• BMB Reports
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• v.31 no.5
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• pp.475-483
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• 1998

Many antibiotics contain partially deoxygenated sugar components that are usually essential for biological activity, affinity, structural stability, and solubility of antibiotics. Gene probes of the biosynthetic genes related with the deoxysugar were obtained from PCR. Primers were designed from the conserved peptide sequences of the known dTDP-D-glucose 4,6-dehydratases, which are the key step enzymes in the biosynthesis of deoxysugar. The primers were applied to amplify parts of dehydratase genes to 27 actinomycetes that produce the metabolites containing deoxysugar as structural constituents. About 180 and 340 bp DNA fragments from all of the actinomycetes were produced by PCR and analyzed by Southern blot and DNA sequencing. The PCR products were used as gene probes to clone the biosynthetic gene clusters for the antibiotic mithramycin, rubradirin, spectinomycin, and elaiophyrin. This method should allow for detecting of the biosynthetic gene clusters of a vast array of secondary metabolites isolated from actinomycetes because of the widespread existence of deoxysugar constituents in secondary metabolites.

• Journal of Microbiology and Biotechnology
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• v.20 no.3
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• pp.480-484
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• 2010

AfsR2 is a global regulatory protein that stimulates antibiotic biosynthesis in both Streptomyces lividans and S. coelicolor. Previously, various afsR2-dependent genes including a putative abaA-like regulatory gene, SCO4677, were identified through comparative DNA microarray analysis. To further identify the putative SCO4677-dependent proteins, the comparative proteomics-driven approach was applied to the SCO4677-overexpressing strains of S. lividans and S. coelicolor along with the wild-type strains. The 2D gel electrophoresis gave approximately 277 protein spots for S. lividans and 207 protein spots for S. coelicolor, showing different protein expression patterns between the SCO4677-overexpressing strains and the wild-type strains. Further MALDI-TOF analysis revealed that only 18 proteins exhibited similar expression patterns in both S. lividans and S. coelicolor, suggesting that the SCO4677 could encode an abaA-like regulator that controls a few cross-species common proteins as well as many species-specific proteins in Streptomyces species.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 2005.06a
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• pp.107-109
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• 2005

• Journal of Microbiology and Biotechnology
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• v.13 no.4
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• pp.564-570
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• 2003

The antibacterial mechanism of enterobacter Salmonella typhimurium was studied. The rfa (Waa) gene cluster of S. typhimurium encodes the core oligosaccharide biosynthesis of lipopolysaccharide (LPS). Among the rfa gene cluster, we recently cloned the rfaE gene, which is involved in ADP-L-glycero-D-manno-heptose biosynthesis. The rfaE mutant synthesizes heptose-deficient LPS, which consists of only lipid A and 3-deoxy-D-manno-octulosonic acid (KDO), thus making an incomplete LPS and a rough phenotype mutant. S. typhimurium deep-rough mutants with the heptose region of the inner core show a reduced growth rate, sensitivity to high temperature, and hypersensitivity to hydrophobic antibiotics such as baicalin isolated from the medicinal herb of Scutellaria baicalensis Georgi. Thus, in this study, the cloned rfaE gene was added to the S. typhimurium rfaE mutant strain SL1102 (rfaE543), which makes heptose-deficient LPS and has a deep-rough phenotype. The complementation created a smooth phenotype in the SL1102 strain. The sensitivity of SL1102 to bacteriophages was also recovered to that of wild-type strain, indicating that LPS is used as the receptor for bacteriophage infection. The permeability barrier of SL1102 to hydrophobic antibiotics such as novobiocin and baicalin was restored to that of the wild-type, suggesting that antibiotic resistance of the wild-type strain is highly correlated with their LPS. Through an agar diffusion assay, the growth-inhibition activity of baicalin was fully observed in the mutant SL1102 strain. However, only a half of the inhibitory activity was detected in the rfaE complemented SL1102 strain. Furthermore, the LPS produced by the rfaE-complemented SL1102 strain was indistinguishable from LPS biosynthesis of smooth strains.

• KSBB Journal
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• v.24 no.3
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• pp.259-266
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• 2009

AfsR2 in Streptomyces lividans, a 63-amino acid protein with limited sequence homology to Streptomyces sigma factors, has been known for a global regulatory protein stimulating multiple antibiotic biosynthetic pathways. Although the detailed regulatory mechanism of AfsK-AfsR-AfsR2 system has been well characterized, very little information about the AfsR2-dependent down-stream regulatory genes were characterized. Recently, the null mutant of afsS in S. coelicolor (the identical ortholog of afsR2) has been characterized through DNA microarray system, revealing that afsS deletion regulated several genes involved in antibiotic biosynthesis as well as phosphate-starvation. Through comparative DNA microarray analysis of afsR2-overexpressed S. lividans, here we also identify several afsR2-dependent genes involved in phosphate starvation, morphological differentiation, and antibiotic regulation in S. lividans, confirming that the AfsR2 plays an important pleiotrophic regulatory role in Streptomyces species.

