• The Plant Pathology Journal
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• v.35 no.1
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• pp.84-89
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• 2019

Two-component systems (TCSs) are critical to the pathogenesis of Xanthomonas oryzae pv. oryzae (Xoo). We mutated 55 of 62 genes annotated as responsive regulators (RRs) of TCSs in the genome of Xoo strain PXO99A and identified 9 genes involved in Xoo virulence. Four (rpfG, hrpG, stoS, and detR) of the 9 genes were previously reported as key regulators of Xoo virulence and the other 5 have not been characterized. Lesion lengths on rice leaves inoculated with the mutants were shorter than those of the wild type and were significantly restored with gene complementation. The population density of the 5 mutants in planta was smaller than that of PXO99A at 14 days after inoculation, but the growth curves of the mutants in rich medium were similar to those of the wild type. These newly reported RR genes will facilitate studies on the function of TCSs and of the integrated regulation of TCSs for Xoo pathogenesis.

• Journal of Applied Biological Chemistry
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• v.61 no.4
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• pp.327-334
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• 2018

Monascus is a source of food colorant with high productivity of the pigment azaphilone. Monascus azaphilone (MAz) is biosynthesized through a single non-reducing polyketide pathway, the major components of which are ankaflavin (1), monascin (2), rubropunctatin (3) and monascorubrin (4); valuable biological activities have been reported for these compounds. Thus, various culture conditions were explored to reduce the cost of culture ingredients, enhance productivity and modulate compound composition. In the present study, we examined an extractive fermentation (EF) method with Diaion HP-20 resin (HP20) in direct comparison to a previously explored method involving Triton X-100 (TX100) to explore the modulated production of the major MAzs. We employed wild-type Monascus purpureus as well as two derivative recombinant strains (${\Delta}mppG$ and ${\Delta}mppE$) that are known to have differential MAz profiles as that of the wild-type strain. The HP20 resin was capable of modulating the MAz profile in favor of orange MAzs 3 and 4, oxidized congeners in this class, as was TX100-a phenomenon not previously observed for TX100 EF with Monascus anka. These finding substantiate that HP20 can be employed for the selective production of oxidized MAz and for diversifying the culture conditions used for Az production.

• Journal of Mushroom
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• v.22 no.3
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• pp.81-86
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• 2024

Cordyceps militaris is widely used in China, Korea, and other Asian countries as both a traditional medicinal ingredient and an edible fungus. This study aimed to optimize the growth conditions and fruiting body production of C. militaris by investigating various culture media and physical parameters such as pH, aeration, illumination, temperature, spawn materials, and oat-sawdust-based substrate formulations. After a 7-day incubation period, oats with a pH of 6.0, under sealed and illuminated conditions at 32℃, demonstrated the most effective mycelial growth. Substrates consisting of 70% oat and 30% sawdust had the shortest incubation time of 30.5 days for fruiting body formation. The basidiospores showed a typical germination pattern where the sporidium produced a single germ tube that elongated, and branched to form monokaryotic primary mycelia. In conclusion, using oats as a substrate in the cultivation of C. militaris could reduce production costs and help protect the environment.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.12
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• pp.7523-7527
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• 2013

Thymidylate synthase (TS) catalyzes the transfer of a methyl group from methylenetetrahydrofolate to dUMP to form dTMP. It is a primary target in the chemotherapy of colorectal cancers and some other neoplasms. In order to obtain pure protein for analysis of structure and biological function, an expression vector TS-pET28b (+) was constructed by inserting wild-type human thymidylate synthase (hTS) cDNA into pET28b (+). Then an expression strain was selected after transformation of the recombined plasmid into Rosetta (DE3). Fusion protein with His-tag was efficiently expressed in the form of inclusion bodies after IPTG induction and the content was approximately 40.0% of total bacteria proteins after optimizing expression conditions. When inclusion bodies were washed, dissolved and purified by Ni-NTA under denatured conditions, the purity was up to 90%. On SDS-PAGE and West-blotting, the protein band was found to match well with the predicted relative molecular mass-36kDa. Bioactivity was 0.1 U/mg. The results indicated that high-level expression of wild-type hTS cDNA can be achieved in prokaryotes with our novel method, facilitating research into related chemotherapy.

