Gyoo Yeol Jung

3D-Printed Microfluidic Device for the Detection of Pathogenic Bacteria Using Size-based Separation in Helical Channel with Trapezoid Cross-Section

A facile method has been developed to detect pathogenic bacteria using magnetic nanoparticle clusters (MNCs) and a 3D-printed helical microchannel. Antibody-functionalized MNCs were used to capture E. coli (EC) bacteria in milk, and the free MNCs and MNC-EC complexes were separated from the milk using a permanent magnet. The free MNCs and MNC-EC complexes were dispersed in a buffer solution, then the solution was injected into a helical microchannel device with or without a sheath flow. The MNC-EC complexes were separated from the free MNCs via the Dean drag force and lift force, and the separation was facilitated in the presence of a sheath flow. The concentration of the E. coli bacteria was determined using a light absorption spectrometer, and the limit of detection was found to be 10 cfu/mL in buffer solution and 100 cfu/mL in milk.

Engineering metabolism and product formation in Corynebacterium glutamicum by coordinated gene overexpression

Single gene overexpression in product pathways such as lysine synthesis has often been employed in metabolic engineering efforts aiming at pathway flux amplification and metabolite overproduction. This approach is limited due to metabolic flux imbalances that often lead to unpredictable physiological responses and suboptimal metabolite productivity. This deficiency can be overcome by the coordinated overexpression of more than one flux controlling genes in a production pathway selected by considering their individual contributions on the cell physiology This concept is demonstrated by the simultaneous overexpression of pyruvate carboxylase and aspartate kinase, two key enzymes in central carbon metabolism and the lysine production pathway in Corynebacterium glutamicum. Contrary to expectations based on the importance of each of these two genes in lysine production, the monocistronic overexpression of either gene results in marginal changes in the overall lysine productivity due to either reduced cell growth or reduced lysine specific productivity. In contrast, the simultaneous amplification of the activities of the two enzymes yielded more than 250% increase of the lysine specific productivity in lactate minimal medium without affecting the growth rate or final cell density of the culture. These results demonstrate that significant flux amplification in complex pathways involving central carbon metabolism is possible through coordinated overexpression of more than one gene in the pathway. This can be achieved either by external, gene expression inducing, controls or controls responding to the physiological cellular state.

Synthetic RNA devices to expedite the evolution of metabolite-producing microbes

An extension of directed evolution strategies to genome-wide variations increases the chance of obtaining metabolite-overproducing microbes. However, a general high-throughput screening platform for selecting improved strains remains out of reach. Here, to expedite the evolution of metabolite-producing microbes, we utilize synthetic RNA devices comprising a riboswitch and a selection module that specifically sense inconspicuous metabolites. Using L-lysine-producing Escherichia coli as a model system, we demonstrated that this RNA device could enrich pathway-optimized strains to up to 75% of the total population after four rounds of enrichment cycles. Furthermore, the potential applicability of this device was examined by successfully extending its application to the case of L-tryptophan. When used in conjunction with combinatorial mutagenesis for metabolite overproduction, our synthetic RNA device should facilitate strain improvement.

Current status of the metabolic engineering of microorganisms for biohydrogen production.

The improvement of H2 production capabilities of hydrogen (H2)-producing microorganisms is a challenging issue. Microorganisms have evolved for fast growth and substrate utilization rather than H2 production. To develop good H2-producing biocatalysts, many studies have focused on the redirection and/or reconstruction of cellular metabolisms. These studies included the elimination of enzymes and carbon pathways interfering or competing with H2 production, the incorporation of non-native metabolic pathways leading to H2 production, the utilization of various carbon substrates, the rectification of H2-producting enzymes (nitrogenase and hydrogenase) and photophosphorylation systems, and in silico pathway flux analysis, among others. Owing to these studies, significant improvements in the yield and rate of H2 production, and in the stability of H2 production activity, were reached. This review presents and discusses the recent developments in biohydrogen production, with a focus on metabolic pathway engineering.

Hydrogen production by a new chemoheterotrophic bacterium Citrobacter sp. Y19

Abstract A newly isolated Citrobacter sp. Y19 grows on organic carbons aerobically and produces hydrogen from carbon monoxide (CO) and water when transferred to anaerobic conditions. Hydrogen production capability of Y19 was studied in serum-bottle and bioreactor cultures. Optimal cell growth was observed at pH 5–8, temperature of 30–40°C, oxygen partial pressure of 0.2– 0.4 atm . Induction of hydrogen production activity could be carried out efficiently under 20% (v/v) CO when the culture was removed to anaerobic conditions at 12 h . Optimal conditions for hydrogen production were 30–40°C and pH 5.5–7.5. The maximum hydrogen production activity was observed as 27.1 mmol / g cell h , which was about three times higher than that of Rhodospirillum rubrum. In bioreactor experiments, a stable hydrogen production along with a high activity of 20 mmol H 2 / g cell h was observed during the continuous operation of 68 h .

