).

Chiral amines are of great interest in pharmaceutical and fine-chemical industries. Transaminases have good characteristics required for industrial biocatalysts such as broad substrate specificity, high enantioselectivity, high turnover number, and no requirement for external cofactor regeneration. Omega-transaminase can be developed to either prepare chiral amines from prochiral compounds (asymmetrization) or from racemic mixtures (kinetic resolution). Pichia pastoris expression system has been used successfully for production of various recombinant heterogous proteins, which offers several advantages such as tight regulation by methanol, easy growth to high cell density, high levels of protein expression at the intra- or extra- cellular level, resistant to organic solvent, and the ability to perform eukaryotic protein modifiacation. In this study, recombinant Pichia pastoris expressing omegatransaminase was used as a whole-cell biocatalyst to kinetic resolve alphamethylbenzylamine (MBA). To overcome product inhibition of omega-TA by acetophenone (deaminated product of alpha-MBA), the reaction condition for endogenous oxidoreductases which catalyze the reduction of acetophenone into noninhibitory 1-phenylethanol was optimized. When the whole-cell reaction was carried out using recombintat P. pastoris in 100 mM Tris/HCl buffer (pH 9.0) containing 2.5 % glucose and 1% methanol, 100 mM alpha-MBA was successfully resolved to (R)-alpha-MBA (>99% ee) at 52.2% conversion.

WSSV caused major viral diseases in shrimp aquaculture industry. Viral envelop proteins of WSSV play important roles on the virus infection. This study was carried out for the identification of mRNA transcriptional level at various WSSV infection stages using Reverse Transcription-PCR (RT-PCR).mRNAs of shrimps were isolated from shrimp organs at various infection stages after WSSV infection. Through the comparison of mRNA transcriptional levels of infected shrimp organs, it was confirmed that the vaccination of shrimp delayed viral gene transcription for 5~ 10 days. However, the general mRNA transcriptional pattern of vaccinated shrimps showed similar trend to that of unvaccinated shrimps. RT-PCR results from infected shrimps indicated that VP28 was not suitable for the analysis of infection stage due to early and high level transcription in every sample. But, VP26 was a useful marker of infection stage owing to infection stage related RT-PCR transcriptional pattern. RT-PCR of VP26 could provide information on the shrimp mortality and virus propagation.In further study, infection patterns will be analyzed using mRNA level via RTPCR from infected shrimps using other primer sets.

Although Saccharomyces cerevisiae is capable of fermenting galactose into ethanol, ethanol yield and productivity from galactose are significantly lower than those from glucose. An inverse metabolic engineering approach was undertaken to improve ethanol yield and productivity from galactose in S. cerevisiae. The characterization of genetic perturbations in the isolated transformants revealed three target genes whose overexpression elicited an enhanced galactose utilization. One confirmatory (SEC53) and two novel targets (SNR84 and a truncated form of TUP1) were identified as overexpression targets that potentially improve galactose fermentation. Moreover, the overexpression of tTUP1 significantly shortened lag periods that occurs when substrate is changed from glucose to galactose. The results presented in this study illustrate that alteration of global regulatory networks through overexpression of the identified targets (SNR84 and tTUP1) is as effective as overexpression of a rate limiting metabolic gene (PGM2) in the galactose assimilation pathway for efficient galactose fermentation in S. cerevisiae. In addition, these results will be industrially useful in the biofuels area as galactose is one of the abundant sugars in marine plant biomass such as red seaweed as well as cheese whey and molasses.

4 kinds of RNPs(Regional Special Natural Products) were used to verify for Anti-obesity effet. In the western blot for 3T3-L1 cell, expression level of PPARγ(peroxisome proliferator-activated receptor γ) was suppressed by different concentraton of 4 RNPs when treated chemical with MDI(IBMX, Dexamethasone, Insulin) at the same time. Also, SREBP-1(Sterol regulatory element binding protein) and PPARγ expression levels were reduced by 4 RNPs in a dose-dependent manner when treated chemicals after differentiation. And adipogenesis was reduced by 7.5%, 14.4%, 18.3% and 30% at different concentration of Anthrisci Radix when performed Oil red O staining. Also, it was reduced by 2%, 4.9%, 9.3% and 38% at different concentration of Psoraleae Semen. When treated with Siegesbeckiae Herba, it was inhibited by 1.4%, 6.4%, 16.4% and 30.1%, respectively. And Corni Fructus was also showed by 0.9%, 6.3%, 13.7% and 33% at same concentration of Siegesbeckiae Herba. As a result, 4 RNPs were expected for a useful material for Anti-obesity and Anti-cancer as well.

