[Show abstract][Hide abstract] ABSTRACT: The CusSR two-component system (TCS) is a copper-sensing apparatus of E. coli that is responsible for regulating the copper-related homeostatic system. The dynamic characteristics of the CusSR network were modified by the introduction of a positive feedback loop. To construct the feedback loop, the CusR, which is activated by the cusC promoter, was cloned downstream of the cusC promoter and reporter protein. The feedback loop system, once activated by environmental copper, triggers the activation of the cusC promoter, which results in the amplification of a reporter protein and CusR expression. The threshold copper concentration for the activation of the modified CusSR TCS network was lowered from 2,476.5 μg/l to 247.7 μg/l, which indicates a tenfold increase in sensitivity. The intensity of the output signal was increased twofold, and was maintained for 16 h. The strategy proposed in this study can also be applied to modify the dynamic characteristics of other TCSs.
[Show abstract][Hide abstract] ABSTRACT: The effect of the reducing power on the reduction of methyl-2-chlorobenzoylformate was evaluated by using carbon substrates with different reducing powers. Glucose, sorbitol, and gluconate regenerated 2, 3, and 1 NAD(P)H during its conversion to pyruvate, respectively. When sorbitol was used as the carbon substrate, complete conversion was achieved in 8 h while it took 12 h and 19 h when glucose and gluconate were used, respectively. The enantiomeric excess (ee) value was 96.7% when sorbitol was used.
Korean Journal of Chemical Engineering 12/2011; 28(12). DOI:10.1007/s11814-011-0124-3 · 1.17 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Two new tetracationic hetero-bimetallacycles were prepared from a bis-pyridine amide ligand and metal (Pd and Pt) acceptors. We found that both self-assembled hetero-bimetallacycles bind and unwind supercoiled DNA as established by photophysical and gel electrophoresis analyses, respectively.
[Show abstract][Hide abstract] ABSTRACT: Gamma-aminobutyric acid (GABA) is a non-essential amino acid and a precursor of pyrrolidone, a monomer of nylon 4. GABA can be biosynthesized through the decarboxylation of L: -glutamate by glutamate decarboxylase. In this study, the effects of glutamate decarboxylase (gadA, gadB), glutamate/GABA antiporter (gadC) and GABA aminotransferase (gabT) on GABA production were investigated in Escherichia coli. Glutamate decarboxylase was overexpressed alone or with the glutamate/GABA antiporter to enhance GABA synthesis. GABA aminotransferase, which redirects GABA into the TCA cycle, was knock-out mutated. When gadB and gadC were co-overexpressed in the gabT mutant strain, a final GABA concentration of 5.46 g/l was obtained from 10 g/l of monosodium glutamate (MSG), which corresponded to a GABA yield of 89.5%.
[Show abstract][Hide abstract] ABSTRACT: Synthetic biological systems are becoming more and more feasible for commercial and medical purposes through the genetic engineering of several components. The simple assembly of a genetic circuit was shown to stimulate the removal of copper by bacteria through the engineering of a two-component system. The CusSR two-component systems is a regulator of Escherichia coli copper homeostatic system. In this system, genetic circuits of CusSR were fused to a cell surface display system for metal adsorption; this system is suitable for the display of a copper binding peptide through outer membrane protein C (OmpC). E. coli ompC codes for an outer membrane pore protein (porin) are induced at high osmolarity and temperature, which can also be used as an anchoring motif to accept the passenger proteins. The bacteria that produce the chimeric OmpC containing the copper binding peptide adsorbed maximum concentrations of 92.2 μmol of Cu(2+)/gram dry weight of bacterial cells. This synthetic bacterial system senses the specific heavy metal and activates a cell surface display system that acts to remove the metal.
[Show abstract][Hide abstract] ABSTRACT: The type III secretion system (T3SS) is a mechanism by which bacteria export proteins from the cytoplasm, through the membranes,
to the extracellular environment. T3SS is made up of more than 20 different proteins, about half of which maintain conserved
sequences. This review summarizes the features of this novel apparatus and discusses the potential of utilizing T3SS to export
recombinant proteins into the external environment, and the impact this system will have on protein production technology.
