• Journal of Microbiology and Biotechnology
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• v.32 no.7
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• pp.911-917
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• 2022

As valuable antibiotics, microbial natural products have been in use for decades in various fields. Among them are polyene compounds including nystatin, amphotericin, and nystatin-like Pseudonocardia polyenes (NPPs). Polyene macrolides are known to possess various biological effects, such as antifungal and antiviral activities. NPP A1, which is produced by Pseudonocardia autotrophica, contains a unique disaccharide moiety in the tetraene macrolide backbone. NPP B1, with a heptane structure and improved antifungal activity, was then developed via genetic manipulation of the NPP A1 biosynthetic gene cluster (BGC). Here, we generated a Streptomyces artificial chromosomal DNA library to isolate a large-sized NPP B1 BGC. The NPP B1 BGC was successfully isolated from P. autotrophica chromosome through the construction and screening of a bacterial artificial chromosome (BAC) library, even though the isolated 140-kb BAC clone (named pNPPB1s) lacked approximately 8 kb of the right-end portion of the NPP B1 BGC. The additional introduction of the pNPPB1s as well as co-expression of the 32-kb portion including the missing 8 kb led to a 7.3-fold increase in the production level of NPP B1 in P. autotrophica. The qRT-PCR confirmed that the transcription level of NPP B1 BGC was significantly increased in the P. autotrophica strain containing two copies of the NPP B1 BGCs. Interestingly, the NPP B1 exhibited a previously unidentified SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) inhibition activity in vitro. These results suggest that the Streptomyces BAC cloning of a large-sized, natural product BGC is a valuable approach for titer improvement and biological activity screening of natural products in actinomycetes.

• Proceedings of the Korean Society of Soil and Groundwater Environment Conference
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• 2003.04a
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• pp.340-343
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• 2003

Soils contaminated by hazardous hydrophobic organic compounds, such as polycyclic aromatic hydrocarbons (PAHs), have become a major environmental issue due to toxic and carcinogenic properties of those compounds. In this work, we investigated effects of various metabolic inducers on phenanthrene biodegradation. Biodegradation tests were peformed with two different Pseudomonads: Pseudononrdia hydrocarboxydans (Gram positive) and Pseudomonas putida (Gram negative). Intermediates of phenanthrene metabolism (1-hydroxy-2-naphthoate, salicylate, catechol, phthalate and protocatechuate) were selected as inducers. The tests indicated that 1-hydroxy-2-naphthoate was the most effective inducer and enhanced the phenanthrene degradation rate up to 5.7 times, even though all the others also had induction ability to some extent. The effective induction could be achieved even at a low concentration of 1-hydroxy-2-naphthoate. Addition of metabolic inducers would be an attractive trick for the successful bioremediation of PAH-contaminated soil.

• Korean Journal of Microbiology
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• v.49 no.2
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• pp.184-190
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• 2013

Cave environment provides special ecosystems for evolution of lives distant from surface environments. We investigated bacterial and archaeal communities of wall biofilm obtained from of a volcanic cave (Daesubee) in Jeju, Republic of Korea. Bacterial and archaeal 16S rRNA genes were PCR-amplified and sequenced using pyrosequencing technologies. Unique prokaryotic communities with low diversities were observed. The main bacterial sequences (ca. 83% of total reads) were affiliated with Pseudonocardia mongoliensis of phylum Actinobacteria and clustered with clones obtained from various caves. Reflection of light on the wall surface of cave might be caused by formation of beads of water caused by hydrophobic filaments of actinobacterial colonies. Main archaeal sequences (ca. 65.7% of total reads) were related with those of I.1a-Associated group of phylum Thaumarchaeota. The sequences were related with that of Candidatus Nitrosotalea devanaterra which was known to oxidize ammonia under acidic condition (ca. pH 5.0). Nutrients leached through volcanic soils contribute formation of unique microbial communities of wall biofilm of cave Daesubee.

• Korean Journal of Clinical Laboratory Science
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• v.48 no.3
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• pp.230-236
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• 2016

Economic growth has increased the living standards around the world. Water pollution, in particular, is a public relations issue because it poses a direct threat to everyone's lives. As of recently, the production of taste and odor (T&O) compounds has been a common problem in the water industry. The adsorption process using granular activated carbon (GAC) has been the most widely used process. The objectives of this study were to evaluate the microorganisms before and after the backwashing of GAC and to identify the species of the microorganisms found. Five dominants microorganisms were confirmed after the microfiltration process from backwashing of GAC, and the dominant bacterial species were found to be ${\beta}$-proteobacterium species, Porphyrobacter donghaensis, Polaromonas rhizophaerae, Hydrogenophaga species, and Pseudonocardia species. However, when UV treatment after microfiltration was performed, Hydrogenophaga species and Psedonocardia species were eliminated. Herein, I conclude that the UV treatment post microfiltration process is more efficient than microfiltration process alone. The findings of this study may provide useful information regarding the management of microfiltration process.