• Archives of Pharmacal Research
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• v.3 no.1
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• pp.7-12
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• 1980

Ampliciline was synthesized from 6-amino-pencillanic acid (6-APA) and D-.alpha. phenylglycine methyl ester by using amplicilin synthesizing enzyme from Peudomonas melanogenum (IAM 1655). The whole cell enzyme was immobilized by entrapping it in the polyacrylamide gel lattices. The polymer used in the enzyme entrapment was made from 150 mg per ml of acrylamide monomer and 8 mg per ml of N, N'-methylenebisacrylamide. About 200 mg/whole cell enzyme was mixed in the polymer for entrapment. The maximal activity retention after immobilization was 56%. The optimal pH values for the whole cell enzyme and the immobilized whole cell enzyme were 6.0 and 5.9, respectively. The optimal temperature for the enzyme activity were the same for both type of preparations. The enzyme stabilities against pH and heat increased for immobilized whole cell enzyme. Immobilized cell was more stable especially in the acidic condition while both type were found to be very suceptible to thermal inactivation at a temperature above 4.deg.C. The kinetic constants obtained from Lineweaver-Burk plot based on two substate reaction mechanism showed somewhat higher value for immobilized whole cell enzyme as compared to the whole cell enzyme : the Km value for 6-APA were 7.0 mM and 12.5 mM while Km values for phenylglycine methyl ester were 4.5 mM and 8.2 mM, respectively. Using the immobilized whole cell enzyme packed in a column reactor, the productivity of ampiciline was studied by varying the flow rate of substrate solution. At the space velocity, SV, 0.14 hr$^{-1}$ the conversion was 45%. Operational stability found in terms of half life was 30 hr at SV = 0.2 hr.

• Korean Journal of Microbiology
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• v.53 no.4
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• pp.329-331
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• 2017

Bacillus velezensis YC7010 is an endophytic bacterium isolated from the rice rhizosphere in Jinju, Republic of Korea, with properties conductive to growth promotion, antibiosis and induced systemic resistance to significant, soil-borne rice fungal and bacterial pathogens. The genome of B. velezensis YC7010 comprises a 3,975,683 bp circular chromosome which consists of 3,790 protein-coding genes (86tRNA and 27rRNA genes). Based on genomic analysis, we identified genes involved in colonization and establishment inside the plant, biosynthesis of antibiotic compounds such as surfactin, plipapastatin, bacillibactin, and bacillaene, as well as the production of the phytohormones and volatile compounds which serve to promote the plants growth and development.

• 한국생물공학회:학술대회논문집
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• 2005.04a
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• pp.420-422
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• 2005

TMC (Tautomycetin) is a liner polyketide immunosuppressive antifungal compound produced by Streptomyces spp. Inhibition of T cell proliferation with TMC was observed highly efficient at 100-fold lower than those needed to achieve maximal inhibition with cyclosporin A. To elucidate the biosynthetic pathway of TMC, a genomic DNA library was constructed using a E. coil-Streptomyces shuttle cosmid vector, pOJ446. The DNA libraries were screened by colony blot hybridization using several polyketide ${\beta}-ketosynthase$ (KS) probes amplified from TMC-producing Streptomyces genomic DNA using polymerase chain reaction (PCR), of which the degenerate primers were designed based on the highly conserved sequences present in KS domains of various type I polyketide synthase genes in Streptomyces species. This library construction and screening approach led to the isolation of several positive cosmid clones representing type I polyketide biosynthetic gene clusters. In addition, a Streptomyces regulatory gene called afsR2 (a global regulatory gene stimulating antibiotic production in both S. coelicolor and S. lividans) was successfully integrated into the TMC-producing Streptomyces chromosome via E. coil-Streptomyces heterologous conjugation mehtod. The more detailed results of production improvement and genetic characterization of TMC-producing Streptomyces spp. will be discussed.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 2003.10a
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• pp.109.1-109
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• 2003

From the PMMV (pepper mild mottle virus)-inducible ESTs differentially expressed in Capsicum chinense PI257284, we isolated a full-length cDNA (CcPHZF： Capsicum chinense phenazine), encoding a phenazine biosynthesis protein which catalyzes the hydroxylation of phenozine-1-carboxylic acid to 2-hydroxyphenazine-1-carboxylic acid. Phenazine compound has been known to exhibit broad-spectrum of antibiotic activity against various species of bacteria and fungus. The entire region of CcPHZF is 879 bp in length and the open reading frame predicted a polypeptide of 292 amino acids. The homolog of CcPHZF is not Present in database except clones of AC004044 and NM100203 from Arabidopsis with 58 and 59％, respectively. Genomic Southern analysis indicated that the pepper genome contains a single copy of CcPHZF. The CcPHZF was strongly induced in the pepper leaves 3 days after PMMV treatment, when HR occurs on the leaf surface. Characterization of CcPHZF is underway to investigate if the CcPHZF is related to disease resistance against pathogens.

• Proceedings of the Microbiological Society of Korea Conference
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• 2001.11a
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• pp.146-156
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• 2001

The biosynthetic gene clusters for bluensomycin and spectinomycin were isolated and characterized from the bluensomycin producer, Streptomyces bluensis ATCC27420 and the spectinomycin producer, Streptomyces spectabilis ATCC27741, respectively. PCR primers were designed specifically to amplify a segment of dTDP-glucose synthase gene based on its conserved sequences of several actinomycete strains. By screening cosmid libraries using amplified PCR fragments, 30-kb and 45-kb DNA fragments were isolated from Streptomyces bluensis and Streptomyces spectabilis, respectively. Sequencing analysis of them revealed that each contains 15 open reading frames (ORFs). Some of these ORFs were turned out to be antibiotic resistance genes (blmA and speN), dTDP-glucose synthase genes (blmD and spcD), and dTDP-D-glucose 4,6-dehydratase genes (blmE and spcE), suggesting that the blm and spec gene clusters are likely involved in the biosynthesis of bluensomycin and spectinomycin, respectively.