Glycosylation of free sterol by whole-cell bioconversion in E. coli

Jae Kyung Sohng,Jae Kyung Sohng 한국당과학회 2011 한국당과학회 학술대회 Vol.2011 No.1

Steryl glucosides play important roles in many physiological and biochemical process in organism such as the heat shock, enhancement of immunological system, etc. The alignment of a putative sterol glucosides isolated from S. tropica CNB-440 has been shown 34%, 42% and 57% in homology with the corresponding ones from Arabidopis thaliana, Avena sativa and Salinispora arenicola CNS-205, respectively. Engineered E. coli host-high level production of the UDP-glucose was used for whole-cell bioconversion of free sterol (cholesterol and β-sistosterol) and the production of glycosylated product was only detected with β-sitosterol as substrate.

Cloning, Sequencing and Expression of dTDP-D-Glucose 4,6-Dehydratase Gene from Streptomyces antibioticus Tu¨99, a Producer of Chlorothricin

Sohng, Jae Kyung,Yoo, Jin-cheol 朝鮮大學校 1997 藥學硏究誌 Vol.18 No.2

DNA fragments, homologous to the dTDP-Dgiucose 4,6-dehydratase gene, obtained from the genomic DNA of Streptomyces antibioticus Tu¨99,a producer of the unusual macrolide antibiotic chlorothricin, were cloned and aequenced. This dehydratase gene was designated as oxil. The coding region of the oxil gene is composed of 987 bp, and analysis of the DNA sequence data reveals sequences for the gene products of 329 amino acids(molecular weight of 36.037). The deduced amino acids are 59% identical to the StrE, dTDP-D-glucose 4,6-dehydratase from the Streptomycin pathway. The oxil's function was examined by expressing it in E. coil using the T7 RNA polymerase/promoter system(pRSET) to produce an active fusion protein including a his tag. This enzyme shows apecificity of substrate, specific only to dTDP-D-glucose.

Biosynthesis of Chlorothricin produced by Streptomyces antibioticus Tu99

Sohng, Jae Kyung 선문대학교 첨단과학기술연구소 1997 첨단과학기술연구소 논문집 Vol.2 No.-

This paper considers a security framework for geographic information System(GIS). The GIS is an information system for supporting fast decision associated spacial problems and the system has a role of infra structure of the information system. The security is also one of the major technology for information system. However, researches on secure GIS are presented little and this paper considers the secure GIS. This paper suggest a framework for the secure GIS based on derived requirements on the secure system. Analysis on security for a serial, parallel and hierarchical secure system is also added.

Production, Isolation and Biological Activity of Nargenicin from Nocardia sp. CS682

Sohng, Jae-Kyung,Yamaguchi, Tokutaro,Seong, Chi-Nam,Baik, Keun-Sik,Park, Seong-Chan,Lee, Hyo-Jeong,Jang, So-Young,Simkhada, Jaya Ram,Yoo, Jin-Cheol 대한약학회 2008 Archives of Pharmacal Research Vol.31 No.10

Culture broth of an actinomycete isolate, Nocardia sp. CS682 showed specifically higher antibacterial activity against methicilin resistant Staphylococcus aureus (MRSA). Purified substance from the organism, CS-682, which is active against MRSA and Micrococcus leuteus, is a $C_{28}H_{37}NO_8$ ($M+H^+$, observed: 516.83) and identified as an unusual macrolide antibiotic, nargenicin. The chemical structure of CS-682 was identified by FT-IR, $^1H$-NMR, $^{13}C$-NMR, and ($^1H-^1H$ and $^1H-^{13}H$) COSY. The anti-MRSA activity of CS-682 was stronger than that of oxacillin, vancomycin, monensin, erythromycin, and spiramycin. Phylogenetic analysis showed that strain CS682 is closely related to Nocardia tenerifensis DSM $44704^T$ (98.7% sequence similarity), followed by N. brasiliensis ATCC $19296^T$ (98.4% sequence similarity). The ability of Nocardia sp. CS682 to produce nargenicin was unique.

Mechanism Study of dTDP-D-Glucose 4,6-Dehydratase: General Base in Active Site Domain

Sohng, Jae-Kyung,Noh, Hyung-Rae,Yoo, Jin-Cheol Korean Society for Biochemistry and Molecular Biol 1999 Journal of biochemistry and molecular biology Vol.32 No.4

dTDP-D-glucose 4,6-dehydratase as an oxidoreductase catalyzes the conversion of dTDP-D-glucose to dTDP-4-keto-6-deoxy-D-glucose, which is essential for the formation of 6-deoxysugars. dTDP-D-glucose 4,6-dehydratase shows remarkable sterochemical convergence in which displacement of the C-6 hydroxyl group by a C-4 hydrogen proceeds intramolecularly with inversion of configuration. The reaction mechanism is known to be oxidation, dehydration, and reduction by bases mediating proton transfer and $NAD^+$ cofactor. In this study, the bases in the active site domain are proposed to be His-79 and His-300 from a comparison of the peptides of the dehydratase and UDP-D-glucose epimerase. His-79 and His-300 were mutated to prepare the mutants H79L (mutation of histidine to leucine at the 79th amino acid) and H300A (mutation of histidine to alanine at the 300th amino acid) by site-directed mutagenesis. The H79L protein was inactive, showing that His-79 participates in the reaction mechanism.