• Journal of Microbiology and Biotechnology
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• v.15 no.1
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• pp.188-191
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• 2005

Abstract Cyclosporins are a family of clinically-important immunosuppressive cyclic peptides produced by Tolypocladium inflatum. The structural modification of cyclosporins via hydroxylation at various positions of N-methyl leucines in cyclosporin A leads to a dramatic change of their bioactive spectra. Among over 100 soil actinomycetes screened, two actinomycetes species, Sebekia benihana and Pseudonocardia autotrophica, were identified to contain superior cyclosporin A hydroxylation activities. A HPLC-based cyclosporin A hydroxylation assay revealed that each strain possesses distinctive hydroxylation specificity and regiospecificity; mono-hydroxylation at the 4th N-methyl leucine of cyclosporin A by S. benihana, and di-hydroxylations at both 4th and 9th N-methyl leucines of cyclosporin A by P. autotrophica. The conversion yields for cyclosporin A hydroxylation by both S. benihana and P. autotrophica were significantly improved from less than 10% and 18% up to 58% and 45%, respectively, in the optimized culture containing molybdenum with 0.05 g/l of cyclosporin A concentration. An ancymidol-specific inhibition of cyclosporin hydroxylation also suggested that the regiospecific cyclosporin hydroxylation might be catalyzed by a putative cytochrome P450 mono-oxygenase enzyme.

• Journal of Microbiology and Biotechnology
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• v.25 no.10
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• pp.1634-1639
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• 2015

Cytochrome P450 hydroxylase (CYP) in actinomycetes plays an important role in the biosynthesis and bioconversion of various secondary metabolites. Two unique CYPs named CYP-sb21 and CYP-pa1, which were identified from Sebekia benihana and Pseudonocardia autotrophica, respectively, were proven to transfer a hydroxyl group at the 4^th or 9^th N-methyl leucine position of immunosuppressive agent cyclosporin A (CsA). Interestingly, these two homologous CYPs showed different CsA regio-selectivities. CYP-sb21 exhibited preferential hydroxylation activity at the 4^th position over the 9^th position, whereas CYP-pa1 showed the opposite preference. To narrow down the CYP domain critical for CsA regio-selectivity, each CYP was divided into four domains, and each domain was swapped with its counterpart from the other CYP. A total of 18 hybrid CYPs were then individually tested for CsA regio-selectivity. Although most of the hybrid CYPs failed to exhibit a significant change in regio-selectivity in the context of CsA hydroxylation, hybrid CYP-pa1 swapped with the second domain of CYP-sb21 showed a higher preference for the 9^th position. Moreover, hybrid CYPsb21 containing seven amino acids from the 2nd domain of CYP-pa1 showed higher preference for the 4^th position. These results imply that the 2nd domain of CsA-specific CYP plays a critical role in CsA regio-selectivity, thereby setting the stage for biotechnological application of CsA regio-selective hydroxylation.

• KSBB Journal
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• v.22 no.4
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• pp.218-221
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• 2007

The polyene antifungal antibiotics, mostly produced by Gram-positive soil actinomycetes, are a family of type I polyketide macrolide ring compounds with 20$\sim$40 carbon backbone contain 3$\sim$8 conjugated double bonds. Using polyene-specific genomic screening strategy, we previously isolated three novel polyene-producing actinomycetes strains from soil, implying the potential application of these strains' spores as microbial pesticides. Here, we report that the sonication is a very efficient method for actinomycetes spore generation with a sonicator power-dependent manner. In addition, these sonication-driven actinomycetes spores retained significant portion of their cell viabilities as well as antifungal activities after freeze-drying procedure, implying the potential application of these strains' spores as microbial pesticides.

• Food Science of Animal Resources
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• v.28 no.4
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• pp.457-462
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• 2008

Lactococcus lactis NK34, isolated from jeotgal (Korean traditional fermented fish), produces bacteriocin against bovine mastitis pathogens such as Staphylococcus aureus 7, S. aureus 8, Staphylococcus chromogenes 10, S. chromogenes 19, Staphylococcus hominis 9, Streptococcus uberis E290, Enterococcus faecium E372, Streptococcus agalactiae ATCC 13813, Pseudonocardia autotrophia KCTC 9455, and Staphylococcus simulans 78. Lacticin NK34 was inactivated by protease XIV but not by protease IX, protease XIII, proteinase K, $\acute{a}$-chymotrypsin, trypsin, and pepsin. Also, lacticin NK34 was stable over a pH range of 2 to 9 for 4 hr and withstood exposure to temperatures of 30-$100^{\circ}C$ for 30 min. Lacticin NK34 showed bactericidal effects against S. simulans 78. This bacteriocin was purified using ammonium sulfate precipitation, ion exchange chromatography, ultrafiltration, and hydrophobic chromatography. Tricin-SDS-PAGE of purified bacteriocin gave the same molecular weight (3.5 kDa) as nisin. The gene encoding this bacteriocin was amplified by PCR using nisin gene-specific primers. It showed similar sequences to this nisin Z gene. These results indicate that lacticin NK34 is a nisin-like bacteriocin, and could be used as an antimicrobial alternative for livestock.

• Microbiology and Biotechnology Letters
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• v.30 no.1
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• pp.79-85
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• 2002

Ecological characteristics of microorganisms in tideland soils were studied by investigation of microbial diversity and population. Twenty soil samples were taken at surface, 10, 20 and 30 cm depth each. Bacteria, actinomycetes and fungi were isolated on each selective isolation medium containing different concentration of NaCl. Actinomycetes were the most isolated from soil samples taken at 10 cm depth and isolated by humic acid-vitamin (HV) medium without sea water or salt. Twenty nine strains of actinomycetes were isolated at surface soil and 74, 39, 37 strains were at 10, 20, and 30 cm depth, respectively. All these isolates were analysed and grouped by random amplified polymorphic DNA (RAPD)-PCR analysis. Many of the isolates were clustered into Microtetraspora and Pseudonocardia. Fungal isolates were highly distributed at the surface soil and isolated well on potato dextrose agar (PDA) medium with sea water. Bacterial isolates were higly distributed at surface soil and isolated well by nutrient medium without sea water or salt. Soil samples taken at 10 cm depth showed the highest microbial diversity and population.