Key wordsType III Secretion System (T3SS)–Secretion–Recombinant Protein
Korean Journal of Chemical Engineering 07/2011; 28(7):1573-1579. DOI:10.1007/s11814-011-0024-6 · 1.17 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Zinc ion plays essential roles in biological chemistry. Bacteria acquire Zn(2+) from the environment, and cellular concentration levels are controlled by zinc homeostasis systems. In comparison with other homeostatic systems, the ZraSR two-component system was found to be more efficient in responding to exogenous zinc concentrations. To understand the dynamic response of the bacterium ZraSR two-component system with respect to exogenous zinc concentrations, the genetic circuit of the ZraSR system was integrated with a reporter protein. This study was helpful in the construction of an E. coli system that can display selective metal binding peptides on the surface of the cell in response to exogenous zinc. The engineered bacterial system for monitoring exogenous zinc was successfully employed to detect levels of zinc as low as 0.001 mM, which directly activates the expression of chimeric ompC(t)--zinc binding peptide gene to remove zinc by adsorbing a maximum of 163.6 μmol of zinc per gram of dry cell weight. These results indicate that the engineered bacterial strain developed in the present study can sense the specific heavy metal and activates a cell surface display system that acts to remove the metal.
[Show abstract][Hide abstract] ABSTRACT: A series of stable arenediazonium camphorsulfonate salts (2a-2j) were synthesized by simple diazotization of several aromatic amines in the presence of sodium nitrite and camphorsulfonic acid. All the new arenediazonium camphorsulfonates, which were characterized by multinuclear (H-1 and C-13) NMR, IR, DSC, and X-ray diffraction analysis (2e and 2f) provide unambiguous proof for the molecular structures of 2e and 2f. The efficient application of these salts in halogenation reactions was studied in solvent and solvent-free conditions and the DNA cleavage activity was also assessed. These arenediazonium camphorsulfonate salts are noticed as efficient DNA cleaving agents.
European Journal of Medicinal Chemistry 01/2011; 69(16). DOI:10.1016/j.tet.2013.02.016 · 3.45 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In this study, we designed and applied molecular biosensors for heavy metals, zinc and copper, for use in bioremediation strategies. Bacteria utilize two component systems to sense changes in the environment by multiple signal components including heavy metals and control gene expression in response to changes in signal molecules. zraP and cusC promoters were selected from a genetic circuit of the ZraSR and CusSR two-component system and were fused to a dual-labeling reporter protein as an interactive biological component for zinc and copper to generate a signal from the constructed biosensor. The biosensor efficiently senses zinc and copper with a calculated detection limit of 16 mu M and 26 mu M, respectively, and was shown to be a sensitive and effective heavy metal monitoring bacterial system. To extend the application of the bacterial biosensor, we assembled a bioadsorption system that can trigger bacteria to sense and adsorb 13 +/- 0.3 mg/L of zinc and 11.4 +/- 0.42 mg/L of copper per gram of dry cell weight with induction at a concentration of 100 mg/L of the respective metal ion.
PROCESS BIOCHEMISTRY 01/2011; 47(5). DOI:10.1016/j.procbio.2012.02.007 · 2.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Methyl (R)-2-chloromandelate, a key intermediate in the synthesis of clopidogrel, was obtained by the reduction of methyl-2-chlorobenzoylformate using whole cells of Saccharomyces cerevisiae. A 100% conversion and 96.1% of enantiomeric excess (ee) value was obtained when 17 methyl-2-chlorobenzoylformate/l was reacted with 8 g S. cerevisiae/l and 83 g glucose/l at pH 7.