• Korean Journal of Microbiology
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• v.45 no.3
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• pp.263-267
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• 2009

An RpoS factor is a transcriptional regulator which participates in numerous biological processes. In this work, we investigated the transcriptional regulation of proBA and proC composing proline biosynthetic pathway in Escherichia coli. While the proBA and proC genes were greatly induced in an exponential growth phase, they were dramatically repressed in a stationary growth phase in the wild type E. coli. Unlike the wild type E. coli, the proBA and proC genes were not repressed even in the stationary growth phase in its isogenic rpoS mutant. These results suggest that the RpoS factor acts as a transcriptional repressor of proBA and proC genes. The production of threonine, methionine, lysine, and arginine in the rpoS mutant were also increased by more than two times compared to its parental wild type, suggesting that the mutant is able to be used as an useful host strain for the amino acid overproduction.

• The Journal of the Korean Society for Microbiology
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• v.35 no.4
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• pp.273-282
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• 2000

dTDP-rhamnose provides L-rhamnose to the bridge-like structure between mycolyl arabinogalactan and peptidoglycan of the mycobacterial cell wall. dTDP-rhamnose is composed of glucose-l-phosphate and dTTP by four enzymes encoded by rmlA-D. To determine the region(s) of RmlC protein essential for its dTDP-4-keto-6-deoxyglucose epimerase activity, we overexpressed both whole (202 amino acids) and three different truncated (N-terminal 106 or 150 or C-terminal 97 amino acids) RmlC proteins of Mycobacterium tuberculosis. The RmlC enzyme activity in the soluble lysates of ${\Delta}rmlC$ E. coli strain $S{\Phi}874$ (DE3 PlysS) expressing the wild type or truncated rmlC genes was initially analyzed by three sequential reactions from dTDP-glucose to dTDP-rhamnose in the presence of purified RmlB and RmlD. All three soluble lysates containing the truncated RmlC proteins showed no enzyme activity, while that containing the wild type RmlC was active. This wild type RmlC was then overexpressed and purified. The incubation of the purified RmlC enzyme so obtained with dTDP-4-keto-6-deoxyglucose resulted in the conversion of dTDP-4-keto-rhamnose. The results show that the truncated regions of the RmlC protein are important for the RmlC enzyme activity in M. tuberculosis.

• IMMUNE NETWORK
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• v.16 no.2
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• pp.140-145
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• 2016

Ophiocordyceps sinensis is a natural fungus that has been valued as a health food and used in traditional Chinese medicine for centuries. The fungus is parasitic and colonizes insect larva. Naturally occurring O. sinensis thrives at high altitude in cold and grassy alpine meadows on the Himalayan mountain ranges. Wild Ophiocordyceps is becoming increasingly rare in its natural habitat, and its price limits its use in clinical practice. Therefore, the development of a standardized alternative is a great focus of research to allow the use of Ophiocordyceps as a medicine. To develop an alternative for wild Ophiocordyceps, a refined standardized extract, CBG-CS-2, was produced by artificial fermentation and extraction of the mycelial strain Paecilomyces hepiali CBG-CS-1, which originated from wild O. sinensis. In this study, we analyzed the in vitro immune-modulating effect of CBG-CS-2 on natural killer cells and B and T lymphocytes. CBG-CS-2 stimulated splenocyte proliferation and enhanced Th1-type cytokine expression in the mouse splenocytes. Importantly, in vitro CBG-CS-2 treatment enhanced the killing activity of the NK-92MI natural killer cell line. These results indicate that the mycelial culture extract prepared from Ophiocordyceps exhibits immune-modulating activity, as was observed in vivo and this suggests its possible use in the treatment of diseases caused by abnormal immune function.

• Korean Journal of Agricultural Science
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• v.50 no.4
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• pp.749-758
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• 2023