In Purple nonsulfur photosynthetic bacteria, carbon dioxide fixation has been intensively investigated, and through the Calvin reductive pentose phosphate pathway by ribulose 1,5-bisphosphate carboxylase/ oxygenase, this organism can produce organic compounds such as sugar from carbon dioxide. However, very little is known about the physicochemical properties and functional potentials of organic compounds produced from carbon dioxide by photosynthetic bacteria. Therefore, this study is focused on analyzing the properties of organic compounds and the impact on physiological activation of other microorganisms by carbon dioxide fixed organic compounds. The organic compounds was hydrolyzed by a mixture of cellulose and b-glucosidase because of its structural complexity, and after supplementation of mixed hydrolytic enzymes, we could get the increased yields of glucose, galactose and arabinose. In addition, the impact of organic compounds produced by carbon dioxide fixation was assessed by the growth rate and fermentation efficiency of Saccharomyces cerevisiae and Clostridium acetobutylicum. We found that physiological activation of two microorganisms was induced under the supplemented condition by organic compounds. The data provide strong evidence that carbon dioxide fixed organic compounds have the multifunctional potential to substantially increase the physiological activity of other organisms.

The hexanoic acid could be converted to valuable chemicals, for example, adipic acid (nylon’s material) by oxidation process and hexanol by hydrogenation. For the isolation of hexanoic acid producing bacteria from cow rumen, RCM (Reinforced clostrdia medium) with glucose as carbon source and hexanoic acid as selection pressure was used. Isolated bacteria was designated Megasphaera sp. nov. BS-4 and its 16s rDNA showed 94% similarity with Megasphaera pauchivorance DSM 16981T. Physiological test is performed by API kit and API 50CH showed that Megasphaera sp. nov. BS-4 uses D, L-arabinose, inositol and fructose. Especially, the isolated bacterium showed the maximum hexanoic acid production, around 4 g/L without pH adjustment, when fructose was used as a carbon source.

Hydrogen production in biological way can be one of solutions for energy crisis. Previously, we demonstrated successful production of biohydrogen by expression of recombinant [NiFe]-hydrogenase 1 in recombinant E. coli. Although [NiFe]-hydrogenase 1 showed relatively high oxygen-tolerance compare to other hydrogenases, we observed that translocation efficiency to the membrane was very low which is important for hydrogen production. In the present work, to increase translocation efficiency, we investigated effects of cultural factors including culture temperature and inducer concentration. In results, we found that cell membrane targeting was significantly increased at lower culture temperature than 37℃, general culture temperature. Optimal IPTG inducer concentration for targeting efficiency was lower than 1 mM. Finally, we also investigated effect of signal sequence on targeting efficiency and hydrogen production by changing Tat signal sequence.

Korean climate condition may not provide enough light condition for outdoor microalgal cultures, although utilization of sun light energy has numerous benefits in aspect of economics. Therefore, it is necessary to investigate if supplementing artificial light in addition to solar radiation can be more effective. Night biomass loss (NBL) was measured under natural light condition without any artificial light supplement. The results have shown that NBL quantity was about 30% compared to the cell concentrations with continuous lighting scheme (the same sun light during the day and supplementing artificial light at night). In order to minimize NBL with minimum energy for the artificial light at night, various artificial lighting schemes at the night were performed including intermittent light effects. While the light and dark ratio was fixed at 1:1, the intermittent lighting cycle periods were varied among 30, 60, and 150 minutes. The average of fresh cell weight obtained under intermittent lights was 2.67 g/L, which was 62% lower compared with natural condition by supplying 50% less energy. For the relative yield from electric power to cell mass, the intermittent light cycle of 150 minutes was the best with 72% higher than that under continuous condition. Furthermore, some fatty acid concentrations such as C18:1, C18:3 found to be affected by the intermittent lighting cycle period.

Precious metals are extensively used as catalysts in a wide range of industrial chemical processes. Gold has been showed to be an active and selective catalyst for numerous reactions. In particular, porous gold is believed to be a good candidate as a substrate for batteries, sensors, and catalysts. This study focused on a new process for making porous fiber forms of gold by incineration of Au-loaded biosorbent fiber from goldcyanide solutions. For preparing Au-loaded fiber, a batch system was used for adsorption of Au(I) ions. The maximum uptake of Au(I) ions was estimated to be 338.96 mg/g at pH 5. Au-loaded biosorbents were freezedried and then incinerated. During incineration, organic constituents of the biosorbent were removed and at the same time, Au(I) was reduced to zerovalent form. The obtained zero-valent gold fibers were around 60μm in diameter. The purity of gold was estimated to be more than 94.8%. SEM analysis showed that the section planes of cutting fibers were observed to be highly porous. Thus, this process may reveal the prospect of an efficient method to make zero-valent porous gold fibers using combined method of biosorption and incineration.