[Show abstract][Hide abstract] ABSTRACT: A single submerged membrane bioreactor (MBR) for nitrification of ammonium and a pre-denitrification MBR process for total nitrogen (TN) removal were investigated in comparison. A single nitrifying MBR was fed with synthetic ammonium wastewater of up to 900mgN/l without organics so that the MBR was maintained as a pure nitrifying system. A high nitrifying capacity around 1.8kgNH4-N/m3/day was achieved while keeping the ammonium oxidation rate above 98%. Sludge volume index (SVI) gradually decreased down to less than 50 indicating good settleability of nitrifying sludge. The increase of suction pressure was less than 5cmHg over 7-months of operation. TN removal efficiency was determined in a pre-denitrification configuration with an anoxic reactor. Synthetic wastewater of 1200mgCOD/l and 200mgN/l was fed to the system at loads of 2.4kgCOD/m3/day and 0.4kgN/m3/day, respectively. As the internal recycle ratio from aerobic to anoxic zone increased from 2 to 6, TN removal efficiency was enhanced from 70±9 to 89±3%. With the sludge concentration of around 12,000mg/l, SVI was highly fluctuated from 60 to 350 indicating the partial deterioration of sludge settleability. The suction pressure after 8 months of operation increased to above 10cmHg which is higher than that in a single nitrifying MBR. The concentration of extracellular polymeric substances (EPS), especially for carbohydrate content, was higher in the operation of a pre-denitrification MBR process than in a single nitrifying MBR. It is likely that the sludge characteristic such as settleability is related with membrane fouling but, further extensive study is needed. The performance of a pre-denitrification MBR process was also verified with real petrochemical nitrogen wastewater.
Journal of Industrial and Engineering Chemistry 07/2010; 16(4):546-550. DOI:10.1016/j.jiec.2010.03.024 · 3.51 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The evolution of living organisms occurs via a combination of highly complicated processes that involve modification of various features such as appearance, metabolism and sensing systems. To understand the evolution of life, it is necessary to understand how each biological feature has been optimized in response to new environmental conditions and interrelated with other features through evolution. To accomplish this, we constructed contents-based trees for two-component system (TCS) and metabolic network to determine how the environmental communication mechanism and the intracellular metabolism have evolved, respectively. We then conducted a comparative analysis of the two trees using ARACNE to evaluate the evolutionary and functional relationship between TCS and metabolism. The results showed that such integrated analysis can give new insight into the study of bacterial evolution.
Journal of Microbiology and Biotechnology 11/2009; 19(11):1301-5. · 1.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In this study, two-component system (TCS) gene profile and metabolic network gene profile based phylogenetic trees were constructed
and compared to each other to evaluate the evolutionary relationship between the bacterial sensing system and metabolism.
The gene profiles of the these systems suggested that bacteria employed different evolutionary strategies to optimize the
two-component system and metabolic network. In addition, comparative analysis revealed that the TCS based tree showed better
family grouping than the metabolic network based tree, which indicated that the TCS and metabolic network have been modified
via self-evolution and recruitment methods, respectively.
[Show abstract][Hide abstract] ABSTRACT: ZnO thin films have been prepared by chemical bath deposition in aqueous/ethanolic solution. The film texture was successfully controlled by varying the volume ratio of water to ethanol. Films consisting of densely oriented nanorod arrays with the c-axis perpendicular to the substrate were fabricated in aqueous solution. The crystals became increasingly tilted as more ethanol was introduced to the solution, resulting in the cracked nanocolumns and the smoothed crystals. The crystal size was decreased with increasing ethanol content, and granular morphology was obtained in films deposited in ethanolic solution. A gradual evolution of the film texture is possibly due to the inhibited crystal growth in solution with higher ethanol content.