Method for Cloning Biosynthetic Genes of Secondary Metabolites Including Deoxysugar from Actinomycetes

Sohng, Jane-Kyung,Oh, Tae-Jin,Kim, Chun-Gyu The Korea Science and Technology Center 1998 BMB Reports Vol.31 No.5

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 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.

Cloning, Sequencing and Expression of dTDP-D-Glucose 4,6-Dehydratase Gene from Streptomyces antibioticus $T\ddot{u}99$, a Producer of Chlorothricin

Sohng, Jae-Kyung,Yoo, Jin-Cheol Korean Society for Biochemistry and Molecular Biol 1996 Journal of biochemistry and molecular biology Vol.29 No.3

DNA fragments, homologous to the dTDP-D-glucose 4,6-dehydratase gene, obtained from the genomic DNA of Streptomyces antibioticus $T\ddot{u}99$, a producer of the unusual macrolide antibiotic chlorothricin, were cloned and sequenced. This dehydratase gene was designated as oxil. The coding region of the oxil gene is composed of 987 bp, and analysis of the DNA sequence data reveals sequences for the gene products of 329 amino acids (molecular weight of 36,037). The deduced amino acids are 59% identical to the StrE, dTDP-D-glucose 4,6-dehydratase from the streptomycin pathway. The oxil's function was examined by expressing it in E. coli using the T7 RNA polymerase/promoter system (pRSET) to produce an active fusion protein including a his tag. This enzyme shows specificity of substrate, specific only to dTDP-D-glucose.

Method for Cloning Biosynthetic Genes of Secondary Metabolites Including Deoxysugar from Actinomycetes

Sohng, Jae-Kyung,Oh, Tae-Jin,Kim, Chun-Gyu Korean Society for Biochemistry and Molecular Biol 1998 Journal of biochemistry and molecular biology Vol.31 No.5

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.

Gene Cloning and Analysis of Biosynthetic Gene of Spectinomycin

Sohng, Jae-Kyung,Oh, Tae-Jin,Liou, Kwang-Kyoung,Lee, Hei-Chan 선문대학교 첨단과학기술연구소 2001 첨단과학기술연구소 논문집 Vol.5 No.-

A biosynthetic gene cluster involved in the production of spectinomyein was cloned from Streptomyes spectabilis cosmid library with dTDP-glucose 4,6-dehydratase and amnino transferase gene probes. The DNA probes were obtained from S. speciabilis genome with primers based on consensus sequences of known enzymes using a polymerase chain reaction. One of the cloned cosmids, pSPM8, contained the homologue of the dTDP-glucose 4,6-dehydratase and amino transferase gene. The DNA sequence analysis reveals four open reading frames and one incomplete reading frame. The putative orf1 protein reveals 29% amino acid sequence homology of putative myo-inositol-2-dehydrogenase from Vibrio anguillamm. The orf2 is about 46% homologous to putative myo-inositol-1-monophosphotase of Mycobacterium leprae. The orf3 showed strong homology (67.4%) to putative export protein (spcT) from s. flavopersicus. The amino acid sequel deduced from orf4 showed a high similarity (70.8%) to a spectinomycin resistance gene (spcN) from S. flavopersicus. Expression of orf4 in E. coli BL21 showed the spectinomycin resistance.

Mechanism Study of dTDP-D-Glucose 4,6-Dehydratase: General Base in Active Site Domain

Sohng, Jae-Kyung,Noh, Hyung-Rae,Yoo, Jin-Cheol The Korea Science and Technology Center 1999 BMB Reports Vol.32 No.4

dTDP-D-glucose 4,6-dehydratase as an oxidoreductase catalyzes the conversion of dTDP-D-glucose to dTDP-4-keto-6-deoxy-D-glucose, which is essential for the formation of 6-deoxysugars. dTDP-D-glucose 4,6-dehydratase shows remarkable stereochemical convergence in which displacement of the C-6 hydroxyl group by a C-4 hydrogen proceeds intramolecularly with inversion of configuration. The reaction mechanism is known to be oxidation, dehydration, and reduction by bases mediating proton transfer and NAD? cofactor. In this study, the bases in the active site domain are proposed to be His-79 and His-300 from a comparison of the peptides of the dehydratase and UDP-D-glucose epimerase. His-79 and His-300 were mutated to prepare the mutants H79L (mutation of histidine to leucine at the 79th amino acid) and H300A (mutation of histidine to alanine at the 300th amino acid) by site-directed mutagenesis. The H79L protein was inactive, showing that His-79 participates in the reaction mechanism.