An understanding of safety problems pursuant to environmental release of GM (Genetically Modified) crops is considered important. Among the recognized safety problems, the possibilities of weediness and ecosystem invasion are constantly being validated. We herein compared the growth characteristics and germination rate of soybeans formed by hybridization with vitamin A-enhanced soybeans carrying an introduced gene that increases β-carotene content. We also examined overwintering, survival, and weed competitiveness to evaluate hybrid ecological impact on long-term unmanaged cultivatable land. These studies revealed that the hybrid soybeans exhibited intermediate growth characteristics and germination rate compared with the vitamin A-enhanced soybeans and wild soybeans, or exhibited traits similar to those of the maternal strain. Overwintering experiments were conducted by planting seeds at depths of 0, 5, 10, and 20 cm and recovering them after three or five months. After five months, all seeds at depths more than 5 cm lost viability. Among seeds recovered after three months, only wild soybeans retained viability at depths of more than 5 cm. Survival and weed competitiveness were assessed by sowing each type of seed and performing no irrigation, or pest or weed control. Quantitative assessment of numbers of individual soybean plants that appeared in the experimental plot revealed that all plants germinated after sowing, but only wild type plants survived overwintering. These studies suggest that both GM soybeans and hybrid soybeans cannot survive in uncultivated land even if they are released into the environment, which indicates less possibility of ecosystem invasion and weediness.

• Journal of Microbiology and Biotechnology
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• v.15 no.3
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• pp.609-615
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• 2005

The safety and immunogenicity of an attenuated recombinant Salmonella vaccine strain, Salmonella enterica serovar Typhimurium llaB, was assessed. This vaccine strain could survive in low pH condition, and its ability of intracellular survival did not differ from that of S. enterica serovar Typhimurium UK1, which is the wild-type of the vaccine strain. The mortality of the mice orally administered with the vaccine strain was $50\%$ at the dose of $10^7$ CFU. All mice administered with $10^5\;or\;10^3$ CFU of the vaccine strain survived for 3 days postinoculation (pi). However, all mice administered with more than $10^3$ CFU of the vaccine strain died within 3 days pi. To examine the protective effect of the vaccine strain, mice were orally immunized with $10^4\;and\;10^6$ CFU of the bacteria. Control mice were given with 0.5 ml of phosphate buffered saline (PBS). After 8 days, the mice were challenged with $10^9$ CFU of S. enterica serovar Typhimurium UK1, and mortality was examined for 5 days. The survival rates of the mice immunized with $10^4\;and\;10^6$ CFU of the vaccine strain were $60\%\;and\;80\%$, respectively, whereas all control mice died within 2 days after challenging. To investigate the immunogenicity of S. enterica serovar Typhimurium llaB, mice were orally immunized with $10^5\;or\;10^6$ CFU ml of the vaccine strain. Five mice of each group were sacrificed at 5 and 12 days after immunization, and results showed that immunization of the vaccine strain led to increases of IgG1, IgG2, and IgM titers against S. enterica serovar Typhimurium UK1 in mouse sera, cytokine expressions such as IL-2, IL-4, IL-6, and IL-10 in spleen, and the lymphocyte proliferation response to mitogens (concanavalin A or LPS) stimulation.

• Journal of Microbiology and Biotechnology
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• v.27 no.9
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• pp.1701-1710
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• 2017

Classical swine fever virus (CSFV) is the etiologic agent of classical swine fever, a highly contagious disease that causes significant economic losses to the swine industry. The lapinized C-strain, a widely used vaccine strain against CSFV, has low growth efficiency in cell culture, which limits the productivity in the vaccine industry. In this study, a recombinant virus derived from C-strain was constructed and subjected to continuous passaging in PK-15 cells with the goal of acquiring a high progeny virus yield. A cell-adapted virus variant, RecCpp80, had nearly 1,000-fold higher titer than its parent C-strain but lost the ability to induce fever in rabbits. Sequence analysis of cell-adapted RecC variants indicated that at least six nucleotide changes were fixed in RecCpp80. Further adaption of RecCpp80 variant in swine testicle cells led to a higher virus yield without additional mutations. Introduction of each of these residues into the wild-type RecC backbone showed that one mutation, M979R (T3310G), located in the C-terminal region of E2 might be closely related to the cell-adapted phenotype. Rabbit inoculation revealed that $RecCpp40_{+10}$ failed to induce fever in rabbits, whereas $RecCpp80_{+10}$ caused a fever response similar to the commercial C-strain vaccine. In conclusion, the C-strain can be adapted to cell culture by introducing specific mutations in its E2 protein. The mutations in RecCpp80 that led to the loss of fever response in rabbits require further investigation. Continuous passaging of the C-strain-based recombinant viruses in PK-15 cells could enhance its in vitro adaption. The non-synonymous mutations at 3310 and 3531 might play major roles in the enhanced capacity of general virus reproduction. Such findings may help design a modified C-strain for improved productivity of commercial vaccines at reduced production cost.