Isolation and characterization of Rhodopseudomonas palustris P4 which utilizes CO with the production of H2

A novel photosynthetic bacterium, Rhodopseudomonas palustris P4, was isolated from an anaerobic wastewater sludge digester by virtue of its ability to utilize CO with the production of H2. P4 grew under light with CO as a sole carbon source with the doubling time of 2 h and produced H2 at 20.7 mmol −1 cell h.

Synergic degradation of phenanthrene by consortia of newly isolated bacterial strains

Three different bacteria capable of degrading phenanthrene were isolated from sludge of a pulp wastewater treatment plant and identified as Acinetobacter baumannii, Klebsiella oxytoca, and Stenotrophomonas maltophilia. Phenanthrene degradation efficiencies by different combinations (consortia) of these bacteria were investigated and their population dynamics during phenanthrene degradation were monitored using capillary electrophoresis-based single-strand conformation polymorphism (CE-SSCP). When a single microorganism was used, phenanthrene degradation efficiency was very low (48.0, 11.0, and 9.0% for A. baumannii, K. oxytoca, and S. maltophilia respectively, after 360 h cultivation). All consortia that included S. maltophilia degraded approximately 80.0% of phenanthrene and reduced lag time to 48 h compared to the 168 h of pure A. baumannii culture. CE-SSCP analysis showed that S. maltophilia was the predominant species during phenanthrene degradation in the mixed culture. The results indicate that mixed cultures of microorganisms may effectively degrade target chemicals, even if the microorganisms show low degradation activity in pure culture.

Ultrarapid detection of pathogenic bacteria using a 3D immunomagnetic flow assay.

We developed a novel 3D immunomagnetic flow assay for the rapid detection of pathogenic bacteria in a large-volume food sample. Antibody-functionalized magnetic nanoparticle clusters (AbMNCs) were magnetically immobilized on the surfaces of a 3D-printed cylindrical microchannel. The injection of a Salmonella-spiked sample solution into the microchannel produced instant binding between the AbMNCs and the Salmonella bacteria due to their efficient collisions. Nearly perfect capture of the AbMNCs and AbMNCs-Salmonella complexes was achieved under a high flow rate by stacking permanent magnets with spacers inside the cylindrical separator to maximize the magnetic force. The concentration of the bacteria in solution was determined using ATP luminescence measurements. The detection limit was better than 10 cfu/mL, and the overall assay time, including the binding, rinsing, and detection steps for a 10 mL sample took less than 3 min. To our knowledge, the 3D immunomagnetic flow assay described here provides the fastest high-sensitivity, high-capacity method for the detection of pathogenic bacteria.

Butyrate production in engineered Escherichia coli with synthetic scaffolds

Butyrate pathway was constructed in recombinant Escherichia coli using the genes from Clostridium acetobutylicum and Treponema denticola. However, the pathway constructed from exogenous enzymes did not efficiently convert carbon flux to butyrate. Three steps of the productivity enhancement were attempted in this study. First, pathway engineering to delete metabolic pathways to by-products successfully improved the butyrate production. Second, synthetic scaffold protein that spatially co-localizes enzymes was introduced to improve the efficiency of the heterologous pathway enzymes, resulting in threefold improvement in butyrate production. Finally, further optimizations of inducer concentrations and pH adjustment were tried. The final titer of butyrate was 4.3 and 7.2 g/L under batch and fed-batch cultivation, respectively. This study demonstrated the importance of synthetic scaffold protein as a useful tool for optimization of heterologous butyrate pathway in E. coli.

Quantitative correlation between mRNA secondary structure around the region downstream of the initiation codon and translational efficiency in Escherichia coli.

Translational efficiency in Escherichia coli is known to be strongly influenced by the secondary structure around the ribosome-binding site and the initiation codon in the translational-initiation region of the mRNA. Several quantitative studies have reported that translational efficiency is attributable to effects on ribosome accessibility predominantly caused by the secondary structure surrounding the ribosome-binding site. However, the influence of mRNA secondary structure around regions downstream of the initiation codon on translational efficiency after ribosome-binding step has not been quantitatively studied. Here, we quantitatively analyzed the relationship between secondary structure of mRNA surrounding the region downstream of the initiation codon, referred to as the downstream region (DR), and protein expression levels. Modified hairpin structures containing the initiation codon were constructed by site-directed mutagenesis, and their effects on expression were analyzed in vivo. The minimal folding free energy (DeltaG) of a local hairpin structure was found to be linearly correlated with the relative expression level over a range of fourfold change. These results demonstrate that expression level can be quantitatively controlled by changing the stability of the secondary structure surrounding the DR.