• The Journal of Korean Medicine
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• v.32 no.3
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• pp.35-43
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• 2011

Objectives: This study was performed to screen target-specific inhibitors of ${\beta}$-catenin/TCF signaling whose functional activation plays an important role in early events in carcinogenesis. Methods: To investigate the activation or suppression of ${\beta}$-catenin/TCF transcription, we established a transiently transfected cell line with a constitutively active ${\beta}$-catenin mutant gene whose product is not degraded. This cell line was also co-transfected with luciferase reporter gene constructs containing either an optimized (TOPflash) or mutant (FOPflash) TCF-binding element. We investigated cytotoxic effects using a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt (MTS) assay. To find effective inhibitors of ${\beta}$-catenin/TCF signaling from medicinal herbs, the crude extracts of 99 types of medicinal herbs were screened using a luciferase assay system in HEK-293 and SH-SY5y cells. Results: At a concentration of $50{\mu}g$/ml, extracts of Angelica koreanae radix, Cannabis sativa semen, Ephedrae intermedia Schrenk radix, and Vitis rotundifolia fruit showed the following inhibitory effects on ${\beta}$-catenin/TCF signaling: $40{\pm}5.6%$, $23{\pm}6.1%$, $8{\pm}5.1%$, and $22{\pm}9.8%$ in ${\beta}$-catenin-activated HEK-293 cells and $9{\pm}4.7%$, $39{\pm}8.1%$, $39{\pm}6.4%$, and $42{\pm}10.1%$ in ${\beta}$-catenin-activated SH-SY5y cells, respectively. Crude extracts of E. radix were isolated by silica gel column chromatography, and two non-polar fractions of these extracts showed inhibitory effects on ${\beta}$-catenin/TCF signaling. Conclusions: In this study, we established a transiently transfected cell line as a screening system and found that various medicinal herb extracts had inhibitory effects on ${\beta}$signaling.

• Journal of Microbiology and Biotechnology
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• v.12 no.6
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• pp.1010-1012
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• 2002

In order to isolate carotenoid-hyperproducing yeast, low-dose gamma irradiation was used as means of mutagenesis. Phaffia rhodozyma was treated by gamma irradiation of less than 10 kGy, which is considered to be a wholesome irradiation condition established by the Food and Drug Administration. Through repeated rounds of gamma irradiation and visual screening, mutant 3A4-8 was obtained. It produced a $3,824{\mu}g$ carotenoid/g yeast, 69% higher content than $2,265{\mu}g/g$ yeast of the unirradiated one. This result indicates that low-dose gamma irradiation could be used as means of mutagenesis to obtain carotenoid-hyperproducing strain of Phaffia rhodozyma, since only carotenoid-hyperproducing yeast survived gamma irradiation by scavenging oxygen radicals generated by radiolysis of water.

• Journal of Microbiology
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• v.43 no.3
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• pp.266-271
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• 2005

In eukaryotic cells, various proteins are anchored to the plasma membrane through glycosylphosphatidylinositol (GPI). To study the biosynthetic pathways and modifications of GPI, various mutant cells have been isolated from the cells of Chinese hamster ovaries (CHO) supplemented with several exogenous genes involved in GPI biosynthesis using aerolysin, a toxin secreted from gram-negative bacterium Aeromonas hydrophila. Alpha toxin from Gram-positive bacterium Clostridium septicum is homologous to large lobes (LL) of aerolysin, binds GPI-anchored proteins and possesses a cell-destroying mechanism similar to aerolysin. Here, to determine whether alpha toxins can be used as an isolation tool of GPI-mutants, like aerolysin, CHO cells stably transfected with several exogenous genes involved in GPI biosynthesis were chemically mutagenized and cultured in a medium containing alpha toxins. We isolated six mutants highly resistant to alpha toxins and deficient in GPI biosynthesis. By genetic complementation, we determined that one mutant cell was defective of the second subunit of dolichol phosphate mannose synthase (DPM2) and other five cells were of a putative catalytic subunit of inositol acyltransferase (PIG-W). Therefore, C. septicum alpha toxins are a useful screening probe for the isolation of various GPI-mutant cells.

• Korean Journal Plant Pathology
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• v.14 no.6
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• pp.606-611
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• 1998

An antagonistic bacterium, Pseudomonas flurorescens MC07 inhibited the mycelial growth of Rhizoctonia solani, Pythium ultimum, Fusarium oxysporum, and Phytophthora capsici in on potato dextrose agan (PDA) and other media. The strain MC07 conlonizes various plant roots and possesses antifungal activity. To determine the role of antifungal activity of the bacterium in disease suppression, a mutant Okm3-4 which lost its activity was isolated after screening 2,500 colonies generated by Omegon-Km insertions. The mutant Okm3-4 showed diminished growth inhibition of R. solani, P. ultimum, F. oxysporum, and Ph. capsici in vitro and had reduced suppressive effects on sesame damping.-off compared to the parental strain. In soils, accumulation of the pathogens by continuous cropping, 90% of sesame plants were killed by natural infection of damping-off whereas, only 29% of plants grown from seeds treated with MC07 were killed. On the other hand, 85% of plants died when sesame seeds were treated with the Okm3-4 cells. This indicated that antifungal activity of MC07 in vitro is directly responsible for the suppression of damping-off disease. Emergence rates of sesame seeds in pots containing diseased soil were 33%. However, MC07 treatments on seeds significantly improved emergence rates, which has similar effects of Benomyl treatment. The mutant Okm3-4 exhibited 53% of emergence rate. This indicated that antifungal activity of MC07 also affects the emergence rate of sesame seeds.