[Show abstract][Hide abstract] ABSTRACT: A biosynthetic pathway for the production of (S)-3-hydroxybutyric acid (S3HB) from glucose was established in recombinant Escherichia coli by introducing the beta-ketothiolase gene from Ralstonia eutropha H16, the (S)-3-hydroxybutyryl-CoA dehydrogenase gene from R. eutropha H16, or Clostridium acetobutylicum ATCC824, and the 3-hydroxyisobutyryl-CoA hydrolase gene from Bacillus cereus ATCC14579. Artificial operon consisting of these genes was constructed and was expressed in E. coli BL21 (DE3) codon plus under T7 promoter by isopropyl beta-D: -thiogalactoside (IPTG) induction. Recombinant E. coli BL21 (DE3) codon plus expressing the beta-ketothiolase gene, the (S)-3-hydroxybutyryl-CoA dehydrogenase gene, and the 3-hydroxyisobutyryl-CoA hydrolase gene could synthesize enantiomerically pure S3HB to the concentration of 0.61 g l(-1) from 20 g l(-1) of glucose in Luria-Bertani medium. Fed-batch cultures of recombinant E. coli BL21 (DE3) codon plus were carried out to achieve higher titer of S3HB with varying induction time and glucose concentration during fermentation. Protein expression was induced by addition of 1 mM IPTG when cell concentration reached 10 and 20 g l(-1) (OD(600) = 30 and 60), respectively. When protein expression was induced at 60 of OD(600) and glucose was fed to the concentration of 15 g l(-1), 10.3 g l(-1) of S3HB was obtained in 38 h with the S3HB productivity of 0.21 g l(-1)h(-1). Lowering glucose concentration to 5 g l(-1) and induction of protein expression at 30 of OD(600) significantly reduced final S3HB concentration to 3.7 g l(-1), which also resulted in the decrease of the S3HB productivity to 0.05 g l(-1)h(-1).
[Show abstract][Hide abstract] ABSTRACT: To adapt to their environment, bacterial strains have developed various environmental signal sensing systems or twocomponent
systems. To evaluate the evolutionary relationship of two-component systems, 246 two-component system genes from KEGG were
analyzed. Phylogenetic tree structure indicated that most two-component systems are strain specific. Most of two-component
system genes have co-evolved, and some two-component system pairs have evolved via recruitment model. By two-component system
gene content analysis, new aspect of cellular metabolism evolution was provided.
[Show abstract][Hide abstract] ABSTRACT: Malic acid is a C4-dicarboxylic acid and an intermediate of tricarboxylic acid (TCA) cycle. It has been widely used in the polymer, food and pharmaceutical industries. Metabolic flux analysis was performed to find a strategy for enhanced malic acid production in Escherichia coli. The simulation results suggested that the amplification of phosphoenolpyruvate (PEP) carboxylation flux allowed increased malic acid production. Since the PEP carboxylase of E. coli converts PEP to oxaloacetate without generating ATP, thus losing the high-energy phosphate bond of PEP, the PEP carboxykinase, which generates ATP during this conversion, was chosen. However, the E. coli PEP carboxykinase catalyzes the reaction that converts oxaloacetate to PEP rather than the desirable opposite reaction. Thus, we cloned the PEP carboxykinase (enconded by the pckA gene) of Mannheimia succiniciproducens, which converts PEP to oxaloacetate as a favorable reaction. The pta mutant E. coli strain WGS-10 harboring the plasmid p104ManPck containing the M. succiniciproducens pckA gene was constructed and cultured at 37 °C. The final malic acid concentration of 9.25 g/L could be obtained after 12 h of aerobic cultivation.
[Show abstract][Hide abstract] ABSTRACT: Succinic acid is a cellular metabolite belonging to the C4-dicarboxylic acid family, and the fermentative production of succinic
acid via the use of recombinant microorganisms has recently become the focus of an increasing amount of attention. Considering
the difficulty inherent to the direct application of natural succinic acid producers to the industrial process, a variety
of systems biology studies have been conducted regarding the development of enhanced succinic acid production systems. This
review shows how the metabolic processes of microorganisms, includingEscherichia coli andMannheimia succiniciproducens, have been optimized in order to achieve enhanced succinic acid production. First, their metabolic networks were constructed
on the basis of complete genome sequences, after which their metabolic characteristics were estimated viain silico computer modeling. Metabolic engineering strategies were designed in accordance with the results ofin silico modeling and metabolically engineered versions of bothE. coli andM. succiniciproducens have been constructed. The succinic acid productivity and yield obtained using metabolically engineered bacteria was significantly
higher than that obtained using wild-type bacteria.