• Journal of Microbiology and Biotechnology
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• v.14 no.3
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• pp.570-575
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• 2004

Three different strains of carotenoid accumulating XantlwphyUomyces dendrorhous mutants, JH1, JH2, and JH3, were isolated by NTG (N-methyl-N'-nitro-N-nitrosoguanidine) mutagenesis, which might potentially be useful for animal feed as well as for studies on the regulation and biosynthesis of astaxanthin. Mutants were selected based on the capability of growth and carotenoid production on the YM agar plate containing chemical inhibitor, $\beta$-ionone. Astaxanthin-overproducing mutant JH1 produced 4.032 mg astaxanthinlg dry cell weight, and this value was about 15-folds higher than that of wild-type. $\beta$-Carotene-overproducing mutant JH2 produced 0.273 mg $\beta$-carotene/g dry cell weight, and this was 4-folds increase from that of wild-type. In contrast, JH3 was a white-colored mutant that was unable to produce carotenoid pigment.

• Journal of Microbiology and Biotechnology
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• v.33 no.10
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• pp.1257-1267
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• 2023

Although rational genetic engineering is nowadays the favored method for microbial strain improvement, building up mutant libraries based on directed evolution for improvement is still in many cases the better option. In this regard, the demand for precise and efficient screening methods for mutants with high performance has stimulated the development of biosensor-based high-throughput screening strategies. Genetically encoded biosensors provide powerful tools to couple the desired phenotype to a detectable signal, such as fluorescence and growth rate. Herein, we review recent advances in engineering several classes of biosensors and their applications in directed evolution. Furthermore, we compare and discuss the screening advantages and limitations of two-component biosensors, transcription-factor-based biosensors, and RNA-based biosensors. Engineering these biosensors has focused mainly on modifying the expression level or structure of the biosensor components to optimize the dynamic range, specificity, and detection range. Finally, the applications of biosensors in the evolution of proteins, metabolic pathways, and genome-scale metabolic networks are described. This review provides potential guidance in the design of biosensors and their applications in improving the bioproduction of microbial cell factories through directed evolution.

• Microbiology and Biotechnology Letters
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• v.19 no.1
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• pp.1-7
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• 1991

A bacterial strain No.71, which produced alkaline protease, was isolated from soil and identified to the genus Bacillus. With the successive mutation, a mutant strain No. M-71, having high alkaline protease productivity, was obtanined from the parental strain No 71. Alkaline protease productivity of mutant strain No. M-71 was about 50 times as much as that of the parental strain No.71. The enzyme preparations showed strong activities toward casein, the optimum pH being 11.0 and the optimum temperature about $55^{\circ}C$.

• Microbiology and Biotechnology Letters
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• v.27 no.4
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• pp.292-297
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• 1999

Overproduction of L-ornithine by mutant strains isolated from Brevibacterium detoglutamicum BK1046 was investigated. The strain was a L-ornithine auxotroph and exhibited culture instability during fermentation. Through a sequential screening effort, a highly stable strain with lless revertant formation was finally selected and designated B. ketoglutamicum BK52 (KCTC0141BP). It prouduced L-ornithine at a high concentration (above 9 g/L) independent of subculture or cultivation time, and also had a very low tendency of revertant formation. In a long-term storage, this strain maintained its cell stability and productivity of L-ornithine to a reasonable range.

• Mycobiology
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• v.37 no.4
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• pp.267-271
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• 2009

A fungal strain producing a high level of cellulase was selected from 320 fungal isolates and identified as Aspergillus sp. This strain was further improved for cellulase production by sequential treatments by two repeated rounds of $\gamma$-irradiation of $Co^{60}$, ultraviolet treatment and four repeated rounds of treatment with N-methyl-N'-nitro-N-nitrosoguanidine. The best mutant strain, Aspergillus sp. XTG-4, was selected after screening and the activities of carboxymethyl cellulase, filter paper cellulase and $\beta$-glucosidase of the cellulase were improved by 2.03-, 3.20-, and 1.80-fold, respectively, when compared to the wild type strain. After being subcultured 19 times, the enzyme production of the mutant Aspergillus sp. XTG-4s was stable.

• Journal of Radiation Industry
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• v.4 no.3
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• pp.247-252
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• 2010

The present study has been performed to select the osmotic tolerant lines using polyethylene glycol (PEG 6000) through an in vitro and in vivo mutagensis with a gamma-ray. During the screening, we selected three mutant lines that seemed to confer elevated osmotic tolerance in high concentrations of PEG 6000. Fruits of these mutants (Os-HK101, Os-HK102 and Os-HK103) were increased to sugar concentration, L-glutamine acid, vitamin C content and lycopine content than those of the wild type. Also the chlorophyll contents were few decreased more in the three mutant lines than the WT plants. Our results suggest that the Os-HK101 is characterized as osmotic stress tolerance considering the sugar concentration and lycopine content. It is expected that the result of this study can be used for breeding more competitive species with respect to contents in sugar or functional chemicals from the selected osmotic resistant lines.